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AMEN-RA’S

AGE INHIBITION REGIMEN (AIR)

AN EXPOSITION ON THE EXPERIMENTAL, THEORETICAL & PRACTICAL


UNDERPINNINGS OF LIFESPAN EXTENTION
& OPTIMAL HEALTH PROMOTION,
A DISCOURSE ON THE METHOD OF GEROSTASIS

VOLUME II
Tractatus I-A of X (Glycation & AGEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED SUGAR

Dr. Nūn Sava-Siva Amen-Ra, Dr.PH, MA, MSW


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AMENTA ARCHIVES
© MMXVII
Gettysburg (Orrtanna), Pennsylvania USA

Bw-Ka-Nun (Buchanan) Valley

ISBN 0-9741469-3-5

Correspondence concerning this publication is accepted at the following address:

Amen-Ra@AmentaEliteAthlete.com

Copies of this publication may be procured at the following website:

www.AmentaEliteAthlete.com

DYSTOPIAN DISCLAIMER: This Treatise does not constitute an attempt to dispense


medical advice. Rather, it is intended to inform individuals devoid of disease how they can
best insure freedom from infirmity and increase their odds of attaining exceptional longevity
and vitality. Information is offered and arguments are advanced in support of logically
coherent claims contained herein. Rational agents are hereby advised to rely principally on
their own reasoning faculties to critically evaluate the veracity of the Author’s statements.
The Protocol presented in the pages of this book is admittedly austere. As such, it will not
appeal to the masses of mankind nor ought it to be attempted by anyone deficient in
discipline and discernment—such efforts are bound to be abortive. There is risk inherent in
anything remotely rigorous and this Regimen is no exception to the rule. If, however, the
attested benefits of the Amen Protocol are substantive, the risk is arguably outweighed
thereby.

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CONTENTS

VOLUME II

Tractatus I-A of X (Glycation & AGEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED SUGAR

Starvation Slows Senescence: The Function of the Fasting Hormone, FGF21

Glucose, Glycation & Protein Pathomorphosis

Glucose, Glycation & DNA Damage

The Glory of Glyoxalase: Guarantor of AGE Attenuation

Defying the ‘Dawn’: ‘Amenistic Entrainment’, Exercise, and Elements of the Amen
Apothecary Attenuate Innate, Ordinary Elevations of Glucose Concentration Commonly
Concordant with Circadian Control

Excessive Circulating Sugar ‘Short-Circuits’ Circadian Cyclicity, Confounds Optimal


Physiological Order, Accelerates Aging, and Compromises the Recuperative Quality of
Sleep:
AIR Opposes these Inimical Effects most Markedly via the Amen Fast

Tea & Other Herbal Infusions Imbibed During the Amen Fast Forestall Glycation and
Accordingly Conduce to Lifespan Extension

Human Longevity is Linked to Lower Levels of Glucose

Metformin Mitigates Glycation and Promotes Lifespan Prolongation

Meli (Honey), Metformin, & Other Anti-Diabetic Agents Mitigate Hyperglycemia & Thereby
Promote Lifespan Prolongation

Metformin May Modulate Lifespan Via an Intermediary Endocrine Hormone, FGF21

Amenistic Mimetics: Molecules that Mimic the Fasting State by Suppressing Glycemia
Consequently Lengthen Lifespan:
Especial Emphasis on the Anti-Aging Agents Chromium & Glucosamine

Insulin: Perils Potentiated by a Potent Pancreatic Product and its Pernicious Partners

Telomeres Tellingly Correlate with Human Longevity and Levels of Circulating Sugar

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________________________________

Tractatus I-B of X (Carbamylation & ACEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED UREA

Carbamylation Increases with Age and Accelerates Cellular Senescence

____________________________

Tractatus I-C of X (Carbonylation & AOEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED OXIDANTS

Carbonylation Increases with Age and Aggravates Protein Aggregation

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AIR VOLUME II
ESSENTIALS OF THE AMEN FAST

Tractatus I-A of X (Glycation & AGEs)

___________________________________________________

“Whenever you fast, do not put on a gloomy face as the hypocrites do, for they
neglect their appearance so that they will be noticed by men when they are fasting.
Truly I say to you, they have their reward in full. But you, when you fast, anoint
your head and wash your face so that your fasting will not be noticed by men, but
by your Father who is in secret; and your Father who sees what is done in secret will
reward you.”

–Lord Jesus (Matthew XII: 16-18)

‘Alas, Ascetic Amenites, the fruits of your fasting shall be slowed senescence,
physical fortitude, psycho-spiritual strength, and mental mastery—I would not tell
you so (nor should you trust my testimony) if I did not manifest these observable
effects myself.’

–Lord Amen-Ra (AIR II:I-A)

______________________________________________________

In this Volume we shall present the scientific and philosophic rationale for what is
undoubtedly the most integral, most fundamental feature of our Regimen—the Amen Fast.
Indeed, the Amen Regimen can rightly be conceived as a perpetual fast punctuated by a brief
daily repast. As the Feeding Phase of the Amen Diet spans a mere hour, the Amen Fast is
accordingly twenty-three hours in duration. Thus, in a week’s time, only seven hours are spent
feeding whilst one hundred sixty-one hours are spent fasting. Stated differently, less than five
percent of the Amen Adherent’s time is spent actively engaged in eating, leaving ninety-five
percent of the aspiring Ascetic’s life left for more prudent, more pensive, perhaps more
pious pursuits. What is more, the customary, twice-daily Exercise Sessions are executed at
the waxing and waning periods of the waking Fast—the Persistence Session in the morning
at roughly the 10th hour and the Resistance Session in the evening at roughly the 20th hour of
abstention. Though we shall have occasion to expound on this in Volume IV, some
preliminary points bear mention presently. To engage in persistence (i.e. aerobic) exercise
after a nocturnal fast depletes the body (specifically the striated muscles and the liver) of
glycogen. For the next twenty hours the Amenite’s sole source of energy originates from
within his or her own body. Then, after a Fast of twenty hours or more, the Anchorite
submits to a bout of rigorous resistance exercises that he or she sustains for upwards of an

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hour or more. The final phase of physical exercise is followed by a phase of ‘metaphysical
exercise’ entailing standing meditation—‘Arew Aha’I in our esoteric idiom.II Only after this
display of discipline does the Adherent deign to dine. Admittedly, the Architekton is inured
to the psychophysical severity of his System after over a decade and a half of dutiful practice.
This apparently immodest admission serves only to illustrate, however, its feasibility as a
sustainable modus vivendi.III The Reader and Incipient Adherent, it is hoped, will derive the
requisite fortitude to adopt the Regimen from a thorough understanding of its scientific and
philosophic foundations and its unsurpassed potential to promote the prolongation of life
and the optimization of health—both physical and mental.

Starvation Slows Senescence: The Function of the Fasting Hormone, FGF21

This section starts with the startling significate “starvation”. Should Amenites accept such a
forbidding descriptor for our defining Fast? It depends decidedly on one’s degree of self-
discipline and the psycho-physical severity that one associates with our System. Neophyte
Initiates to the Amen Regimen may well consider a 23-hour fast synonymous with starvation
while inveterate Adherents who have practiced the Protocol for many years may merely
regard it as rather restrictive. Some (such as the Author) who have endured this Ascetic
intervention for over a decade and a half positively embrace the vacuity and vitality it
invariably engenders. However one conceives of it, it is admittedly austere. Quite probably, no
other Order, organization, or indeed, individual, has ever voluntarily adopted and consistently
practiced a protocol as abstemious, as ascetic, as rigorous as our Regimen. Moreover, if it is
accurate to adjudge the Amen Fast as amounting to starvation, it is scientifically sound
starvation, as we shall soon see.
It is with unconcealed eagerness that we commence our analysis with one of the
most momentous indications of the scientific soundness of the structure of the Amen Fast.
This welcomed scientific service is rendered by a study published in the aptly named journal
eLIFE. Preliminarily, it should be explained that fasting induces multiple molecular
alterations in organisms, enabling them to adapt metabolically to prolonged cessation of
sustenance. Most of these alterations impact adipose and skeletal muscle in such ways as to
effectuate the exploitation of energy embedded in these tissues. Individual organs are also
important in this respect, such as the ghrelin-giving gut and the insulin-exuding pancreas.
Apart from the hypothalamus, hidden in the deep recesses of the brain and exerting its

I Irw aha [Phonetically Arew Aha] (Ancient Egyptian, “Standing Form”)


II For extended sessions of meditation, Amenites oftener assume the recumbent posture of Irw Sdjr

[Phonetically Arew Sedjer, “Lying Form”, rendered hieroglyphically as . Less oftener still is
Arew Hmsw “Sitting Form” assumed by Amen Adherents. Though the principal reason for this postural
preference is esoteric, it can be revealed that the standing state is more austere and the recumbent state more
reminiscent of death. From the philosophical perspective of an Ascetic Osiriologist therefore, both
aforementioned ideal postures—Aha and Sedjer—are symbolically and sacredotally superior. However, the
‘Purgatorial Posture’ of Arew Hmsw is powerful when properly executed and can conduce to the state of
Supreme Isolation (Kevalya in Sanskrit) as completely as our Canonical Amenite Asanas.
III[Classical Latin, ‘way of living’.]

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overarching influence as the ‘Metabolic Master’, the organ most essential to the regulation of
intermediate metabolism is the liver. The liver, it has been found, enlists an important
endocrine hormone in its energy adjusting agenda. This hormone is called fibroblast growth
factor 21 (FGF21). In diverse ways the liver is able to detect dietary deficits and “determine”
the appropriate compensatory response. Glucose monitoring is a principal part of the liver’s
dietetic detection design. When levels of glucose plummet to a particular point indicative of
prolonged fasting, the liver releases FGF21 (among other agents). The purpose is to prompt
the body to pry the polymer glycogen apart into glucose, to fashion neutral fats into fatty
acids, and (lastly) to convert amino acids into chemicals called ketones. The aforementioned
agents—glucose, fatty acids, and ketones—embody energy that can be used to fuel the
body’s myriad metabolic processes. This explanatory simplification conceals complex
phenomena whose precise formulation would fulsomely fill pages of text. That, thankfully,
shall not be necessary to advance our understanding. For our present purposes it is
important to appreciate that the attenuation of energy intake instated by fasting forces the
body to function most efficiently and this increased efficiency translates into the inhibition
of aging, the lengthening of lifespan. The very complexity of the chemical cascade catalyzed
by chronic fasting means that many interventions that agonize an element of the fasted state
might have the potential to prolong life. This is so with FGF21. We shall begin our
examination of the exceptional study conducted by Dr. Yuan Zhang and colleagues with a
presentation of their pithy prologue. It is recommended that the Reader not attend
excessively to unfamiliar terms, especially those included in Abstracts. Abstracts of scientific
studies are intended to concisely encapsulate information to audiences expected to have an
overall understanding of the area under consideration. The Architekton assumes the task of
translating any technical terms or obscure ideas the elucidation of which will contribute to
our Agenda of integrating all pertinent data pursuant to the prolongation of lifespan or the
optimization of health. No comprehensive dietetic or lifestyle regimen is remotely as
scientifically sound and theoretically thorough as the Amen Protocol and the sheer
multiplicity of studies substantiating our System makes the employment of Abstracts
especially efficient. I ask the Adherent’s indulgence in this regard as the Author has
endeavored to include full Abstracts of only the most lucid, most informative variety or
those whose exclusion would necessitate excessive exegesis. With respect to the ensuing
Abstract from eLIFE, three terms utilized by the investigators will be seen to have
substantial significance in subsequent sections of this and other Volumes of AIR—mTOR,
ADP and NAD. The Amenite is advised to ignore these for now, for a fuller explanation is
forthcoming:

Fibroblast growth factor-21 (FGF21) is a hormone secreted by the liver


during fasting that elicits diverse aspects of the adaptive starvation response.
Among its effects, FGF21 induces hepatic fatty acid oxidation and
ketogenesis [formation of ketones], increases insulin sensitivity, blocks
somatic growth and causes bone loss. Here we show that transgenic
overexpression of FGF21 markedly extends lifespan in mice without
reducing food intake or affecting markers of NAD+ metabolism or AMP
kinase and mTOR signaling. Transcriptomic analysis [i.e. analysis of the
transcripts or products of genes] suggests that FGF21 acts primarily by
blunting the growth hormone/insulin-like growth factor-1 signaling pathway
in liver. These findings raise the possibility that FGF21 can be used to extend
lifespan in other species.

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Indeed it is possible that appropriate application of the information offered in the study
under analysis “can be used to extend lifespan in other species”. Our interest is, of course,
the human species.
As will undoubtedly become evident throughout our investigative journey, published
research in the realms of biogerontology and epidemiology often entail multiple separate
studies skillfully subsumed into one. The publication before us is a fine example of this. The
investigators operated on several fronts. They identified the gene in the liver of the mouse
that manufactures the FGF21 protein. They then amplified its expression using standard
genetic engineering techniques. They verified that the genetic manipulation effectuated the
intended augmentation of the protein. They permitted the population of experimental
animals to live out their lifespan and compared their survival to a control group,
conventionally fed and comparable to the experimental group in every observable way
excepting the intervention (i.e. FGF21 amplification). As the curves and chart in the
accompanying illustration convey (Figure II:I-I), lifespan was lengthened significantly by the
intervention. As delineated in their Discussion:

In this report, we demonstrate that chronic exposure of mice to the


starvation hormone, FGF21, increases median survival time by ∼30% and
∼40% in males and females, respectively, without decreasing food intake.
The increase in lifespan extension is comparable to that achieved by caloric
restriction. While the FGF21-mediated increase in longevity is less than the
50–70% increase seen in pituitary loss-of-function Ames mice and GH
receptor/GH binding protein-knockout mice, it is similar to that seen in
other loss-of-function dwarf models including hypopituitary Snell mice and
mice lacking pregnancy-associated plasma protein-A (PAPP-A), a protease
that increases IGF-1 activity. The FGF21-Tg [i.e. genetically engineered]
mice share other phenotypic similarities with long-lived dwarf mice including
small size, reduced circulating insulin and IGF-1 concentrations, increased
circulating adiponectin levels and female infertility. Like the Ames and Snell
dwarf mice, female FGF21-Tg mice live longer than males. These similarities
together with our previous finding that FGF21-Tg mice are GH resistant
suggest that FGF21 may increase lifespan by inhibiting the GH/IGF-1
signaling pathway. Because FGF21 also regulates other pathways that impact
metabolism, it is not surprising that some of the effects that are seen in dwarf
mice, such as increased adiposity in GH receptor-knockout mice, are not
recapitulated in FGF21-Tg mice.

While we shall have opportunities to discuss the implications of many of the model
organisms identified above, let us focus on the central finding: Augmentation of a hormone
associated with fasting is able to extend lifespan in an experimental mammal to a degree
comparable to caloric restriction. This is compelling. Amenites are interested in the ability of
an intervention—any prudent, practical intervention—to aid us in our aim of life extension
however. How can the study in question contribute to this? Now we come to the
significance of the constituent, corollary studies comprising the experimental undertaking.
The investigators formulated two additional experimental groups. One group was subjected
to caloric restriction amounting to 40% of the caloric intake of the control group—that is,

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60% CR. Another group was subjected to a single session of 24-hour fasting. The principal
purpose of these interventions was to compare their capacity to induce FGF21 expression.
Importantly, conventional caloric restriction was altogether ineffective in inducing FGF21
expression. The day-long fast did promote the production of FGF21. Engineered
amplification of the FGF21 gene augmented expression of the protein to levels 5 to 10 times
greater than that of fasting however. These facts tell us several critical things and
concomitantly cause us to question the conceptual creativity of the scientists, brilliant though
they may be. First it tells us that despite the pleiotropic power of caloric restriction to
promote lifespan extension in multiple species and strains, it is not singular as an anti-aging
intervention—CR must share the spotlight with fasting as a suppressor of senescence. This
argues strongly for the appropriateness of the composite nature of the Amen Protocol—it
includes caloric restriction as well as fasting. The finding that the DGM (discrete genetic
modification) was five to ten times more effective in amplifying FGF21 production permits
of qualification however. The investigators, we must reiterate, subjected the experimental
animals to simply a single session of daylong fasting. What if they had imposed ‘doses’ of
daylong fasting (e.g. ~22-23 hours) punctuated by a prandial period (~1-2 hours) over an
extended interval of time—that is, what if they had imposed an experimental intervention
approximating the Amen Protocol? Alas, we cannot be certain. Such is the very purpose of
empirical investigation, to bring us closer to scientific certitude by confirming or
disconfirming conjectures, hypotheses, and theories through amassing, analyzing, and
interpreting data. We cannot be condemned for suggesting that if a single session of daylong
fasting were able to induce appreciable expression of FGF21, an ‘Amenesque’ fast,
constituting a continuous cycle of daylong fasting punctuated by brief feeding periods,
would promote the production of FGF21 to a degree considerably greater than that
observed after a single day.

Excursus: The Architekton has in fact communicated this contention to the study’s
authors. Time will tell if they accept the soundness of my suggestion and are
inclined to undertake an investigation to ascertain its truth or falsity. If they are not
so inclined, we may resolve to pursue the project ourselves, impel more obliging
investigators to undertake it, or conceptually content ourselves with the quite
compelling conclusions that can be drawn from the information already available to
us as well as that which will undoubtedly emerge in the future.

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Figure II:I-I: Anti-Aging effect of genetically augmented FGF21 expression.

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It is important to appreciate that many of the accompanying effects of FGF21 are known.
Not surprisingly, these effects accord with those commonly observed under fasting
conditions. Most importantly for our purposes, and in keeping with the purport of Part I of
the present Volume, FGF21 is associated with suppressed circulating sugar. An informative
study in Cell Metabolism sheds additional light on the ‘gluco-regulatory’ relations of the fasting
hormone. A molecule of critical importance in the modulation of adiposity has been found
to require the recruitment of FGF21 in its gluco-regulatory role. As indicated in the
Abstract:

Peroxisome proliferator-activated receptor α (PPARα) regulates the


utilization of fat as an energy source during starvation and is the molecular
target for the fibrate dyslipidemia drugs. Here, we identify the endocrine
hormone fibroblast growth factor 21 (FGF21) as a mediator of the
pleiotropic actions of PPARα. FGF21 is induced directly by PPARα in liver
in response to fasting and PPARα agonists. FGF21 in turn stimulates lipolysis in
white adipose tissue and ketogenesis in liver. FGF21 also reduces physical activity and
promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves
energy. These findings demonstrate an unexpected role for the PPARα-
FGF21 endocrine signaling pathway in regulating diverse metabolic and
behavioral aspects of the adaptive response to starvation. [Emphasis added
by the Author in italics.]

We welcome this mechanistic insight afforded by Dr. Inagaki and colleagues concerning the
intercessional role of FGF21 mainly in lipid metabolism. We are even more appreciative of
their investigations into the influence of fasting duration on the expression of FGF21,
knowledge of which the aforecited eLIFE article failed to furnish us. As delineated in their
Discussion:

Since PPARα plays a prominent role in the starvation response, we next


examined whether FGF21 is regulated by fasting. FGF21 mRNA levels were
increased 28-fold after a 12 hr fast. Refeeding for 12 hrs reduced FGF21 mRNA to
prefasting levels. While induction of FGF21 by fasting was greatly diminished in
PPARα−/− mice, fasting still caused a 5-fold increase in FGF21 mRNA.
These data demonstrate that FGF21 is induced during fasting by PPARα but
that other pathways also contribute. [Emphasis added by the Author in
italics.]

The above information has important implications for the Amen Fast. It informs us that
fasting for 12 hours effectuates a nearly thirty-fold increase in FGF21. It further informs us
that a comparable period of feeding is necessary to nullify the fasting induced increase in
FGF21. Importantly, the Amen Protocol does not permit such a protracted period of
feeding. Quite the contrary, our Feeding Phase spans a mere hour or less (in Amen Adepts
assuredly, a little longer in Neophytes). Thus, it is likely that levels of the life-extending
endocrine hormone remain high in the hepatic tissue and serum of the Amenite.
More mechanistic insight is available regarding the means by which FGF21 may
mitigate mortality and it corresponds closely to the conceptual content of this Tractate.
Specifically, it seems to suppress the process of glycation as such. Though we are already

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aware that reduced circulating sugar induces the endocrine hormone and that hypoglycemic
states lessen the extent of glycation, there is evidence that elevated FGF21 itself interferes
with the formation of AGEs. This indeed may be among its most important anti-aging
effects. The following Abstract from Pharmacology, Biochemistry, & Behavior explains thusly:

Fibroblast growth factor 21 (FGF21) is a hormone secreted predominantly in


the liver, pancreas and adipose tissue. Recently, it has been reported that
FGF21-Transgenic mice [exhibit extended] lifespan compared with wild type
counterparts. Thus, we hypothesize that FGF21 may play some roles in aging
of organisms. In this study d-galactose (d-gal)-induced aging mice were used
to study the mechanism [whereby] FGF21 protects mice from aging. The
three-month-old Kunming mice were subcutaneously injected with d-gal (180
mg/kg/d) for 8weeks and [supplemented] simultaneously with FGF21 (1, 2
or 5mg/kg/d). Our results showed that administration of FGF21
significantly improved behavioral performance of d-gal-treated mice in water
maze task and step-down test, reduced brain cell damage in the
hippocampus, and attenuated the d-gal-induced production of MDA, ROS
and advanced glycation end products (AGEs). At the same time, FGF21 also
markedly renewed the activities of superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (GPx) and total anti-oxidation capability (T-
AOC), and decreased the enhanced total cholinesterase (TChE) activity in
the brain of d-gal-treated mice. The expression of aldose reductase (AR),
sorbitol dehydrogenase (SDH) and [membrane]-anchored receptor for AGEs
(RAGE) declined significantly after FGF21 treatment. Furthermore, FGF21
suppressed inflamm-aging by inhibiting I-κβα degradation and NF-κβ p65
nuclear translocation. The expression levels of pro-inflammatory cytokines,
such as TNF-α and IL-6, decreased significantly. In conclusion, these results
suggest that FGF21 protects the aging mice brain from d-gal-induced injury
by attenuating oxidative stress damage and decreasing AGE formation.I

While it may not be technically correct to characterize this as a sort of “negative feedback
loop”, it is edifying to appreciate the essence of this phenomenon: Suppressing sugar
concentration via fasting simultaneously suppresses glycation and induces the anti-aging
hormone FGF21 which, in turn, suppress glycation. If not nominally “negative” sensu stricto,
it is certainly a ‘Salutary Cycle’.

Though this entire Volume is devoted to delineating the details of the Amen Fast
(while Volume III is directed at the descriptive dispensation of the Amen Diet), the exquisite
interrelationship among each element of our Agenda impels some overlap in exposition. In
this respect, it is important to introduce evidence that dietetic factors reinforce the effect of
fasting on FGF21. Amenites who have intellectually assimilated the all-important edifice of
the Tetractys of Anti-Aging Interventions are aware that Methionine Restriction (MR)
independently promotes mammalian maximum lifespan prolongation. As shall be discussed
in Volume III, Part II (on the Main Meal of the Amen Diet), multiple mechanisms are

IYu Y, Bai F, Wang W, Liu Y, Yuan Q, Qu S, Zhang T, Tian G, Li S, Li D, Ren G. Fibroblast growth factor 21
protects mouse brain against d-galactose induced aging via suppression of oxidative stress response and
advanced glycation end products formation. Pharmacology, Biochemistry, & Behavior. 2015; 133:122-31.

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implicated in MR-mediated mammalian life extension. Investigators from the University of
Aberdeen ascertained that MR exerts its ameliorative anti-aging effect partly by promoting
production of FGF21. As their informative Abstract in Aging Cell indicates:

Methionine restriction (MR) decreases body weight and adiposity and


improves glucose homeostasis in rodents. Similar to caloric restriction, MR
extends lifespan, but is accompanied by increased food intake and energy
expenditure. Most studies have examined MR in young animals; therefore,
the aim of this study was to investigate the ability of MR to reverse age-
induced obesity and insulin resistance in adult animals. Male C57BL/6J mice
aged 2 and 12 months old were fed MR (0.172% methionine) or control diet
(0.86% methionine) for 8 weeks or 48 h. Food intake and whole-body
physiology were assessed and serum/tissues analyzed biochemically.
Methionine restriction in 12-month-old mice completely reversed age-
induced alterations in body weight, adiposity, physical activity, and glucose
tolerance to the levels measured in healthy 2-month-old control-fed mice.
This was despite a significant increase in food intake in 12-month-old MR-
fed mice. Methionine restriction decreased hepatic lipogenic gene expression
and caused a remodeling of lipid metabolism in white adipose tissue,
alongside increased insulin-induced phosphorylation of the insulin receptor
(IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited
increased circulating and hepatic gene expression levels of FGF21,
phosphorylation of eIF2a, and expression of ATF4, with a concomitant
decrease in IRE1α phosphorylation. Short-term 48-h MR treatment increased
hepatic FGF21 expression/secretion and insulin signaling and improved whole-body
glucose homeostasis without affecting body weight. Our findings suggest that MR
feeding can reverse the negative effects of aging on body mass, adiposity, and
insulin resistance through an FGF21 mechanism. These findings implicate
MR dietary intervention as a viable therapy for age-induced metabolic
syndrome in adult humans.I [Emphasis added in italics by the Author.]

This research reveals that restricting the ingestion of methionine reduces the rapidity of
aging such that middle-aged mice mirror immature ones in indices of health. This
improvement was effectuated in spite of increased energy intake among the experimental
animals. Most importantly from our present perspective, the investigation established the
FGF21-eliciting effect of methionine restriction.

Excursus: Among the evident functions of FGF21 is to facilitate efficient


assimilation of ingested nutrients upon feeding. A factor in this facilitation is
improved insulin sensitivity. Though “insulin sensitivity” is often uttered in
scientific circles, it is seldom explained in an intelligible manner and its meaning for
the mitigation of mortality is even less frequently elaborated. The Author shall fill
this intellectual lacuna for the Acolyte forthwith. Insulin sensitivity is simply an
expression and indication of the efficiency with which circulating sugar is absorbed
from the serum. Such absorption is effectuated by adequate elaboration and activity

ILeesEK, Król E, Grant L, Shearer K, Wyse C, Moncur E, Bykowska AS, Mody N, Gettys TW, Delibegovic
M. Methionine restriction restores a younger metabolic phenotype in adult mice with alterations in fibroblast
growth factor 21. Aging Cell. 2014; 13(5):817-27.

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of insulin. The imaginative Amenite can already conceive how the efficiency of this
sugar/insulin interplay influences aging: Optimal clearance of glucose keeps it from
causing damage to tissues. The preceding Abstract indicated that methionine
restriction results in improved insulin signaling and ‘gluco-stasis’ and, further, that
FGF21 is operative in this effect. What the Author wishes to relate in this Excursus
is that Meal Feeding (MF)—a modality of lifespan maximization among mammals—
similarly improves insulin signaling/sensitivity. So, clearly we are witnessing a
welcomed ‘Mechanistic Convergence’ suggestive of Selective
Synergism/Summation. The study from which this supposition stems is summarized
as follows:

Meal-fed (MF) rats with access to food for only 4 consecutive hours during
the light cycle learn to eat large meals to maintain energy balance. MF animals
develop behavioral and endocrine changes that permit glucose tolerance
despite increased meal size. We hypothesized that enhanced activity of the
enteroinsular axis mediates glucose homeostasis during MF. Cohorts of rats
were allocated to MF or ad libitum (AL) regimens for 2-4 wk. Insulin
secretion and glucose tolerance were determined after oral carbohydrate and
intraperitoneal (ip) and intravenous (iv) glucose. MF rats ate less than AL in
the first week but maintained a comparable weight trajectory thereafter. MF
rats had decreased glucose excursions after a liquid mixed meal (AUC: MF 75
± 7, AL 461 ± 28 mmol·l⁻¹·min, P < 0.001), with left-shifted insulin secretion
(AUC(0-15): MF 31.0 ± 4.9, AL 9.6 ± 4.4 pM· min, P < 0.02), which peaked
before a significant rise in blood glucose. Both groups had comparable
fasting glucagon levels, but postprandial responses were lower with MF.
However, neither intestinal expression of proGIP and proglucagon mRNA
nor plasma incretin levels differed between MF and AL groups. There were
no differences in the insulin response to ip or iv glucose between MF and AL
rats. These findings demonstrate that MF improves oral glucose tolerance
and is associated with significant changes in postprandial islet hormone
secretion. Because MF enhanced β-cell function during oral but not
parenteral carbohydrate administration, and was not accounted for by
changes in circulating incretins, these results support a neural mechanism of
adaptive insulin secretion.I

Thus, via a neurological mechanism mediated by the “entero-insular axis” of the


nervous system, Meal Feeding enhances glucose homeostasis or improves the
glycemic state of subjects. This so-called “cephalic” feature of cyclic fasting is
furthered by FGF21 and it is for this reason that the Author elected to intersperse
this informative Abstract in the midst of this section.

A study by Dr. Stone and colleagues quantified the catalyzing effect of MR on FGF21. The
details of their study is delineated in an Abstract appearing in Diabetes:

IVahl TP, Aulinger BA, Smith EP, Drazen DL, Ulrich-Lai Y, Seeley RJ, Woods SC, D’Alessio DA. Meal
feeding improves oral glucose tolerance in male rats and causes adaptations in postprandial islet hormone
secretion that are independent of plasma incretins or glycemia. American Journal of Physiology: Endocrinology &
Metabolism. 2014; 307(9):E784-92.

15
To understand the physiological significance of the reduction in fasting
insulin produced by dietary methionine restriction (MR), hyperinsulinemic-
euglycemic clamps were used to examine the effect of the diet on overall and
tissue-specific insulin sensitivity in mice. The steady-state glucose infusion
rate was threefold higher in the MR group and consistent with the 2.5- to
threefold increase in 2-deoxyglucose uptake in skeletal muscle, heart, and
white adipose tissue. Dietary MR enhanced suppression of hepatic glucose
production by insulin, enhanced insulin-dependent Akt phosphorylation in
the liver, and increased hepatic expression and circulating fibroblast growth
factor 21 (FGF-21) by fourfold. Limitation of media methionine
recapitulated amplification of Akt phosphorylation by insulin in HepG2 cells
but not in 3T3-L1 adipocytes or C2C12 myotubes. Amplification of insulin
signaling in HepG2 cells by MR was associated with reduced glutathione....
In contrast, FGF-21, but not restricting media methionine, enhanced insulin-
dependent Akt phosphorylation in 3T3-L1 adipocytes. These findings
provide a potential mechanism for the diet-induced increase in insulin
sensitivity among tissues that involves a direct effect of methionine in liver
and an indirect effect in adipose tissue through MR-dependent increases in
hepatic transcription and release of FGF-21.I

We hasten to highlight the fourfold enhancement of FGF21 expression induced by


methionine minimization. This amidst a mitigation of liver glucose output, an effect that
facilitates the anti-glycative aim of the Amen Fast.
Apart from their overall scientific significance (which is substantial), Amenites
should attend to the aforecited findings on FGF21 for additional, more practical purposes.
First, our Protocol is intentionally and inherently methionine deficient. This is so by virtue of
its vegetal nature: methionine mainly predominates in meat and eggs and is present in
comparatively low concentrations in vegetation. This first point relates to the second—
specifically, Selective Synergism/Summation. Note that the investigators identified
physiological effects of MR in several distinct domains: Glucose Regulation, Adiposity, and
Autophagy. Each of the aforementioned domains is an interventional element of the
Tetractys. In short, such findings as these indicate that interventions known to extend
lifespan often have overlapping effects and that the Amen Agenda, by integrating
multifarious such anti-aging interventions, is poised to potentially promote lifespan
prolongation more potently than any isolated approach.
Yet another aspect of the Amen Protocol that potentiates FGF21 production is
exercise. Though an entire Volume of AIR is devoted to delineating the details of the Amen
Exercise Regimen, we would be remiss to refrain from reflecting on a particularly relevant
investigation at this juncture. Dr. Kook Kim and colleagues conducted a concise study
confirming the capacity of exercise to promote increased production of the fasting protein
FGF21. As expressed in their Abstract:

Fibroblast growth factor 21 (FGF21) plays an important role in the


regulation of energy homeostasis during starvation and has an excellent
therapeutic potential for the treatment of type 2 diabetes in rodents and

IStone KP, Wanders D, Orgeron M, Cortez CC, Gettys TW. Mechanisms of increased in vivo insulin sensitivity
by dietary methionine restriction in mice. Diabetes. 2014; 63(11):3721-33.

16
monkeys. Acute exercise affects glucose and lipid metabolism by increasing
glucose uptake and lipolysis. However, it is not known whether acute
exercise affects FGF21 expression. Here, we showed that serum FGF21 level
is increased in mice after a single bout of acute exercise, and that this is
accompanied by increased serum levels of free fatty acid, glycerol and ketone
body. FGF21 gene expression was induced in the liver but not in skeletal
muscle and white adipose tissue of mice after acute exercise, and further, the
gene expression levels of hepatic peroxisome proliferator-activated receptor
α (PPARα) and activating transcription factor 4 (ATF4) were also increased.
In addition, we observed increased FGF21 level in serum of healthy male
volunteers performing a treadmill run at 50 or 80% VO2max. These results
suggest that FGF21 may also be associated with exercise-induced lipolysis in
addition to increased catecholamines and reduced insulin.I

It is appropriate to emphasize the exquisite order that the Amen Protocol entails: the Amen
Fast promotes heightened production of the life-extending protein FGF21; the calorically
and temporally restricted Amen Diet ensures that feeding does not diminish FGF21
exceedingly; the Amen Exercise Regimen, executed twice or thrice daily during the Fasting
Phase, further increases the induction of FGF21. Thus, three distinct domains of our
Protocol promote lifespan extension via the FGF21 mechanism. Again, this serves as a
prominent example of Selective Synergism/Summation.

I
Kim KH, Kim SH, Min YK, Yang HM, Lee JB, Lee MS. Acute exercise induces FGF21 expression in mice
and in healthy humans. PLoS One. 2013; 8(5): e63517.

17
The Author’s execution of the ‘Persistence Phase’ of the Amen Exercise Regimen promotes
production of the anti-aging endocrine hormone FGF21.

18
The initial sentence of the above Abstract alludes to the prospect of employing the
endocrine protein FGF21 as a pharmaceutical agent. This project is indeed already
underway. Though direct application of the protein has reportedly proven effective in some
investigations, oral administration of the agent is limited by the fact that proteins such as
FGF21 are subject to proteolytic cleavage from enzymes exuded by the intestines of all
animals. As the Abstract below indicates, pharmaceutical scientists are seeking to circumvent
this proteolytic problem by producing a modified FGF21 analog less likely to be degraded
during passage through the digestive tract:

Fibroblast growth factor 21 is a novel hormonal regulator with the potential


to treat a broad variety of metabolic abnormalities, such as type 2 diabetes,
obesity, hepatic steatosis, and cardiovascular disease. Human recombinant
wild type FGF21...has been shown to ameliorate metabolic disorders in
rodents and non-human primates. However, development of FGF21 as a
drug is challenging and requires re-engineering of its amino acid sequence to
improve protein expression and formulation stability. Here we report the
design and characterization of a novel FGF21 variant, LY2405319. To enable
the development of a potential drug product with a once-daily dosing profile,
in a preserved, multi-use formulation, an additional disulfide bond was
introduced in FGF21 through Leu118Cys and Ala134Cys mutations [i.e.
alterations in the sequence of the amino acids appearing in the protein—
specifically, leucine, cysteine, and alanine]. FGF21 was further optimized by
deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP)
[abbreviations of the amino acids histidine, proline, and isoleucine], which
was subject to proteolytic cleavage. In addition, to eliminate an O-linked
glycosylation site in yeast a Ser167Ala [i.e. serine/alanine] mutation was
introduced, thus allowing large-scale, homogenous protein production in
Pichia pastoris [a single-celled eukaryotic organism widely employed in genetic
engineering]. Altogether re-engineering of FGF21 led to significant
improvements in its biopharmaceutical properties. The impact of these
changes was assessed in a panel of in vitro and in vivo assays, which confirmed
that biological properties of LY2405319 were essentially identical to FGF21.
Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-
induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of
plasma glucose coupled with a 10–30% reduction in body weight. Thus,
LY2405319 exhibited all the biopharmaceutical and biological properties
required for initiation of a clinical program designed to test the hypothesis
that administration of exogenous FGF21 would result in effects on disease-
related metabolic parameters in humans.I

While reading such technical treatises is tedious, it illustrates an important point: prominent
scientists in the employ of powerful pharmaceutical companies are inclined to invest
considerable quantities of capital to create drugs that, at best, merely mimic what the Amen

I
Kharitonenkov A, Beals JM, Micanovic R, Strifler BA, Rathnachalam R, Wroblewski VJ, Li S, Koester A, Ford
AM, Coskun T, Dunbar JD, Cheng CC, Frye CC, Bumol TF, Moller DE. Rational design of a fibroblast
growth factor-21-based clinical candidate, LY24o5319. PLoS One. 2013; 8(3): e58575.

19
Protocol produces at no monetary cost and with much material-saving owing to its
sustainability. This is a point that Amenites should be proud to promulgate.

The Amen Fast promotes the production of the protein FGF21, which has proven to
lengthen lifespan in a mammalian model organism. The brevity of the Feeding Phase
prescribed by the Amen Diet enhances the effect of the Amen Fast in facilitating FGF21
induction. As empirical investigations indicate that exercise augments FGF21 production,
the Amen Exercise Regimen accordingly enhances expression of the life-extending
endocrine hormone.

Glucose, Glycation & Protein Pathomorphosis

As mentioned in the preceding Volume of AIR, glucose is the molecule into which most
ingested food is converted. The energy embedded in this sugary substance is then
systematically and sequentially “funneled” into the formation of the chemical bonds
contained in the body’s chief fuel currency, ATP. When food is transformed into glucose, it
circulates freely in the bloodstream, accessible to the cells of the body. Glucose, however, is
not innocuous in all its guises. In the previous section we learned that lifespan lengthening is
possible through fasting-facilitated augmentation of FGF21 and that levels of this endocrine
agent are associated, in turn, with levels of glucose—sugar, in short, suppresses secretion of
the lifespan-lengthening fasting hormone, FGF21. We shall presently recapitulate several
important points regarding the risks posed by chronically elevated glucose in the body—
such repetition is necessitated, in our estimation, in light of the evident centrality of glycation
in the etiology of aging. Moreover, the avoidance of this deleterious condition serves as a
chief justification for the Amen Fast, the central topic of this Volume. Among the most
momentous findings that we should bear in mind in this context is a seminal study which
convincingly confirmed that limiting the level of glycation in the body exerts an independent
effect on longevity—that is, inhibiting glycation increases lifespan independently of caloric
intake or intermittent fasting. The superb study of which we speak found that feeding fewer
glycation products to animals decreased the levels of such agents in their bodies and
inhibited aging such that maximum lifespan was significantly extendedI. Appropriately, we
shall reserve a fuller discussion of the interesting details of this investigation for the tripartite
Volume III encompassing the three elements of the Amen Diet. Two additional findings
warrant mention presently. It has been established that a commonly prescribed anti-diabetic
drug of the class called biguanides, extends maximum lifespan in strains of murine mammals.
This effect is evidently attributable to its anti-glycative, gluco-suppressant properties. Similar
effects on the combined and correlated reduction of glycemia and glycation are apparently
responsible for the attested life extending potential of a surprisingly simple substance—the
metal chromium. Interestingly, the life-extending effect of chromium was confirmed over a
half century ago in a study conducted by a capable researcher and published in a highly
reputable journal—The Journal of NutritionII. Despite this auspicious origin, however, the
extraordinary finding (which was confirmed in a subsequent study by the same scientists) has
fallen largely on deaf ears, with few gerontology researchers referring to it or indicating its
implications.

20
Excursus: It must be mentioned that investigators in this, the 2nd decade of
the 21st century have established the ability of chromium to extend the
lifespan of the Zucker Fatty ratI. This animal exhibits a mutation in its
receptor for leptin. Leptin is a hormone exuded by adipocytes, which exerts
extensive effects on myriad metabolic processes. Zucker rats are rendered
hyperphagic by their impaired leptin signaling, being ostensibly unresponsive
to satiety signals. Consequently they consume food voraciously and their
excessive eating eventuates in obesity. The condition caused by the mutation
is characterized by cardiovascular and endocrine abnormalities including
impaired insulin and glucose metabolism. Given the established insulin
sensitizing and glucose normalizing effects of chromium, it is not surprising
that introducing this metal into the murine mutants’ diet mitigates mortality.
Indeed, investigators found that supplementing the experimental diet of the
Zucker rat with a form of chromium chemically bonded to the vitamin niacin
increased both median and maximum lifespan by more than 20%. Our
enthusiasm for this momentous finding is moderated only by the
interpretative caution that must be counseled. Less cryptically conveyed: Can
the Zucker Fatty rat be considered “normal”? We are interested primarily in
interventions that establish the ability of agents or actions to extend
maximum lifespan in any “ordinary” mammalian species, though it is
admittedly arguable how “ordinary” are animals reared in captivity and
confined to cages. This modest criterion enables us to extrapolate empirical
findings more easily to the human mammal. This being said, however, an
animal that eats to excess, expends energy at a reduced rate, and accumulates
fat with increasing age is perhaps more comparable to the common Man in
modern advanced societies than a “normal” rodent whether in a laboratory
or at large. Hence, chromium deserves consideration as an anti-aging
compound and is accordingly included in the Amen Apothecary.

It is understandable that early research into the lifespan-lengthening effect of chromium


languished so long in obscurity insofar as ours is the first attempt to identify and elucidate all
known anti-aging interventions, discuss in detail their implications and integrate them, in
principle if not in fact, into a practical protocol.
The three interventions at which our attention is presently directed, though they
induce similar effects, are compositionally diverse—foods low in glycation concentration, an
organic molecule in the case of the biguanide drug, and a simple inorganic metal in the case
of chromium. We refer to each of them here for an important and relevant reason: they
unambiguously illustrate our guiding principle of Selective Synergism/Selective Summation.
Our reasoning is as follows. Insuring low levels of glycation in the body attenuates aging and
facilitates the attainment of maximum lifespan potential; the Amen Fast is especially suited
to the suppression of circulating sugar; elevated circulating sugar is the chief catalyst of

IISchroeder HA, Balassa JJ, Vinton WH. Chromium, cadmium and lead in rats: effects on life span, tumors and
tissue levels. Journal of Nutrition. 1965;86:51-66.
ICai W, He C, Zhu L, Chen X, Wallenstein S, Striker GE, Vlassara H. Reduced oxidant stress and extended

lifespan in mice exposed to a low glycotoxin diet: Association with increased AGER1 expression. The American
Journal of Pathology 2007; 170(6): 1893-1902.
IPreuss HG, Echard B, Clouarte D, Bagchi D, Perricone NV. Niacin-bound chromium increases life span in

Zucker Fatty Rats. Journal of Inorganic Biochemistry. 2011;105(10):1344-9.

21
glycation; the Amen Fast therefore independently potentiates our goal of lifespan
prolongation—it adds to the independent life extending effect of the Amen Diet (entailing,
as it does, inherent caloric restriction). As to why sugar and its several surrogates are so
central in aging and pathophysiology we shall see presently. Lastly, it seems that each of the
ten anti-aging interventions comprising the Tetractys—all those measures that extend
maximum lifespan in one or more mammalian species—induce reductions, directly or
indirectly, in the circulating concentration of glucose or primarily promote the protection of
proteins from the pathologic effects of glycation. This is true of all the anti-aging
interventions entailing dietary restriction, including caloric restriction, alternate-day feeding,
meal-feeding, protein restriction, and methionine restriction. To expatiate, this is true of
glycation restriction insofar as this intervention reduces the ultimate end-products of
elevated glucose—advanced glycation compounds. This is similarly true of those anti-aging
interventions that exert their effect through the augmentation of autophagy—autophagy is
directed primarily at those proteins damaged from excessive exposure to glucose. In a related
manner this also extends to those intriguing interventions that enhance longevity by
extirpating adipose or inhibiting the synthesis and storage of fat—dispensing with fat forces
the body to renew its proteinaceous tissues more rapidly and effectively averts the accretion
of glucose-damaged proteins. Finally, the several anti-aging interventions entailing discrete
genetic modification exert their effects, in part, by suppressing circulating glucose or
augmenting autophagy. Thus, the ability of the Amen Fast to optimize autophagy and safely
ensure reductions in glucose below the physiologic norm for humans is a major reason for
our attestation that the Amen Protocol is an anti-aging regimen par excellence.

Among the more pernicious properties of glucose is its tendency to bind aberrantly
to the proteinaceous structures of the body. When glucose binds thuswise to proteins, it
alters their structure and impairs their activity. Moreover, such deranged ‘pathomorphic’
proteins are resistant to the normal mechanisms of tissue degradation and renewal. As a
result, these ‘glycated’ proteins accumulate in the body and their accretion, it is surmised,
results in pathology and accelerated aging. Such debilitating diseases as diabetes, Alzheimer’s,
and amyotrophic lateral sclerosis (ALS) are thought to be potentiated or exacerbated by glycation.
The latter two disorders, tellingly, are localized to the nervous system. Because neurons are
limited in their capacity for regeneration, they are correspondingly more susceptible to
glycation-induced damage. Consequently, glycated proteins can be expected to accumulate
more precipitously in nervous tissue than in other tissues. [Muscle tissue, because its
constituent cells do not normally divide, is also more susceptible to glycation than mitotic
(dividing) tissues.] To put this threat into perspective we reiterate that the tissues of the body
are constituted principally by protein and, further, that every metabolic reaction is dependent
upon enzymes, which are likewise constituted preponderantly by protein. Nearly ninety
percent of the organic element of the body is protein—protein that is susceptible to assault
and eventual impairment by excessive exposure to sugar. Glycation is therefore a serious
(though woefully under-appreciated) threat to optimal health and longevity. This
appreciation deficit extends to experts as well as laymen. It is not our intention to remedy
this unfortunate state of affairs, rather it is the Author’s ambition merely to inform my
worthy Reader of this matter.
That glycation is involved in the pathophysiology of aging is suggested by facts that
bear reiteration: (1) it accumulates with increasing age (2) it is substantially suppressed by
caloric restriction and (3) several separate anti-aging interventions—dietary glycation
restriction, chromium consumption, and administration of biguanide drugs—all act directly

22
or indirectly to reduce the burden of glycation in the body. Indeed, averting or attenuating
glycation is virtually equivalent to the inhibition of aging itself inasmuch as the major source
of protein derangement is attributable to glycation and it is this very derangement of proteins
that properly defines the process of aging. Not only does glycation constitute the most
conspicuous molecular manifestation of aging, it retards rectification by debarring the body
from regenerating and renewing its tissues. As the following excerpt from an article in
Biogerontology explains:

…[B]iological aging mainly depends on a deleterious accumulation of


insoluble inert protein that has escaped physiological proteolytic degradation.
Intracellularly, a three-dimensional network of polypeptide chains may
exclude macromolecular structures and organelles from part of the cytosolic
water. In this way vital intracellular transport mechanisms may be obstructed,
providing a rationale to several observations linked to agingI.

In other words, accretion of deranged proteins interferes with normal cellular physiological
functions, thereby precipitating pathological outcomes operative in aging. Glycation is a
central player in this pernicious process of protein-precipitated cytotoxicity. An instructive
article published in the journal Brain Research Reviews further informs us that:

The cytotoxicity of glycation results from the following three mechanisms:


(1) inhibition of specific functions of proteins; (2) cross-linkage, aggregation,
and precipitation of proteins; and (3) production of reactive oxygen species
[free radicals]….Through glycation, specific functions of protein are altered,
i.e. the function is lost or its level is reduced. In addition to functional
changes at active sites [of enzymes], changes in functions are also caused by
accelerated or delayed degradation of glycated proteins.II

It is essential to appreciate that proteins play many important roles in the body
besides constituting the main molecular components of tissues. Proteins serve as receptors,
chemical carriers, and effecters of enzymatic reactions. This last role is particularly important
given the centrality of enzymes in every aspect of physiological functioning. When enzymes
sustain glycative damage, the effects are insidious for the reactions rendered by these crucial
catalysts are accordingly compromised. Consider the antioxidant enzyme superoxide
dismutase, which catalyzes the conversion of the highly reactive radical superoxide into
oxygen. Researchers from Osaka University Medical School in Japan found that superoxide
dismutase from erythrocytes exhibited an age-related increase in glycative damageIII.
Moreover, they found that patients with the accelerated aging phenotype characteristic of
Werner’s syndrome sustained greater damage to their superoxide dismutase enzymes than
did normal controls. This is an important finding insofar as glycative and oxidative damage
are known to be operative in aging and disease. The fact that glycative damage to antioxidant
enzymes can result in increased free radical damage clarifies to some extent the
interrelationship between glycation, oxidation, aging and degenerative diseases. Glycation is
probably primary in the acceleration of aging as it inhibits the effectiveness of antioxidant

IHallen A. Accumulation of insoluble protein and aging. Biogerontology 2002; 3: 307-315.


IIKikuchiS, Shinpo K, Takeuchi M, Yamagishi S, Makita Z, Sasaki N, Tashiro K. Glycation—a sweet tempter
for neuronal death. Brain Research Reviews 2003; 41: 306-323.

23
enzymes and by so doing facilitates free radical damage. Oxidative damage from free
radicals, in turn, exacerbates tissue derangement in a vicious cycle that feeds on its own
destruction.

Quite obviously, limiting the intake of calories will have the salutary effect of
reducing the extent of glycation-induced damage as less food will be converted into
glucose—less glucose, less glycation. But the duration over which the body’s tissues are
exposed to glucose is also of signal importance—a longer period of exposure means a larger
number of glycation reactions and more damage to tissues. Thus, the quantity of calories
ingested is of evident import but, to reiterate, the span of time over which glucose circulates in
the blood is also a decisive determinant of the degree to which glycation damages the body.
Herein lies one of the many benefits of the Amen Fast. Indeed, it is the fasting component
of our Regimen that makes the Amen Protocol vastly superior to caloric restriction alone. As
was explained in the previous Volume, many CR studies employ methodologies wherein
animals are given daily fractional quantities of food relative to a freely fed control group,
whereas the other frequently employed CR methodology effectuates caloric curtailment by
restricting the feeding times of experimental subjects to alternating days. In terms of
glycative potential, the former, continuous CR protocol is more deleterious, potentially and
theoretically, for it provides animals with a continuous (though calorically restricted)
allotment of food (for, say, an 8 to 12-hour duration), which would be expected to induce a
continuous elevation of glucose (relative to fasted animals). Such continuously elevated
glucose would facilitate greater glycative damage. In the case of alternate day CR protocols,
animals are allowed to consume a quantity of food equivalent to that which libitum animals
ingest over a 1-day diurnal (waking) period or simply given free access to food on alternating
days. Such animals are customarily given access to food over a period of 8-12 hours, every
other day. Thus, over a 2-day interval, animals on a continuous CR protocol feed for a
period of ~20 hours whereas those animals on an alternate day CR protocol feed for a
period of ~10 hours in the span of 2 days. Clearly the latter protocol is superior in terms of
inferred inhibition of glycation. Let us then compare this relatively superior, alternate day
(AD) CR regimen to the Amen protocol. In terms of absolute feeding time, animals
subjected to the standard scientific AD-CR protocol feed (discontinuously) for upwards of
12 hours over a 2-day (48-hour) period. Conversely, over a 2-day period, the Amen Fast
prescribes a feeding period of only 2 hours. Over a 1-week (168-hour) period, the absolute
feeding time under standard AD-CR conditions is upwards of 42 hours, whereas over a
comparable 1-week period, Amen practitioners feed for a mere 7 hours. A six-fold weekly
difference in feeding duration such as this can have considerable consequences over the span
of a lifetime. Discrete instances of feeding should also be considered. On a standard AD-CR
protocol an animal can consume food on multiple occasions in the 8-12 hour interval over
which feeding is permitted. On the Amen Fast, only 7 discrete feeding sessions are
permissible in the span of a week. Consider the potential physiological consequences of this
distinct dietary divergence between standard CR and the Amen Protocol, the latter
ostensibly effectuating a substantially greater temporal attenuation of glycation. Necessarily,
such a difference will result in less glycation-induced tissue damage and less precipitous
aging. In a qualitative, somewhat superficial sense, this means that the Amen Protocol has

IIITaniquchiN, Kinoshita N, Arai K, Iizuka S, Usi M, Naito T. Inactivation of erythrocyte Cu-Zn-superoxide


dismutase through nonenzymatic glycolsylation. Progress in Clinical & Biological Research 1989; 304: 277-290.

24
the capacity to induce a more juvenescent phenotype, a more youthful appearance. It has
been established that the external appearance of the integument is an accurate index of
aging—that is, the superficial appearance of skin is significantly suggestive of the celerity of
senescence. Interesting studies have shown that it is possible to predict or estimate the ages
of individuals with a considerable degree of accuracy by simply looking at their faces or
photos thereof. Moreover, such subjective studies have objectively linked these observations
to levels of glucose circulating in the sera of the subjects. Consider a compelling study
published in the journal Age. The Abstract of the study conducted by Dr. Noordam and
colleagues explains that:

Estimating perceived age by facial photographs is a good estimate of health


in elderly populations. Previously, we showed that familial longevity is
marked by a more beneficial glucose metabolism already at middle age. As
glucose is also related to skin aging, this study aimed to investigate the
association between glucose metabolism and perceived age. Perceived age
was assessed using facial photographs and non-fasted glucose and insulin
were measured in 602 subjects from the Leiden Longevity Study. Non-
diabetic subjects (n=569) were divided in three strata according to their
glucose levels, and diabetic subjects (n=33; as a proxy of long-term
hyperglycemic exposure) were included as a forth stratum. Considered
confounding factors were gender, chronological age, current smoking, body
mass index, photo-damage score, and insulin levels. Perceived age was
increased from 59.6 years (SE [standard error] = 0.3) in the first stratum to
61.2 years (SE = 0.6) in diabetic subjects (p for trend = 0.002). In non-
diabetic subjects only, perceived age was increased from 59.6 years (SE =
0.3) in the first stratum to 60.6 years (SE = 0.3) in the third stratum (p for
trend = 0.009). Continuously, perceived age increased 0.04 years (SE = 0.14,
p = 0.006) per 1 mmol/L increase in glucose level in non-diabetic subjects.
The present study demonstrates that, also among non-diabetic subjects,
higher glucose levels are associated with a higher perceived age. Future
research should be focused on elucidating mechanisms linking glucose levels
to perceived age.I

This study has both aesthetic and gerontologic significations. It is of aesthetic significance
because the appearance of one’s skin and the estimation of one’s age are subjective signifiers
of beauty. It is of gerontologic significance because the study shows that circulating levels of
glucose correlate positively with perceived age; perceived age has previously been found to
correlate closely with actual age and ultimate mortality. As to the mechanism linking levels of
glucose to overall organismal aging, we shall amass evidence illustrating the central role of
glycation (and, relatedly, autophagy) throughout the present Volume. Volume VIII will deal
explicitly with the Amen Aesthetic, including an explication of how the elements of the AIR
promote general juvenescence.
In a weightier, more fundamental sense, the Amen Protocol, by exerting a potent
glucose-suppressing, glycation-inhibiting effect, has the theoretical potential to extend

INoordam R, Gunn DA, Tomlin CC, Maier AB, Mooijaart SP, Slagboom PE, Westendorp RG, de Craen AJ,
van Heemst D; Leiden Longevity Group. High serum glucose levels are associated with a higher perceived age.
Age. 2013; 35(1): 189-95.

25
lifespan to a greater extent than the conventional alternate day feeding regimen. To reiterate,
because food is ingested over an interval of only 1 hour each day on the Amen Fast, the level
of glucose circulating in the bloodstream is depressed, the length of time tissues are
susceptible to sugar-mediated sabotage is truncated, and the potential for glycation-induced
damage is thereby reduced. Though caloric restriction is clearly efficacious in the mitigation
of glycation, the Amen Protocol summates or synergizes this effect by tremendously
truncating the time allotted for feeding.
Considering the brief feeding interval prescribed by the Amen Protocol, it is
imperative that nutrients be ingested in a highly degraded, highly simplified, easily assimilable
form in order to ensure adequate intake of essential nutrients. As we shall see in the
forthcoming Volume III (Part 1), the Amen Diet is specifically suited to augment
assimilation of nutrients and conduce to the conservation of metabolic energy.

The Amen Fast protects the body from precipitous aging and degeneration by
minimizing the exposure of its tissues to glucose and its metabolites, thereby facilitating the
efficient recycling and renewal of bodily proteins.

Glucose, Glycation & DNA Damage

Considering that proteins are the most prevalent macromolecules comprising the body,
considering that damage to proteins defines the degree to which one has actually aged in a
functional, fundamental sense, and considering that glycation is the most marked
manifestation of protein damage, we are understandably intently interested in averting
glycative damage to bodily proteins. However, as indicated in Volume I, it is information
that enables the construction of our bodies and enables the orchestration of its functions. In
the main, this information is genomic in nature. Though many molecules convey
information in the body, it is only the genome that serves as the source of our unique
structure and orchestrates the expression of genes in accordance with information emerging
from inside and outside the body. When this information is corrupted, health is
compromised and longevity is limited. Thus, the fact that the genome is subject to glycation
is a serious matter for mortality mitigation.

The aim of this study was to assess the changes of markers of DNA damage
by glycation and oxidation in patients with type 2 diabetes and the
association with diabetic nephropathy.

Methodology: DNA oxidation and glycation adducts were analysed in plasma


and urine by stable isotopic dilution analysis liquid chromatography-tandem
mass spectrometry. DNA markers analysed were as follows: the oxidation
adduct 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-OxodG) and glycation
adducts of glyoxal and methylglyoxal-imidazopurinones GdG, MGdG, and
N2-(1,R/S-carboxyethyl)deoxyguanosine (CEdG).

Results: Plasma 8-OxodG and GdG were increased 2-fold and 6-fold,
respectively, in patients with type 2 diabetes, with respect to healthy
volunteers. Median urinary excretion rates of 8-OxodG, GdG, MGdG, and

26
CEdG were increased 28-fold, 10-fold, 2-fold, and 2-fold, respectively, in
patients with type 2 diabetes with respect to healthy controls. In patients with
type 2 diabetes, nephropathy was associated with increased plasma 8-OxodG
and increased urinary GdG and CEdG. In a multiple logistic regression
model for diabetic nephropathy, diabetic nephropathy was linked to systolic
blood pressure and urinary CEdG.

Conclusion: DNA oxidative and glycation damage-derived nucleoside adducts


are increased in plasma and urine of patients with type 2 diabetes and further
increased in patients with diabetic nephropathy.I

As the Author indicated in Volume I, it is the alteration in the expression of genes that
largely determines the rapidity with which we age and indeed explains the ability of various
experimental anti-aging interventions (CR, CF, DGR, DGM, etc.) to slow senescence. We
therefore accord especial importance to the ensuing Abstract in Biogerontology explaining the
manner in which identifiable AGEs alter gene expression.

The Maillard reaction and its end products, AGEs (Advanced Glycation
End-products) are rightly considered as one of the important mechanisms of
post-translational tissue modifications with aging. We studied the effect of
two AGE-products prepared by the glycation of lysozyme and of BSA, on
the expression profile of a large number of genes potentially involved in the
above mentioned effects of AGEs. The two AGE-products were added to
human skin fibroblasts and gene expression profiles investigated using
microarrays. Among the large number of genes monitored the expression of
16 genes was modified by each AGE-preparation, half of them only by both
of them. Out of these 16 genes, 12 were more strongly affected, again not all
the same for both preparations. Both of them upregulated MMP and serpin-
expression and downregulated some of the collagen-chain coding genes, as
well as the cadherin and fibronectin genes. The BSA-AGE preparation
downregulated 10 of the 12 genes strongly affected, only the serpin-1 and
MMP-9 genes were upregulated. The lysozyme-AGE preparation upregulated
selectively the genes coding for acid phosphatase (ACP), integrin chain
alpha5 (ITGA5) and thrombospondin (THBS) which were unaffected by the
BSA-AGE preparation. It was shown previously that the lysozyme-AGE
strongly increased the rate of proliferation and also cell death, much more
than the BSA-AGE preparation. These differences between these two AGE-
preparations tested suggest the possibility of different receptor-mediated
transmission pathways activated by these two preparations. Most of the gene-
expression modifications are in agreement with biological effects of Maillard
products, especially interference with normal tissue structure and increased
tissue destruction.

IWaris S, Winklhofer-Roob BM, Roob JM, Fuchs S, Sourij H, Rabbani N, Thornalley PJ. Increased DNA
dicarbonyl glycation and oxidation markers in patients with type 2 diabetes and link to diabetic nephropathy.
Journal of Diabetes Research. 2015; 2015:915486.

27
AGEs accelerate cell death in neurons, eventuating in Alzheimer’s-associated aggregation of
proteins and consequent patterns of pathology. Aberrant chromosomal copying (called
hyperploidy) is one consequence of such AGE-induced aggregation, though this pathological
process, the study established, can be arrested by antioxidants. This is what the Abstract in
Neurobiology of Aging indicated:

Neurons that reenter the cell cycle die rather than divide, a phenomenon that
is associated with neurodegeneration in Alzheimer's disease (AD).
Reexpression of cell-cycle related genes in differentiated neurons in AD
might be rooted in aberrant mitogenic signaling. Because microglia and
astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque
components or factors secreted from plaque-activated glia induce neuronal
mitogenic signaling. Advanced glycation end products (AGEs), protein-
bound oxidation products of sugar, might be one of those mitogenic
compounds. Cyclin D1 positive neurons are colocalized with AGEs or
directly surrounded by extracellular AGE deposits in AD brain. However, a
direct proof of DNA replication in these cells has been missing. Here, we
report by using fluorescent in situ hybridization that consistent with the
expression of cell cycle proteins, hyperploid neuronal cells are in
colocalization with AGE staining in AD brains but not in nondemented
controls. To complement human data, we used apolipoprotein E-deficient
mice as model of neurodegeneration and showed that increased oxidative
stress caused an intensified neuronal deposition of AGEs, being
accompanied by an activation of the MAPK cascade via RAGE. This
cascade, in turn, induced the expression of cyclin D1 and DNA replication.
In addition, reduction of oxidative stress by application of α-lipoic acid
decreased AGE accumulations, and this decrease was accompanied by a
reduction in cell cycle reentry and a more euploid neuronal genome.

Conceptually speaking, the above investigation is pertinent to our present topic insofar as it
identifies a pathological association among glycation, oxidation, and aberrant genomic
regulation of cell cycle control, ultimately increasing Alzheimer’s risk in this case. Since an
entire Volume is devoted to dementia and diverse mental and neurological conditions that
can be ameliorated by the Amen Protocol, we shan’t elaborate at length here. Let us merely
reflect upon the fact that glycation can compromise the function of DNA in the brain and
catalyze a condition that is among the most miserable imaginable: Alzheimer’s. The ability of
the Amen Regimen to avert this peril is important and no ostensible anti-aging intervention
is able to effect such attenuation of Alzheimer’s risk in a manner remotely comparable to
ours.

Let us opportunistically add to our understanding of glycation-induced DNA damage


at this juncture. The Reader is reminded that the DNA nestled in our nuclei is not naked but
enmeshed in protective proteins that perform complex regulatory routines rather than
merely draping the genetic material. Histones are the proteinaceous components of
chromosomes of which we speak. It has been found that glycative damage to histone
proteins promote aberrant structural alteration of chromosomes, the instability of which may

28
compromise organismal integrity and catalyze aging and disease. From Free Radical Biology &
Medicine we read the following:

A number of oxidative stress agents cause DNA and protein damage, which
may compromise genomic integrity. Whereas oxidant-induced DNA damage
has been extensively studied, much less is known concerning the occurrence
and fate of nuclear protein damage, particularly of proteins involved in the
regulation and maintenance of chromatin structure. Protein damage may be
caused by the formation of reactive carbonyl species such as glyoxal, which
forms after lipid peroxide degradation. It may also result from degradation of
early protein glycation adducts and from methylglyoxal, formed in the
process of glycolytic intermediate degradation. Major adducts indicative of
protein damage include the advanced glycation end product (AGE)
carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the
formation of CML and argpyrimidine protein adducts represents potential
biomarkers for nuclear protein damage deriving from a variety of sources.
The purpose of this study was to identify and quantify AGE adducts formed
in vivo in a nuclear protein, specifically histone H1, using CML and
argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus
collected immediately after slaughter under conditions designed to minimize
AGE formation before isolation. Using antibodies directed against oxidative
protein adducts, we identified CML, argpyrimidine, and protein crosslinks
present in the freshly isolated histone H1. Detailed mass spectroscopy
analysis of histone H1 revealed the presence of two specific lysine residues
modified by CML adducts. Our results strongly suggest that glycation of
important nuclear protein targets such as histone H1 occurs in vivo and that
these oxidative changes may alter chromatin structure, ultimately
contributing to chronic changes associated with aging and diseases such as
diabetes.

There is much of interest in this investigation for the attentive Amenite and Observer. First,
it informs us that the proteins “intended” to protect DNA are susceptible to glycative attack
and that a consequence of such sugar-mediated assault is the origination of advanced
glycation end-products that aberrantly attach to the genomic material itself; this adulteration
of DNA structure can conceivably lead to deleterious mutations and adversely alter the
expression of genes in such a way as to potentially limit lifespan. The latter logical linkages
are speculative, though scientifically sound. Less speculative is the existence of specific
compounds created by the combination of reactive carbonyl species and components of
nuclear (i.e. genomic) proteins: carboxymethyllysine (CML) and argpyrimidine. These
molecules may have more meaning to Adherents once they read Volume III:I and when they
appreciate that the inclusion of specific amino acids in our Amen Amino Aliksir are able to
bind to sugar and reactive carbonyl compounds and accordingly inhibit the attachment of
these agents to proteins comprising the body and proteins protecting DNA. More
specifically, lysine (conducing to carboxymethyllysine) creation and arginine (conducing to
argpyrimidine) creation, are added to our Aliksir and enhance the efflux of these inimical
agents from the body. While most of the dozen different isolated amino acids comprising
our ‘Sacred Shake’ similarly achieve this anti-glycative effect, the nitrogenous amino acids
indicated above are especially effective owing to particular properties of their structure and

29
specific so-called “functional groups”. We shall have far more to say on this important topic
in the Aliksir Tractate (Volume III:I).

Lifespan is decidedly determined by DNA; damage thereto therefore compromises


longevity. Glycation contributes considerably to DNA damage. Thus, the ability of the
Amen Fast to forestall excessive glycation is an additional exemplification of the myriad
means by which our Regimen mitigates aging and optimizes health.

The Glory of Glyoxalase: Guarantor of AGE Attenuation

An entire century has elapsed since scientists initially embarked upon an understanding of
the enzyme system integral to the inhibition of glycation: the Glyoxalase Complex. The
complex, contained in every eukaryotic cell, consists of two essential enzymes—Glyoxalase I
& II. The “aim” of these enzymes is to convert the glycative agents glyoxal and
methylglyoxal into the innocuous metabolite lactate. The Reader is reminded that the
metabolism of glucose (and other simple sugars) eventuates in chemicals containing carbonyl
groups that are considerably reactive and particularly pernicious in their interactions with
protein residues, amino acids, lipid-laden membranes, and the nucleotides of genetic
material. Glyoxal and methylglyoxal are merely two of the most numerous and nefarious
such reactive carbonyl compounds that catalyze glycation. Given the significance of sugar as
a source of energy in animal metabolism, and given the inevitable generation of glycating
compounds catalyzed by the conversion of sugar into usable energy, it is understandable why
evolution would elect to originate an enzyme system such as the Glyoxalase Complex. It is
also understandable why it is observed that the activity of this important enzyme system
declines with age. Though this age-related reduction in glyoxalase has been established by
several empirical investigations, a study published in the journal Gerontology is particularly
poignant. Aging is commonly characterized by a compromised capacity to heal, recuperate,
and renew one’s tissues rapidly and robustly. Dr. Fleming found that a factor in this faulty
regeneration that often accompanies aging is an accumulation of advanced glycation
compounds. This age-associated accumulation of AGEs is evidently enabled by attenuated
expression of glyoxalase enzymes, especially that of isoform I. Let us learn from their
illuminating Abstract:

Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase


1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased
GLO1 activity in situ has been shown to result in an accumulation of MG and
increased formation of advanced glycation endproducts, both of which can
accumulate during physiological aging and at an accelerated rate in diabetes
and other chronic degenerative diseases. To determine the physiological
consequences which result from elevated MG levels and the role of MG and
GLO1 in aging, wound healing in young (≤ 12 weeks) and old (≥ 52 weeks)
wild-type mice was studied. Old mice were found to have a significantly
slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ±
1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated

30
with decreases in GLO1 transcription, expression and activity. The
importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct
application of MG to the wounds of young mice, decreased wound healing
by 24% compared to untreated mice, whereas application of BSA [bovine
serum albumin] modified minimally by MG had no effect. Treatment of
either young or old mice with aminoguanidine, a scavenger of free MG,
significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p
<0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day
6. As a result of the aminoguanidine treatment, the overall rate of wound
healing in the old mice was restored to the level observed in the young mice.
These findings were confirmed in vitro, as MG reduced migration and
proliferation of fibroblasts derived from young and old, wild-type mice. The
data demonstrate that the balance between MG and age-dependent GLO1
downregulation contributes to delayed wound healing in old mice.

To reiterate: Aging impairs regenerative ability; such impairment is attributable to AGE


accumulation; AGE accumulation is augmented by glyoxalase attenuation; impairment of
regenerative wound healing can be improved by administering an agent (i.e. aminoguanidine)
which inhibits AGE formation. The Reader should be informed that fibroblasts form the
fundamental cellular structure of connective tissues comprising cartilage, ligaments, tendons,
skin, bones, blood, and adipose. The investigators demonstrated that direct exposure of
fibroblasts to the glycative compound methylglyoxal mitigated the movement and mitotic
multiplication of these rejuvenate cells. If such cells are prevented from replicating and
precluded from perambulating to sites of tissue damage, destruction and degeneration of
tissue cannot be rectified and the physiological conditions that characterize aging intensify
unremittingly. This explains why individuals afflicted with diabetes heal so slowly: Excessive
AGE accumulation impairs the activity of their fibroblasts. This also explains the
physiological similarities between senescence and the disease of diabetes: Both states reflect
the results of pervasive, protracted glycative damage. We can conceivably capitalize on the
crucial information conferred by this and comparable studies and it is the Author’s intent to
explain how.

Given what Amenites now know about the age-inhibiting aspects of our
Intervention, it is perhaps predictable that the principal components of our Protocol—
fasting, caloric restriction, and specific bioactive agents encompassing the ‘Amen
Apothecary’—retard reductions in the activity of a system so central to aging, specifically the
Glyoxalase Complex. Let us start with a simple synopsis that shall be unstintingly restated:
fasting facilitates systemic suppression of circulating sugar and, in so doing, substantially
spares the body the damage done by exorbitant and even “ordinary” amounts of glucose.
The study to which we will direct our immediate attention established the capacity of
concentrated glucose to curtail the activity of glyoxalase in the eukaryote Caenorhabditis elegans
and, consequently, compromise its longevity. Further, the German investigators from
Heidelberg University found that engineered overexpression of the enzymes increased
maximum lifespan in C. elegans, ostensibly by attenuating glycation and aberrant oxidation.
This is what their descriptive Abstract in Diabetes delineated:

Objective: Establishing Caenorhabditis elegans as a model for glucose toxicity-


mediated life span reduction.

31
Research Design & Methods: C. elegans were maintained to achieve glucose
concentrations resembling the hyperglycemic conditions in diabetic patients.
The effects of high glucose on life span, glyoxalase-1 activity, advanced
glycation end products (AGEs), and reactive oxygen species (ROS)
formation and on mitochondrial function were studied.

Results: High glucose conditions reduced mean life span from 18.5 ± 0.4 to
16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4
days....The formation of methylglyoxal-modified mitochondrial proteins and
ROS was significantly increased by high glucose conditions....Overexpression
of the methylglyoxal-detoxifying enzyme glyoxalase-1 attenuated the life-
shortening effect of glucose by reducing AGE accumulation (by 65%) and
ROS (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and
maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of
glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days)
and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days)....

Conclusions: C. elegans is a suitable model organism to study glucose toxicity, in


which high glucose conditions limit the life span by increasing ROS
formation and AGE modification of mitochondrial proteins....Most
importantly, glucose toxicity can be prevented by improving glyoxalsase-1-
dependent methylglyoxal detoxification or preventing mitochondrial
dysfunction.I

A few points warrant reinforcement. First, the scientists sought to simulate the state of
diabetes by boosting the sugar concentration of the in vitro cellular system—thuswise they
suppressed the function of glyoxalase, augmented AGE formation, and doomed the exposed
organism to early death. Intentional induction of the enzyme glyoxalase effected an
extension of lifespan by both averting AGE accumulation and free radical formation. This is
a powerful study supporting the scientific soundness of our abstemious System from a new
standpoint. Similarly, a study in a separate species—Podospora anserina—established that
engineered enhancement of glyoxalase expression significantly suppressed senescence
specifically in an environment of artificially elevated sugar concentration, though not
substantially so when sugar levels were comparatively low and only when both isoforms of
the enzyme (i.e. I & II) were elevated.II Admittedly, this evidence is more indicative of the
inherently inimical, age-accelerating effect of elevated sugar than of the independent anti-
aging efficacy of glyoxalase. But, alas, boosting glyoxalase activity is undoubtedly “insurance”
against elevated sugar and such sinister sugar surrogates as the AGEs glyoxal and
methylglyoxal.
While we appreciate this entire evidentiary schema concerning the glyoxalase
complex, we seek an even sturdier foundation. Empirically, we know that fasting suppresses
ISchlottererA, Kukudov G, Bozorgmehr F, Hutter H, Du X, Oikonomou D, Ibrahim Y, Pfisterer F, Rabbani
N, Thornalley P, Sayed A, Fleming T, Humpert P, Schwenger V, Zeier M, Hamann A, Stern D, Brownlee M,
Bierhaus A, Nawroth P, Morcos M. C. elegans as model for the study of high glucose- mediated life span
reduction. Diabetes. 2009; 58(11):2450-6.
IIScheckhuber CQ, Mack SJ, Strobel I, Ricciardi F, Gispert S, Osiewacz HD. Modulation of the glyoxalase

system in the aging model Podospora anserina: effects on growth and lifespan. Aging. 2010; 2(12):969-80.

32
sugar in multifarious organisms including Man, that substantial suppression of glyoxalase
results from excessive exposure to sugar in the organism C. elegans and that this glyoxalase
reduction results in AGE-induced decrements in maximum lifespan. Logically, it is legitimate
to expect that extension of lifespan is possible in Man owing to elevations in glyoxalase
activity. We want to know whether there is direct evidence that glyoxalase activity can be
experimentally augmented or enhanced in Man and (in accordance with the intellectual
agenda of this segment of the AIR) whether the Amen Fast is likely to effectuate such
augmentation or enhancement of glyoxalase. The short answer to this question is “probably,
though not definitively”. Amenites ought not be entirely surprised however. It must be
appreciated that the Glyoxalase Complex evolved to avert the damaging effects of excessive
circulating sugar. One would therefore expect sugar to induce instead of attenuate expression
of glyoxalase. This is indeed what certain experimental evidence indicates. Fasting,
alternatively, ought to exert no appreciable effect on the expression or activity of glyoxalase.
Despite such discursive deductions, South Korean investigators demonstrated that starvation
(or what Amenites would rather regard as fasting) resulted in increased expression of a
glyoxalase analog, the activation of which attenuates AGE accumulation. As Dr. Lee and
colleagues indicate in the ensuing terse tractate:

Caenorhabditis elegans DJR-1.2, a homolog of human DJ-1, has recently been


demonstrated to be a novel glyoxalase and protects worms against glyoxals.
Here, we report that expression of DJR-1.2 is substantially increased during
starvation as well as in the dauer stage. The induction of DJR-1.2 led to
increased glyoxalase activity in the dauer state, thereby increasing protection
against glyoxals. The induction is regulated by DAF-16, a transcriptional
regulator implicated in oxidative stress and normal lifespan.I

Perhaps the preceding tractate is too terse for the taste of the Reader. Let us accordingly
elaborate on its significance. The multi-cellular metazoan model C. elegans expresses a protein
that serves the same purpose as its counterpart in Man: it acts as a glyoxalase, deterring the
destructive effects of the glycating compounds collectively called glyoxals. Most significantly,
starving the specimens prompted the activation of the glyoxalase analog and protected them
from the pernicious effects of the AGE precursors. In a separate study, the South Korean
scientists performed another service for us. Their prior paper published in the journal Human
Molecular Genetics describes an additional function of the human homologue of the elegans
glyoxalase enzyme: protection from Parkinson’s disease and the protein damage that
precipitates the neuropathological condition. Though their Abstract is admittedly inelegant,
the Amenite may opine, it is assuredly informative:

Human DJ-1 is a genetic cause of early-onset Parkinson’s disease (PD),


although its biochemical function is unknown. We report here that human
DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types
of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid,
respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit
typical Michaelis-Menten kinetics [a quantification of chemical catalysis
characterizing the interaction of an enzyme and some substrate or reactant],

ILeeJY, Kim C, Kim J, Park C. DJR-1.2 of Caenorhabditis elegans is induced by DAF-16 in the dauer state. Gene.
2013; 524(2):373-6.

33
which were abolished completely in the mutants of essential catalytic
residues, consisting of cysteine and glutamic acid. The presence of DJ-1
protected mouse embryonic fibroblast and dopaminergically derived SH-
SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1,
a DJ-1 homolog expressed primarily in the intestine, protected worms from
glyoxal-induced death. Sub-lethal doses of glyoxals caused significant
degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2,
another DJ-1 homolog expressed primarily in the head region, including
neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl
species may provide a new insight into the causation of PD.I

Again, some interpretation (and justification) is in order. Parkinson’s is a condition


characterized by the aberrant accumulation of protein in particular regions of the brain. In
part, the purpose of DJ-1 is to prevent such protein pathology. Prevention seems contingent,
in part, on the protection conferred by the glyoxalase activity of DJ-1. This illustrates an
integral link between glyoxalase, glycation, progressive protein damage, and the protective
potential of augmented autophagy. That is, augmented autophagy can correct the damage
done by excessive glycation; fasting dualistically augments the activity of the glyoxalase
enzyme that averts glycation and (as we shall explore more fully in Part III of the present
Volume) enhances activity of the autophagic apparatus that prevents protein damage typical
of Parkinson’s, Alzheimer’s, and other age-related degenerative ailments.

It is important to appreciate that a crucial constituent of the glyoxalase complex is


the sulfur-containing compound glutathione. Glutathione serves several physiological
functions centering upon the attenuation of oxidative and glycative damage. Indeed, there is
evidence that glutathione is the most efficacious endogenous anti-glycative agent—this
according to an analysis published by Polish investigators in the journal General Physiology &
Biophysics. The researchers from the University of Rzeszów report that:

...Numerous exogenous compounds have been tested for their anti-glycating


activity. The aim of this study was to answer the question, which endogenous
compounds at physiological concentrations can effectively prevent glycation.
A set of endogenous compounds has been tested for the ability to protect
albumin from glucose-induced glycation in vitro at a concentration of 1 mM
and in a physiological concentration range. Only glutathione was found to
protect significantly against glycation at physiological concentrations.
Glutathione depletion increased the rate of hemoglobin glycation in
erythrocytes incubated with high glucose concentrations. These results
indicate that the level of glutathione is the main determinant of glycation of
intracellular proteins.II

Particularly pertinent to our present focus is the fact that glutathione is depleted when it is
employed in the rectification of oxidative and glycative damage. Because fasting inhibits the

ILee JY, Song J, Kwon K, Jang S, Kim C, Baek K, Kim J, Park C. Human DJ-1 and its homologs are novel
glyoxalases. Human Molecular Genetics. 2012; 21(14):3215-25.
IIGaliniak S, Bartosz G, Sadowska-Bartosz I. Glutathione is the main endogenous inhibitor of protein

glycation. General Physiology & Biophysics. 2016 (February 20).

34
damaging oxidative reactions arising from hyperglycemic (and indeed normoglycemic) states,
it effectively increases the amount of glutathione available for antioxidant defense and
enables it to be employed in the glyoxalase pathway. Moreover, there is evidence that fasting
actively augments glutathione levels, thereby optimizing the operation of glyoxalase. This is
what we desire, this is what we wish to exploit. We have evidence of a means by which
fasting can facilitate the induction or enhancement of an agent (i.e. glutathione) that
thereupon participates in a pathway (i.e. the glyoxalase pathway) which plausibly promotes
lifespan extension. Evidence for the ability of fasting to enhance glutathione formation is
found in several scientific studies. One such study is particularly persuasive insofar as the
intensity of fasting fell far short of the Amenite ideal of abstemiousness. The form of fasting
to which We refer is the Ramadan fast of the Muslim faith. An Arabian investigator, Dr. Al-
Shafei established that fasting during Ramadan resulted in an overall improvement in the
lipid profiles of patients with hypertension as well as among fasting controls. Of greater
interest to us is the observed effect of fasting on glutathione—it was elevated appreciably.
Dr. Al-Shafei’s Abstract delineates the design and outcome of his investigation:

Background & Aims: Effects of Ramadan fasting on health are important. Its
effects on arterial pulse pressure (PP), lipid profile and oxidative stress were
characterized in hypertensives.

Methods: PP, indices of lipid profile and oxidative stress were measured pre-,
during and post-fasting in equal (40 each), sex- and age-matched groups (age
55 ± 5 years) of hypertensives (HT) and controls (C).

Results: Fasting reduced PP significantly by 17.2% and insignificantly by 9.3%


in the HT and C groups, respectively. Total cholesterol (TC) was lowered
insignificantly by 11.7% and 4.7% in the HT and C patients, respectively.
Triglycerides (TG) and malondialdehyde (MDA) were significantly lowered
by: TG: 24.5% and 22.8%; MDA: 45.6% and 54.3%; while glutathione
(GSH) elevated by 56.8% and 52.6% in the HT and C groups, respectively.
High-density lipoproteins (HDL) were raised significantly by 33.3% and
insignificantly by 6.7%, whereas low-density lipoproteins (LDL) decreased
significantly by 17.7% and insignificantly by 4.0% in the HT and C groups,
respectively. At 6 weeks post-fasting, MDA remained significantly lower than
the pre-fasting level by 24.3% and 25.7%, and GSH higher by 30.2% and
26.3% in the HT and C groups, respectively, while PP and TC returned to
pre-fasting values in both groups. The post-fasting, HDL was significantly
higher by 20.3% and LDL lower by 12.0% than the fasting levels in the HT
patients.

Conclusion: Fasting improves PP and lipids profile and ameliorates oxidative


stress in hypertensives.I

Thus the moderate Ramadan fast resulted in an increase in glutathione of more than 50
percent among hypertensives and control participants alike. Impressively, this elevation

IAl-Shafei AI. Ramadan fasting ameliorates arterial pulse pressure and lipid profile, and alleviates oxidative
stress in hypertensive patients. Blood Pressure. 2014; 23(3):160-7.

35
persisted six weeks after the cessation of fasting, being 30 and 26 percent higher in
hypertensives and controls, respectively. Dr. Al-Safei followed up this study on the effects of
fasting on hypertension with an exploration of its effects on diabetes. The Abstract,
appearing in the European Journal of Nutrition, is as follows:

Purpose: The effects of Ramadan fasting on public health are important. The
present study characterized the metabolic effects of Ramadan fasting and
evaluated its influence on oxidative stress in diabetic patients.

Methods: The current study was carried out in the city of Benha, Egypt, during
the period from July 12, 2012 to October 4, 2012. This corresponds to 22
Shaban 1433 to 18 Dhul Al-Qi’dah 1433 in the Islamic Calendar. Two equal,
sex- and age-matched groups (n = 40 each; age 55 ± 5 years) of non-diabetic
subjects (ND group) and diabetic patients (D group) were recruited for this
study. Parameters of glycemic control, lipid profile, and oxidative stress were
measured pre-, during and post-fasting.

Results: Ramadan fasting reduced fasting blood glucose (FBG) insignificantly


by 5.8% and significantly by 23.0% in the (ND) and (D) groups, respectively.
Serum triglycerides (TG) and malondialdehyde (MDA) were lowered
significantly by: TG (22.8, 22.1%), MDA (54.3, 46.6%), and total cholesterol
(TC) and low-density lipoproteins (LDL) insignificantly by: TC: (4.7, 6.1%),
LDL: (4.0, 5.1%), whereas high-density lipoproteins (HDL) were raised
significantly by 6.7% and insignificantly by 2.2%, and blood glutathione
(GSH) significantly by 52.6 and 59.4%, in the (ND) and (D) groups,
respectively. At 6 weeks post-fasting FBG, TG, TC, HDL, and LDL returned
to levels indistinguishable from their baseline values in both groups, while
MDA was maintained significantly lower by (25.7, 22.7%), and GSH
significantly higher by (26.3, 31.3%), in the (ND) and (D) groups,
respectively.

Conclusions: Ramadan fasting improves glycemic control and lipids profile and
alleviates oxidative stress in diabetics.I

These findings accord with Al-Shafei’s earlier investigation establishing increased glutathione
concentration secondary to fasting, substantial sustainment of this increase after several
weeks of fasting cessation and significant suppression of fasting glucose concentration
among the diabetic patients. Readers are reminded that the fast of Ramadan is not nearly as
rigorous as the Fast of the Amen Order. Limited to refraining from feeding during daylight,
the duration of the Ramadan fast (with the region of Earth’s equator as a reference) may
amount to merely 12 hours, roughly half the length of the Amen Fast. Thus, if the Ramadan
fast is efficacious in elevating glutathione, the Amen Fast may be expected to be
exceptionally effective in increasing the concentration of this glycation-averting agent that is
integral to the operation of the glyoxalase system.

IAl-Shafei AI. Ramadan fasting ameliorates oxidative stress and improves glycemic control and lipid profile in
diabetic patients. European Journal of Nutrition. 2014; 53(7):1475-81.

36
Evolution has equipped eukaryotic organisms with a defensive system that degrades
deleterious AGEs—it is called the glyoxalase complex. Because boosting levels of this
enzyme system leads to lengthened lifespan in lower organisms, the ability of the Amen Fast
to induce this same effect is impressive and worth underscoring.

Defying the ‘Dawn’: ‘Amenistic Entrainment’, Exercise, and Elements of the Amen
Apothecary Attenuate Innate, Ordinary Elevations of Glucose Concentration Commonly
Concordant with Circadian Control

It was explained in Volume I of AIR that ensuring strict, strenuous, systematic circadian
entrainment facilitates lifespan lengthening in multiple mammalian models and that Meal
Feeding and Cyclic Fasting of a form analogous to the Amen Regimen aids entrainment and,
ex hypothesi, heightens lifespan potential. Now Dr. Nun deems it necessary to nuance this
knowledge. For one such phasic feature of fasting and circadian rhythmicity is an
understandable, adaptive increase in glucose production and utilization upon arising with
‘Ra’, our Sacred Solar Orb. It is activity as such that makes this molecular measure sensible:
sugar simply provides the energy that makes movement and mentation possible. As
Amenites are abundantly aware however, simple sugar simultaneously stimulates glycation,
conducing to the creation of AGEs—AGEs, ultimately accelerate aging. The Author shall
affirm the obvious fact that maintaining a moderate, adequate amount of circulating sugar in
the fasted state is essential and not inimical to our aim of life prolongation. As such, our
mission is to mitigate the magnitude of sugar elevation associated with what
Endocrinologists call the ‘Active Phase’ in animals and, accordingly, attenuate AGE
formation. Before the Architekton elaborates the ways in which our System effectuates such
inhibition, let us explore the evidence upon which our understanding of this sugar-related
segment of the circadian cycle is based. In this we are ‘blessed’ with brilliant research
exhibited especially in two endocrinologic studies. Fittingly enough, the first is found in the
journal entitled Endocrinology. Its Abstract informs us that:

Fasting blood glucose (FBG) and hepatic glucose production are regulated
according to a circadian rhythm. An early morning increase in FBG levels,
which is pronounced among diabetic patients, is known as the dawn
phenomenon. Although the intracellular circadian clock generates various
molecular rhythms, whether the hepatic clock is involved in FBG rhythm
remains unclear. To address this issue, we investigated the effects of phase
shift and disruption of the hepatic clock on the FBG rhythm. In both
C57BL/6J and diabetic ob/ob mice, FBG exhibited significant daily rhythms
with a peak at the beginning of the dark phase. Light-phase restricted feeding
altered the phase of FBG rhythm mildly in C57BL/6J mice and greatly in
ob/ob mice, in concert with the phase shifts of mRNA expression rhythms
of the clock and glucose production-related genes in the liver. Moreover, the
rhythmicity of FBG and Glut2 [i.e. an isoform of a glucose transport portal]
expression was not detected in liver-specific Bmal1-deficient mice.
Furthermore, treatment with octreotide suppressed plasma growth hormone
concentration but did not affect the hepatic mRNA expression of the clock
genes or the rise in FBG during the latter half of the resting phase in

37
C57BL/6J mice. These results suggest that the hepatic circadian clock plays a
critical role in regulating the daily FBG rhythm, including the dawn
phenomenon.I

The scientists say a great deal that is superfluous in this Summary of theirs, so let us seize
upon what is most significant from our vantage. Animals experience an increase in the
amount of circulating glucose as they awake and prepare to engage in energetically
demanding activity. The preparatory period, particularly pronounced in diabetics (owing to
pathological impairments) is known as the “dawn phenomenon”. Not only is the
phenomenon known in animals, it exists among normal, non-diabetic humans as the ensuing
excerpt indicates:

To ascertain whether the dawn phenomenon occurs in nondiabetic


individuals and, if so, whether it is due to an increase in glucose production
or a decrease in glucose utilization, we determined plasma concentrations of
glucose, insulin, C-peptide, and counterregulatory hormones, as well as rates
of glucose production, glucose utilization, and insulin secretion at one-half-
hourly intervals between 1:00 and 9:00 a.m. in eight normal volunteers. After
5:30 a.m., plasma glucose, insulin, and C-peptide concentrations all increased
significantly; rates of glucose production, glucose utilization, and insulin
secretion also increased (all P less than 0.05). Plasma cortisol, epinephrine,
and norepinephrine increased significantly from nocturnal nadirs between
4:00 and 6:30 a.m. Plasma growth hormone, which had increased episodically
between 1:00 and 4:30 a.m., decreased thereafter nearly 50% (P less than
0.05). Plasma glucagon did not change significantly throughout the period of
observation. These results indicate that a dawn-like phenomenon, initiated by
an increase in glucose production, occurs in nondiabetic individuals. Thus,
early morning increases in plasma glucose concentrations and insulin
requirements observed in IDDM and NIDDM [i.e. Insulin-Dependent and
Non-Insulin Dependent Diabetes Mellitus, respectively] may be an
exaggeration of a physiologic circadian variation in hepatic insulin sensitivity
induced by antecedent changes in catecholamine and/or growth hormone
secretion.I

So, Ascetic Students, if excessive sugar accelerates aging, if sugar seems to “spike” at specific
times, if such times can be anticipated in accordance with circadian cyclicity, then it is
possible to purposefully protect proteins and other tissues from the potential damage done
by the “dawn phenomenon”. The engineered order of the Amen Regimen provides such
protection in the following ways. First and foremost it confers protection via the Amen Fast.
Though this is ‘Second Nature’ to Inveterate Amenites, it is important to appreciate that by
abstaining from eating upon awaking and eschewing food for the entire diurnal day,
Practitioners of the Amen Protocol necessarily spare their tissues from the damaging effects
of elevated sugar, unlike ordinary individuals inclined to eat animalistically whenever edible,

IAndo H, Ushijima K, Shimba S, Fujimura A. Daily fasting blood glucose rhythm in male mice: A role of the
circadian clock in the liver. Endocrinology. 2015 Dec 10.
IBolli GB, De Feo P, De Cosmo S, Perriello G, Ventura MM, Calcinaro F, Lolli C, Campbell P, Brunetti P,

Gerich JE. Demonstration of a dawn phenomenon in normal human volunteers. Diabetes. 1984; 33(12):1150-3.

38
enticing food finds itself in the vicinity of such voluptuously visceral votaries of Vice.
Secondly, the Amen Exercise Regimen (Resistance and Persistence Phases alike) defies the
Dawn dualistically—that is, by proximately reducing circulating sugar concentration after
exercise is executed and, distally, by decreasing the quantity of stored fat while concomitantly
increasing the proportion of skeletal muscle, an effect which enables more efficient
metabolism of sugar and, as such, suppression of “spiking”. Thirdly, the Amen Apothecary
(as is discussed in detail in AIR V) contains copious compounds that curtail the
concentration of sugar as well as many molecules that more directly mitigate glycation by
blocking the combination of glucose, AGEs, and active carbonyls with the constitutive and
catalytic proteins of the body. Fourth and finally, (as elaborated in AIR II:IV ‘On Autophagy
& The Amen Fast), the autophagy-enhancing effects of the Amen Regimen (of which there
are several) effectuate the elimination of impaired proteins accrued as a consequence of
oxidation, glycation, or other chemically detectable damage. As the Adherent shall come to
apprehend, the circadian cyclicity of autophagy (especially under fasting conditions) is
complemented by cardinal components of the Amen Regimen that amplify this effect,
illustrating again the ideality of ‘Amenistic Entrainment and Order’.

Sugar concentration surges considerably and with circadian regularity upon waking.
Each element of AIR, especially the Amen Fast, opposes this ‘dextrose-dominated Dawn
Phenomenon’. The ability of AIR to avert the deleterious effects of the Dawn Phenomenon
substantiate the gerostatic status of our System.

Excessive Circulating Sugar ‘Short-Circuits’ Circadian Cyclicity, Confounds Optimal


Physiological Order, Accelerates Aging, and Compromises the Recuperative Quality of
Sleep: AIR Opposes these Inimical Effects most Markedly via the Amen Fast

Amenites are now enlightened about the deleterious effects of the ‘Dawn Phenomenon’ and
understand the degree to which our ‘unnatural’ Regimen resists this natural state of circadian
sugar elevation. The Architekton now wishes to refine this understanding. To this end, more
mechanistic material shall be presented that is pertinent to the linkages among longevity,
glycemia, glycation, entrainment, circadian conformity, sound slumber and the specific
molecular entities operative in the regulation of these intricate interactions. With this added
insight we shall deepen our appreciation for the ideality of AIR. First, let us be reminded of
the ‘Mupa Mouse’—that magnificent murid model whom we first met in Fascicle I of
Volume I.
In the last decade of the last millennium, Israeli investigators introduced us to a
genetically engineered mouse model whose mortality was reduced by amplifying the
expression of a urokinase enzyme especially in the brains of the experimental subjects.
Secondary to the augmentation of this enzyme several physiological effects were seen, the
most salient of which offer an explanation for the observed increase in longevity among
these animals: reduced food intake, reduced core body temperature, increased proteolytic
activity (indicative of increased autophagy), reduced glycemia, and increased circadian
regularity.I Of the aforecited effects, we are currently most concerned with the latter two—
glycemia and circadian cyclicity. The pertinence of these phenomena to our Protocol is
patently obvious. For we have here a Discrete Genetic Modification (DGM) whose

39
maximization of mammalian lifespan is linked to lessened glycemia and strengthened
circadian cyclicity alike. We shall see presently that this sugar/circadian connection seems to
have clinical significance in humans. A manuscript in Metabolism convincingly makes this
momentous case:

Nearly all mammalian cells express a set of genes known as clock genes.
These regulate the circadian rhythm of cellular processes by means of
negative and positive autoregulatory feedback loops of transcription and
translation. Recent genomewide association studies have demonstrated an
association between a polymorphism near the circadian clock gene CRY2
and elevated fasting glucose. To determine whether clock genes could play a
pathogenetic role in the disease, we examined messenger RNA (mRNA)
expression of core clock genes in human islets from donors with or without
type 2 diabetes mellitus. Microarray and quantitative real-time polymerase
chain reaction analyses were used to assess expression of the core clock
genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets.
Insulin secretion and insulin content in human islets were measured by
radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were
significantly lower in islets from donors with type 2 diabetes mellitus. To
investigate the functional relevance of these clock genes, we correlated their
expression to insulin content and glycated hemoglobin levels: mRNA levels
of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ =
0.37, P = .0047) correlated positively with insulin content. Of these genes,
expression of PER3 and CRY2 correlated negatively with glycated
hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore,
in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA
level was reduced in human islets exposed to 16.7 mmol/L glucose per 1
mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in
human islets. The data suggest that perturbations of circadian clock
components may contribute to islet pathophysiology in human type 2
diabetes mellitus. [Architekton’s Note: ρ, the Greek letter whose phoneme or
fuller grapheme is ‘rho’, symbolizes and connotes correlativity or the extent
to which two variables are statistically related; it is commonly signified as
simply “r” in epidemiological jargon.]I

Let us not be lured into the lamentable (il)logic that diabetics are destined to develop that
devastating disease due to their genetic endowment alone—an inheritance impairing
circadian symmetry and strength. The results presented in this paper do not permit such a
postulation. Conversely, the data do indicate that individuals with diabetes exhibit an altered
expression of molecules mediating circadian cyclicity and, perforce, that the pathophysiology
of diabetes may be precipitated in part by adverse circadian regulation of sugar circulation.
The researchers reinforced this reasoning by resorting to an in vitro analysis wherein they
adroitly established that elevated sugar concentration considerably curtailed the expression
of core clock/circadian genes, specifically in cells of the endocrine pancreas, which produce
insulin. In an effort to further establish what they correctly characterized as the “functional
relevance” of this phenomenon, it was ascertained that the expression of clock genes
correlated inversely with the extent of glycation of the molecule hemoglobin. In a

40
subsequent study by separate scientists the same inverse correlativity between circadian
expressivity and glycation was reaffirmed. The study in question focused more concertedly
on a specific subset of diabetes that dawns during pregnancy and plagues particularly
corpulent matrons—so called gestational diabetes. The Athenian obstetricians offer their
findings in the aptly named journal, Gynecological Endocrinology:

Dysfunction of the circadian clock genes is involved in the development of


obesity and type 2 diabetes (T2D). Since gestational diabetes mellitus (GDM)
and T2D share common genetic and phenotypic features, in the present
study, we investigated the status of the circadian clock in a cohort of 40
Greek pregnant women with GDM, four with T2D and 20 normal controls.
Peripheral blood mRNA transcript levels of 10 clock genes (CLOCK1,
BMAL1, PER1, PER2, PER3, PPARΑ, PPARD, PPARG, CRY1 and CRY2)
were determined by real-time quantitative PCR. GDM patients expressed
significantly lower transcript levels of BMAL1, PER3, PPARD and CRY2
compared to control women (p < 0.05). No significant difference was
documented between GDM women maintained either under insulin
treatment or diet. A positive correlation was found between the expression
of BMAL1 versus CRY2 (r = 0.45, p = 0.003) and BMAL1 versus PPARD (r
= 0.43, p = 0.004). Further investigation on the functional relevance of these
clock genes, disclosed that expression of PER3 correlated negatively with
HbA1C levels (r = -0.36, p = 0.022). These data document for the first time
that the expression of BMAL1, PER3, PPARD and CRY2 genes is altered in
GDM compared to normal pregnant women and support the notion that
deranged expression of clock genes may play a pathogenic role in GDM.I

Very well. What is the relevance of this research to the Amen Regimen and how can
Adherents capitalize upon it? Confessedly, we can capitalize conceptually by compounding
our confidence that our intellectual analyses and overall Anti-Aging Agenda is unassailably
sound. That is, we know that the main modalities of AIR advance a phenomenon
unambiguously associated with maximum lifespan extension: strict circadian cyclicity. We
know that such circadian cyclicity is linked to the lessening of glycemia and glycative damage.
We know, moreover, that deficient circadian cyclicity conduces to one of the defining
diseases of aging—diabetes—and that diabetics display a particular pathophysiological
phenotype marked by attenuated clock gene expression. In the estimation of the
Architekton, this places Amen Protocol Practitioners in quite a privileged position
theoretically, empirically, and most importantly, practically.
Fittingly, we shall finalize our investigative foray into the conceptual convergence of
circadian cyclicity and the pathophysiology of hyperglycemia with a segment on slumber.
Commoners can spend upwards of a third of their existence in this inert state and Amenites
in optimal health can pass fully a fifth of life in a somnolent state. Assuredly slumber is
salubrious and it helps us heal, recuperate, and integrate our thoughts. But the salubriousness
of sleep circuitously depends upon a salutary state of being. People are often perplexed
(perhaps even incredulous or envious) that the Architekton requires an average of 4.5 hours
of sleep per night and rarely sleeps more than 5 hours. This coming from one who values
the vitalizing quality of sleep and is far from shunning the inactivity it entails. Intriguingly,
there is evidence that my ability to optimally function with comparatively little sleep (of a

41
confessedly high quality) may be attributable to my chronic curtailment of circulating sugar
and the strict synchronicity of my circadian state. For this revelation Shanghai scientists are
responsible and their research is deserving of discussion. This is what is reported in their
Abstract in the International Journal of Endocrinology:

We investigated whether poor sleep quality is associated with both dawn


phenomenon and impaired circadian clock gene expression in subjects with
diabetes. Methods. 81 subjects with diabetes on continuous glucose
monitoring were divided into two groups according to the Pittsburgh Sleep
Quality Index. The magnitude of dawn phenomenon was quantified by its
increment from nocturnal nadir to prebreakfast. Peripheral leucocytes were
sampled from 81 subjects with diabetes and 28 normal controls at 09:00.
Transcript levels of circadian clock genes (BMAL1, PER1, PER2, and PER3)
were determined by real-time quantitative polymerase chain reaction. Results.
The levels of HbA1c and fasting glucose and the magnitude of dawn
phenomenon were significantly higher in the diabetes group with poor sleep
quality than that with good sleep quality. Peripheral leucocytes from subjects
with poor sleep quality expressed significantly lower transcript levels of
BMAL1 and PER1 compared with those with good sleep quality. Poor sleep
quality was significantly correlated with magnitude of dawn phenomenon.
Multiple linear regression showed that sleep quality and PER1 were
significantly independently correlated with dawn phenomenon. Conclusions.
Dawn phenomenon is associated with sleep quality. Furthermore, mRNA
expression of circadian clock genes is dampened in peripheral leucocytes of
subjects with poor sleep quality.I

Of incidental interest, this is how the investigators operationalized sleep quality:

A total of 81 subjects with type 2 diabetes were divided into two groups
according to the Pittsburgh Sleep Quality Index (PSQI), including a diabetes
group with poor sleep quality and a diabetes group with good sleep quality.
PSQI is a self-reported questionnaire that assesses sleep quality and
disorders. It contains seven component scores: subjective sleep quality, sleep
latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of
sleeping medication, and daytime dysfunction. The sum of the scores for
these seven components determined sleep quality classification: more than 7
points indicated poor sleep quality and less than or equal to 7 points
indicated good sleep quality (the threshold of poor sleep quality was 7 points
in Chinese people as reported. (Ibidem).

Impressively, the investigators determined the degree to which diabetics displayed definitive
signs of the Dawn Phenomenon; this they defined as exhibiting a difference from the
nocturnal nadir (i.e. maximum) of glucose concentration to the morning pre-meal minimum

IHuang Y, Wang H, Li Y, Tao X, Sun J. Poor Sleep Quality Is Associated with Dawn Phenomenon and
Impaired Circadian Clock Gene Expression in Subjects with Type 2 Diabetes Mellitus. International Journal of
Endocrinology. 2017.

42
of greater than 20 mg/mL. Those subjects with a ‘Dawn Delta’ (∆ or ∂) exceeding this
threshold tellingly exhibited higher fasting sugar levels, greater glycative damage and poorer
quality of sleep. Further, these ‘Dawn-doomed diabetics’ evidenced alterations in the
expression of the main genes that modulate circadian cycles: BMAL1, PER1, PER2,
and PER3. It should not now surprise us that compared to non-diabetic controls, those with
the disease exhibited attenuated expression of the cardinal clock genes.
Now Amenites, the Architekton asks whether the technical information herein
analyzed is actionable? The answer is ‘not necessarily to a prudent Practitioner of our
Protocol’. Nevertheless, the information is illuminating and confirmatory. That is, it confirms
our consistent contention that our quality of life is appreciably improved especially by
adhering to the Amen Fast—for frankly, the quality of one’s sleep contributes considerably
to the quality of one’s life. It also provides an empirically validated explanation for why
Incipient Amenites almost invariably experience a simultaneous suppression of sleep
duration and an improvement of sleep quality upon commencing the Regimen. It is the
regularity of our Regimen and the reduction in glycemia and glycation that concomitantly
contribute to close circadian control and qualitative improvements in sleep; the convergence
of these conditions clearly conduces to life prolongation and optimization.

Excessive circulating sugar during the dawn is characteristic of the age-accelerating


condition of diabetes, is associated with disruptions in circadian synchronicity and
compromises the quality of sleep. By averting glycemic excess, the Amen Fast antagonizes
the aforementioned impairments and potentiates prolongation of life.

Tea & Other Herbal Infusions Imbibed During the Amen Fast Forestall Glycation and
Accordingly Conduce to Lifespan Extension

It is perhaps an opportune time to explain why the Amen Protocol prescribes the imbibition
of teas and herbal infusions instead of simply water during the course of our Divine Diurnal
Fast and our discourse shall be characteristically didactic. The Author thus inquires of the
Adherent, ‘What is the uppermost aim of our Agenda?’ ‘Life extension’, the Acolyte utters.
‘What is a main mechanistic means by which the Amen Fast facilitates this aim?’
‘Suppression of circulating serum sugar and consequent curtailment of glycation’ is the
informed response. ‘Is water alone able to induce such an anti-AGE effect’? ‘Negative’ the
Neophyte rightly responds. ‘Alternatively, are there not diverse drinks that demonstrate such
a salutary sugar-suppressing, anti-AGE effect?’ ‘Indeed there are, Grand Inquisitor.’ ‘Can
you name some Novice?’ ‘Assuredly Architekton: Honeybush, Hibiscus, and Red, Black, and
Green teas of various types to which multifarious medicinal spices may be added to enhance
the anti-aging effect.’ ‘Most excellent elaboration Incipient Amenite.’ [Though the Author
might mention His coveted concoction called ‘Tiba’s Trisula Tea’ to more favored Followers
or add Chamomile, Blackberry Leaf and Licorice infusions to enliven and enlarge the list of
likely lifespan lengthening beverages.] In part, this protracted preamble was prompted by the

IPappa KI, Gazouli M, Anastasiou E, Iliodromiti Z, Antsaklis A, Anagnou NP. Circadian clock gene expression
is impaired in gestational diabetes mellitus. Gynecol Endocrinol. 2013 Apr;29(4):331-5.

43
practice (persistent and puerile if not especially pervasive) of what is called “water fasting”.
There are those who regard it as rigorous; the Author regards it as absurd, irrational,
inconsistent and unnecessary. For many individuals who avow the practice of water fasting
are similarly seeking to suppress senescence. So, why would they eschew ingesting non-
calorific items exhibiting properties that potentiate lifespan extension? No sensible answer
has ever been offered to this unambiguous inquiry. Our argument is equally poignant when
we ponder the other mechanisms by which the Amen Fast promotes life prolongation:
namely, enhancement of autophagy, opposition of adiposity, augmentation of antioxidant
defense, induction of neurotrophins, improvement of immune function and attenuation of
inflammation, and (most concordant with claims of our wayward ‘water fasters’)
enhancement of systemic detoxification. Each of the Ten Tractates comprising the present
Volume shall expound briefly upon the manner in which one or more teas imbibed during
the Amen Fast facilitate the effect under analysis in the respective Fascicle—i.e. on
Autophagy, Adiposity, Antioxidant Defense, Detoxification, etc. In this section we shall
confine our discussion to Glycemia/Glycation.
It is fitting to focus first on a study featured in Phyotomedicine. For this analysis affirms
the ability of Aspalathus to extend lifespan in C. elegans in a manner analogous to
amplification of the glyoxalase enzyme in an environment of elevated glucose as exposited
above:

Rooibos leaves and fine stems (Aspalathus linearis; Fabaceae) are increasingly
enjoyed as herbal tea, largely in fermented (oxidised) red-brown form, but
also in unfermented (unoxidised) green form. Rooibos is rich in antioxidant
polyphenols, with the dihydrochalcone, aspalathin, as a major active
ingredient. We used Caenorhabditis elegans as a model organism to investigate
the effect of rooibos extracts against oxidative stress in vivo. In a high glucose
environment, C. elegans treated with rooibos extract exhibited an extended
lifespan. Furthermore, green rooibos was a more potent antioxidant than red
rooibos, probably due to its substantially higher aspalathin content. In
addition, rooibos decreased acute oxidative damage caused by the superoxide
anion radical generator, juglone, with aspalathin playing a major role in
improving the survival rate of C. elegans. Quantitative real-time PCR results
demonstrated that aspalathin targets stress and ageing related genes, reducing
the endogenous intracellular level of ROS. These findings suggest that
rooibos increases stress resistance and promotes longevity under stress,
probably mediated via a regulation of the DAF-16/FOXO insulin-like
signaling pathway, supporting some of the health claims put forward for
rooibos tea.I

Once again this report reminds us of the important interrelationship between oxidation and
glycation, with the active agent aspalathin inhibiting both pernicious processes. Further,
though the fermented form is fine, the unfermented variety is more medicinal in mitigating
glycation and oxidation owing to elevated amounts of aspalathin therein. As we shall see
forthwith, the same salubrious superiority is evident in the other exemplary South African
tea, Honeybush, the unoxidized form having a higher efficacy than the oxidized form. This,

IChen W, Sudji IR, Wang E, Joubert E, van Wyk BE, Wink M. Ameliorative effect of aspalathin from rooibos
(Aspalathus linearis) on acute oxidative stress in Caenorhabditis elegans. Phytomedicine. 2013 Feb 15;20(3-4):380-6.

44
moreover, may be due to more mangiferin and hesperidin being extant in the unoxidized
varieties of the tea:

A variety of biological pro-health activities have been reported for mangiferin


and hesperidin, two major phenolic compounds of Honeybush (Cyclopia sp.)
tea extracts. Given their increasing popularity, there is a need for
understanding the mechanisms underlying the biological effects of these
compounds. In this study, we used real-time cytotoxicity cellular analysis of
the Cyclopia sp. extracts on HeLa cells and found that the higher hesperidin
content in non-fermented “green” extracts correlated with their higher
cytotoxicity compared to the fermented extracts. We also found that
mangiferin had a modulatory effect on the apoptotic effects of hesperidin.
Quantitative PCR analysis of hesperidin-induced changes in apoptotic gene
expression profile indicated that two death receptor pathway members,
TRADD and TRAMP, were up regulated. The results of this study suggest
that hesperidin mediates apoptosis in HeLa cells through an extrinsic
pathway for programmed cell death.II

Though the above investigation was aimed at exploring the effects of Honeybush on
apoptosis instead of glycation, its identification of hesperidin as the active agent enables us
to extrapolate accordingly and enlist other studies such as the one presented presently in
PLoS One:

Aim: Diabetic neuropathy (DN) is one of the most common long-term


complications of diabetes mellitus and clinically can be characterized by an
elevated nociceptive response with electrophysiological conduction
abnormalities. The present investigation was designed to evaluate the
neuroprotective effect of hesperidin against STZ induced diabetic
neuropathic pain in laboratory rats.

Materials & Methods: DN was induced in Sprague-Dawley rats (150-200 g) by


intraperitoneal administration of streptozotocin (STZ) (55 mg/kg, p.o.
[orally]). Rats were divided into various groups, namely, STZ control
(vehicle), hesperidin (25, 50, and 100 mg/kg, p.o.), insulin (10 IU/kg, s.c.
[subcutaneously]), and combination of hesperidin (100 mg/kg, p.o.) with
insulin (10 IU/kg, s.c.) for 4 weeks. Various behavioral (allodynia [i.e.
aberrant pain produced by a normally non-noxious stimulus] and
hyperalgesia), biochemical parameters (oxido-nitrosative stress, Na-K-
ATPase, aldose reductase (AR)), and molecular changes (TNF-α and IL-1β)
along with hemodynamic changes were determined.

Results: Rats treated with hesperidin (50 and 100 mg/kg, p.o., 4 weeks)
significantly reduced (p < 0.05) hyperglycemia and its metabolic

IIBartoszewski R, Hering A, Marszałł M, Stefanowicz Hajduk J, Bartoszewska S, Kapoor N, Kochan K,


Ochocka R. Mangiferin has an additive effect on the apoptotic properties of hesperidin in Cyclopia sp. tea
extracts. PLoS One. 2014 Mar 14; 9(3):e92128.

45
abnormalities induced by intraperitoneal administration of STZ. The
decreased nociceptive threshold, motor nerve conduction velocity (MNCV)
and sensory nerve conduction velocity (SNCV), serum insulin as well as Na-
K-ATPase activity were significantly increased (p < 0.05) by hesperidin (50
and 100 mg/kg, p.o.) treatment. It significantly attenuated (p < 0.05) elevated
glycated hemoglobin, AR activity, oxido-nitrosative stress, neural calcium,
and pro-inflammatory cytokines (TNF-α and IL-1β) levels. Histological
aberration induced after STZ administration was restored by administration
of hesperidin (50 and 100 mg/kg, p.o.).

Conclusion: In combination with insulin, hesperidin not only attenuated the


diabetic condition but also reversed neuropathic pain via control over
hyperglycemia as well as hyperlipidemia to down-regulate generation of free
radicals, release of pro-inflammatory cytokines as well as elevation in
membrane bound enzyme.I

While the above Abstract does not indicate that the hesperidin alone, in the absence of
insulin, exerts an ameliorative effect in the in vivo experiment, the ensuing Abstract in
Pharmaceutical Biology does indeed demonstrate the in vitro antiglycative effect of hesperidin. It
reads as follows:

...Objective: To evaluate the inhibitory potential of hesperidin, its derivatives


and their stereoisomers against advanced glycation end-products (AGEs)
formation.

Materials & Methods: Hesperidin and hesperetin were chirally separated and
the inhibitory effects of 1:1 mixture of (2S)- and (2R)-hesperidin (1), (2S)-
hesperidin (2), (2R)-hesperidin (3), 1:1 mixture of (S)- and (R)-hesperetin (4),
(S)-hesperetin (5), (R)-hesperetin (6), and monoglucosyl hesperidin (7) [1:1
mixture of (2S)-glucosyl hesperidin (8) and (2R)-glucosyl hesperidin (9)] at a
concentration of 1 mM on protein glycation reaction have been revealed
using the newly constructed RNase A-methylglyoxal (MGO) assay for the
early stage and the bovine serum albumin (BSA)-glucose assay for the late
stage of Maillard reaction.

Results: This study has demonstrated that hesperidin and its derivatives
possessed relatively strong activity against the formation of AGEs. (S)-
Hesperetin (5) possessed the highest inhibitory rate up to 57.4% in BSA-
glucose assay, 38.2% in RNase A-MGO assay.

Discussion & Conclusion: The new RNase A-MGO assay system could be used
for the screening of AGE inhibitors and hesperidin, and its derivatives could
be promising candidates adjuvants for the treatment of diabetes
complication, and age-related chronic diseases.

IVisnagri A, Kandhare AD, Chakravarty S, Ghosh P, Bodhankar SL. Hesperidin, a flavanoglycone attenuates
experimental diabetic neuropathy via modulation of cellular and biochemical marker to improve nerve
functions. Pharmaceutical Biology. 2014; 52(7):814-28.

46
While Amen Adherents may not be especially interested in the novel assay employed by the
investigators to ascertain hesperidin/hesperetin’s anti-glycative efficacy, we are appreciative
of their findings and the Author confessedly finds interest in the fact that the scientists saw
fit to submit their study to the journal Pharmaceutical Biology, indicating their interest in
developing an additional drug for the disease of diabetes. This is admirable, for such drugs
are desperately needed by more and more of mankind, since the scourge is spreading
stepwise with the waistlines of the wealthy and even among the impoverished who see
overindulgence at every opportunity as their sole secure solace in an otherwise insecure
existence. But is it not better to employ such an agent as hesperidin to help prevent
pathological conditions such as diabetes? Is it not better to integrate hesperidin-rich
Honeybush into a rational regimen that reduces the risk of premature mortality and diverse
diseases among which diabetes is perhaps one of the most devastating? This, at least, is the
enlightened outlook of an Inveterate Amenite.
Now we turn to another anti-glycative tea also commonly cultivated on the African
continent—Hibiscus.

Excursus: The beauty and ubiquity of this popular plant causes it to be coveted and
claimed by many cultures, it being emblematic of the Divine Goddess Kali, honored
by the Atlantic isle of Haiti, and held by Hawaiians as symbolic of their paradisiacal
state. While we recognize the reverence that Koreans and Malaysians have for the
flower as well, we wish to appreciate its etymological origin from the ancient Greek,
ιβισκος (hibiskos).

The ensuing Abstract from the Journal of Agricultural & Food Chemistry enumerates and
explicates the impressive health effects elicited by Hibiscus:

H. sabdariffa polyphenolic extract (HPE) was demonstrated to inhibit high


glucose-stimulated cellular changes. In this study, we analyzed the
composition of HPE and used a type 2 diabetic rat model to test its
protective effect. At least 18 phenolic compounds were found in HPE.
Treatment with HPE reduced hyperglycemia and hyperinsulinemia, especially
at the dose of 200 mg/kg. HPE decreased serum triacylglycerol, cholesterol,
and the ratio of low density lipoprotein/high density lipoprotein
(LDL/HDL). Diabetes promoted plasma advanced glycation end product
(AGE) formation and lipid peroxidation, while HPE significantly reduced
these elevations. Immunohistological observation revealed that HPE
inhibited the expression of connective tissue growth factor (CTGF) and
receptor of AGE (RAGE), which was increased in type 2 diabetic aortic
regions. Furthermore, HPE recovered the weight loss found in type 2
diabetic rats. In conclusion, we demonstrated the anti-insulin resistance
properties of HPE and its effect on hypoglycemia, hypolipidemia, and
antioxidation. HPE has the potential to be an adjuvant for diabetic therapy.I

The hue of Hibiscus, its rich ruddiness verging on vermillion or scarlet when strongly steeped,

I Peng CH, Chyau CC, Chan KC, Chan TH, Wang CJ, Huang CN. Hibiscus sabdariffa polyphenolic extract
inhibits hyperglycemia, hyperlipidemia, and glycation-oxidative stress while improving insulin resistance. J Agric

47
is as exquisite as its floral fragrance and tart taste, especially when enhanced with other
botanical embellishments such as stevia and various fruit essences as is the Author’s wont.
Observe, Amenites, the three-fold efficacy of this fine flower; it inhibits
glycation/hyperglycemia, attenuates insulin, and reduces free radical damage as particularly
evidenced by protection against membrane lipid peroxidation. Of course it exerts other
ameliorative effects. But, as ever, Amenites are most interested in those effects that accord
with experimental lifespan extension, and the three aforementioned modalities manifestly
modulate mortality in multiple model systems, mammalian and otherwise. Direct evidence of
the anti-aging efficacy of Hibiscus is available from an exhaustive investigation utilizing C.
elegans as the experimental system and exposing this organism to numerous natural agents
including our adored anthos I

Enhanced blood glucose levels are a hallmark of diabetes and are associated
with diabetic complications and a reduction of lifespan. In order to search
for plant extracts that display preventive activities in such a scenario, we
tested 16 extracts used in human nutrition for their survival enhancing
activities in the nematode Caenorhabditis elegans. Nematodes were exposed for
48 h to 10 mM glucose in the absence or presence of 0.1% extract.
Thereafter, survival was measured at 37 °C. Extracts made from coffee, kola,
rooibos and cinnamon, did not influence the glucose-induced reduction of
survival. Those made from ginseng, chamomile, lime blossom, paraguay tea,
balm, rhodiola, black tea, or knotgrass all extended the lifespan of the
glucose-treated nematodes significantly but did not rescue survival
completely. Extracts from the leaves of blackberries, from hibiscus,
elderberries, or jiaogulan completely countered the glucose-induced survival
reduction. A potent activation of the proteasome was shown for the most preventive
extracts suggesting a more efficient degradation of proteins impaired by glucose. In
conclusion, we present a simple animal model to screen for plant extracts
with potency to reverse glucose toxicity. Extracts from blackberry leaves,
hibiscus, elderberries, and jiaogulan were identified as very potent in this
regard whose exact mechanisms of action appear worthwhile to investigate at
the molecular level. [Emphasis added by the Author in italic font.]

We Amenites would, admittedly, be ever more elated were other items in our august
Apothecary (such as cinnamon, rooibos, and coffee) similarly found to extend C. elegans
lifespan in this study. But we must be content that, along with Hibiscus, our beloved black tea
and ginseng lengthened lifespan in these sugar-saturated subjects. Further, we must make
mention of the mechanisms reportedly responsible for the reduction in mortality: the
simultaneous suppression of protein damage due to sugar and enhanced degradation of such
damaged proteins by an enzymatic cellular system called the proteasome. Because this latter
effect shall occupy appreciable attention and space in our expositions on autophagy and the
Amen Fast, we shan’t elaborate upon it presently. But we wish to stress that it is an
important observation and arms us with the ability to enhance this effect. Lastly, we must
appreciate that the above investigation is a single study, employing a single experimental
environment and assessing agents in isolation from each other; conceivably, different

Food Chem. 2011 Sep 28;59(18):9901-9.


Iανθος anthos, Gk. flower.

48
conditions and combinations of compounds could collectively conduce to lifespan extension
even in this specific system in a manner mirroring ‘Selective Synergism’. This postulate is
particularly plausible if the protection of proteins proffered thereby is potent, pervasive, and
persistent enough—precisely what the Amen Protocol is predicated upon producing.

Continuing our discourse with


another commodity originating from
Africa, we commence with coffee. This
discussion demands some exegesis
however. For while we shall have no
difficulty defending our imbibition of
coffee in the context of our analyses of
autophagy, adiposity, antioxidant defense,
and especially detoxification enhancement,
there is a conspicuous characteristic of
coffee that superficially makes it seem
contrary to the aim of curtailing glycation:
coffee contains an appreciable amount of
AGEs. This is so simply as a consequence
of cooking—coffee beans being routinely
roasted, a preparatory practice that
inevitably results in the creation of
advanced glycation end-products. Do we
not endeavor to avoid these inimical
agents? Why then would we willingly ingest
them, especially during the interval of our
Regimen wherein the suppression of
circulating sugar and the inhibition of
glycation are integral aims (i.e. in the hours of the Amen Fast)? The most important aspect
of our argument is that the uppermost intent of the Amen Protocol is lifespan prolongation
and there is convincing evidence (as we shall see in subsequent segments of our
Encyclopedia) that compounds in coffee conduce to significant life extension. Further, the
complexity of coffee is such that, though it contains AGEs outright, it also contains agents
that lower glucose concentration and inhibit glycation directly. This in addition to the
properties pertinent to other aspects of the Amen Fast and our overall Regimen generally—
namely, coffee’s capacity to attenuate adiposity, induce autophagy, enhance detoxification,
and promote proliferation and protection of brain cells.
Amenites and Observers can count themselves fortunate that the popularity of
coffee has compelled scientists to conduct copious investigations into the effects of this
bold, black beverage on commonly considered health effects, the most important of which
being mortality itself. Our intellectual fortune is furthered by the fact that, due to the
diversity of studies on the epidemiological impact of coffee intake, it was deemed desirable
to essentially synthesize such studies in a systematic way in order to ascertain more incisively
its overall effect in multiple populations over a protracted period of time. The type of

49
epidemiological study to which the Author refers is known as a Meta-Analysis. In essence, it
amounts to a study of studies, systematically selected for their similarity, and statistically
summed in such a way as to paint a picture or provide a quantified, numerical assessment of
a phenomenon in aggregate. In the context of coffee, such a study was conducted by Drs. Je
and Giovanunucci and published in the British Journal of Nutrition. This is how they
summarized their ‘Meta-Synthetic’ study:

Coffee consumption has been shown to be associated with various health


outcomes, but no comprehensive review and meta-analysis of the association
between coffee consumption and total mortality has been conducted. To
quantitatively assess this association, we conducted a meta-analysis of
prospective cohort studies. Eligible studies were identified by searching the
PubMed and EMBASE databases for all articles published through June
2013 and reviewing the reference lists of the retrieved articles. Pooled relative
risks (RR) with 95% CI were calculated using a random-effects model. We
identified twenty studies of coffee consumption and total mortality, including
129,538 cases of deaths among the 973,904 participants. The RR of total
mortality for the high v. low category of coffee consumption was 0.86 (95%
CI 0.80, 0.92). The pooled RR for studies using ≥ 2-4 cups/d as a cut-off for
the high category was similar to that for studies using ≥ 5-9 cups/d as the
cut-off. By geographical region, the inverse association tended to be stronger
for the eight studies conducted in Europe (RR 0.78, 95% CI 0.70, 0.88) and
three studies carried out in Japan (RR 0.82, 95% CI 0.73, 0.92) than for the
nine studies conducted in the USA (RR 0.92, 95% CI 0.84, 1.00). The inverse
association was similar for men (RR 0.81, 95% CI 0.73, 0.90) and women
(RR 0.84, 95% CI 0.79, 0.89). A weak, but significant, inverse association was
found with moderate coffee consumption (1-2 cups/d; RR 0.92, 95% CI
0.87, 0.98). High decaffeinated coffee consumption was also found to be
associated with a lower risk of death, but the data are limited. Our findings
indicate that coffee consumption is associated with a reduced risk of total
mortality.I

Permit the Author to underscore several particularly important points concerning the above
investigation. (I) It was prospective—that is, it involved studies in which individuals were
followed from a particular point, thence onward into the future, thereby rendering the
imputation of a causal effect of coffee consumption more defensible; (II) It was large,
involving almost a million participants (or more accurately, data derived therefrom); (III) It
was geographically diverse, involving information extracted from Asians, Americans, and
Europeans; and (IV) The most momentous finding: A dose-response relationship was
discernible, with the rate of death decreasing with increased intake of coffee. As shall be
indicated elsewhere in our Encyclopedia when discussing epidemiological investigations,
though causality cannot be definitively determined by any non-experimental study, several
factors enable us to reasonably assume that an observed association is indeed causal (instead
of simply incidental) in nature: apart from the mere magnitude of the statistical association,
these include temporality, mechanistic plausibility, an apparent dose-response relationship.

I Je Y, Giovannucci E. Coffee consumption and total mortality: a meta-analysis of twenty prospective cohort
studies. Br J Nutr. 2014 Apr 14;111(7):1162-73.

50
The Meta-Analysis before us fits each of these ‘quasi-causal’ criteria as we may rightly
characterize it. A less important point that nonetheless warrants mention is the finding that
decaffeinated coffee was also associated with a reduction in overall mortality risk, though the
effect was far less substantial. Clearly this means that compounds other than caffeine (and
other agents eliminated by the decaffeination process) exert an ameliorative effect on
mortality. Thus, those inclined to abstain from ingesting coffee due to its considerable
caffeine content (ostensibly endeavoring to avoid over-stimulation) may enjoy the mortality
mitigating effect of the dark drink devoid of this energizing element that may be too much
for many.

Now that we have discussed death and its diminution deriving from coffee
consumption, we turn more directly to the narrower topic of this specific Tractate: AGE
inhibition. Compelling evidence of the capacity of coffee to curtail glycation (and
consequently conduce to conditions leading to lifespan lengthening) is to be found in the
following Abstract from the Archives of Pharmacal Research. More specifically, a substance
present in coffee—chlorogenic acid—exceeded the ability of aminoguanidine to inhibit
glycation; this agent is often employed by investigators as the gold standard, the exemplar of
effective AGE-inhibitors. Dr. Kim and colleagues inform us that:

...Chlorogenic acid (CGA) is a phenolic compound formed by the


esterification of caffeic and quinic acids. In this study, we evaluated the
inhibitory effects of CGA against the formation of AGEs and AGEs protein
cross-linking in vitro. An in vitro assay for glycation of bovine serum
albumin by high glucose showed that CGA inhibited AGEs formation with
an IC(50) value of 148.32 µM and was found to be more effective than
aminoguanidine, a well-known AGEs inhibitor (IC(50); 807.67 µM). In an
indirect AGE-ELISA assay, the CGA exhibited more potent inhibitory
activity on the cross-linking of AGEs to collagen than aminoguanidine. In
addition, the inhibitory effects of CGA on AGEs formation and on its cross-
linking with collagen might be caused by its interactions with reactive
dicarbonyl compounds, such as methylglyoxal. These results suggest that
CGA could be beneficial in the prevention of AGEs progression in patients
with diabetes because CGA can attenuate AGEs deposition in glucose.I

Not only can substances in coffee curtail collagen cross-linkage (consequently conferring
protection to the joints of which they largely compose) and suppress circulating sugar
concentration, the chemicals composing coffee undergo conversion in the colon and these
induce independent ameliorative effects according to the ensuing study. The Italian
investigators from the University of Modena inform us that:

...Dietary flavonoids and allied phenolic compounds are thought to be


beneficial in the control of diabetes and its complications, because of their
ability to inhibit oxidative stress, protein glycation and to act as
neuroprotectants. Following ingestion by humans, polyphenolic compounds
entering the large intestine undergo extensive metabolism by interaction with

IKim J, Jeong IH, Kim CS, Lee YM, Kim JM, Kim JS. Chlorogenic acid inhibits the formation of advanced
glycation end products and associated protein cross-linking. Archives of Pharmacal Research. 2011; 34(3):495-500.

51
colonic microbiota and its metabolites and catabolites of the parent
compounds that enter the circulatory system. The aim of this study was to
investigate the inhibitory activity of some colonic microbiota-derived
polyphenol catabolites against advanced glycation endproducts formation in
vitro and to determine their ability, at physiological concentrations, to
counteract mild oxidative stress of cultured human neuron cells.

Methods & Results: This study demonstrated that ellagitannin-derived


catabolites (urolithins and pyrogallol) are the most effective antiglycative
agents, whereas chlorogenic acid-derived catabolites (dihydrocaffeic acid,
dihydroferulic acid and feruloylglycine) were most effective in combination
in protecting neuronal cells in a conservative in vitro experimental model.

Conclusion: Some polyphenolic catabolites, generated in vivo in the colon,


were able in vitro to counteract two key features of diabetic complications,
i.e. protein glycation and neurodegeneration. These observations could lead
to a better control of these events, which are usually correlated with
hyperglycemia.I

Let us look, and mayhap marvel, at how closely certain components of the Amen Protocol
comport with the insights offered above. The Author often consumes his coffee in the form
of crude grounds pulverized into a powdery, espresso-like consistency (to which pure cocoa
and stevia are added). The crudeness of the concoction ensures that a considerable portion
of the coffee preparation passes into the colon. As we shall discuss in Volume III:I,
mutualistic microbes are added to the Amen Amino Aliksir and additional encapsulated
probiotic pills are ingested during the Amen Fast. Elevated amounts of benign microbes in
the intestines enhance the production of polyphenol catabolites of coffee. As these anti-
glycative agents are absorbed by the intestinal enterocytes and enter the systemic circulation,
they continually contribute to the curtailment of glycation throughout the body. This
synergistic effect is impressive indeed.
Now that we know at least one mechanism by which coffee, its chemical
constituents, and its colonic metabolites may mitigate mortality—that is, by preventing
protein damage deriving from glycation—it is appropriate to analyze yet more
epidemiological evidence indicating its pro-longevity potential. Before recounting this
research, I ask the Reader to recall one important criterion by which epidemiological studies
may intimate a causal connection between independent and dependent variables: namely,
mechanistic or biologic plausibility. Quite simply it is plausible, given what we know about
the influence of glycation upon aging and the capacity of coffee to curtail glycation, that an
observed effect of coffee on the extension of lifespan is likely attributable in part to the
inhibition of AGE-induced tissue damage; the development of diabetes is determined by the
extent of glycative damage incurred by an individual. The epidemiological evidence that we
shall explore forthwith established the existence of an inverse relationship between the
extent of daily coffee intake and the development of diabetes in Athenians observed over a

IVerzelloniE, Pellacani C, Tagliazucchi D, Tagliaferri S, Calani L, Costa LG, Brighenti F, Borges G, Crozier A,
Conte A, Del Rio D. Antiglycative and neuroprotective activity of colon-derived polyphenol catabolites.
Molecular Nutrition & Food Research. 2011; 55 Suppl 1:S35-43.

52
decade. This is how the scientists summarized their study:

Background/Objectives: The purpose of this work was to investigate the


association between coffee drinking and diabetes development and potential
mediation by oxidative stress and inflammatory biomarkers.

Subjects/Methods: In 2001–2002, a random sample of 1514 men (18–87 years


old) and 1528 women (18–89 years old) were selected to participate in the
ATTICA study (Athens metropolitan area, Greece). A validated food-
frequency questionnaire was used to assess coffee drinking (abstention,
casual, habitual) and other lifestyle and dietary factors. Evaluation of
oxidative stress and inflammatory markers was also performed. During
2011–2012, the 10-year follow-up of the ATTICA study was carried out. The
outcome of interest in this work was incidence of type 2 diabetes, defined
according to American Diabetes Association criteria.

Results: During follow-up, 191 incident cases of diabetes were documented


(incidence 13.4% in men and 12.4% in women). After various adjustments,
individuals who consumed 250 ml of coffee (≈1.5cup) had 54% lower odds
of developing diabetes (95% confidence interval: 0.24, 0.90), as compared
with abstainers. A dose-response linear trend between coffee drinking and
diabetes incidence was also observed (P for trend=0.017). When controlling
for several oxidative stress and inflammatory biomarkers, the inverse
association between habitual coffee drinking and diabetes was found to be
mediated by serum amyloid-A levels.

Conclusions: This work highlights the significance of long-term habitual coffee


drinking against diabetes onset. The anti-inflammatory effect of several
coffee components may be responsible for this protection.I

Does the Disciple need reminding that diabetes is a disease wherein those afflicted display
accelerated aging, showing those signature signs of senescence that uncontrolled
concentrations of sugar incessantly assail their cells, tissues and organs and impair almost
every physiological function occurring in their body and brain? It is for this reason that we
can rightly regard certain clinical and epidemiological investigations of diabetes in humans as
approximations of life-long aging studies in animals, though the latter sort of study is
substantively superior. For interventions that inhibit the development of diabetes can
generally be considered to exhibit an inherent anti-aging effect, especially since serum sugar
regulation is so central to the aging process as such. And this is exactly what the above
investigation found. Explicitly, in a sizeable, randomly selected sample of the Athenian
citizenry, ingesting a quarter-liter of coffee correlated with an approximately fifty percent
reduction in the incidence of diabetes relative to individuals who abstained therefrom. And
this effect was ascertained prospectively over a decade-long interval, exhibiting a discernible
dose response relationship—that is, the more coffee imbibed the more the diabetic malady

IKoloverou E, Panagiotakos DB, Pitsavos C, Chrysohoou C, Georgousopoulou EN, Laskaris A, Stefanadis C.


The evaluation of inflammatory and oxidative stress biomarkers on coffee-diabetes association: results from the
10-year follow-up of the ATTICA Study (2002-2012). Eur J Clin Nutr. 2015 Jul 1.

53
was mitigated. Importantly, though the diagnosis of diabetes is definitively determined by the
degree of fasting sugar elevation and commonly corroborated or reinforced by recourse to
the quantification of glycated hemoglobin, the investigators additionally assessed and
identified two other indicators that they deemed to be operative in coffee’s anti-diabetic
(and, perforce, anti-aging) effect: inflammation inhibition and antioxidant defense. So,
sympathetic Reader, be not dismayed should this same study be cited in subsequent sections
of Volume II, wherein the features of the Amen Fast are discussed in the context of the
aforementioned phenomena—inflammation and oxidation—specifically, in Fascicles VI and
IX.

The second line of evidence is afforded by a study published in the Journal of


Epidemiology. Practitioners should ponder that the study was, similar to the previous,
prospective and longitudinal, enabling investigators to analyze the effects of differential
coffee intake on death over time, nearly a decade indeed—an interval identical to the prior
study. Interestingly, in a land legendary for its laudation of traditional tea, it was instead the
‘Black Beverage’ from the “Black Land’ that mitigated mortality amongst the population of
rural Japanese from which the subjects were selected. This is what the Summary states:

We conducted a cohort study to investigate the effects of coffee and green


tea consumption on all-cause mortality in a rural Japanese population. Data
were obtained from 2,855 men and women aged 40-79 years in 1989, and
during the subsequent 9.9 years of follow-up. Using the Cox regression
model to adjust for potential confounding factors, we calculated the
multivariate hazard ratios of death from all causes separately for men and
women. The multivariate hazard ratio of mortality for men who consumed
two or more cups of coffee per day, compared with those who consumed
less than half a cup per day, was 0.43 (95% confidence interval, 0.30-0.63),
and the ratio for those who consumed half to one cup of coffee per day was
0.70 (95% confidence interval, 0.52-0.94). Exclusion of subjects with less
than 5 years of follow-up did not substantially change the findings. No other
statistically significant associations were identified between consumption of
the two beverages and all-cause mortality. For men, multivariate hazard ratios
of death from apoplexy showed a significant inverse association with
increasing coffee consumption. The effects of habitual coffee consumption
and its related factors on health in Japan need to be studied in greater detail.

Momentously, mortality risk was reduced by more than half in individuals imbibing two or
more cups of coffee per day relative to those ingesting a minimal amount (i.e. under a cup).
This is quite consistent with the conclusion of the Meta-Analysis with which our discourse
on coffee commenced. Tellingly, green tea consumption caused no (or more correctly
expressed, was not correlated with) comparable curtailment of mortality in this case. The
scientists understandably state their standard assessment that this should be “studied in
greater detail”. Perhaps so, especially as I thoroughly enjoy integrating actionable, important
information into our Regimen. However, we already know enough about the salubrious
quality of coffee to drink it daily and duly implant it into the Amen Apothecary and the
Amen Fast.

We are confident that Camellia senensis (i.e. conventional varieties of tea) can

54
undoubtedly conduce to lifespan lengthening in certain circumstances. Our confidence is
reinforced by several lines of research that can be concisely characterized as follows: (I)
Traditional tea or its chemical constituents independently lengthen lifespan in several
metazoan species; (II) Tea components mitigate mortality in mammals made to serve as
models for specific states (such as accelerated senescence) and (III) Tea, in combination with
two other polyphenols (to be discussed in Volume III:III), augments the lifespan-
lengthening effect of intermittent fasting in a murid model.I Moreover, numerous studies
have sought to establish in a mechanistic sense which substances in tea are responsible for its
age-reducing effects and how such age inhibition is effectuated. True to the topic of this
Tractate, reduced circulating sugar and inhibition of glycation are operative in tea’s
multifarious effects.
First we shall focus on the lifespan-lengthening effects of tea—this is demonstrated
in Drosophila, Caenorhabditis, and among mammalian models of the family Muridae. In the
ensuing Summary we are informed that an extract of green tea (with a quantified catechin
content) significantly extended the lifespan of Drosophila and that this lengthening of life was
linked to a lessening of oxidative damage to the metazoan’s membranes that was enabled, in
turn, by tea-induced increases in antioxidant enzyme expression.

Chinese Longjing green tea is an excellent source of polyphenol antioxidants.


HPLC [high-performance liquid chromatography] analysis revealed that
Longjing green tea catechin extract (GTC) contained 62% epigallocatechin
gallate (EGCG), 19% epigallocatechin (EGC), 9% epicatechin gallate (ECG),
and 7% epicatechin (EC). Investigating the effect of GTC on the lifespan of
Drosophila melanogaster, we observed that a 10 mg GTC/mL diet could
prolong its 50% survival time by 36% and mean lifespan by 16%. This was
consistent with 17% reduction in total body lipid hydroperoxide (LPO) level
in GTC-treated flies compared to the control group. Supplementation of 10
mg GTC/mL diet increased the survival time only in wild type Oregon-R-C
(OR) but not in two mutant fly lines, SOD(n108)/TM3 (gene for superoxide
dismutase (SOD) was knocked out) and Cat(n1)/TM3 (gene for catalase was
knocked out), when the flies were challenged with paraquat or hydrogen
peroxide. Accordingly, SOD and catalase activities in OR wild type increased
by 40 and 19%, respectively. RT-PCR analysis indicated that the genes for
copper-zinc containing SOD (CuZnSOD), manganese containing SOD
(MnSOD), and catalase were upregulated. It was concluded that prolonging
lifespan by GTC in D. melanogaster was influenced, among others, by
upregulation of endogenous antioxidant enzymes.II

The Reader is reminded, and shall be reminded in similar instances, that the experimental
induction of the antioxidant enzyme catalase through direct genetic modification has been
found to independently increase maximum lifespan in a mammalian model. This renders the
results of the study even more relevant and significant from our evaluative standpoint. The

IAires DJ, Rockwell G, Wang T, Frontera J, Wick J, Wang W, Tonkovic-Capin M, Lu J, E L, Zhu H, Swerdlow
RH. Potentiation of dietary restriction-induced lifespan extension by polyphenols. Biochimica et Biophysica Acta.
2012 Apr; 1822(4):522-6.
IILi YM, Chan HY, Huang Y, Chen ZY. Green tea catechins upregulate superoxide dismutase and catalase in

fruit flies. Molecular Nutrition & Food Research. 2007; 51(5):546-54.

55
next study is comparably compelling, for it found that feeding fruit flies fulsome fat
foreshortened their lives by damaging the lipids of their membranes and that exposing the
organisms to extracts of tea (as well as broccoli) inhibited this inimical effect of fat and
maximized the lifespan of these lipid-laden metazoans. From Dr. Li and colleagues we learn
that:

Dietary fat accelerates the ageing process and causes a greater mortality by
accumulating lipid hydroperoxide (LPO) in Drosophila melanogaster. The
present study found that the life span of D. melanogaster was shortened from
54 to 6 days in a dose-dependent manner when fat in diet increased from 0%
to 25%. The results showed that supplementation of both green tea catechins
(GTC) and broccoli extract (BE) reversed partially the fat-induced mortality.
The maximum life span was 44 days for the control group fed with a 5% fat
diet, whereas it increased to 50 and 59 days in the GTC- and BE-
supplemented groups, respectively. The 50% survival time for the control
flies fed with a 5% fat diet was 30 days. In contrast, it increased to 32 and 48
days when GTC and BE were supplemented in the diet. This was consistent
with a significant reduction in total body LPO level in D. melanogaster
maintained on the GTC- and BE-supplemented diet. Accordingly, catalase
and superoxide dismutase (SOD) activities increased significantly in the flies
fed with a GTC or a BE diet compared with those fed with a control 5% fat
diet. Reverse transcriptase-polymerase chain reaction analysis indicated that
the increase in enzymatic activities of catalase and SOD was accompanied by
up-regulation of genes for catalase, copper-zinc containing SOD and
manganese-containing SOD. It was concluded that GTC and BE reversed
the fat-induced mortality in D. melanogaster, most likely but not necessarily
solely, by up-regulation of endogenous antioxidant enzymes.I

The documented dose-dependent increase in the rate of death in Drosophila with increasing
assimilation of fat is instructive in itself and is indirectly related to the topic of the present
Tractate on the anti-glycative efficacy of the Amen Fast. For the fats forming membranes are
similarly subject to glycative attack and the ingestion of excessive amounts of fat stimulates
cellular multiplication—simply stated, membrane multiplication means the manufacture of
physiologically superfluous molecules susceptible to glycative and oxidative attack. Clearly,
this line of reasoning reinforces the rationality of making carbohydrates the compositional
cornerstone of a prudent, prolongevity protocol. But do ‘Dietetic Deviants’ exist even
among the disciplined Adherents of Amenism? Do some such Ascetics of our Ilk ingest
more fat than they should? Certainly so, though no confession shall be forthcoming from
the Architekton. It is therefore gratifying to find that ingesting green tea and its constituent
catechins in concentrated form can counteract the age-accelerating effects of super-optimal
fat intake.
Now let us learn what research reveals about the effects of tea in that other model
metazoan, Caenorhabditis. A pithy Abstract in Planta Medica explains that exposing strains of
C. elegans to an antioxidant compound contained in C. sinensis significantly extended the
lifespan of the organism by inhibiting oxidative damage therein. Thusly they state:

ILiYM, Chan HY, Yao XQ, Huang Y, Chen ZY. Green tea catechins and broccoli reduce fat-induced mortality
in Drosophila melanogaster. The Journal of Nutritional Biochemistry. 2008; 19(6):376-83.

56
Epigallocatechin gallate (EGCG) is a major green tea polyphenol with
pronounced antioxidative activity. The effects of EGCG on lifespan and
stress resistance in wild-type N2 and transgenic strains of Caenorhabditis elegans
[HSP-16.2/GFP, MEV-1(KN1), FEM-1(HC17)] were investigated. The
expression of HSP-16.2 (induced by the pro-oxidant juglone) and the
intracellular levels of H2O2 were inhibited by EGCG treatment. Daily
administration of 220 µM EGCG increased the mean lifespan by 10.14% and
14.27% in N2 and FEM-1(HC17) strains, respectively, and 55 µM EGCG
increased the mean lifespan in MEV-1(KN1) by 16.11%. The survival rate
was also increased under lethal oxidative stress by 65.05%. These findings
suggest that the increased mean lifespan and stress resistance in C. elegans
apparently depend, among other factors, on the antioxidant properties of
EGCG.I

Apart from EGCG, another chemical constituent of tea likewise effectuates lifespan
extension in C. elegans, in the absence as well as in the presence of a potent pro-oxidant
agent. The study was summarized thusly in the pages of The European Journal of Nutrition:

Purpose: Compounds that delay aging in model organisms may be of


significant interest to anti-aging medicine, since these substances potentially
provide pharmaceutical approaches to promote healthy lifespan in humans.
We here aimed to test whether pharmaceutical concentrations of L-theanine,
a putative anti-cancer, anti-obesity, blood pressure-lowering, and
neuroprotective compound contained in green tea (Camellia sinensis), are
capable of extending lifespan in a nematodal model organism for aging
processes, the roundworm Caenorhabditis elegans.

Methods: Adult C. elegans roundworms were maintained on agar plates, were


fed E. coli strain OP50 bacteria, and L-theanine was applied to agar to test
(1) whether it may increase survival upon paraquat exposure and (2) whether
it may promote longevity by quantifying survival in the presence and absence
of the compound.

Results: L-Theanine increases survival of C. elegans in the presence of paraquat


at a concentration of 1 micromolar. L-theanine extends C. elegans lifespan
when applied at concentrations of 100 nM, as well as 1 and 10 micromolar.

Conclusions: In the model organism C. elegans, L-theanine is capable of


promoting paraquat resistance and longevity suggesting that this compound
may as well promote healthy lifespan in mammals and possibly humans.II

As the ensuing excerpt from the researchers’ Results section states, the lengthening of

I Abbas S, Wink M. Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress
resistance in Caenorhabditis elegans. Planta Med. 2009 Feb;75(3):216-21. doi: 10.1055/s-0028-1088378.
II Zarse K, Jabin S, Ristow M. L-Theanine extends lifespan of adult Caenorhabditis elegans. Eur J Nutr. 2012

Sep;51(6):765-8.

57
lifespan linked to L-theanine was also observed in the absence of the oxidizing agent. This is
of more interest, intellectually and practically, as it enables us to exclude the possibility that a
particular effect of the oxidant (i.e. paraquat) is especially inhibited by theanine. The capacity
of the compound to extend the lives of normal nematodes not exposed to a noxious agent
permits us to presume that it can conceivably have a comparable effect in “normal” humans
such as ourselves. Thus they state:

While the following apparently does not apply in all cases, increased
resistance against paraquat stress suggests that L-theanine may exert effects
on C. elegans lifespan. Applying this compound at three different
concentrations (100 nM [nanomolar], 1 and 10 micromolar) to C. elegans
using the above-mentioned methods extends lifespan significantly
(P < 0.001, P = 0.008 and P = 0.002, respectively). This effect appears not to
be strictly dose-dependent, since no such correlation could be seen for ROS
defense capabilities while all concentrations evaluated had a lifespan-
extending effect. The maximum observable effect on mean lifespan and
maximum lifespan (80th percentile) was 0.8 days and 1.1 days, respectively,
which occurred at a concentration of 100 nM. (Ibidem)

We Amenites and Observers can also appreciate the investigators’ practical perspective, their
opining that the ability of this agent, extracted from tea, to mitigate mortality in a metazoan
automatically means that it has the plausible potential to lengthen lifespan in Man, our
ultimate aim. So state the scientists:

Since the current study has been performed in the model organisms C.
elegans, it is unclear whether our results can be extrapolated to mammals or
even humans. Hence, further studies will have to show whether L-theanine
has any effect on mammalian or human health span and/or longevity.
However, other compounds that have been identified using a similar,
metazoan-based approach have been shown to be effective in rodents. Taken
together, these findings indicate that L-theanine extends C. elegans lifespan
suggesting that this compound may be worth evaluating in mammals and
potentially humans in regard to prevention of aging and age-associated
diseases. (Ibidem)

With this conservative conclusion we Amenites concur.

Now we move on to mammals and that most mentally masterful (and mayhap most
miserable) member of the Class, Man. As is our wont, we are inclined to ascertain whether
any experimental evidence indicates that an intervention or agent independently extends
lifespan in a mammalian model. The Author already alluded to that seminal study, to which
we will turn in Volume III:III, establishing the ability of green tea along with two other plant
polyphenols to lengthen the lives of murids undergoing intermittent fasting. Despite the
profundity and practical positivity of this finding, it is not an independent effect. Rather, it is
an aggregate, collective, composite effect—an exquisite exemplification of Selective
Synergism/Summation. We shall also see a study cited in Volume II:III which inclines us,

58
tentatively, to accept that tea can lengthen lifespan independently in a mammalian model via
induction of autophagy. Though the following investigation did not determine that tea
constituents can lengthen lifespan in a “normal” mouse model, it is so applicable to such a
sizable segment of the human population that its importance may exceed that of a “normal”
investigation. For the investigation in question employed ‘age-accelerated’ animals whose
psycho-physical stress was experimentally exacerbated by the creation of a confrontational
social condition. I sincerely ask the Reader, is this state not identical or analogous to the
existence of innumerable men and women? Sadly, this is so for the Author in his vocational
capacity as an Ascetic Analyst. I imagine it is so for some of my fellow Amenites. The
potential of a simple, single compound from tea, to mitigate mortality, mental deficits,
emotional distress, and indices of neurological damage in such unfortunate animals and
(‘extrapolatively’) to morbid masses of Mankind, makes the ensuing study momentous
indeed. The thorough Summary in Free Radical Research recounts the relevant findings:

To evaluate the psychosocial effect on lifespan and cognitive function, this


study investigated the effect of confrontational housing on mice because
conflict among male mice is a psychosocial stress. In addition, it investigated
the anti-stress effect of theanine (γ-glutamylethylamide), an amino acid in tea.
Mice were housed under confrontation. That is, two male mice were
separately housed in the same cage with a partition for establishing the
territorial imperative in each mouse. Then, the partition was removed and
mice were co-housed confrontationally (confront-housing) using a model
mouse of accelerated-senescence (SAMP10) that exhibited cerebral atrophy
and cognitive dysfunction with ageing. It was found that mice began to die
earlier under confront-housing than group-housed control mice.
Additionally, it was found that cerebral atrophy, learning impairment and
behavioural depression were higher in mice under the stressed condition of
confront-housing than age-matched mice under group-housing.
Furthermore, the level of oxidative damage in cerebral DNA was higher in
mice housed confrontationally than group-housed control mice. On the
other hand, the consumption of purified theanine (20 µg/ml, 5-6 mg/kg)
suppressed the shortened lifespan, cerebral atrophy, learning impairment,
behavioural depression and oxidative damage in cerebral DNA. These results
suggest that psychosocial stress accelerates age-related alterations such as
oxidative damage, lifespan, cognitive dysfunction and behavioural
depression. The intake of theanine might be a potential candidate for
suppression of disadvantage under psychosocial stress.I

Attenuation of atrophy in the brain, diminishment of depressive symptomatology, inhibition


of impairments in learning: it is not surprising that an agent able to induce these impressive
effects should also lengthen lifespan. For as we shall sadly see in Volume VI of our
Encyclopedia, major psychological disorders do more than make their victims suffer socially
and emotionally—still further, they foreshorten their lives. This is yet another aspect of the
AIR that accords with the AAI—the Amen Anti-Addiction/Anxiety/Depression

IUnno K, Fujitani K, Takamori N, Takabayashi F, Maeda K, Miyazaki H, Tanida N, Iguchi K, Shimoi K,


Hoshino M. Theanine intake improves the shortened lifespan, cognitive dysfunction and behavioural
depression that are induced by chronic psychosocial stress in mice. Free Radic Res. 2011 Aug;45(8):966-74.

59
Intervention. This is why the imbibition of tea during the course of the Amen Fast factors as
a fundamental therapeutic component of the AAI (and ‘gerostatic’ element of the AIR): tea
compounds curtail the corollary neuro-cognitive damage that commonly accompanies most
major mental disorders including addiction. Such studies as this make the power of our
Protocol perspicuous to any objective Observer.
Though experimental evidence does not enable us to definitively declare that
increased tea intake attenuates mortality in Man, impressive observational (i.e.
epidemiological) evidence does indeed permit us to propound that tea plausibly exhibits life-
prolonging potential in Man. As in the case of coffee, we are indebted to investigators
employing the conceptually powerful epidemiological tool called Meta-Analysis. Similar to
the aforesaid coffee study, the scientists’ findings are featured in the British Journal of Nutrition.
The researchers relate that:

Epidemiological studies have demonstrated inconsistent associations


between tea consumption and mortality of all cancers, CVD and all causes.
To obtain quantitative overall estimates, we conducted a dose-response meta-
analysis of prospective cohort studies. A literature search in PubMed and
Embase up to April 2015 was conducted for all relevant papers published.
Random-effects models were used to calculate pooled relative risks (RR) with
95 % CI. In eighteen prospective studies, there were 12 221, 11 306 and 55
528 deaths from all cancers, CVD and all causes, respectively. For all cancer
mortality, the summary RR for the highest v. lowest category of green tea
and black tea consumption were 1·06 (95 % CI 0·98, 1·15) and 0·79 (95 %
CI 0·65, 0·97), respectively. For CVD mortality, the summary RR for the
highest v. lowest category of green tea and black tea consumption were 0·67
(95 % CI 0·46, 0·96) and 0·88 (95 % CI 0·77, 1·01), respectively. For all-
cause mortality, the summary RR for the highest v. lowest category of green
tea and black tea consumption were 0·80 (95 % CI 0·68, 0·93) and 0·90 (95
% CI 0·83, 0·98), respectively. The dose-response analysis indicated that one
cup per d increment of green tea consumption was associated with 5 % lower
risk of CVD mortality and with 4 % lower risk of all-cause mortality. Green
tea consumption was significantly inversely associated with CVD and all-
cause mortality, whereas black tea consumption was significantly inversely
associated with all cancer and all-cause mortality.

Thus, this complex compilation of diverse data established that black and green tea intake
inhibited mortality rates prospectively in pooled populations by ten and twenty percent,
respectively. This reduction in overall mortality was also evident when attention was fixed on
those clinical conditions that cause the most death and chronic morbidity in modern Man:
cardiovascular disease and cancers. Concerning cardiovascular disease, the Author is
compelled to concentrate rather more attention, insofar as he naturally identifies with
African Americans, an ethno-racial group that suffers disproportionately from this disease.
Black Americans are approximately twice as likely to die of complications of CVD.
Hypertension heightens the risk of developing the disease and dying therefrom. This
condition can be characterized as abnormally elevated systemic pressure caused by
constriction of the various vessels of the body owing often to excessive deposition of
cholesterol and other lipid-linked metabolites lining the lumen of the vasculature and
consequently obstructing the flow of blood. It is easy to imagine the effect of this impaired

60
circulation on an organ as sensitive and central as the brain. This conceptualization renders
the research we shall review presently all the more compelling. In essence, the scientists
established that African American individuals with hypertension exhibit increased cognitive
and functional deficits that correlate with increased mortality from all causes. This is how
Dr. Hajjar and colleagues describe their findings in the American Journal of Hypertension:

Background: Subjective cognitive and functional limitations are early markers


of future dementia and physical disability. Hypertension may increase the risk
of dementia; however, the magnitude and significance of subjective
limitations in the hypertensive US population are unknown, particularly in
African Americans who bear the greatest burden of hypertension. Our
objectives were to assess the prevalence and racial disparity of subjective
cognitive and functional limitations and their impact on mortality in the
hypertensive US population.

Methods: We analyzed data from the National Health and Nutrition


Examination Survey (NHANES) collected between 1999 and 2010 (N =
28,477; 31% with hypertension; 11% African American), which included
blood pressure measurement, self-reported cognitive and functional (physical
and non-physical) limitations, and all-cause mortality. Complex survey
regression models were used.

Results: In the United States, 8% of the hypertensive population reported


cognitive and 25% reported functional limitations (vs. 5.7% and 15%
respectively in the non-hypertensive population, P < 0.0001). Hypertensive
African Americans carried the highest burden of cognitive (11%, P = 0.01)
and functional (27%, P = 0.03) limitations compared to non-hypertensive
African Americans and to non-African Americans. All-cause mortality was
significantly higher in hypertensive individuals who reported cognitive or
functional limitations (P < 0.0001 for both) relative to those without either.

Conclusions: The prevalence of cognitive and functional disability is larger in


the US hypertensive population compared to the non-hypertensive
population. African Americans with hypertension carry a disproportionate
burden of these limitations. Individuals with hypertension who report
cognitive or functional symptoms have higher all-cause mortality and query
about these symptoms should be part of hypertension evaluation.I

Because of the cross-sectional, ‘snap-shot’ nature of the aforecited study it cannot be clearly
determined that hypertension causes cognitive and functional decline—it may merely be
correlated therewith. But given the dependence of cognition and general physiological
integrity on adequate circulation and considering the degree to which hypertension
compromises circulation it is certainly defensible to presume the existence of a causal
connection. The next nexus of knowledge ensconced in the journal Nutrients is therefore
interesting and actionable. For it informs us that:

IHajjar I, Wharton W, Mack WJ, Levey AI, Goldstein FC. Racial Disparity in Cognitive and Functional
Disability in Hypertension and All-Cause Mortality. Am J Hypertens. 2015 Jul 1.

61
Hypertension and arterial stiffening are independent predictors of
cardiovascular mortality. Flavonoids may exert some vascular protection. We
investigated the effects of black tea on blood pressure (BP) and wave
reflections before and after fat load in hypertensives. According to a
randomized, double-blind, controlled, cross-over design, 19 patients were
assigned to consume black tea (129 mg flavonoids) or placebo twice a day for
eight days (13 day wash-out period). Digital volume pulse and BP were
measured before and 1, 2, 3 and 4 h after tea consumption. Measurements
were performed in a fasted state and after a fat load. Compared to placebo,
reflection index and stiffness index decreased after tea consumption
(p<0.0001). Fat challenge increased wave reflection, which was counteracted
by tea consumption (p<0.0001). Black tea decreased systolic and diastolic BP
(-3.2 mmHg, p<0.005 and -2.6 mmHg, p<0.0001; respectively) and
prevented BP increase after a fat load (p<0.0001). Black tea consumption
lowers wave reflections and BP in the fasting state, and during the
challenging haemodynamic conditions after a fat load in hypertensives.
Considering lipemia-induced impairment of arterial function may occur
frequently during the day, our findings suggest regular consumption of black
tea may be relevant for cardiovascular protection.I

Now, Beloved Amenites, I ask: ‘When is the last time you’ve seen a Black person partaking
of black tea (apart from our Indian brethren who hail from the Subcontinent)?’ Though this
study is small, it established that a week-long interval of ingesting black tea twice daily was
sufficient to significantly suppress blood pressure both in the fasted state and after the
subjects were served lipid-laden meals. This modest measure has the capacity to curtail
cardiovascular complications and, consequently, premature mortality. Ignoring such
straightforward, simple evidence is unacceptable from a Public Health perspective, especially
owing to its economic nature; consuming this quantity of black tea costs next to nothing
monetarily or temporally. Ostensibly, the only obstacle to ensuring the implementation of
this information is its dutiful dissemination. Accordingly, I am disseminating it to you
Amenites and interested Observers.

Lest we forget that our focus in this Fascicle is the Amen Fast and how the features
thereof inhibit glycation, we turn to evidence that tea is able to effectuate this aim in
mammals. It is fitting, therefore, that we avail ourselves of information from investigators of
Japanese extraction whose evidence incorporates a particularly potent type of tea that is a
personal favorite of the Author’s and is integral to one of our several Aliksirs: matcha. Dr.
Yamabe and colleagues summarize their findings in the Journal of Medicinal Food thusly:

Matcha, a powdered green tea produced by grinding with a stone mill, has
been popularly used in the traditional tea ceremony and foods in Japan.
Matcha is well known to be richer in some nutritional elements and
epigallocatechin 3-O-gallate than other green teas. In our previous study,

I Grassi D, Draijer R, Desideri G, Mulder T, Ferri C. Black tea lowers blood pressure and wave reflections in
fasted and postprandial conditions in hypertensive patients: a randomised study. Nutrients. 2015 Feb
4;7(2):1037-51.

62
epigallocatechin 3-O-gallate exhibited protective effects against renal damage
in a rat model of diabetic nephropathy. In the present study, we investigated
the preventive effects of Matcha (50, 100, or 200 mg/kg/day) on the
progression of hepatic and renal damage in type 2 diabetic Otsuka Long-
Evans Tokushima Fatty (OLETF) rats. OLETF rats were orally administered
Matcha for 16 weeks, and we assessed biochemical parameters in the serum,
liver, and kidney and expression levels of major products of advanced
glycation end products (AGEs), N(6)-(carboxylmethyl)lysine (CML) and
N(6)-(carboxylethyl)lysine (CEL), receptor for AGE (RAGE), and sterol
regulatory element binding proteins (SREBPs)-1 and -2. Serum total protein
levels were significantly increased by Matcha administration, whereas the
serum albumin and glycosylated protein levels as well as the renal glucose and
triglyceride levels were only slightly or not at all affected. However, Matcha
treatment significantly lowered the glucose, triglyceride, and total cholesterol
levels in the serum and liver, renal AGE levels, and the serum thiobarbituric
acid-reactive substances levels. In addition, Matcha supplementation resulted
in decreases in the renal CML, CEL, and RAGE expressions as well as an
increase in hepatic SREBP-2 expression, but not that of SREBP-1. These
results suggest that Matcha protects against hepatic and renal damage
through the suppression of renal AGE accumulation, by decreases in hepatic
glucose, triglyceride, and total cholesterol levels, and by its antioxidant
activities.I

At several junctures throughout the day, especially after extended sessions of Irew Sedjer (i.e.
our recumbent meditation), we ritualistically imbibe sparing servings of matcha sweetened
with Stevia. Thus we incur all the ameliorative effects associated with the savory Asiatic
agent enumerated by the investigators above: (I) Reduction of circulating serum sugar (II)
Reduction of serum and hepatic triglycerol and cholesterol concentration (III) Reduction of
renal AGEs and (IV) Reduction of various indices of glycation, oxidation, and inflammation.
These effects, especially amplified in the fasted state, aggregately advance our aim of life
extension. Let us cheerfully continue with this systematic, circadian theme, the melding of
matcha and Maat (i.e. Order). For our extended intervals of meditation mainly follow our
exercise sessions. Ingesting tea of any type plentiful in the polyphenol EGCG during this
interval attenuates the glycation and inflammation that often impair joint integrity even in
avid exercisers as they age. This is what the following study in Arthritis Research & Therapy
established:

The major risk factor for osteoarthritis (OA) is aging, but the mechanisms
underlying this risk are only partly understood. Age-related accumulation of
advanced glycation end products (AGEs) can activate chondrocytes and
induce the production of proinflammatory cytokines and matrix
metalloproteinases (MMPs). In the present study, we examined the effect of
epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-
induced activation and production of TNF alpha and MMP-13 in human OA
chondrocytes.

I
Yamabe N, Kang KS, Hur JM, Yokozawa T. Matcha, a powdered green tea, ameliorates the progression of
renal and hepatic damage in type 2 diabetic OLETF rats. J Med Food. 2009 Aug;12(4):714-21.

63
Methods: Human chondrocytes were derived from OA cartilage by enzymatic
digestion and stimulated with in vitro-generated AGE-BSA. Gene expression
of TNF-α and MMP-13 was measured by quantitative RT-PCR. TNF-α
protein in culture medium was determined using cytokine-specific ELISA.
Western immunoblotting was used to analyze the MMP-13 production in the
culture medium, phosphorylation of mitogen-activated protein kinases
(MAPKs), and the activation of NF-κβ. DNA binding activity of NF-κβ p65
was determined using a highly sensitive and specific ELISA. I-κβ kinase
(IKK) activity was determined using an in vitro kinase activity assay. MMP-13
activity in the culture medium was assayed by gelatin zymography.

Results: EGCG significantly decreased AGE-stimulated gene expression and


production of TNF-α and MMP-13 in human chondrocytes. The inhibitory
effect of EGCG on the AGE-BSA-induced expression of TNF-α and MMP-
13 was mediated at least in part via suppression of p38-MAPK and JNK
activation. In addition, EGCG inhibited the phosphorylating activity of IKK
in an in vitro activity assay and EGCG inhibited the AGE-mediated activation
and DNA binding activity of NF-κβ by suppressing the degradation of its
inhibitory protein...in the cytoplasm.

Conclusions: These novel pharmacological actions of EGCG on AGE-BSA-


stimulated human OA chondrocytes provide new suggestions that EGCG or
EGCG-derived compounds may inhibit cartilage degradation by suppressing
AGE-mediated activation and the catabolic response in human
chondrocytes.II

Reducing the degradation of joint cartilage catalyzed by glycation by simply imbibing tea is a
facile yet potent adjuvant of the Amen Exercise Agenda and Amen Fast that not only
promotes the prolongation of lifespan but preserves the functional capacity of the Amenite,
enabling her to engage in physically demanding activities that, for many of us, make life far
more enjoyable than it otherwise would be.
We now turn to ‘Ra’s Rheum/Rebaudiana’ (R2) Tea. The Reader will soon see why this
salubrious solution warrants regard as an ideal Amen Adjuvant. Though Ra’s R2 Tea contains
two constituents, we shall profit by focusing our attention on the “Rheum” element alone, for
the second—Stevia rebaudiana—is accorded its own distinct discussion space and indeed is
added to every Aliksir and tea of which the Architekton partakes. Amenites are undoubtedly
accustomed to the Architekton’s evaluative criteria concerning supplements and staples—
criteria encompassing the capacity of items to collaboratively or independently induce
increases in lifespan, augment antioxidant defense, suppress glycemia/glycation, optimize
autophagy, antagonize adiposity, inhibit inflammation, enhance immunity, increase
detoxification, improve aspects of neurophysiology, and promote muscular integrity. We
shall selectively see how constituents of R2 factor in that phenomenon which is the central
concern of this section—specifically, suppression of glycemia/glycation.

II Rasheed Z, Anbazhagan AN, Akhtar N, Ramamurthy S, Voss FR, Haqqi TM. Green tea polyphenol
epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-
alpha and matrix metalloproteinase-13 in human chondrocytes. Arthritis Res Ther. 2009;11(3):R71.

64
Rheum emodi is used as a culinary plant across the world and finds an eminent
role in the Ayurvedic and traditional Chinese systems of medicine. The plant
is known to principally contain 1,8-dihydroxyanthraquinones (DHAQs) like
rhein, aloe emodin, emodin, chrysophanol and physcion that possess diverse
pharmacological and therapeutic actions. The present work deals with
developing a platform technology for isolation of these DHAQs and
evaluating their anti-diabetic potential. Herein, we report the anti-
hyperglycemic activity and alpha glucosidase (AG) inhibitory actions of five
isolated DHAQs from R. emodi. All the five isolated DHAQs showed good
anti-hyperglycemic activity with aloe emodin exhibiting maximum lowering
of blood glucose in an oral glucose tolerance test. However, on evaluation of
the AG inhibitory potential of the DHAQs only emodin exhibited potent
intestinal AG inhibition (93 ± 2.16%) with an IC50 notably lower than
acarbose. Subsequent kinetic studies indicated a mixed type of inhibition for
emodin. In vivo studies using oral maltose load showed almost total
inhibition for emodin when compared to acarbose. Molecular docking
studies revealed the presence of an allosteric topographically distinct
‘quinone binding site’ and showed that interaction with Ser 74 occurs
exclusively with emodin, which is vital for AG inhibition. The net benefit
from the glucose lowering effect and mixed type inhibition by emodin would
enable the administration of a small dosage that is safe and non-toxic in the
case of prolonged use in treating diabetes.

Apart from the mention of the molecule emodin as an agent especially efficacious in the anti-
diabetic action of the rhubarb plant (also extant in aloe), the article importantly establishes its
dualistic anti-diabetic character—particularly, its potential to avert hyperglycemia and its
ability to inhibit the enzyme α–glucosidase. Because this Fascicle is on the Amen Fast it is
fitting that we should stress the former feature—i.e. suppression of sugar concentration as
such. For this, Amenites understand, refines the rigor and heightens the healthiness of our
Holy Fast insofar as the imbibition of R2 reduces the concentration of sugar circulating in
the system of the Amen Ascetic even more than merely abstaining from feeding. However,
we cannot casually postpone the latter point to Parts II & III of Volume II. The Reader is
asked to recall our reasoning that alpha-glucosidase inhibition is most effective upon feeding,
particularly coincident with the Minor Meal or in the immediate post-absorptive phase when
the Anchorite has consumed a considerable amount of carbohydrates and can afford to
curtail excessive sugar absorption—this is precisely what the inhibition of the enteric enzyme
effectuates. [The Architekton confessedly counts Himself among those Amen Adherents
who are honorably hedonic and he unapologetically indulges His “sweet tooth” to a
dietetically defensible degree.] The astute Amenite will realize another reason for the Lord’s
lingering on the latter effect: Emodin was found to be more effective than acarbose in
inhibiting the enzyme alpha-glucosidase. Recall, dear Disciple, that acarbose independently
extends maximum lifespan in an animal model. It should enthuse the Amenite that a plant as
pervasive and prolific (particularly in the Author’s own Bw-Ka-Nun Valley) as
Rheum/rhubarb potentially promotes life prolongation as potently as the pharmacologic
drug acarbose. It surely animates and edifies the Architekton. It is interesting to further note
that –glucosidase facilitates FGF-21 release: the Acolyte assuredly intuits the significance of

65
this fortuitous effect. Admirably, it was even apparent to the investigators who undertook an
exceptionally integrative and insightful study of the molecular linkages among several life-
extending interventions. In the archives of Aging Cell the researchers relate that:

ATF4, a DNA-binding factor that modulates responses to amino acid


availability and ribosomal function, has been shown to be altered in both
liver and fibroblasts from two strains of long-lived mice, i.e. Snell dwarf and
PAPP-A knockout mice. New data now show elevated ATF4 levels, and
elevation of ATF4-dependent proteins and mRNAs, in liver of mice treated
with acarbose or rapamycin, calorically restricted mice, methionine-restricted
mice, and mice subjected to litter crowding. Elevation of ATF4, at least in
liver, thus seems to be a shared feature of diets, drugs, genes, and
developmental alterations that extend maximum lifespan in mice.I

As central as the obscure transcription factor ATF4 is to the investigators’ exposition, it has
more general relevance to the topic that interests us presently—acarbose-mediated
modulation of FGF-21. An illuminating excerpt from the said study states that:

Acarbose (Aca) inhibits α-glucosidases in the intestine, thereby slowing the


breakdown of complex polysaccharides to sugars and thus blunting the spike
in blood glucose levels that follows a meal. It is an FDA-approved agent for
treatment of human diabetes. Food intake is typically increased in Aca-
treated mice. The NIA Interventions Testing Program (ITP) has found that
Aca increases median lifespan in genetically heterogeneous UM-HET3 mice
by 22% in males (P < 0.0001) and by 5% in females (P = 0.01). Body weight
was reduced, at 12 months of age, by 14% in males and by 22% in females,
suggesting that the sexual dimorphism in longevity response does not parallel
effects on body weight. Aca lowers fasting insulin levels in males but not in
females, lowers IGF-1 levels in both sexes, and leads to a significant increase
in blood levels of FGF-21, a mediator that declines dramatically in mice on a
calorie-restricted (CR) diet. Maximum lifespan, or more specifically the
likelihood of survival to the 90th percentile age of the joint survival
distribution, is increased in both males and females (P < 0.0001).I

May Drs. Li, Li, and Miller enjoy long lives for affording Amenites this momentous lesson in
the multifarious molecular underpinnings of aging and its intentional antagonism.
As we are well aware, inhibition of glycation can be effectuated indirectly or directly.
Suppressing circulating sugar is the indirect avenue; complimentarily, averting (or reversing)
the binding of glycative metabolites (e.g. simple sugars, reactive carbonyl compounds, and
AGEs as such) directly reduces and rectifies glycative damage to the body’s subcellular
structures. Impressively, emodin does both. We have seen evidence of its sugar-suppressing
(thus indirect anti-glycative) ability. Presently, the Anchorite shall be apprised of its direct anti-
glycative capacity. This evidence is afforded by a study published in the pages of the Biological
& Pharmaceutical Bulletin. The Korean scientists selectively isolated several substances from

I Li W, Li X, Miller RA. ATF4 activity: a common feature shared by many kinds of slow-aging mice. Aging Cell.
2014 Dec;13(6):1012-8.
IIbidem.

66
the seeds of a species of Cassia. Among nine such Cassia compounds emodin exhibited the
most ameliorative inhibitory effects against glycation. Moreover, as the investigators
observantly underscore, the efficacy of emodin exceeded that of aminoguanidine—a “gold
standard” anti-glycative agent. An informative excerpt from their Results section reads as
follows:

A number of natural inhibitors of AGE formation have been


reported....[M]any of them are flavonoids with IC50 values ranging from 90 to
200 µM. In this study, emodin (6) and obtusifolin (8) exhibited much
stronger inhibitory activity on AGE-BSA formation (IC50 [a quantification of
the capacity for chemical inhibition of an enzyme or reaction] values of 118
and 28.9 µM, respectively) than aminoguanidine (IC50 value of 961 µM), a
well known glycation inhibitor, while others were found to be inactive. The
inhibitory effects of emodin (6) and obtusifolin (8) on AGE-BSA formation
in the assay were reconfirmed by a specific AGE-ELISA (absorbance of 450
nm)....[C]ompounds 6 and 8 showed a dose-dependent inhibitory effect, the
percentage inhibition of compounds 6 and 8 being more potent than that of
aminoguanidine. CML-BSA formation was also inhibited by compounds 6 and
8 [in] a dose-dependent manner. AGE adducts such as pyrraline, pentosidine
and CML have been found at elevated levels in diabetics. AGE inhibitors
such as pyridoxamine and aminoguanidine prevent development of
complications in experimental diabetes. We have previously reported that
magnolol showed an in vitro inhibitory effect on the formation of AGEs and
prevented the progression of diabetes and diabetic nephropathy in animal
models of both type 1 and type 2 diabetes. Emodin (6) was recently reported
as a potent inhibitor of AGE formation from the Rhei Rhizoma.II [Emphasis
added in italics by the Architekton.]

Moreover, molecules resident in Rheum other than emodin exert a hypoglycemic effect and
accordingly oppose diabetes and aging alike. Regarding the roots (or rhizome) of rhubarb,
Korean researchers inform us in the following Abstract liberally lifted from Life Sciences:

Extracts from Rhei Rhizoma (RR) have been reported to attenuate metabolic
disorders such as diabetic nephropathy, hypercholesterolemia and platelet
aggregation. With this study we investigated the anti-diabetic action of 70%
ethanol RR extract in streptozotocin-induced diabetic mice, and determined
the action mechanism of active compounds of RR in vitro. In the diabetic
mice, serum glucose levels at fasting and post-prandial states and glucose area
under the curve at modified oral glucose tolerance tests were lowered
without altering serum insulin levels, indicating that RR contained potential
anti-diabetic agents. The fractions fractionated from RR extracts by [chemical
separation] revealed that 60%, 80% and 100% methanol fractions enhanced
insulin sensitivity and inhibited alpha-glucoamylase activity. The major
compounds of these fractions were sennosides, rhein and rhaponticin.

IIJangDS, Lee GY, Kim YS, Lee YM, Kim CS, Yoo JL, Kim JS. Anthraquinones from the seeds of Cassia tora
with inhibitory activity on protein glycation and aldose reductase. Biological & Pharmaceutical Bulletin. 2007;
30(11):2207-10.

67
Rhaponticin and rhein enhanced insulin-stimulated glucose uptake in 3T3-L1
adipocytes. Rhaponticin increased adipocytes with a differentiating effect
similar to pioglitazone, but rhein and sennoside B decreased triglyceride
accumulation. Sennoside A and B inhibited alpha-glucoamylase activity as
much as acarbose. In conclusion, a crude extract of RR improves glucose
intolerance by enhancing insulin-stimulated glucose uptake and decreasing
carbohydrate digestion via inhibiting alpha-glucoamylase activity. Rhein and
rhaponticin are potential candidates for hypoglycemic agents.I

Several observations in the above excerpt are of interest to us. First, facilitating the influx of
sugar into fat cells should serve to suppress levels circulating in the serum—this is good.
Further, such augmented uptake of sugar by adipocytes did not apparently increase their
synthesis of fat—indeed, the opposite effect was observed. Also, the agents extant in Rheum
reduced alpha-amylase activity. Alpha-amylase is expressed and exuded orally as well as
intestinally. Carbohydrate catabolism commences first in the oral cavity and its curtailment
therein likely lessens the amount of sugar available for assimilation—a welcome result
whenever sugar is consumed in excess, ‘sweet-toothery’ being a predilection to which even
Ascetic Amen Practitioners are disposed. Alpha-amylase initiates early digestion of sugar in
the mouth and the inhibition of this enzyme can consequently conduce to tighter glycemic
control. Lastly, like emodin, the substances styled “sennosides” suppressed the
carbohydrate-catabolizing action of enzymes as effectively as acarbose. Impressive indeed.

Excursus: The Architekton shall (reluctantly) reveal His simple method of preparing
R2. Select a sturdy stalk of rhubarb rather ruddy in color. Slice these into segments
sufficiently small for processing within a mechanical blender. Pulse the pieces until a
puree is produced. Place pure stevia powder (~1/8 teaspoon) in a tea infuser along
with the mashed measure of Rheum. Pour boiled water atop these two items and
permit to steep to a desirable taste and temperature. I declare that the Disciple shall
be delighted with this divine herbal infusion. [Feature photos—one from Mike’s
garden perhaps.]

Mint is a mundane yet manifestly marvelous herb. It is peppermint of which I more


particularly speak and more specifically still the Author shall employ its Latinate appellation
to avoid any confusion amongst the more learned; suchwise, it is Mentha piperita. By its being
included in this Tractate the Reader properly presumes that peppermint is either a potent
suppressor of sugar or an effective inhibitor of glycation. This is certainly so. However, did I
not disclose that peppermint is partly included herein because it is delectable and rivals
Rheum as the Architekton’s favorite infusion, I would not be the candid Counselor that I am
justly considered by my devoted Amenites. But banish, Brethren, all thought of teabags
when conceiving of peppermint at its highest culinary quality. The full measure of its
magnificence is only discernible when one selects the freshest shoots swaying in the setting
sun, shears the delicate though sturdy stems, plucks the prickly leaves, grinds them into a
greenish mash, sprinkles thereupon a sweetening mite of stevia powder, pours pure boiled
water over the twain, lets the leaves infuse for three minutes or more and enjoys its volatile,
invigorating aroma, mouth-watering taste, and beholds the light-emerald color in a clear glass
IChoi SB, Ko BS, Park SK, Jang JS, Park S. Insulin sensitizing and alpha-glucoamylase inhibitory action of
sennosides, rheins and rhaponticin in Rhei Rhizoma. Life Sciences. 2006;78(9):934-42.

68
through which its beauty can shine forth.

Merrily and mindfully harvesting peppermint in the pre-dusk period on a picturesque Pennsylvania
day in late Spring.

Assured of its agreeable taste and aroma the Amenite should be eager for evidence of its
efficacy in averting glycation and ultimately extending lifespan. She shall be so obliged. Only
in a certain context can we claim that peppermint prolongs lifespan. That context is currently
confined to an aquatic, multicellular metazoan model inoculated with an infectious agent.
Summarily, several species of commercially captured fish are susceptible to a hemolytic
(blood cell-bursting) infection with an intestinal bacterium called Yersinia ruckeri. Iranian
researchers exposed groups of trouts infected with the organism to defined concentrations
of peppermint extract.

69
This study was aimed to assess the potential effects of Mentha piperita on the
hemato—immunological and biochemical parameters, skin antibacterial
activity and protection against Yersinia ruckeri infection—in rainbow trout
Oncorhynchus mykiss. Fish were divided into 4 groups before being fed diets
supplemented with 0, 1, 2 and 3% of Mentha piperita (MP) plant extract for 8
weeks. Dose-dependent increases in immune (both skin mucus and blood
serum) and hematological parameters (number of red and white cells,
hematocrit and hemoglobin contents), as well as in respiratory burst activity,
total protein, albumin, and neutrophil levels in fish fed supplemented diets
[were observed] compared to the control fish. Furthermore, dietary MP plant
extract supplements have no significant effect on blood biochemical
parameters and enzymatic activities of liver [so signifying, no deleterious,
toxic effects exerted by peppermint upon hepatic parameters] determined in
serum of rainbow trout.... Eight weeks [after] the cessation of feeding with
MP plant extract, survival rates of 54.4%, 63.6% and 75.2% were recorded in
groups which received 1, 2 and 3% of MP plant extract of feed, respectively,
compared to 34.6% survivals in the control. This study underlying several
positive effects of dietary administration of MP plant extract to farmed fish.I

Thus, not only did peppermint alleviate the immunologic pathology produced by the
microbe, it lengthened the lifespan of the affected organisms and did so in a manner
especially appealing to an Epidemiologist—in a distinctive, dose-dependent manner. That is,
increasing amounts of peppermint extract extended lifespan more markedly. More
momentously still, the highest concentration of the herbal extract (a substantial 3% of the
fishes’ fare) doubled survival relative to the control group. We could conservatively elect to
limit the intellectual extension of this evidence to ordinary organisms, arguing that those of
us unaffected by Yersinia infection cannot hope to benefit ‘gerostatically’ by ingesting
peppermint, particularly in proportions approximating those employed in the above
investigation. But the immune-enhancing effects of peppermint are not so narrow and (as
shall be made evident in Tractate IX of Volume II) optimal immune function is patently an
operative, independent avenue of lifespan modulation. And must we make a meal of mint in
order to modulate our mortality in some measure as suggested by the science? Certainly not.
The Amen Regimen is integrated and multi-factorial for a reason: each integral element may
exert an additive effect on longevity and optimal health. Thus, an agent that lengthens
lifespan in a metazoan via an immune mechanism is, ex hypothesi, a gerostatic substance and is
a suitable provisional Amen Adjuvant. Fortunately, I needn’t underscore the folly of
consuming fish for my faithful Followers. However, there are decent, respectable Readers
who have yet to relinquish meat and fish from their diets as they rationally weigh the merits
of the Orthodox Amen Regimen. Though I suggest that such Seekers consult Evolutionary
Nutrition for an exhaustive exposition upon the dietetic dangers of flesh/fish eating, such
studies as the one above illustrate one such danger: exposing oneself to the innumerable
infectious organisms inhabiting the carcasses of crustaceans, fishes, fowl, and mammals.

IAdel M, Pourgholam R, Zorriehzahra J, Ghiasi M. Hemato - Immunological and biochemical parameters, skin
antibacterial activity, and survival in rainbow trout (Oncorhynchus mykiss) following the diet supplemented
with Mentha piperita against Yersinia ruckeri. Fish & Shellfish Immunology. 2016 Aug;55:267-73.

70
Mint may militate against infection with some such pathogens but it is not worth the risk in
our estimation and the injury to sentient organisms is not justifiable ethically by anyone’s
culinary whims.
Before presenting the study that most persuasively demonstrates the anti-AGE
property of peppermint, we shall take this opportunity to appreciate the thorough manner in
which the authors of the investigation explain the basic nature of the assay they (and other
scientists) employ to determine the degree to which substances suppress glycation. As
elaborated in their Methods section:

The fluorescence index due to glycation produced in presence of BSA and


glucose was represented as 100% glycation which is the same as 0%
inhibition in absence of tested extracts and controls. Any sample giving
fluorescence equal to the fluorescence of BSA/glucose implied that there
was no inhibition of glycation, whereas, any sample giving fluorescence lower
to that of BSA/glucose indicated that there was inhibition of glycation by the
extract present....[T]he percentage inhibition of glycation of each of the
control and plant extract has been calculated as follows:I

Fluorescence of specimen – Fluorescence of BSA/Glucose


__________________________________________ (x 100)

Fluorescence of BSA/Glucose

The investigators from the island of Mauritius studied the suppressive effects of ten
common culinary plants upon glycation and oxidation. Of course it is interesting and
edifying that all ten herbs exhibiting inhibitory effects are integral elements of the Amen
Apothecary and Diet. How potent is peppermint in this respect? Well, mint is middling.
Along with several other herbs, it is approximately half as potent as the pharmacologic
standard aminoguanidine at disrupting the glycation of protein. As reported in their Results
section:

The positive control aminoguanidine inhibited formation of fluorescent


AGEs (P<0.05) by 75.9 %.....In vitro glycation assays demonstrated that the
ten extracts (garlic, ginger, thyme, parsley, curry leaves, pepper mint,
turmeric, onion, green onion scallion and coriander) exerted marked
inhibition of fluorescent AGEs formation....When glycation was monitored
over 2 weeks the percentage inhibition was found to be: garlic (26.1%),
ginger (25.7%), thyme (42.3%), parsley (41.2%), curry leaves (40.9%),
peppermint (39.8%), turmeric (39.3%), onion (11.9%), scallion (27.9%) and
coriander (38.8%). (Ibidem)

IRamkissoon JS, Mahomoodally MF, Ahmed N, Subratty AH. Antioxidant and anti-glycation activities
correlates with phenolic composition of tropical medicinal herbs. Asian Pacific Journal of Tropical Medicine. 2013;
6(7):561-9.

71
This, as indicated in the Abstract, was an in vitro study. In contrast to in vitro studies, in vivo
studies are performed upon living organisms. One is not necessarily superior to the other in
informational content. However, in terms of practical import, we would rather know how an
intervention affects organisms that are alive. We are therefore thankful for the following
study for its focus on living organisms and for its contextualization of the capacity of
peppermint and other herbs to curtail cardiovascular disease deaths by virtue of its many
medicinal properties. The Iranian investigators opine in their Abstract that:

Cardiovascular diseases, with an incidence of approximately 50%, are the


main causes of death in most advanced countries and an increasing trend in
the developing world as well. The World Health Organization estimates that
12 million people per year worldwide die from cardiovascular diseases.
Cardiovascular diseases are becoming an increasing problem worldwide and
hypercholesterolemia has been correlated for coronary heart diseases. Nearly
all lipoproteins are involved in this process including cholesterol carried by
very low density lipoproteins (VLDL), remnant lipoproteins and low density
lipoproteins (LDL). Currently, available hypolipidemic drugs have been
associated with the number of side effects. Herbal treatment for
hyperlipidemia poses no side effects and is relatively cheap and locally
available. In view of this, the present study was carried out to investigate the
effect of Mentha piperita on serum lipid levels of albino rats. Mentha
piperita aqueous extract (100 mg/Kg, 250 mg/Kg p.o. daily) was fed for 3
weeks on fructose-fed rats and the levels of glucose, cholesterol, triglycerides,
very low density lipoprotein, low density lipoprotein, and atherogenic index
was measured. Twenty-four male Sprague Dawley rats were divided into four
groups (6 per group). The results of present study indicate that Mentha
piperita had significant beneficial effects against fructose-induced
hyperlipidemia and showed good antioxidant activity. The aqueous extract of
the plant produced a significant decrease (p < 0.05) in elevated levels
of glucose, cholesterol, triglycerides, very low density lipoprotein, low density
lipoprotein and atherogenic index and also increased the high density
lipoprotein cholesterol levels and HDL-ratio without affecting serum insulin
levels in fructose-fed rats.I

The most relevant result of the above research was the reduction in glucose—more relevant
to our fundamental focus on glycation. What also adds to the significance of the study is its
simplicity. Disability was induced dietetically in the rodents—the same manner in which
most maladies materialize in Man. Medicinal molecules were extracted from Mentha piperita
with pure water—obviously an abundant aqueous agent much more accessible than such
solvents as the ethers and alcohols ordinarily employed in chemical assays. Now, Neophytes,
we can confidently claim that consuming our minty concoction can curtail hyperglycemia
and glycation and, perforce, promote longevity; more importantly, Proselytes, we can profit
from this prolongevity property of peppermint.

IMani Badal R, Badal D, Badal P, Khare A, Shrivastava J, Kumar V. Pharmacological Action of Mentha piperita
on Lipid Profile in Fructose-Fed Rats. Iranian Journal of Pharmacological Research. 2011 Fall;10(4):843-8.

72
We continue our discourse in this section with a consideration of compounds
comprising the chamomile plant in addition to the leaves of blackberry. Though evidence of
their ability to independently or unambiguously extend lifespan in any organism is not evidently
extant at the time of this Encyclopedia’s authorship, the capacity of compounds extractable
from these prolific plants that abound on the beautiful Atlantic Coast of the North
American continent to suppress sugar concentration certainly merits mention.

Excursus: We are already aware that blackberry and camomile/chamomile can


conduce to mortality curtailment in C. elegans in the midst of excessive sugar
exposureI but whether this can be extended to “ordinary” conditions is less clear.
However, as the Architekton characteristically comments, commoners consume
food so frequently that investigative conditions entailing obesity or elevated sugar
are arguably more applicable to modern Man than many more conventionally
“controlled” or “naturalistic”, “non-clinical” studies. Thus, this must be borne in
mind by Amenites as we engage in our analyses.

The North African investigators who undertook the ensuing study showed that chamomile
lowered serum glucose concentration in animals chemically induced to exhibit Diabetes as
well as lowering levels of the sugar in control specimens devoid of Diabetes. This latter point
is particularly important from an Amenistic perspective as the apparent efficacy of the agents
in the plant is not necessarily limited to or discernible only among diseased, disordered, or
otherwise afflicted individuals. Rather, the results are applicable to “ordinary” individuals
seeking to suppress senescence via strict, scrupulous sugar control. Hence does Dr. Eddouks
and colleagues inform us in their Abstract in Diabetes Research & Clinical Practice that...

The purpose of this study was to investigate the effect of both a single dose
and daily oral administration for 15 days of the aqueous extract of the aerial
part of Chamaemelum nobile (C. nobile) at a dose of 20mg/kg body weight on
blood glucose concentrations and basal insulin levels in normal and
streptozotocin-induced diabetic rats (STZ). Single oral administration of C.
nobile aqueous extract reduced blood glucose levels from 6.0 +/- 0.3 mmol/l
to 4.9 +/- 0.09 mmol/l (P < 0.05) 6h after administration in normal rats and
from 21.1 +/- 1.3 mmol/l to 14.5 +/- 0.9 mmol/l (P < 0.001) in STZ
diabetic rats. Furthermore, blood glucose levels were decreased from 6.1 +/-
0.06 mmol/l to 4.6 +/- 0.17 mmol/l (P < 0.01) and from 21.1 +/- 1.31
mmol/l to 13.7 +/- 0.9 mmol/l (P < 0.01) in normal and STZ diabetic rats,
respectively, after 15 days of treatment. Basal plasma insulin concentrations
remain[ed] unchanged after treatment in both normal and STZ diabetic rats
so the mechanism of this pharmacological activity seems to be independent
of insulin secretion. We conclude that the aqueous extract of C. nobile
exhibits a significant hypoglycaemic effect in normal and STZ diabetic rats
without affecting basal plasma insulin concentrations and support, therefore,
its traditional use by the Moroccan population.II

IFitzenberger E, Deusing DJ, Wittkop A, Kler A, Kriesl E, Bonnländer B, Wenzel U. Effects of plant extracts
on the reversal of glucose-induced impairment of stress-resistance in Caenorhabditis elegans. Plant Foods & Human
Nutrition. 2014; 69(1):78-84.

73
Not merely Moroccans but modern Man more generally may mitigate mortality via the
hypoglycemic efficacy of the mundane camomile plant (more so if consumed in a manner
mirroring the Amen Regimen). Moreover, in ‘mature’ females of Mexican ancestry,
epidemiological evidence points to the potential prolongevity potency of Chamaemelum nobile.
This was established in a study published in the journal Gerontologist.

Purpose: Approximately 20% of adults use some kind of herbal; however, little
data exists from population-based [studies] or clinical trials to support the
effectiveness of most herbal products. Chamomile is a commonly used herb
among older adults of Mexican origin. We examined the effects of herbal
chamomile consumption on mortality among older adults of Mexican origin.

Methods & Design: A sample from the Hispanic Established Populations for
Epidemiologic Study of the Elderly, a population-based study of
noninstitutionalized Mexican Americans aged 65 and older from five
Southwestern states (Texas, California, New Mexico, Colorado, and
Arizona). We included all men and women from 2000 to 2007 (n = 1,677).

Results: Chamomile was used by 14% of the sample. Cox proportional


hazards regression analyses showed that chamomile was associated with a
decreased risk of mortality in the total sample (hazard ratio [HR] 0.71, 95%
confidence interval [CI] 0.55-0.92) and for women (HR 0.67, 95% CI 0.49-
0.92) but not for men. In models adjusted for sociodemographic variables,
health behaviors, and chronic conditions, chamomile remained significantly
associated with reduced mortality in women (HR 0.72, 95% CI 0.53-0.98).

Implications: The use of chamomile shows protective effects against mortality


in this sample of older adults of Mexican origin for women. Further research
is warranted in other populations to determine if these effects are consistent.I

Now, might the Mexican men among this sample have inhibited the anti-aging effects of
imbibing the herbal infusion by ingesting or imbibing unnamed inimical agents in amounts
exceeding mexicanas (i.e. Mexican matrons)? Maybe. But such are the logistical limitations of
epidemiological investigations, especially those reliant upon “recall/recollection” and
“report”. Nevertheless, the available evidence is valuable to us and is conceptually
strengthened by its comportment with our knowledge of the centrality of glycemic
suppression in the deceleration of senescence, especially in a population predisposed
(environmentally and, perhaps, genotypically) to the age-accelerating disease of Diabetes.
The correctitude of the tentative conclusion that chamomile consumption might conceivably
conduce to lifespan prolongation is rendered even more rational when we are made aware
that among the mechanisms by which the herb inhibits hepatic damage is that of boosting
catalase concentration (antagonistically toward toxin exposure more exactly). Amenites have

IIEddouks M, Lemhadri A, Zeggwagh NA, Michel JB. Potent hypoglycaemic activity of the aqueous extract of
Chamaemelum nobile in normal and streptozotocin-induced diabetic rats. Diabetes Research & Clinical Practice. 2005;
67(3):189-95.
IHowrey BT, Peek MK, McKee JM, Raji MA, Ottenbacher KJ, Markides KS. Chamomile Consumption and

Mortality: A Prospective Study of Mexican Origin Older Adults. Gerontologist. 2015.

74
been told by the Architekton that targeted genetic induction of this antioxidant enzyme has
led to lifespan lengthening in a mammalian model. Moreover, magnification of glutathione
expression is associated with increased longevity among organisms in which this antioxidant
enzyme is augmented by various agents—many of which are included in the Amen
Apothecary (among them the root of Rhodiola rosea)I. Thus we are informed in this Abstract
from the journal Drug Research that:

Paraquat (PQ), an effective and widely used herbicide, has been proven to be
safe when appropriately applied to eliminate weeds. However, PQ poisoning
is an extremely frustrating clinical condition with a high mortality and with a
lack of effective treatments in humans. PQ is known to induce injury via a
redox cyclic reaction. The purpose of this study was to investigate the effect
of [an] aqueous extract [of] Matricaria chamomilla. L (M. chamomilla) against
PQ-induced liver injury in association with its antioxidant activity. The male
rats were treated by gastric gavage daily with PQ (5 mg/kg/day) and M.
chamomilla (50 mg/kg/day) were administered alone or in combination for 7
days. After treatments, total antioxidant capacity (TAC), total thiol molecules
(TTG) [with the otherwise cryptic ‘G’ in the TTG acronym apparently
indicating the antioxidant agent ‘glutathione’, characterized by the sulfur-
featuring ‘thiol’ functional group] levels and catalase (CAT) activity in liver
tissue were measured. At the end of the experiment, plasma and lung tissue
of the animals was separated. The activity of enzymatic scavengers such as
CAT, TAC and TTG were measured in liver homogenate. In this sample, the
TAC and TTG were lower in the PQ group as compared with control group.
Co-administration of PQ with M. chamomilla extract increased TAC and TTG
in liver tissue as compared with the PQ group. In conclusion, M. chamomilla
as a natural antioxidant may be considered beneficial for the protection
oxidative liver injury in PQ poisoning.II

Excursus: Quite confusingly, chamomile/camomile is a common name for a couple


of medicinal plants popularly cultivated by Man for many millennia; Matricaria and
Chamaemelum being biologically distinct genera that are sufficiently similar in
superficial appearance for their flowers to fool some into subsuming them under a
single, incorrect appellation. More importantly from an Amenistic aspect is the fact
that both specimens exert anti-senescent, anti-hyperglycemic effects which
hypothetically heighten the healthiness of the Amen Fast. And lest the Architekton
appear somewhat condemnatory of the cladistic misclassifications made by
misinformed amateurs, as an admitted enthusiast for the enjoyable practice of
harvesting edible wild plants, confusion is common (and often intellectually
entertaining) as it adds to the fun of chronicling instances of apparent convergent
evolution or simply stimulates admiration for the delightful diversity of plant
adaptations and modes of living. Alas, Biology is beautiful—but prithee Practitioner
not to confuse the roots of wild carrots for the heinous hemlock that killed both our
philosophical brethren, Master Socrates and the Stoic Sage Seneca!

IChen C, Song J, Chen M, Li Z, Tong X, Hu H, Xiang Z, Lu C, Dai F. Rhodiola rosea extends lifespan and
improves stress tolerance in silkworm, Bombyx mori. Biogerontology. 2016 Apr;17(2):373-81.
IITavakol HS, Farzad K, Fariba M, Abdolkarim C, Hassan G, Seyed-Mostafa HZ, Akram R. Hepatoprotective

effect of Matricaria chamomilla L in paraquat induced rat liver injury. Drug Research. 2015 Feb;65(2):61-4.

75
Licorice is last on our list of longevity-promoting plants whose herbal infusions we
ingest whilst we follow our daily Fast. And like the preceding infusions we have enumerated
in this section, we shall stress the sugar-suppressing property of licorice primarily but not
exclusively. For first we must affirm the manner in which licorice is linked to life
prolongation and this connection compels the Architekton to address autophagy and the
aggregation of proteins. It is the ability of agents in licorice to lessen protein aggregation in a
metazoan model of Alzheimer’s that illustrates its anti-aging efficacy. This is illustrated in an
investigation appearing in Antioxidant & Redox Signaling, the Abstract of which is as follows:

...Proteasomes are constituents of the cellular proteolytic networks that


maintain protein homeostasis through regulated proteolysis of normal and
abnormal (in any way) proteins. Genetically mediated proteasome activation
in multicellular organisms has been shown to promote longevity and to exert
protein antiaggregation activity. In this study, we investigate whether
compound-mediated proteasome activation is feasible in a multicellular
organism and we dissect the effects of such [an] approach in aging and
Alzheimer's disease (AD) progression.

Results: Feeding of wild-type Caenorhabditis elegans with 18 α-glycyrrhetinic acid


(18α-GA; a previously shown proteasome activator in cell culture) results in
enhanced levels of proteasome activities that lead to a skinhead-1- and
proteasome activation-dependent life span extension. The elevated
proteasome function confers lower paralysis rates in various AD nematode
models accompanied by decreased Aβ deposits, thus ultimately decelerating
the progression of AD phenotype. More importantly, similar positive results
are also delivered when human and murine cells of nervous origin are
subjected to 18α-GA treatment.

Innovation: This is the first report of the use of 18α-GA, a diet-derived


compound as prolongevity and antiaggregation factor in the context of a
multicellular organism.

Conclusion: Our results suggest that proteasome activation with downstream


positive outcomes on aging and AD, an aggregation-related disease, is
feasible in a nongenetic manipulation manner in a multicellular organism.
Moreover, they unveil the need for identification of antiaging and
antiamyloidogenic compounds among the nutrients found in our normal
diet.I

Certainly we shall revisit this research in Tractate IV of this Volume (‘On Autophagy & the
Amen Fast’). However, its relevance should be clear from various vantages. Firstly, if an
Amen Adjuvant independently extends the life of an organism this must be mentioned

IPapaevgeniou N, Sakellari M, Jha S, Tavernarakis N, Holmberg CI, Gonos ES, Chondrogianni N. 18α-
Glycyrrhetinic Acid Proteasome Activator Decelerates Aging and Alzheimer's Disease Progression in
Caenorhabditis elegans and Neuronal Cultures. Antioxidant & Redox Signaling. 2016 Mar 30.

76
before matters of molecular mechanism are explored—this the Author has done. Secondly,
Alzheimer’s, like Diabetes, is a disease of aging and any intervention that averts or attenuates
these infirmities is inherently of interest to us; the study above established the ability of the
licorice agent (18α-glycyrrhetinic acid) to ameliorate experimental Alzheimer’s. Thirdly, the
mechanism of action of the agent involved alleviation of the pathological protein aggregation
characteristic of the condition of Alzheimer’s. Lastly, and most materially relevant to our
present Tractate, sugar-stimulated glycation is a catalyst of protein aggregation; thus, the
ability of licorice to lower glycemia/glycation is a factor in its anti-aggregation/anti-aging
efficacy. Direct evidence of this latter phenomenon is found in a study conducted by
Malayan scientists from Monash University. They inform us that...

Beneficial effects of glycyrrhizic acid (GA), a bioactive extract of licorice


root, in the prevention of metabolic syndrome have been consistently
reported while advanced glycation end products (AGE) and receptor for
advanced glycation end product (RAGE) are the leading factors in the
development of diabetes mellitus. The aim of this study was to investigate
the effects of GA on the AGE-RAGE axis using high-fat/high-sucrose
(HF/HS) diet-induced metabolic syndrome rat models. Twenty four male
Sprague-Dawley rats were randomly assigned into three groups for 4 weeks:
(1) Group A, normal diet with standard rat chow; (2) Group B, HF/HS diet;
(3) Group C, HF/HS diet and oral administration of 100 mg/kg GA per day.
The results showed that HF/HS diet elevated the fasting blood glucose level
and insulin resistance index which was prevented by GA supplementation.
GA treatment significantly lowered the circulating AGE independent of its
glucose-lowering effect. HF/HS diet also triggered RAGE upregulation in
the abdominal muscles while GA administration downregulated RAGE
expression in the abdominal muscles, aorta and subcutaneous adipose tissues.
In conclusion, HF/HS diet could cause glucose intolerance, insulin resistance
and upregulation of RAGE expression while GA ameliorated the metabolic
dysregulation besides exhibiting inhibitory effects on the AGE-RAGE axisII.

Excursus: As a young lad I loved to chew on the woody roots of licorice given me by
my eccentric, ‘African Herbsmen’ uncles on my weekend retreats to the Louisiana
Bayou. Understandably, I was regarded as rather weird by my straight-laced New
Orleanean classmates when I weekly (and reluctantly) returned to my Catholic
school. But I was likely (if unbeknowingly) lengthening my lifespan whilst gleefully
gnawing my Glycyrriza root. Though my grandfather is now a nonagenarian, he is
unfortunately afflicted with Alzheimer’s. Though we shared many things, he did not
mirror my love of licorice. He would have chewed and probably pretended to like
licorice had I known enough to encourage the habit in him. Perhaps the progression
of his malady may have been halted or perhaps even prevented altogether, especially
had he adopted the entirety of the Amen Regimen. That was more than three
decades ago. We know far more now and we have a solemn duty to deal deliberately
and intelligently with our knowledge. That is my mission.

IICheng HS, Kong JM, Ng AX, Chan WK, Ton SH, Abdul Kadir K. Novel Inhibitory Effects of Glycyrrhizic
Acid on the Accumulation of Advanced Glycation End Product and Its Receptor Expression. Natural Products
& Bioprospecting. 2014 Dec;4(6):325-33.

77
Glycation accelerates aging. Heightened sugar concentration intensifies glycation and,
consequently, aging. The Amen Fast minimizes glycemia and glycation and, by so doing,
suppresses the celerity of aging. This age-inhibitory effect of the Amen Fast is enhanced by
agents imbibed during the Fast. Among these are coffee, chamomile, conventional Camellia
teas of various types, and botanical infusions such as Hibiscus, Honeybush, Rooibos, Mentha
and Rheum/rhubarb.

Human Longevity is Linked to Lower Levels of Glucose

Among the methodological modalities employed in epidemiological investigations is the


case-control study. Such investigations involve analyzing separate samples of people for the
presence (or absence) of particular health-related states, phenomena, or indices. In one
sample the state is present—these are the “cases”. In the other sample the state is absent—
these are the “controls”. The task before the epidemiologist or investigator is to ascertain
what differs between the groups, what essentially explains the presence of the condition or
state in one group and its absence in another. Consider an example. Suppose we wanted to
ascertain whether glycemia was associated with maximum lifespan in humans. To investigate
this issue directly is difficult. We could, however, make certain simplifying approximations.
We could consider nonagenarians to be proxies for humans who have approached maximum
human lifespan. We could construct a group consisting of such nonagenarians and compare
these to a comparable group, say their spouses. Measuring various phenomena related to
glycemia (especially fasting glucose concentration) we could then compare mean fasting
glucose in the two groups. Using certain statistical analyses involving the comparison of
sample means, we could determine whether the nonagenarians (or siblings sharing a similar
genetic propensity for longevity) exhibited significantly different levels of glucose during
fasting relative to the spousal comparison group. If the siblings of nonagenarians exhibited
significantly lower levels of serum glucose than their genetically dissimilar spouses, this
would suggest an association (not causal connection necessarily) between sugar and the
speed of senescence. Whether maintaining low levels of serum sugar during fasting promotes
prolongation of life or whether long-lived (or potentially long-lived) persons are able to keep
concentrations of serum glucose consistently low owing to some specific physiological
attribute under genetic control cannot be ascertained through case-control studies however.
The scenario described herein is a general synopsis of a study published in The Journal of the
American Geriatrics Society.I In actuality, the study in question is a sub-study of sorts, one of
several separate investigations into aging enabled by the amassing of data derived from a
lengthy analytical examination known as the Leiden Longitudinal Study of Aging. Though
the Author has endeavored to explain the undertaking with a high degree of fidelity, a more
detailed description is provided in the Introduction to one of its many offshoots:

The Leiden Longevity Study was designed to identify genetic and phenotypic
markers related to longevity. A total of 421 families were recruited consisting
of long-lived Caucasian siblings together with their offspring and partners

IRozig MP, Westendorp RG, et al. Favorable glucose tolerance and lower prevalence of metabolic syndrome in
offspring without dibetes mellitus of nonagenarian siblings: The Leiden Longevity Study. Journal of the American
Geriatrics Society. 2010; 58(3):564-569.

78
thereof. Inclusion was only performed when at least two long-lived siblings
were still alive and fulfilled the age criteria of 89 years in case of males and
91 years for females. The siblings were not selected on health conditions or
demographics. Because proper controls at high age are lacking, the offspring
from these nonagenarian siblings were asked to participate and serve as cases,
because they have an increased propensity to reach an old age. The partners
of the offspring were asked to participate in the study as controls of
comparable age.

As an epidemiologist, I can only admire the methodological elegance of this investigation


and the thoughtful, meticulous manner in which the researchers sought to control for the
confounding factor of finding a suitable group to which to compare the cases of
nonagenarians. Settling on spouses sure seems simple, but for a variety of admittedly
technical reasons such a selection mitigates many limitations. Returning our attention to the
case-control study of sugar concentration, let us look at the Abstract:

Objectives: To explore measures of metabolic syndrome and glucose


metabolism in families with exceptional longevity.

Design: Case-control study.

Setting: A university hospital in Leiden, the Netherlands.

Participants: One hundred twenty-one offspring of nonagenarian siblings, who


were enriched for familial factors promoting longevity, and 113 of their
partners. No subject had diabetes mellitus.

Measurements: Prevalence of metabolic syndrome was determined according to


the criteria of the Third Report of the National Cholesterol Education
Program. Glucose tolerance was assessed according to a 2-hour oral glucose
tolerance test.

Results: The offspring of nonagenarian siblings had a lower prevalence of


metabolic syndrome (P=.03), similar body composition, lower mean fasting
blood glucose levels (4.99 vs. 5.16 mmol/L; P=.01), lower mean fasting
insulin levels (5.81 vs 6.75 mU/L; P=.04), a higher mean homeostasis model
assessment of insulin sensitivity (0.78 vs 0.65; P=.02), and a more favorable
glucose tolerance…than their partners. No significant differences were
observed between the offspring and their partners in beta-cell function….

Conclusion: Despite similar body composition, the offspring of nonagenarian


siblings showed a lower prevalence of metabolic syndrome and better
glucose tolerance than their partners, centralizing the role of favorable
glucose metabolism in familial longevity.

This finding would seem to suggest that our genetic endowment influences our ability to
regulate glucose concentration and accordingly exerts an appreciable effect on our rate of

79
aging and eventual mortality. Are we then to despair of intervening advantageously to
lengthen our lives? Certainly not. Such information simply illustrates the unambiguous
importance of glycemia as a determinant of lifespan potential. The crucial question is “Can
we favorably alter glycemia?” or more pointedly “Can we reduce our circulating glucose to a
degree that diminishes the damage to our tissues and thereby permit prolongation of our
lifespan?” The answer is a resounding “yes”.

Epidemiological evidence indicates that longevity is linked to levels of circulating


glucose which individuals are exposed to over a lifespan. Interventions (such as the Amen
Fast) which are capable of curtailing the concentration of sugar in the blood may accordingly
enable individuals to attain longer lifespans.

Metformin Mitigates Glycation and Promotes Lifespan Prolongation

It has been established that chronic administration of biguanide drugs—buformin,


phenformin, and metformin—extend maximum lifespan in mice.I The latter of the three
drugs is widely prescribed as an anti-diabetic agent, the main molecular effect of which is the
inhibition of glycation. In an important study conducted by Russian scientists at the N. N.
Petrov Research Institute of Oncology in St. Petersburg, female mice of the SHR strain were
given dosages of metformin (100 mg/kg daily—a lower concentration than that commonly
prescribed for humans) from the age of 3 months. This treatment was found to increase
maximum lifespan by a statistically significant 10 percent [It likewise increased average
lifespan by nearly 40 percent]. Interestingly, this impressive augmentation of longevity was
not associated with reductions in circulating glucose concentration. Though we have been
extolling the Amen Regimen and other anti-aging interventions for their ability to reduce
glucose and thereby promote longevity, the fact that lifespan prolongation secondary to
metformin treatment is not necessarily mediated by hypoglycemia is in fact a welcomed
finding. For if it is possible to increase longevity by a mechanism wholly or partially
independent of glucose suppression, it is logically possible to lengthen lifespan by multiple
means. One such means is direct inhibition of glycation. Several studies—the most insightful
of which were published in the journals Biochemical Pharmacology and Diabetes & Metabolism—
have demonstrated that, aside from the established insulin sensitizing effect of metformin,
the anti-diabetic drug directly blocks the binding of glycating agents to tissue proteins. This it
does by dint of its guanidine-like chemical structure, which is particularly reactive towards
such common glycating agents as glyoxal and methylglyoxal. By binding with these agents
metformin essentially spares bodily structures from being assailed thereby. As we shall see in
Volume III (Part I), this mechanism informs an important element of the rationale for one
of the supplemental components of the Amen Diet—to wit, the ingestion of excess amino
acids as ‘sacrificial’ substrates for sugar binding and glycation reactions so as to spare the
tissues of the body therefrom. This salubrious, sacrificial action is what the Amen Amino
Aliksir is chiefly intended to effectuate.

80
The pharmacologic molecule metformin is able to extend lifespan by virtue of its
glycation-inhibiting effect. Because the Amen Fast is effective in the mitigation of glycation
through glucose suppression, the findings related to metformin-mediated lifespan extension
increase our confidence in the likely life-extending efficacy of the Amen Protocol.

Meli (Honey), Metformin, & Other Anti-Diabetic Agents Mitigate Hyperglycemia & Thereby
Promote Lifespan Prolongation

Investigative inconsistency does not necessarily imply either error or contradiction, especially
in intellectual arenas as complex as biochemistry, biogerontology, and physiology. A
miniature manifestation of this fact is to be found in the forthcoming study. In the preceding
section we saw that associated with the lifespan lengthening effect of metformin was also
associated a lowering of detectable glycation products in the absence of any discernible
decrement in circulating sugar concentration. Obversely, the ensuing study shows that the
same substance suppresses glucose concentration (and elevated insulin) while evidently
leaving levels of a particular glycation end-product unchanged. How are we to reconcile this
seemingly conflicting information? Perhaps honey shall help dispel our perplexity. The
Author’s obvious levity masks a measure of gravity nevertheless. For it has been found that
though metformin does not invariably inhibit glycation, its combination or co-administration
with honey heightens its latent anti-glycative effect. In an investigation overseen by Dr.
Erejuwa and elaborated in the International Journal of Biological Science, it was shown that
supplementation with honey reduced serum sugar concentration, glycation, and heightened
insulin sensitivity in rats rendered diabetic and did so to a degree far superior to the drugs
metformin and glibenclamide alike. Moreover, metformin ‘monotherapy’ was unable to
mitigate glycation but was effective at inhibiting this pernicious process when wedded with
honey. Here is how the scientists themselves summarize their study:

Diabetes mellitus is associated with deterioration of glycemic control and


progressive metabolic derangements. This study investigated the effect of
honey as an adjunct to glibenclamide or metformin on glycemic control in
streptozotocin-induced diabetic rats. Diabetes was induced in rats by
streptozotocin. The diabetic rats were randomized into six groups and
administered distilled water, honey, glibenclamide, glibenclamide and honey,
metformin or metformin and honey. The animals were treated orally once
daily for four weeks. The diabetic control rats showed hypoinsulinemia (0.27
± 0.01 ng/ml), hyperglycemia (22.4 ± 1.0 mmol/L) and increased
fructosamine (360.0 ± 15.6 µmol/L). Honey significantly increased insulin
(0.41 ± 0.06 ng/ml), decreased hyperglycemia (12.3 ± 3.1 mmol/L) and
fructosamine (304.5 ± 10.1 µmol/L). Although glibenclamide or metformin
alone significantly (p < 0.05) reduced hyperglycemia, glibenclamide or
metformin combined with honey produced significantly much lower blood
glucose (8.8 ± 2.9 or 9.9 ± 3.3 mmol/L, respectively) compared to
glibenclamide or metformin alone (13.9 ± 3.4 or 13.2 ± 2.9 mmol/L,

ITo the Author’s knowledge these findings are attested in several separate strains of female mice. See the
following: Anisimov VN, Berstein LM, Egormin PA, et al. Metformin slows down aging and extends lifespan
of female SHR mice. Cell Cycle 2008; 7(17):2769-2773.

81
respectively). Similarly, glibenclamide or metformin combined with honey
produced significantly (p < 0.05) lower fructosamine levels (301.3 ± 19.5 or
285.8 ± 22.6 µmol/L, respectively) whereas glibenclamide or metformin
alone did not decrease fructosamine (330.0 ± 29.9 or 314.6 ± 17.9 µmol/L,
respectively). Besides, these drugs or their combination with honey increased
insulin levels. Glibenclamide or metformin combined with honey also
significantly reduced the elevated levels of creatinine, bilirubin, triglycerides,
and VLDL cholesterol. These results indicate that combination of
glibenclamide or metformin with honey improves glycemic control, and
provides additional metabolic benefits, not achieved with either
glibenclamide or metformin alone.I

Allow the Author to elaborate and illustrate the elegance of the above investigation and its
ideality in the context of the overall Amen Regimen and in the framework of the Amen Fast
more specifically. Among the chief effects of our Fast is the facilitation of a salubrious state
of hypoglycemia. Such hypoglycemia results in reduced levels of glycation. Reduced
glycation correspondingly promotes lifespan prolongation. None can accuse the Architekton
of excessive complexity in explaining this phenomenon methinks. Now all that is necessary
is to “layer” our logic to speak loosely. At the end of our daily Fast we eat for an hour.
Obviously, any ingested item inclusive of appreciable sugar will result in an elevation of
serum sugar and so promote the process of glycation. The issue is not whether this
unavoidable effect will occur; rather, it is a matter of its magnitude, a question of whether it
can be curtailed. The affirmative answer is precisely what the preceding study proves—
principally, that honey suppresses the surge in serum sugar that should expectedly arise from
ingesting an equivalent amount of alternative calorific sweeteners and concomitantly protects
bodily proteins from glycative attack. Thus, honey magnifies the myriad ‘molecularly
munificent’ effects of the Amen Fast—most manifestly and materially, moreover, glucose
reduction and glycation inhibition.

Hearken honorable Anchorite, meliII merely amounts to an amalgam of simple sugars


and a mixture of medicinal molecules, among which Royal Jelly and propolis are most
prominent. Assuredly, simple sugars are assumed to be agents of glycation. So why, asks the
inquisitive Amenite, do not the constituent sugars of honey heighten AGE formation; why,
obversely, does honey inhibit AGEs from forming fructosamine and other deleterious
compounds characteristic of diabetes and indicative of advanced aging? Basically, because
bitIII abounds in beneficial agents that entirely offset the AGE-inducing effects of its integral
sugars in the amounts analyzed in investigations of which the Author is aware. So, this
saccharine substance is, like stevia, similarly sacred, so appreciably does it augment the
indulgence and happiness of otherwise abstemious, Ascetic, Orthodox Amenites nonetheless

IErejuwa OO, Sulaiman SA, Wahab MS, Sirajudeen KN, Salleh MS, Gurtu S. Glibenclamide or metformin
combined with honey improves glycemic control in streptozotocin-induced diabetic rats. International Journal of
Biological Science. 2011; 7(2):244-52.
IImel, Latin; µελι meli Greek, “honey”.

III Bit (more fully, Nsw Bit, is the Architekton’s rendering in Ancient Egyptian Hieroglyphs of
that most salubrious constituent of honey, Royal Jelly—bit being the literal term for “honey” and nsw denoting
“royal”. Amenites are assured of an interesting read when the Author treats of the fascinating findings
regarding this substance in our Tractate on the Minor Meal of the Amen Diet (AIR Volume III:III).

82
avowedly afflicted with that innocuous ailment that many are inclined to whimsically call a
“sweet tooth”. But unlike stevia which, being devoid of metabolizable energy, Amenites may
enjoy amidst our extended Fast, meli is manifestly a source of substantial energy, so it is
accordingly reserved for and restricted to the Dietary Phase of our Regimen. So why, retorts
the rebellious Amenite, recount the positive properties of this product in this Part of our
Encyclopedia explicitly dedicate to the Fast? Because our Feeding Phase finishes fast (far too
fast for some of us). While the sugars extant in honey and other items of our Diet are
digested and assimilated with alacrity, many medicinal molecules are metabolized more
slowly and linger in circulation for a substantially longer period. This is a welcomed result,
for it allows such agents to exert their ameliorative effects during the Feeding Phase, during
the immediate Post-Absorptive Phase and, most importantly for our present purposes, into
our Fasting Phase. In this respect it is evident why the Architekton elected to highlight the
aforecited study on honey. It shows that honey enhances the hypoglycemic, acute insulin-
inducing, and AGE-inhibiting effects of potent anti-diabetic agents. Among such agents,
metformin is especially important from the Amenistic vantage insofar as we are aware that
this pharmaceutical drug demonstrably lengthens maximum mammalian lifespan. If miel
maximizes this mortality-mitigating effect of metformin, what shall we consider our Holy
Honey but a boon and ideal Amen Adjuvant that fortuitously facilitates the anti-glycative,
anti-aging intent of the Amen Fast?

Excursus: There is another matter concerning meli that may be of material interest to
men and indeed to the matrons that love them. The damage done by diabetes and
excessive glycation is especially deleterious to sperm production and the pathways of
sperm production. Thuswise, cyclic fasting and caloric restriction can facilitate long-
term fertility for females (as elaborated in Evolutionary Nutrition) and of males. This
Excursus offers evidence that honey exerts a similarly salubrious sperm-sparing,
procreation-preserving effect. The language employed by the Author in the
preceding sentence is important: To those who deem biological posterity desirable,
the power to protract one’s procreative potency is patently positive. Investigators
expositing in the Iranian Journal of Reproductive Medicine inform us that…

...The global prevalence of diabetes mellitus is on rise. Diabetes-induced


oxidative stress has been known to affect liver, pancreas, kidney and
reproductive organs pathologically. Honey is a natural product of bees
[displaying] antioxidant properties.

Objective: [The] current study aimed to analyze the protective effects of


Metformin (MF) alone and MF+ natural honey co-administration on
diabetes-induced histological derangements in testis of rats.

Materials & Methods: Thirty six, mature male Wistar rats were randomly
divided into six groups including; control, honey-dosed non-diabetic,
diabetes-induced (65 mg/kg, single dose), honey-administered diabetic (1.0
g/kg/day), Metformin-received diabetic (100 mg/kg/day), Metformin and
honey-co-treated diabetic which were followed 40 days. The animals were
anesthetized by diethyl ether and the blood samples were collected. The
serum levels of testosterone, Insulin, LH and FSH analyzed using antibody

83
enzyme immunoassay method. The testicular tissues were dissected out and
underwent to histological analyses.

Results: The biochemical analyses revealed that diabetes resulted in


significantly reduced testosterone (p<0.01), LH and FSH (P<0.01, 0.001)
levels in serum. Light microscopic analyses showed remarkable (p<0.01)
reduction in seminiferous tubules diameter (STD), spermiogenesis index
(SPI) and thickness of the epithelium in the diabetic group versus control and
co-treated groups. Simultaneous administration of the honey with MF could
fairly up-regulate testosterone, LH and FSH levels. The animals in metformin
and honey-treated group exhibited improved tubules atrophy, elevated
spermiogenesis index and germinal epithelium thickness.

Conclusion: Our data indicated that co-administration of Metformin and honey


could inhibit the diabetes-induced damages in testicular tissue. Moreover, the
simultaneous administration of metformin and honey up-regulated the
diabetes-reduced insulin, LH, FSH and testosterone levels. This article [was]
extracted from M.Sc. thesis. (Ozra Nasrolahi).I

Attend also, my most manly Amenites, to the finding that honey heightened
testosterone concentration or counteracted the hormone’s depletion due to diabetes.
What might this mean? More muscle maintenance of course. And more muscle
maintenance maximizes longevity independently and indirectly by lessening glycemia
and glycation. Again, such fortuitous effects are good grounds for mentioning meli in
this Tractate on the anti-glycative efficacy of the Amen Fast. Recollect, Readers, the
relationship between oxidation and glycation. Oxidative damage accompanies
glycation and the curtailment of such damage lessens the extent of AGE formation.
Further, antioxidant enzymes and molecules—specifically comprising the
glutathione complex—are evolutionarily aimed at attenuating glycation. As the
ensuing study established, meli does not merely suppress fasting sugar
concentration, it also augments levels of antioxidant enzymes, including glutathione
peroxidase, transferase, and reductase, respectively in rats rendered diabetic. So
summarized, the study’s Abstract informs us that…

...Oxidative stress plays a crucial role in the development of diabetic


complications. The aims of this study were to investigate whether honey
could reduce hyperglycemia and ameliorate oxidative stress in kidneys of
streptozotocin-induced diabetic rats.

Methods: Diabetes was induced by a single dose of STZ (60 mg/kg; i. p.).
Diabetic rats were randomly grouped and administered distilled water (0.5
mL/day) and honey (0.2 g/kg/day, 1.2 g/kg/day and 2.4 g/kg/day) by oral
gavage for four weeks. Each group consisted of six rats.

Results: Total antioxidant status (TAS), activities of catalase (CAT),


glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-
transferase (GST) were significantly reduced, while superoxide dismutase

INasrolahi O, Khaneshi F, Rahmani F, Razi M. Honey and metformin ameliorated diabetes-induced damages in
testes of rat; correlation with hormonal changes. Iranian Journal of Reproductive Medicine. 2013; 11(12):1013-20.

84
(SOD) activity was up-regulated in kidneys of diabetic rats. Lipid
peroxidation (TBARS) and fasting plasma glucose (FPG) were significantly
elevated while body weight was reduced in diabetic rats. Honey significantly
increased body weight, TAS, activities of CAT, GPx, GR, and GST in
diabetic rats. It significantly restored SOD activity, and reduced FPG and
TBARS levels in diabetic rats. Histopathological examinations of the kidneys
revealed that mesangial matrix expansion and thickening of glomerular
basement membrane were reduced in the honey-treated diabetic rats.

Conclusions: Honey exerts a hypoglycemic effect and ameliorates oxidative


stress in kidneys of streptozotocin-induced diabetic rats.I

Knowing now, Neophyte, that our Holy Honey inhibits glycation and that its ameliorative
effects likely extend far into our Fasting Phase, the Architekton is compelled to concentrate
on those compounds that are consumed specifically during the course of our diurnal Fast.
Again, though we risk recapitulating some of what shall be stated in our special Volume on
the Essentials of the Amen Apothecary (AIR V), such repetition is neither repugnant to the
Postulant nor wasted thereupon.

Honey heightens the effects of the Amen Fast by dint of its diminishment of
circulating sugar particularly in the presence of the pro-longevity medicine metformin. Its
ability to augment antiglycative, antioxidant enzymes makes it a model Amen Adjuvant.

Metformin May Modulate Lifespan Via an Intermediary Endocrine Hormone, FGF21

As explained earlier in the present Tractate, fasting induces increased expression of an


endocrine hormone called FGF21. This hormone has been found to lengthen lifespan when
its levels are augmented upon genetic amplification. As the abstract below indicates,
exposure of human hepatocytes (liver cells) to metformin promoted expression of the
fasting-associated FGF21:

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator of


glucose and lipid metabolism; however, the exact mechanism of action and
regulation of FGF21 is not fully understood. Metabolic status plays an
important role in the regulation of FGF21, and we therefore examined
whether metformin, an indirect AMPK-activator, regulates FGF21
expression in hepatocytes. FGF21 mRNA and protein expression were
determined after incubation of primary cultured rat and human hepatocytes
with metformin for 24 hours. To study the role of AMPK in the putative
regulation of FGF21, hepatocytes were incubated with Compound C (an
AMPK inhibitor) in the presence of metformin. A strong dose-dependent

IErejuwa OO, Gurtu S, Sulaiman SA, Ab Wahab MS, Sirajudeen KN, Salleh MS. Hypoglycemic and
antioxidant effects of honey supplementation in streptozotocin-induced diabetic rats. International Journal of
Vitamin & Nutritional Research. 2010; 80(1):74-82.

85
increase in FGF21 expression was observed in both rat and human
hepatocytes treated with metformin. This effect was blocked by addition of
the AMPK-inhibitor Compound C. The study shows that metformin is a
potent inducer of hepatic FGF21 expression and that the effect of
metformin seems to be mediated through AMPK activation. As FGF21
therapy normalizes blood glucose in animal models of type 2 diabetes, the
induction of hepatic FGF21 by metformin might play an important role in
metformin’s antidiabetic effect.

Of what importance is this information from the Amenite vantage, the Adherent may ask?
First, it accords with evidence that anti-glycative agents such as metformin are able to
lengthen lifespanI—that is, insofar as it increases expression of an endogenous agent
(FGF21) whose ability to extend lifespan is independently established. This correspondingly
increases our confidence that glycation is operative in aging and, ipso facto, that inhibiting
glycation is a probable pathway of lifespan extension. Fasting is, of course, a principal means
by which glycation can be inhibited. Thus, we are increasingly confident that the Amen Fast
is, independently, an authentic, efficacious avenue of lifespan extension.

Metformin increases in vitro expression of the fasting-facilitated endocrine hormone


linked to lifespan extension, FGF21. This linkage supports the centrality of fasting and
glycation inhibition to life extension.

Amenistic Mimetics: Molecules that Mimic the Fasting State by Suppressing Glycemia
Consequently Lengthen Lifespan: Especial Emphasis on the Anti-Aging Agents Chromium
& Glucosamine

In this Tractate on the anti-glycative features of the Amen Fast, it is fitting to focus attention
on agents that exert an anti-aging effect owing to their ability to inhibit hyperglycemia. In a
manner of thinking, molecules that mitigate hyperglycemia mimic the most characteristic
concomitant of cyclic fasting—that is, suppression of circulating sugar. What makes a
molecule an acceptable ‘Amen Mimetic’ moreover is (I) its ability to independently augment
longevity in a mammalian species (II) its ability to favorably modulate those molecular
mechanisms known to mediate the aging process and (III) its ability to promote lifespan
prolongation in a manner that does not entail excessive, adverse, undesirable, physiological
or psychological effects. Metformin may meet these criteria, though it is generally defensible
to deem pharmaceuticals as potentially more problematic for preventative purposes than the
normally non-noxious, nutrient-like neutraceuticals. Both glucosamine and niacin-bound
chromium conform to these conservative criteria. The study that the Author shall soon cite
showed that the administration of the benign neutraceutical glucosamine lengthened lifespan
not only in a nematode but, more momentously to us Amenites, in a mammalian model—
the mouse. Moreover, maximum lifespan was lengthened in the murids even though the
agent was introduced at an advanced age. Further, in keeping with our concern for cognizing

ITo the Author’s knowledge these findings are attested in several separate strains of female mice. See the
following: Anisimov VN, Berstein LM, Egormin PA, et al. Metformin slows down aging and extends lifespan
of female SHR mice. Cell Cycle 2008; 7(17):2769-2773.

86
the fundamental physiological factors underpinning a given anti-aging intervention, the
investigators established that attenuated amounts of sugar (and apparently augmented
autophagy) is an operative element in glucosamine-linked lifespan extension. Their Abstract
reads as follows:

D-Glucosamine (GlcN) is a freely available and commonly used dietary


supplement potentially promoting cartilage health in humans, which also acts
as an inhibitor of glycolysis. Here we show that GlcN…extends
Caenorhabditis elegans life span by impairing glucose metabolism [which]
activates AMP-activated protein kinase (AMPK/AAK-2) and increases
mitochondrial biogenesis. Consistent with the concept of mitohormesis,
GlcN promotes increased formation of reactive oxygen species (ROS)
culminating in increased expression of the nematodal amino acid-transporter
1 (aat-1) gene. Ameliorating mitochondrial ROS formation or impairment of
aat-1 expression abolishes GlcN-mediated life span extension….Unlike other
calorie restriction mimetics, such as 2-deoxyglucose, GlcN extends life span
of aging C57BL/6 mice, which show an induction of mitochondrial
biogenesis, lowered blood glucose levels, enhanced expression of several
murine amino acid transporters, as well as increased amino acid catabolism.
Taken together, we provide evidence that GlcN extends life span in
evolutionary distinct species by mimicking a low-carbohydrate diet.I

Curiously, a commonly consumed carbohydrate chemical (i.e. glucosamine) can considerably


increase lifespan by lowering levels of a kindred chemical compound (i.e. glucose), an effect
ostensibly attributable to the amino moiety in the former molecule and its modulation of
sugar metabolism in the body.
Benignly bound to the B vitamin niacin, chromium has the capacity to increase
maximum mammalian lifespan in a manner similar to glucosamine—that is, by suppressing
circulating glucose concentration. This finding was furnished by physiologists from
Georgetown University Medical Center in Washington, DC. Appropriately published in the
Journal of Inorganic Biochemistry (in keeping with the inorganic chemical character of chromium
and the biochemical effects it exerts in organisms), the study Abstract informs us that:

Avoiding insulin resistance (IR) associated with aging might lengthen life
span based on previous studies using calorie-restricted animals. We assessed
whether consuming niacin-bound chromium (NBC) alone or in a formula
containing other so-called ‘insulin sensitizers’ would overcome various
manifestations of aging and extend life span in Zucker Fatty Rats (ZFR). We
compared many metabolic parameters of ZFR fed NBC alone (n=12) or
NBC in a unique formula (n=10) to a control group (n=10). In addition to
NBC, the formula contained Allium sativum, Momordica charantia, Trigonella
foenum-graecum and Gymnema sylvestre. The formula group received roughly ½
as much NBC daily as the NBC group. At week 44, all rats still lived, and no
abnormalities in blood count (CBC), renal, or liver functions were found. In

I
Weimer S, Priebs J, Kuhlow D, Groth M, Pribe S, Mansfeld J, Merry TL, Dubis S, Laube B, Pfeiffer AF,
Schulz TJ, Guthke R, Platzer M, Zamboni N, Zarse K, Ristow M. D-Glucosamine supplementation extends
life span of nematodes and of ageing mice. Nature Communications. 2014 Apr 8; 5:3563.

87
the two treatment groups compared to control, circulating glucose levels
were significantly lower, with a trend toward lower HbA1C [i.e. glycated
hemoglobin]. Relatively elevated cholesterol and triglyceride concentrations
occurred in the formula group. Compared to control, the NBC group had
increased average lifespan (21.8%), median lifespan (14.1%), 30th percentile
survival (19.6%), and maximum lifespan (22%). Despite similar beneficial
effects on the glucose and blood pressure systems, a difference in aging was
also found when the NBC group was compared to the formula group. When
all rats in the other two groups had died, four in the NBC group continued
to live at least a month longer. We attribute lack of a similar aging effect in
the formula group to either lower dosing of NBC and/or that various
ingredients in the formula counteracted the antiaging effect(s) of NBC.I

Sure, the researchers ‘recruited’ rodents rendered obese by an inborn, engineered inclination
to overeat but this very propensity for hyperphagia makes the murid model hypothetically
homologous to humans or, more momentously, to modern Man in America and much of
the Western world. Plainly, a twenty plus percent increase in the maximum lifespan of a
mammal owing to an intervention as innocuous as chromium is impressive indeed. A caveat
however. Crude chromium must be ‘complexed’ with the chemical niacin in order to
neutralize its natural toxicity and ensure its salubrious, senescence suppressing effect. Dr.
Preuss, the principal investigator of the previously cited study, provided us with this
important insight in collaboration with colleagues from Creighton University in Nebraska,
USA. Telling too, the study was published in the journal Toxicology. Its Abstract informs us
that:

Chromium exists mostly in two valence states in nature: hexavalent


chromium (chromium (VI)) and trivalent chromium (chromium (III)).
Chromium (VI) is commonly used in industrial chrome plating, welding,
painting, metal finishes, steel manufacturing, alloy, cast iron and wood
treatment, and is a proven toxin, mutagen and carcinogen. The mechanistic
cytotoxicity of chromium (VI) is not completely understood; however, a large
number of studies demonstrated that chromium (VI) induces oxidative
stress, DNA damage, apoptotic cell death and altered gene expression.
Conversely, chromium (III) is essential for proper insulin function and is
required for normal protein, fat and carbohydrate metabolism, and is
acknowledged as a dietary supplement. In this paper, comparative
concentration- and time-dependent effects of chromium (VI) and chromium
(III) were demonstrated on increased production of reactive oxygen species
(ROS) and lipid peroxidation, enhanced excretion of urinary lipid
metabolites, DNA fragmentation and apoptotic cell death in both in vitro
and in vivo models. Chromium (VI) demonstrated significantly higher
toxicity as compared with chromium (III). To evaluate the role of p53 gene
[involved in the inhibition of carcinogenesis], the dose-dependent effects of
chromium (VI) were assessed in female C57BL/6Ntac and p53 mice on

IPreussHG, Echard B, Clouartre D, Bagchi D, Perricone NV. Niacin-bound chromium increases life span in
Zucker Fatty Rats. Journal of Inorganic Biochemistry. 2011; 105(10):1344-9.

88
enhanced production of ROS, lipid peroxidation and DNA fragmentation in
hepatic and brain tissue. Chromium (VI) induced more pronounced oxidative
damage in multiple target organs in p53 deficient mice. Comparative studies
of chromium (III) picolinate and niacin-bound chromium (III), two popular
dietary supplements, reveal that chromium (III) picolinate produces
significantly more oxidative stress and DNA damage. Studies have implicated
the toxicity of chromium picolinate in renal impairment, skin blisters and
pustules, anemia, hemolysis, tissue edema, liver dysfunction, neuronal cell
injury, impaired cognitive, perceptual and motor activity, enhanced
production of hydroxy radicals, chromosomal aberration, depletion of
antioxidant enzymes, and DNA damage. Recently, chromium picolinate has
been shown to be mutagenic and [the] picolinic acid moiety appears to be
responsible as studies show that picolinic acid alone is clastogenic [i.e. causes
chromosomal breakage]. Niacin-bound chromium (III) has been
demonstrated to be more bioavailable and efficacious and no toxicity has
been reported. In summary, these studies demonstrate that a cascade of
cellular events including oxidative stress, genomic DNA damage and
modulation of [the] apoptotic regulatory gene p53 are involved in chromium
(VI)-induced toxicity and carcinogenesis. The safety of chromium (III) is
largely dependent on the ligand, and adequate clinical studies are warranted
to demonstrate the safety and efficacy of chromium (III) for human
consumption.I

Thus, consume chromium complexed with niacin, not picolinic acid, and aging can be
attenuated and untoward effects such as excessive oxidative damage can be avoided.
So, superimposed on our sugar-suppressing System of Cyclic Fasting are salubrious
substances such as glucosamine and niacin-complexed chromium, each of which
independently extend lifespan. This assuredly can be seen as signifying Selective
Synergism/Summation.

Glucosamine and niacin-bound chromium promote lifespan prolongation presumably


by suppressing sugar concentration, thereby mimicking the effect of fasting. These agents are
accordingly adjudged ‘Amen Adjuvants’ or ‘Amen Mimetics’ and included in our
Apothecary.

Insulin: Perils Potentiated by a Potent Pancreatic Product and its Pernicious Partners

Generally speaking, sugar does not flow freely into the cells of the body. Entry into cells is
secured through the mediation of a molecular gatekeeper of sorts—insulin. Insulin, produced
by a select group of cells in the pancreas, is secreted in response to provisioning of glucose
(and secondarily to circulating amino acids). [As obvious as it seems, I ask the Amenite to
bear in mind that glucose and amino acids are, of course, products of food digestion.]
Insulin binds to embedded cellular receptors and facilitates the uptake of glucose by

IBagchiD, Stohs SJ, Downs BW, Bagchi M, Preuss HG. Cytotoxicity and oxidative mechanisms of different
forms of chromium. Toxicology. 2002; 180(1):5-22.

89
individual cells. As is true of glucose, however, insulin is not so innocuous in all its myriad
roles. In one such nefarious guise, insulin is a potent mitogen—that is, it induces cells to
initiate mitosis or cell division. This induction of cellular replication is problematic insofar as
augmented cell division and consequent tissue growth (hyperplasia) are harbingers of
neoplasia—that is, cancer. There is a surprisingly straightforward explanation for this
phenomenon. The genomes of cells mutate routinely as a result of random replicative errors.
These mundane mutations are exasperated by exposure to noxious chemical and physical
stimuli and various nutritional factors. In fact, cancerous and quasi-cancerous cells reside in
your body as you read this passage and in mine as I write the same. Fortunately, our bodies
possess endogenous agents that detect and correct incipient mutational maladies and many
such deviant cells are marked for death each day through a process of stereotyped cellular
suicide known as apoptosis. However, this protection is only partially effective. Clearly, any
agent or process that induces cells—any and all cells without due discretion—to divide can
potentially precipitate the origination of colonies of cancerous cells or further the deviant
designs of an already entrenched aggregation of genetically altered cells by magnifying their
mitotic potential. Insulin is such an agent. Hyperinsulinemia (elevated circulating insulin) has
been linked to the etiology of several chronic diseases including cardiovascular disease and
certain cancers, particularly pancreatic cancer. More potent than insulin in inducing mitosis,
however, is insulin-like growth factor I or IGF-I.I This peptide is released in response to the
presence of particular amino acids—mainly those that predominate in meat.

Excursus: The attentive Amenite may adjudge it ironic that those indicated amino
acids that abound in meat are explicitly branched chain amino acids (BCAAs). The
irony of this revelation will be more apparent in AIR III:I when we review research
indicating that ingesting isolated amino acids of this very variety extend lifespan in
several species inclusive of mammals. Why, therefore, do we deem meat dietetically
deleterious and the Amen Diet divine? It is a question of quantity, quality, and
‘chronicity’. The Amen Amino Aliksir contains a few grams of the amino acids
isoleucine, leucine, and valine—the BCAAs. These amino acids are ingested in
isolated form, thereby ensuring alacrity or rapidity of uptake. This resultant alacrity
of uptake ostensibly minimizes the mitogenic effect of these amino acids as they are
rapidly recruited for the re-synthesis and strengthening of skeletal muscle that is
stimulated during our rigorous Resistance Exercise Session immediately preceding
the evening Amen Meal. Unquestionably both insulin and IGF-I are accordingly
induced by our ingestion of the Amen Amino Aliksir, the influx of amino acids into
our muscles cells doing precisely what we wish to effectuate—hormonally aid
muscular hypertrophy and/or preserve lean muscle tissue. But it must be stressed
that this salubrious effect is acute, transient. The crudity of corpses dictates that they
are digested over a protracted period of time such that the resultant increase in
amino acids is chronically elevated along with insulin and IGF-I. When this
wayward result is multiplied by feeding on flesh multiple times in a given day, the
inimical effect on aging is correspondingly magnified. As we shall see in subsequent
sections, other considerations illustrate the inadvisability of including meat in any
optimal diet and demands its exclusion from an Anti-Aging Regimen such as the
ascetically and scientifically singular AIR.

As previously stated, mitogenesis potentiates carcinogenesis and it is quite telling that dietary
practices and interventions that suppress levels of IGF-I—veganism and caloric

90
restriction—are associated with reductions in the incidence of cancerI. Indeed, the fact that
fasting, independent of reductions in caloric intake, is able to suppress circulating glucose,
insulin, and IGF-I is quite compelling. Intermittent abstention from eating may thus confer
benefits (comparable to those of dietary restriction) on those who possess the requisite
discipline to fast but find it rather more difficult to curtail their caloric intake appreciably.
In addition to contributing to carcinogenesis, insulin and IGF-I are operative in the
etiology of aging. The most important evidence establishing the veracity of this contention
issues from several sources: (1) studies linking lifelong levels of somatotrophic agents to
longevity (2) studies of the effects of spontaneous or direct disruption of somatotrophic
genes on aging and (3) studies of established anti-aging interventions which quantify the
magnitude of somatotrophic suppression in animals so subjected.
The first line of evidence involves an investigation whose analysis was still underway
at the time of this Treatise’s undertaking. Published in the journal Aging Cell, the longitudinal
study in question had a simple aim: to correlate murine mortality with levels of endocrine
hormonesI. The investigators did this by collecting a colony of nearly two thousand
genetically heterogeneous mice, analyzing various aspects of their endocrine physiology and
ascertaining whether any of these endocrine indices were correlated with lifespan. One such
index was: IGF-I. Though the investigators endeavored to execute more extensive analyses
yet incomplete at the time of this writing, their preliminary findings are clear and convincing:
the lower the levels of IGF-I, the longer the animals live. This conclusion is consistent with
several studies which indicate that levels of IGF-I and/or other somatotrophic agentsII are
altered in organisms that experience extension of lifespan secondary to selective or
spontaneous genetic mutation.
In the arena of biogerontologicalIII science, genetically altered animal models play an
inestimably important part. Because it is possible to isolate individual genes, amplify,
attenuate or eliminate their activity and observe the consequent effects on phenotype (i.e. the
physiological manifestation of an organism’s genetic constitution) invaluable information can
be acquired regarding the role of these respective genes in the regulation of lifespan among
other outcomes. For instance, it is undoubtedly important to our understanding of aging that
several genetic models that have led to lifespan extension have involved the so-called
somatotrophic/insulin axis. Let us consider the so-called Ames dwarf mouse, the first
mammalian organism to exhibit extension of maximum lifespan secondary to a single gene
mutation. This murine mammal is mutated in a manner that impairs its ability to produce
GH and IGF-I (as well as other endocrine hormones). As explained in the introductory
paragraphs of an interesting study of this organism published in the Journal of Gerontology:

IMcCarty MF. Suppression of dolichol synthesis with isoprenoids and statins may potentiate the cancer-
retardant efficacy of IGF-I down regulation. Medical Hypotheses 2001; 56(1): 12-16.

I Yuan R, Tsaih S, Petkova SB, et alia. Aging in inbred strains of mice: study design and interim report on
median lifespans and circulating IGF1 levels. Aging Cell 2009; 8(3): 277-287.
IIInsulin, IGF-I, and growth hormone can be collectively conceived as somatotrophic substances insofar as

they support bodily growth [somato- f. Gk σοµα body; -trophic f. Gk τροφε nourishment]. In proper
physiological parlance however somatotrophic cells sensu stricto reside in the anterior aspect of the pituitary
gland and produce somatotrophin solely whereas insulin is exuded by the exocrine pancreas and IGF-I is
elaborated by the liver.

III[Bio- Gk βιοσ bios; geront- Gk γεροντ-, γερων old (spec. old man); -ology Gk λογοσ reasoning, discourse.]

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The actions of GH in the liver stimulate production of IGF-1, which is the
main mediator of GH actions on growth and development. Reduced GH and
IGF-1 signaling affects insulin sensitivity through multiple mechanisms.
Although GH does not bind to the insulin receptor (IR), several signaling
events are shared between GH and IGF-1 and insulin. The GH and IGF-1
system has complex feedback mechanisms that, in general, promote insulin
resistance. Deficiency of GH is associated with increased insulin sensitivity,
decreased insulin secretion, and reduced glucose levels, which are
determinants of extended longevity.I

Of particular importance is the last sentence of the above exposition which links lower levels
of insulin and glucose to lengthened lifespan. Indeed, Ames dwarfs live more than 50%
longer than their normal “wild-type” littermates. If the physiological factors responsible for
this profound promotion of longevity have in fact been correctly characterized, the
importance of insulinemia/glycemia is accordingly underscored—low levels of glucose and
insulin likely attenuate the rapidity of aging.

The Snell Dwarf Mouse model, which also incurs impaired pituitary development and
consequent suppression of somatotrophic substances, likewise exhibits lower levels of
glucose relative to normal mice. This characteristic condition of hypoglycemia is considered
to be crucial in conducing to the animal’s substantially extended lifespan (as well as its
decreased susceptibility to cancer).II As ascertained in an important study published in The
Journal of Biological Chemistry, it is especially during the fasting phase that the long-lived Snell
specimen exhibits low levels of circulating glucose. For at least two reasons, inter-prandialIII
glycemia (serum glucose concentration) is arguably more important than prandial glycemia.
First, the chief function of feeding is to furnish the body’s cells with nutrients and energy
and the primary source of such energy is glucose. Thus, it is appropriate that levels of
glucose in the bloodstream should sharply rise during feeding. Indeed, in the Snell dwarf the
eating-induced increase in glucose is more precipitous than in normal control mice.II
However, given the damage that can be done by chronically elevated glucose and the evident
etiological role that hyperglycemia plays in aging and disease, it is instructive (though not
entirely surprising) that such long-lived specimens as the Snell dwarf mouse are characterized
by chronic fasting hypoglycemia. Further, this fasting hypoglycemia is evidently sufficient to
ensure substantial reduction in protein damage as evidenced by the inhibition of an index
called collagen crosslinkage [to be explained below].IV This brings us to our second point

ILouisA, Bartke A, Masternak MM. Effects of growth hormone and thyroxine replacement on insulin signaling
in Ames dwarf mice. Journal of Gerontology 2010; 65A(4): 344-352.

IIAlderman JM, Flurkey K, Brooks NL et alia. Neuroendocrine inhibition of glucose production and resistance
to cancer in dwarf mice. Experimental Gerontology 2009;44(1-2): 26-33.
III Classical Latin prandium meal (spec. that eaten around midday).
IICoschigano KT, Holland AN, Riders ME, et alia. Deletion, but not antagonism, of the mouse growth

hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and
increased life span. Endocrinology 2003; 144(9): 3799-3810.
IVFlurkey K, Papaconstantinou J, Miller Ra, Harrison DE. Lifespan extension and delayed immune and

collagen aging in mutant mice with defects in growth hormone production. Proceedings of the National Academy of
Sciences (USA). 2001; 98(12):6736-41.

92
regarding the relative importance of inter-prandial versus prandial glycemia. Animals spend
most of their lives in the fasted state. Therefore, from a purely quantitative perspective, the
physiological conditions that characterize the fasting state assume an import in accordance
with its duration. Over a lifetime, the conditions that predominate during the fasting phase
undoubtedly determine, to a great extent, the physiological state and fate of the organism. It
is this consideration that sheds some light on the following excerpt from the aforecited
study:

The link between low glucose utilization and longevity is supported by the
observation that fasting glucose utilization in mice, rats, dogs, and humans,
respectively, 20, 12, 4, and 2 mg/kg/min [milligrams per kilogram per
minute] correlates with the difference in longevity between these species.I

Though the preceding statement is sufficiently simple, we shall elaborate on it slightly in


respectful recognition of its gravity. The level of glucose to which an animal is exposed over
a lifetime (or, more specifically, the amount of glucose it utilizes over time as a function of
its own size) correlates inversely with its lifespan. Conceivably, the maximum length of life
characteristically attained by a given mammalian species is mediated by the degree to which
its tissues are damaged by ambient serum glucose and the extent of such damage is dictated
decisively by the concentration/utilization of glucose ordinarily extant or ensuing in an
animal’s body. Generally, because the concentration of glucose necessarily descends to
minimal levels during fasting, the more frequently one fasts and the longer the duration of
fasting, the longer one is likely to live. Of course, energy and essential nutrients are
indispensable to the optimal functioning of an organism and the levels thereof must be
meticulously maintained. As such, the Amen Protocol endeavors to achieve precisely this
end and the means by which it does so shall be a chief concern in the following Volume.

Excursus: Interestingly, it has been demonstrated that the process of protein


turnover termed autophagy generally declines with age, especially in that all-
important organ, the liver.II Since glycation is among the chief protein pathologies
addressed by autophagy, it would be interesting to discern whether an inter-species
correlation exists between autophagic activity and maximum lifespan, as is true in
the case of glycation. Formally, the Author herein hypothesizes that a positive
correlation exists between autophagic activity (or capacity) and maximum lifespan
potential among mammalian species. It is hoped that an experimentally oriented
investigator will test this hypothesis furnished by a humble Theorist.

We shall now consider a third mouse model of lifespan extension: the Growth
Hormone Knockout Organism (GHKO). This organism incurs ablation of its growth hormone
receptor, the results of which are reduced body size, decreased pancreatic production of
insulin, inhibited hepatic production of IGF-I and—particularly pertinent to our present
discussion—substantial suppression of circulating glucose. A single study published in the

IBrooks NL, Trent CM, Raetzsch CF, Flurkey K, Boysen G, Perfetti MT, Jeong YC, Klebanov S, Patel KB,
Khodush VR, Kupper LL, Carling D, Swenberg JA, Harrison DE, Combs TP. Low utilization of circulating
glucose after food withdrawal in Snell dwarf mice. The Journal of Biological Chemistry. 2007;282(48):35069-77.
IIDonati A, Cavallini G, Bergamini E. Effects of aging, antiaging calorie restriction and in vivo stimulation of

autophagy on the urinary excretion of 8OHdG in male Sprague-Dawley rats. Age. 2012 Feb 16.

93
journal Endocrinology established all of the aforementioned findingsIII. More specifically, they
found that the long-lived GHKO mice exhibited a lifelong reduction in fasting glucose
concentration in the range of 14-35%. This is instructive information. First, it is reasonable
to presume that the resultant reduction in glucose concentration is responsible in large
measure for the extension of lifespan given that this physiological effect was among the
most conspicuous alterations observed. Therefore, reasoning simplistically, if an individual
can judiciously replicate such a reduction in fasting glucose concentration, it is likely that the
lifespan of such an individual will be lengthened analogously. Importantly, a study published
in the Proceedings of the National Academy of Sciences (USA) found that both conventional caloric
restriction (~40% CR) and alternate-day feeding in mice resulted in similar reductions in
fasting glucose concentration of approximately 33%I. It will be noted that this is the upper
range of reduction observed in the long-lived GHKO mutants. It is therefore entirely
possible to achieve appreciable prolonged reduction in serum glucose concentration
comparable to mutant models of lifespan extension through either a routine regimen of caloric
restriction or intermittent fasting. However, given that eschewing eating outrightly protects
the body from the pernicious effects of elevated glucose for a longer period of time, there is
reason to believe that fasting is a more efficacious means of averting glycation and other
glucose-induced damage. Moreover, it must be remembered that the Amen Regimen entails
both quantitative caloric restriction and temporal caloric restriction—that is both reduced
caloric intake and prolonged cyclic fasting. Hence the Amenite need not choose between
two efficacious means of ensuring healthful hypoglycemia but enthusiastically embraces
both. Here is yet another instance of Selective Synergism/Selective Summation that we
would be remiss not to repeat and reflect upon.
It is instructive to consider another mouse model of lifespan extension at this
juncture: the Little Dwarf Mouse. In contrast to the aforementioned GHKO model whose
receptor for growth hormone is ablated, the little mouse is mutated in such a way that it
lacks receptors on its pituitary gland for the binding of growth hormone releasing hormone
(GHRH). GHRH is released by the hypothalamus of the brain. The binding of GHRH by its
receptors on the pituitary prompt release of GH proper. As a consequence of GHRH
receptor ablation, “little mice” exhibit attenuated levels of circulating growth hormone and,
in a manner analogous to the GHKO model, the organism attains an extension of maximum
lifespan on the order of 15%. Like the previous study, this finding was published in
prestigious Proceedings of the National Academy of Sciences (USA).I Unlike the previous study,
however, we are not made aware of whether hypoinsulinemia or hypoglycemia is a
characteristic physiological feature of the “little mouse”. The investigative team, which was
led by Dr. Kevin Flurkey, does provide us with a molecular measure of equal or even greater
value. They determined that the extent of collagen crosslinkage in “little mice” was
considerably lower than in controls. Collagen is a long-lived protein that invests the
extracellular matrices of diverse tissues and is perhaps the most important structural,
supportive material in mammalian organ engineering. Collagen is among the most common
sites of glycation and it is, of course, glucose which catalyzes this pathological effect. In
terms of aging, elevated glucose is the proximate deleterious factor whereas advanced
IIICoschigano KT, Holland AN, Riders ME, et alia. Deletion, but not antagonism, of the mouse growth
hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and
increased life span. Endocrinology 2003; 144(9): 3799-3810.
IAnson RM, Guo Z, de Cabo R, et alia. Intermittent fasting dissociates beneficial effects of dietary restriction

on glucose metabolism and neuronal resistance to injury from calorie intake. Proceedings of the National Academy of
Sciences (USA). 2003; 100(10): 6216-6220.

94
glycation is the distal deleterious consequence. Thus, the fact that the most conspicuous
physiological effect (so far ascertained) of the GHRH receptor mutation is a reduction in
collagen crosslinkage argues forcefully for the involvement of glycation in the etiology of
aging or at least suggests that the suppression of the protein damage characteristically
promoted by glucose is sufficient to secure substantial lifespan extension.

Excursus: Some ambiguity is introduced into the above investigation by the fact that
the diet of the “little mice” was composed of a chow formulated to contain a
considerably reduced quantity of fat—4 percent. This measure was reportedly
introduced to prevent the obesity previously observed in this model. Given the
salubrious association of growth hormone with leanness—it is known to promote
the preservation of lean muscle especially amidst energy restriction—it is not
entirely surprising that the attenuation of GH eventuates in obesity. It can be
claimed that the curtailment of fat intake contributed to the observed extension of
lifespan, confounding the relationship between the genetic intervention and its
effect on aging. While this interpretive limitation cannot be denied, the study and
the model still provide important abstract and practical information.

The examples expounded above derive from studies conducted on animals. Such
studies may be rigorous, relevant, and revelatory but despite our undeniable evolutionary
kinship with murine mammals, we are men not mice. As such, whenever human studies exist
in a particular area of investigation these are preferable, if comparable in quality, to their
animal analogues. Our present preoccupation, to be entirely explicit, is to demonstrate that
hypoglycemia is a characteristic consequence of several, independent, intentionally induced,
mutations that eventuate in maximum lifespan extension in animals. Subsequently we saw
that similar reductions in serum glucose concentration are effectuated in animals subjected
to CR and IF. Presently, I shall cite a study which indicates that CR also induces reductions
in circulating glucose in humans. The study in question, which, like the two previous ones
we have been considering, was published in the Proceedings of the National Academy of Sciences
(USA), compared people who had purportedly practiced some form of caloric restriction for
a protracted period with individuals who had not.I Making an effort to match the nominally
restricted group with individuals comparable in age, gender and other attributes, the
researchers examined various indices of health, chief among which was fasting glucose
concentration. Not surprisingly, they found a statistically significant difference in this
measure between the two groups with the restricted group exhibiting an average fasting
glucose concentration of ~80 milligrams per deciliter (mg/dl) [or equivalently expressed in
mg/100 milliliters] and the control group exhibiting a value of 95 mg/dl. It is worth noting
that this 15% difference is within the lower limit of the difference discerned between the
GHKO mice and their normal counterparts. For several reasons, however, the study leaves
much to be desired. Limiting ourselves to merely two such reasons, the recruitment of the
“CR subjects” was such that the duration of restriction could not be made uniform—that is,
individuals had undergone dietary restriction for varying periods of time. The second
criticism is more telling and it illustrates an important issue introduced in Volume I—that is,
the investigation employed no rigorous way of quantifying or defining the degree of dietary
restriction adopted by the experimental subjects. Evincing an effort to eat less may be

IFlurkey K, Papaconstantinou J, Miller Ra, Harrison DE. Lifespan extension and delayed immune and collagen
aging in mutant mice with defects in growth hormone production. Proceedings of the National Academy of Sciences
(USA). 2001; 98(12):6736-41.

95
laudable but does not convincingly count as caloric restriction in the scientific sense. Given
the gluttony of the general populace, eating appreciably less than the average person may
merely mean eating a “normal” quantity of food. This is why Amen Adherents are counseled
to compute their estimated energy expenditure as a reliable reference for their conventional
caloric requirement and express their degree of restriction relative thereto. This renders the
term “caloric restriction” meaningful, operational, comparable and scientifically sound.
Returning to the study under consideration, it is interesting to note that while the values for
fasting glucose concentration expectedly differed between the groups, the restricted group
displayed a mean value that is higher than would be expected under conditions of
considerable caloric restriction. Indeed, the mean value of the restricted group—80 mg/100
ml—is precisely what one would expect to see in a normal person in the fasted state,
whereas the value observed in the control group—95 mg/100 ml—exceeds what one would
expect to find in a normal person in the fasted state. As explain in Guyton and Hall’s
Textbook of Medical Physiology:

In a normal person, the blood glucose concentration is narrowly controlled,


usually between 80 and 90 mg/100 ml of blood in the fasting person….This
concentration increases to 120 to 140 mg/100 ml during the first hour or so
after a meal, but the feedback systems for control of blood glucose return the
glucose concentration rapidly back to the control level usually within 2 hours
after the last absorption of carbohydrates. Conversely, in starvation [more
properly after prolonged abstention from feeding], the gluconeogenesis
function of the liver provides the glucose that is required to maintain the
fasting blood glucose level.I

For comparative purposes, I have disclosed the approximate value of my own fasting
glucose concentration, which I have regularly measured over the course of the last decade
and a half or so. As the ensuing image illustrates, this value ranges from ~50 to 60 mg/100
ml, a median value of 55 mg/100 ml.

IFontana L, Meyer TE, Klein S, et alia. Long term calorie restriction is highly effective in reducing the risk of
atherosclerosis in humans. Proceedings of the National Academy of Sciences (USA). 2004; 101(17):6659-6663.
IGuyton AC & Hall JE. Textbook of Medical Physiology, 10th Edition. Philadelphia, PA: W.B. Saunders, 2000: 971.

96
Figure II-1 B. Fasting serum glucose concentration at roughly the midpoint of the Author’s
23-hour fast; circa Spring 2011.

97
This value is ~35% less than the median value of fasting glucose concentration cited above
as appropriate for a normal person. The Reader will note further that this value is within the
upper range of reduction in fasting glucose concentration observed in longevous GHKO
mice and comparable to the reduction in glucose concentration induced in experimental
animals undergoing caloric restriction and intermittent fasting protocols. This corroborates
several of our contentions: To wit, (1) conventional caloric restriction in humans (even in
research settings) is inadequate and falls far short of the scientific standard (2) given the
finding that several life extending mutations, caloric restriction, and intermittent fasting each
reduce fasting glucose concentration to similar degrees, this physiological factor is likely of
integral importance in aging and (3) the Amen Protocol plainly promotes lifespan extension
insofar as it induces reductions in fasting glucose concentration comparable to CR, IF, and
life-extending genetic mutations.

Chart IIA. Single Gene Mutation Models Effecting Lifespan Extension through Presumptive
Glucose/Glycation Suppression.

Gene Modification/Model Designation Presumed Mechanism(s) of Action

Ames Dwarf ↓Glucose, ↓Insulin, ↓GH, ↓IGF-I


Snell Dwarf ↓Glucose, ↓Insulin, ↓GH, ↓IGF-I, ↓Collagen Crosslinkage
Little Dwarf ↓GH, ↓Collagen Crosslinkage
GHKO [Growth Hormone Knockout] ↓Glucose, ↓Insulin, ↓GH, ↓IGF-I

Several animal models of lifespan extension bear mutations that affect one or more of
three principal parameters: insulin, IGF-I or growth hormone. The chief physiological
consequence of these mutations is substantial reduction in fasting glucose concentration.
Similarly, animals undergoing caloric restriction or alternate-day feeding exhibit fasting
hypoglycemia. Thus, reduced fasting glucose is ostensibly a sufficient condition for
maximum lifespan extension. Inference from experiential evidence indicates that the Amen
Protocol elicits reductions in fasting glucose analogous to all three aforementioned life-
extending interventions. Hence, this supports the supposition that the Amen Protocol is an
anti-aging regimen that operates through mechanisms similar to scientifically established life-
extension measures.

Telomeres Tellingly Correlate with Human Longevity and Levels of Circulating Sugar

In the ensuing Volume we shall exhaustively explore the topic of telomeres and their
association with the aging process. We introduce an aspect of this discussion presently
because a particular finding of substantial significance to our present focus (i.e. fasting
induced reduction of glycemia) pertains to telomeres. Essentially, epidemiological
explorations have established the existence of a tripartite correlation between glucose

98
concentration, lifespan, and the length of telomeres. As to what telomeres are, an
informative excerpt from the study in question explains that:

Telomeres are noncoding repeat sequences at the ends of


chromosomes…that function to stabilize the chromosome during mitosis,
prevent aberrant recombination, and protect the chromosome from end-
degrading enzymes. In somatic cells, telomere repeats are lost at each division
due to the inability of DNA polymerase to fully replicate chromosome ends,
leading to declines in telomere length with age. When telomere length
reaches a critically short length in one or more chromosomes (the “Hayflick
limit”), the cell is signaled to arrest replication and become senescent, with
eventual apoptosis. Thus, telomere attrition is a fundamental aspect of
senescence at the cellular level. Telomere length varies greatly between
individuals at birth, partially reflecting their inherited genetic potential related
to replicative senescence….I

In their cross-sectional analysis of several hundred individuals ranging in age from 8 to 80


years old, the investigators found that telomere length was lower in the aged—an
observation that was not unexpected. Further, they found that fatness was associated with
reduced telomere length. Finally, and presently most pertinent for our purposes, they found
that fasting glucose concentration was inversely correlated with telomere length. That is, the
higher the serum concentration of sugar, the shorter the telomeres; correspondingly, the
lower the fasting concentration of sugar, the longer the telomeres. In Volume I the Author
opined that any intervention capable of increasing maximum lifespan in an organism must
entail a fundamental alteration of gene structure or function. This example illustrates such an
alteration. We now see that the ability of the Amen Fast to lessen circulating sugar
concentration can plausibly promote the preservation of telomere length and consequently
potentiate lifespan extension. In Part 2 of the present Volume we shall explore additional
avenues and mechanisms by which the Amen Fast and the metabolic conditions it creates
catalyzes the lengthening or mediates the maintenance of telomeres.

Telomere length declines with age and serves as an index of the rate of aging.
Epidemiological evidence has established an inverse relationship between fasting serum
sugar concentration and telomere length. The Amen Fast, by virtue of its ability to suppress
sugar concentration, facilitates lifespan extension by promoting telomere preservation.

ILee M, Martin H, Firpo MA, Demerath EW. Inverse association between adiposity and telomere length: The
Fels Longitudinal Study. American Journal of Human Biology. 2011; 23(1): 100-106.

99
AMEN-RA’S
AGE INHIBITION REGIMEN (AIR)
AN EXPOSITION ON THE EXPERIMENTAL, THEORETICAL &
PRACTICAL UNDERPINNINGS OF LIFESPAN EXTENTION
& OPTIMAL HEALTH PROMOTION,
A DISCOURSE ON THE METHOD OF GEROSTASIS

VOLUME II
Tractatus I-B of X (Carbamylation & ACEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED UREA

Dr. Nūn Sava-Siva Amen-Ra, Dr.PH, MA, MSW

100
If you have understood, Incipient Anchorite, much of what has been related in the sections
preceding this segment, you have undoubtedly become convinced of the centrality of sugar
concentration as a predictor of the pace of aging, the efficacy of the Amen Fast as a means
of suppressing sugar concentration and glycation, and the degree to which protection of the
proteome from glycative damage conduces to lifespan prolongation. There is little merely
theoretical about our understanding: the central experimental anti-aging interventions—CR
and CF unambiguously attenuate hyperglycemia and accordingly inhibit AGE formation;
epidemiological evidence indicates a correlation between serum sugar concentration and
longevity; experimentally reducing the rate of AGE formation with pharmacologic agents
(i.e. biguanides) lengthens maximum lifespan in mammals; lastly, experimentally lessening
the ingestion of AGEs eventuates in life extension. Thus, to preserve one’s proteins from
the pernicious effects of elevated sugar is to suppress senescence and, perforce, to promote
the prolongation of one’s lifespan. But there are other important ways that our constitutive
proteins can incur age-inducing damage and (fortunately) ways in which such damage can be
diminished. This topic shall be the focus of the second segment of our three-fold Fascicle.
More specifically, we shall concern ourselves with carbamylation. At present (circa second
decade of the 21st century), carbamylation is second in import only to glycation as a causal
factor or correlate in the acceleration of aging. And this import is the reason why its
explication warrants an Addendum to the originally unitary Tractatus on Glycation and the
Amen Fast.

Carbamylation Increases with Age and Accelerates Cellular Senescence

Readers are aware that restricting the intake of protein prolongs lifespan in multiple models.
It should be appreciated that this effect is observed independently of energy intake—indeed,
if this were not so, it could be argued that the phenomenon is simply an expected
consequence of caloric restriction. To the contrary, it is the chemical composition of
proteins and their metabolism in mammals that makes their minimization conducive to
longevity. The molecular mechanisms involved in the anti-aging effect of Protein Restriction
(PR) (and, relatedly, Methionine Restriction, MR) are multifarious. In this segment of the
Tractatus the Architekton shall selectively focus on one: consequent catalyzation of
carbamylation. Simply stated, when proteins and amino acids are ingested in excess of what
is required for the rather rapid resynthesis of proteinaceous structures, the remnants must be
metabolized mainly as energy and partly as waste. It is the nitrogenous moiety of amino acids
that is ultimately excreted as waste. However, the ammonia originating from the process of
de-amination is toxic to tissues and organs—especially the all-important encephalon (i.e.
brain). The toxicity of ammonia is rectified by converting it enzymatically into the chemical
called urea. Alas, although the nitrogenous metabolite urea is less nefarious than ammonia, it
is not altogether innocuous. Indeed, it is the affixation of urea to constitutive and catalytic
proteins that results in the deleterious, age-related process called carbamylation. It is time to
enlist the aid of experimentalists to enlighten us further. To this end, let us analyze an
informative Abstract from the Proceedings of the National Academy of Sciences:

Aging is a progressive process determined by genetic and acquired factors.


Among the latter are the chemical reactions referred to as nonenzymatic
posttranslational modifications (NEPTMs), such as glycoxidation, which are

101
responsible for protein molecular aging. Carbamylation is a more recently
described NEPTM that is caused by the nonenzymatic binding of isocyanate
derived from urea dissociation or myeloperoxidase-mediated catabolism of
thiocyanate to free amino groups of proteins. This modification is considered
an adverse reaction, because it induces alterations of protein and cell
properties. It has been shown that carbamylated proteins increase in plasma
and tissues during chronic kidney disease and are associated with deleterious
clinical outcomes, but nothing is known to date about tissue protein
carbamylation during aging. To address this issue, we evaluated
homocitrulline rate, the most characteristic carbamylation-derived product
(CDP), over time in skin of mammalian species with different life
expectancies. Our results show that carbamylation occurs throughout the
whole lifespan and leads to tissue accumulation of carbamylated proteins.
Because of their remarkably long half-life, matrix proteins, like type I
collagen and elastin, are preferential targets. Interestingly, the accumulation
rate of CDPs is inversely correlated with longevity, suggesting the occurrence
of still unidentified protective mechanisms. In addition, homocitrulline
accumulates more intensely than carboxymethyl-lysine, one of the major
advanced glycation end products, suggesting the prominent role of
carbamylation over glycoxidation reactions in age-related tissue alterations.
Thus, protein carbamylation may be considered a hallmark of aging in
mammalian species that may significantly contribute [to] the structural and
functional tissue damage encountered during aging.I

Clearly there is some kinship between carbamylation and glycation. Crucially and quite
concerningly, however, carbamylation may prove more problematic physiologically for this
derangement of proteins proceeds at a rate more rapid than glycation and consequently
accumulates to a greater degree with increasing age. Further, given its correlation with
clinical conditions characteristic of aging (i.e. atherosclerosis, diabetes, and chronic kidney
disease) it is conceivable that concerted curtailment of carbamylation might mitigate
mortality and, moreover, maximize longevity. This conceptually compelling conjecture is of
course the motivation behind the Architekton’s inclusion of the corpus of evidence
concerning carbamylation in our Anti-Aging Encyclopedia. Consider another commonality
linking the kindred ‘proteinopathies’: both carbamylation and glycation exert an especially
injurious effect upon particularly long-lived proteins (e.g. collagen) that form the foundation
of our most abundant constitutive tissues [indeed, collagen comprises roughly half of the
protein of which our bodies are composed]. And comparable to the case of glycation,
animals with divergent species-specific maximum lifespans display differential degrees of
carbamylation accumulation in a manner suggestive of causality—that is, animals with longer
maximum lifespan potential exhibit lower levels of carbamylation. Certainly the system-wide
senescence that shows as aging has its origins in the molecular alterations incurred by our
cells. As my more remarkable middle-school students state with the clarity and confidence of
collegiate scholars, ‘the fundamental units of all living things is the cell’. Revealing research
has shown that it is the cell that is clearly compromised by carbamylation, that the molecular

IGorisse L, Pietrement C, Vuiblet V, Schmelzer CE, Köhler M, Duca L, Debelle L, Fornès P, Jaisson S, Gillery
P. Protein carbamylation is a hallmark of aging. Proceedings of the National Academy of Sciences (USA). 2016;
113(5):1191-6.

102
damage done to the cell slows the regenerative capacity of progenitor cells, that the
regenerative reduction is due to genomic damage and that uremic damage to lipoproteins can
contribute to conditions conducing to cardiovascular disease—the very malady that exacts
the greatest toll in terms of lives in the modern Western World. The ensuing Abstract
explains these allegiant effects admirably:

Carbamylated low-density lipoprotein (cLDL) plays a role in atherosclerosis.


In this study we evaluate the effect of uremia on LDL carbamylation and the
effect of cLDL and oxidized LDL (oxLDL; 200 µg/ml) on number,
function, and genomic stability of endothelial progenitor cells (EPCs)
obtained from healthy volunteers. cLDL was generated after incubation of
native LDL (nLDL) with uremic serum from patients with chronic kidney
disease (CKD) stages 2-4. Oxidative stress was measured by flow cytometry
and fluorescent microscopy, mitochondrial depolarization by flow cytometry,
senescence by β-galactosidase activity and telomere length, and DNA
damage by phosphorylated histone H2AX (γH2AX). The percentage of
cLDL by uremic serum was related to the severity of CKD. Compared with
nLDL, cLDL induced an increase in oxidative stress (62±5 vs. 8±3%,
P<0.001) and cells with mitochondrial depolarization (73±7 vs. 9±5%,
P<0.001), and a decrease in EPC proliferation and angiogenesis. cLDL also
induced accelerated senescence (73±16 vs. 12±9%, P<0.001), which was
associated with a decrease in the expression of γH2AX (62±9 vs. 5±3%,
P<0.001). The degree of injury induced by cLDL was comparable to that
observed with oxLDL. This study supports the hypothesis that cLDL
triggers genomic damage in EPCs, resulting in premature senescence. We
can, therefore, hypothesize that EPCs injury by cLDL contributes to an
increase in atherosclerotic disease in CKD.I

So far scientific evidence does not enable us to say definitively that repressing the rise in
carbamylation can conduce to maximum lifespan extension but we are in an intriguing,
introductory stage in this investigative arena. The circumstantial scientific evidence is
sufficient to impel action, to compel conscientious ‘Djetitarians’ and Amenites to pursue
practical measures to mitigate carbamylation and, most likely, potentiate further lifespan
prolongation by prudent preemptive practices. What might such practices entail? We shall
see forthwith fellow Ascetics.
The first course to consider in the interest of curtailing carbamylation clearly
concerns dietetic composition. Fortunately, a central feature of the Amen Diet already
actuates this aim. The Amen Protocol explicitly prescribes Protein Restriction, PR being an
integral element of the Tetractys of Anti-Aging Interventions. Amenites are encouraged to
consistently ingest a quantity of protein sufficient simply to serve synthetic purposes, not
energetic purposes—that is, our aim is to promote the daily resynthesis of labile proteins
and/or the strengthening of stimulated skeletal muscle proteins. Minimal intake of protein
permits such a state of affairs without providing superabundant protein to serve as energy

ICarracedo J, Merino A, Briceño C, Soriano S, Buendía P, Calleros L, Rodriguez M, Martín-Malo A, Aljama P,


Ramírez R. Carbamylated low-density lipoprotein induces oxidative stress and accelerated senescence in human
endothelial progenitor cells. FASEB Journal. 2011; 25(4):1314-22.

103
owing either to dietary excess or, conversely, exceedingly lengthy fasting that might thuswise
conduce to catastrophic catabolism of constitutive proteins. This is one welcome effect of
our distinctive, day-long Amen Fast: it is optimally catabolic, abstemious, and ascetic.
Complementarily, our distinctive Diet is adequately anabolic, providing proper proteinaceous
nutrients (at an ideal circadian interval) to promote protein preservation without marked
metabolic waste arising from urea production.
But lo, Loyal Adherents, both CF and CR certainly increase the expression of
enzymes which yield urea. Further, the formation of the nitrogenous molecule increases
markedly over the course of a fast. The evolutionary adaptiveness of this effect is altogether
evident. If an organism is dietetically deprived to an appreciable degree or for an extended
duration, access to energy is demanded and when such fuel is forestalled from without, it
must be acquired from within. And after considerable quantities of carbohydrate and adipose
are catabolized, precious proteins must next be metabolized. Not muscle, in the main, but
more labile proteins lurking in the liquid sera must subserve this metabolic mandate.
Moreover, when animals are experimentally adapted to endure regular intervals of extended
dietary deficits or cycles of ‘semi-starvation’, their ability to undergo autophagy is enhanced.
Such ‘increased autophagic facility’ is reflected in augmentation of the efficiency of the urea
cycle. But this urea need not be long-lived, for it is subject to chemical (re)conversion when
requisite nutrients are next available. Such nutrients indeed become newly available every
evening on the Amen Regimen at exactly the interval when urea markedly rises. Yes,
Amenites, there is Order even in this intricate aspect of physiology. Empirical evidence adds
insight into this order. It has been shown that urea is responsive to the circulating
concentration of corticosteroids (i.e. cortisol) and that both chemicals conform to circadian
cyclicity. This was impressively established by a study published in The British Journal of
Nutrition. The investigators subjected rodent specimens to a regimen of temporally restricted
feeding for durations of either one or two days. Among the endocrine and enzymatic effects
they observed were levels of urea, concentrations of urea-yielding enzymes, and the patterns
of expression of such enzymes. An excerpt from their Results section reveals some of their
findings:

Glucocorticoid hormones are some of the principal endocrine regulators of


the induction and activity of the enzymes underlying the urea cycle....In
agreement with the temporal pattern of circulating corticosterone and nuclear
and cytoplasmic liver GCR [Glucocorticoid Receptor], circulating urea in the
ad libitum rats depicted a peak in the transition between the light and the
dark periods. Differently, DRF [dietetically-restricted feeding] promoted high
levels of urea preceding food access (at 11.00 hours), which decreased to very
low levels in response to mealtime (14.00 hours). After 17.00 hours, the
circulating urea was enhanced to reach a peak (08.00 and 11.00 hours) just
before feeding. In spite of the dissimilar rhythmicity, there was no change in
the average values of blood urea between the ad libitum and DRF groups, or
in the values measured in the light and in the dark periods. CPS1 is the rate-
limiting enzyme of the urea cycle. It had a rhythm in the ad libitum group,
with higher values (about 44 %) in the darkness. CPS1 rhythm was lost in the
DRF group, but food restriction caused significantly higher CPS1 activity
during the light period (about 72 %).I

104
So, predictably, rodents receiving restricted access to food portrayed a peak in the
production of urea prior to feeding. However, the average amount of urea exuded by the
restricted and replete rodents, respectively, did not differ significantly. Ostensibly, this
suggests that the degree of deprivation was not so severe as to result in a net loss of labile
proteins yet was sufficient to stimulate increased activity of urea-eliciting enzymes and,
perhaps more importantly, sufficient to promote replacement and replenishment of proteins.
Interestingly, the suppressive effect of feeding on urea production was somewhat diminished
in the fasted specimens. As the investigators explain....

Urea in blood was responsive to the feeding condition, decreasing by about


28 % and by about 44 % by refeeding after 1 and 2 days of fasting,
respectively. The effect associated with food access showed a more
accentuated response in the groups under the DRF protocol (reduction of
about 62 %). The activity of CPS1 did not show any change comparing the
fasted and the fed states in all conditions (1 and 2 days of fasting–refeeding,
and DRF at 11.00 hours and 14.00 hours). However, it was evident that the
activity of CPS1 in the groups under the DRF protocol decreased by more
than 50 % in comparison with their control groups of feeding condition.

This may mean that consistent cyclic fasting may enable any ammonia resulting from excess
ingestion of protein to be detoxified with greater efficiency, thereby protecting the brain and
other organs more effectively. Just as it is a good thing that feeding does not completely
suppress urea synthesis, we will see (in AIR II:IV) that complete inhibition of autophagy is
not necessary to promote desirable muscle preservation.
The fact that day-long fasting does not evidently increase urea above the level
observed among amply fed animals is a welcomed result. However, being an Idealistic
Extremist in some aspects, the Architekton is intent on ascertaining whether it is possible
and optimal to reduce urea levels even more markedly in the interest of lifespan
prolongation. Assuredly the Amenite will have guessed that this is so. ‘How?’ asks the
inquisitive Acolyte. In this analysis we must invoke the Amen Apothecary and the abundant
herbal infusions that are imbibed during our Divine Fast. Preliminarily, it has been
established that Camellia sinensis, Chamomile, Ginseng (North American, Panax quinquefolium),
Hibiscus—four teas commonly consumed by fasting Amenites—each suppress serum
concentration of urea. Moreover, one agent in the arsenal of the Amen Apothecary is
particularly potent in its inhibition of exorbitant urea: It is the antioxidant abundant in
tomatoes and watermelon, namely lycopene. The ensuing summary in a Scandinavian scientific
journal assessed its urea-reducing ability relative to two nutrients—ascorbate (vitamin C) and
tocopherol (vitamin E). The Abstract informs us thusly:

Previous studies have shown that the increase of carbamylated LDL (cLDL),
a product of nonenzymatic modification of LDL in human serum by urea-
derived cyanate, may cause cardiovascular complications in patients with
chronic renal insufficiency. This study examined the inhibitory effect of
ascorbic acid, alpha-tocopherol and lycopene on LDL carbamylation in an in

ILuna-Moreno D, García-Ayala B, Díaz-Muñoz M. Daytime restricted feeding modifies 24 h rhythmicity and


subcellular distribution of liver glucocorticoid receptor and the urea cycle in rat liver. The British Journal of
Nutrition. 2012; 108(11):2002-13.

105
vitro model system.

Methods: After isolation of LDL from plasma using an ultracentrifuge


technique, cyanate was added to it and then LDL; carbamylation was
measured in both the absence and presence of ascorbic acid, alpha-
tocopherol and/or lycopene by the colorimetric method at 530 nm.

Results: The findings indicated that these vitamins inhibit LDL carbamylation
and the most effective vitamin of the three is lycopene. Moreover, the effect
of lycopene on this process increased in the presence of ascorbic acid and
alpha-tocopherol.

Conclusion: This study indicated that ascorbic acid, alpha-tocopherol and


lycopene with antioxidant activity can probably inhibit LDL carbamylation
and therefore may have a role in ameliorating atherosclerotic risk of patients
with kidney failure. However in vitro and in vivo investigations are required
to confirm the exact effects of these vitamins on patients suffering from
uremic disorders.I

It is prescribed that Practitioners partake of a purple pill of pure lycopene at approximately


the mid-point of their Fast, at that period when stored sugars are duly depleted, when fat is
furnishing substantial fuel, and when this energetic stockpile is soon to be supplemented
with labile proteins. For it is when this protein-procuring process is underway that urea
accumulates. For those Amenites that elect to engage in a bout of afternoon exercise, this
lycopenic prescription is particularly important insofar as exercise, especially in the fasted
state in which Ascetics of our Order alone execute it, magnifies metabolism of protein and
accelerates the de-amination of amino acids that heightens urea production. Relatedly, the
evening Repast is replete with dietetic and supplementary sources of each of the three
vitamins demonstrated to be most efficacious in the inhibition of carbamylation—namely,
lycopene, ascorbic acid (vitamin C) and tocopherol (vitamin E).
We now turn to teas and other herbal infusions that exert urea-reducing effects that
can curtail carbamylation. Let us patronize any American patriots among us by beginning
with Panax.

American ginseng (Panax quinquefolium) is an obligate shade perennial plant


that belongs to Araliaceae ginseng species, and is native to eastern USA and
Canada. Ginseng proteins are reported to have several pharmaceutical
properties. However, such properties of American ginseng proteins (AGP)
have seldom been reported. Also, anti-fatigue properties of AGP have not
been studied. Therefore, we examined the anti-fatigue effects of AGP in
mice.

Materials & Methods: The molecular weight and protein contents of AGP
were determined by SDS-PAGE, while the amino acid composition was

IGhaffari MA, Shanaki M. In vitro inhibition of low density lipoprotein carbamylation by vitamins, as an
ameliorating atherosclerotic risk in uremic patients. Scandanavian Journal of Clinical Laboratory Investigation. 2010;
70(2):122-7.

106
analyzed by HPLC. The mice were divided into four groups. The control
group was administered distilled water by gavage every day for 28 days. The
other groups, designated as AGP treatment groups, were administered 125,
250 and 500 mg/kg of body weight, respectively of AGP by gavage every day
for 28 days. Anti-fatigue activity was estimated using forced swimming test,
and biochemical indices were determined using available kits.

Results: The subunit molecular weight of AGP ranged from 8-66 kD and the
protein content measured by Bradford assay was 1.86 mg/mL. The forced
swimming time of low, intermediate and high groups were found to be
longer as compared to the control group. AGP significantly decreased blood
lactate (BLA) and serum urea nitrogen (SUN) levels, and increased hepatic
glycogen (GLU) level. Additionally, AGP lowered malondialdehyde (MDA)
content and increased the levels of glutathione peroxidase (GPx) and
superoxide dismutase (SOD).

Conclusion: AGP shows anti-fatigue activity in mice, as measured by the


physiological indices for fatigue.I

Importantly, this study indicates that AGP or, the Author’s preferred acronym, NAG,
(North American Ginseng) suppresses serum urea under conditions of physical exhaustion
when energy is being actively depleted and whilst, presumably, proteins are being employed
to stem the shortfall. Though these animals had not been subjected to an ‘Amenesque’
regimen, the study findings are especially applicable to those Adherents whose execution of
the Amen Exercise Regimen is exceptionally arduous. The anti-fatigue effect of NAG,
evidenced in part by reduced urea concentration, is also demonstrated by an active agent in
green tea. As the Abstract in Pharmacognosy Magazine inform us....

...Epigallocatechin-3-gallate (EGCG) is the most abundant of the green


tea polyphenols that exhibit a variety of bioactivities. The objective of this
study was to evaluate the anti-fatigue effect of EGCG by forced swimming
exercise.

Materials & Methods: The mice were divided into one control group and three
EGCG-treated groups. The control group was administered with distilled
water and EGCG-treated groups were administered with different dose of
EGCG (50, 100, and 200 mg/kg) by oral gavage for 28 days. On the last day
of experiment, the forced swimming exercise was performed and
corresponding biochemical parameters were measured.

Results: The data showed that EGCG prolonged exhaustive swimming time,
decreasing the levels of blood lactic acid, serum urea nitrogen, serum creatine
kinase and malondialdehyde, which were accompanied by corresponding
increase in liver and muscle glycogen contents, and superoxide dismutase,
catalase, and glutathione peroxidase activities.

IQi B, Liu L, Zhang H, Zhou GX, Wang S, Duan XZ, Bai XY, Wang SM, Zhao DQ. Anti-fatigue effects of
proteins isolated from Panax quinquefolium. Journal of Ethnopharmacology. 2014; 153(2):430-4.

107
Conclusion: This study indicated that EGCG had an anti-fatigue effect.

Summary: EGCG significantly prolonged exhaustive swimming time and


decreased the levels of BLA, SUN [serum urea nitrogen], SCK and MDA,
which were accompanied by corresponding increases in liver and muscle
glycogen contents, and SOD, CAT, and GPx activities. EGCG can be used
to design nutraceutical supplements aimed to facilitate recovery from fatigue
and attenuate exhaustive exercise-induced oxidative damage.

Abbreviations used: EGCG: (-)-Epigallocatechin-3-gallate, ROS: reactive


oxygen species, BLA: blood lactic acid, SUN: serum urea nitrogen, SOD:
superoxide dismutase, GPx: glutathione peroxidase, CAT: catalase, SCK:
serum creatine kinase, MDA: malondialdehyde, C: control, LET: Low-dose
EGCG-treated, MET: Middle-dose EGCG-treated, HET: High-dose
EGCG-treated, GTE: green tea extract.I

It is interesting that the investigators envision the catechin component (EGCG) in tea as a
promising ingredient in a neutraceutical compound capable of curtailing “exhaustive
exercise-induced oxidative damage” and lessening muscle fatigue. Of course, the Amen
Apothecary already includes this measure and additional catechin-containing tea is imbibed
throughout the Amen Fast.
Two additional agents included in the Amen Apothecary and ingested during our
Fast are cinnamon and the powdered leaves of the olive tree. Both agents exert an inhibitory
effect on urea concentration and the latter (olive) also exerts an inhibitory effect on a related
protein derangement to which our attention shall turn in the final Part of this triumvirate
Tractate—it is called carbonylation. For now, let us continue our focus upon carbamylation
but bearing in mind the fortuitous finding that many of the agents in the Amen Apothecary
are impressive Adjuvants, acting trebly to forestall the three principal ‘proteo-senescent’
processes of glycation, carbamylation, carbonylation and indeed other injurious physiological
effects outside of the purview of the present Tractate. Thus does The Journal of Pharmacy and
Pharmacology inform us in the following Abstract concerning cinnamon and olive leaf:

The effects of cinnamon bark and olive leaf have been investigated on
streptozotocin-induced tissue injury, and some biochemical and
haematological changes in rats. The effects on glycaemia were also evaluated.
Long-term administration of olive leaf caused significant improvement in
tissue injury induced by streptozotocin treatment; the effect
of cinnamon bark was less [potent]. No effects on blood glucose levels were
detected. However, significant decreases in some increased biochemical and
haematological parameters of streptozotocin-treated rats were observed.
Aspartate aminotransferase, urea and cholesterol levels were significantly
decreased by treatment with both plant materials, and alanine
aminotransferase by treatment with olive leaf. Cinnamon bark also caused a

ITeng YS, Wu D. Anti-Fatigue Effect of Green Tea Polyphenols (-)-Epigallocatechin-3-Gallate (EGCG).


Pharmacognosy Magazine. 2017; 13(50):326-331.

108
significant decrease in platelet counts. In addition, any visible toxicity, except
decrease in body weight gain, attributable to the long-term use of plant
materials was not established in normal rats. The data indicate that long-term
use of olive leaf and cinnamon bark may provide benefit against diabetic
conditions. Determination of underlying mechanism(s) of beneficial effects,
toxicity to other systems and clinical assessments of related plant materials
are major topics requiring further studies.I

It is fitting that we shall finalize our discourse on the diminishment of urea with a study
establishing this effect in Hibiscus. For infusions of this African herb comprise the largest
proportion of the tea that the Architekton ingests during his daily Fast, during the evening
Resistance Session, and even during and after the Evening Meal. The Acolyte need not adopt
the personal preferences of the Author and may proudly partake of her own favorite tea, but
it helps to know why Hibiscus is so salubrious in my estimation and according to the available
evidence. The study in question explored the antibacterial and hepatoprotective properties of
both green tea and Hibiscus. We shall concentrate our focus primarily upon the latter. First
the authors afford us a very brief bio-geographical and pharmacological primer on the plant,
particularly appealing to an avowed Egyptophile with religio-philosophical affinities for Ta-
Meri ( ) and Ta-Nehesi ( )—Kemit (Egypt) and Nubia
(Sudan), respectively:

Hibiscus sabdariffa calyces are a famous plant cultivated and used widely in
Africa, especially Egypt and Sudan as this region has a long history of the use
of this drink. Hibiscus tea is known to be effective in lowering blood
pressure and cholesterol. In addition, researchers propose that consuming
hibiscus could aid in the prevention of human cancer. Hibiscus
sabdariffa calyces contain different components including mucilage,
polysaccharides, pectins, polyphenols, organic acids, ascorbic acid, citric,
malic and tartaric acids.II

When mice were exposed to the toxic antimicrobial agent, gentamycin, the drug did
demonstrable and predictable damage to the liver and concomitantly elevated urea (and
creatinine) concentration. As indicated in the study’s Results section, Hibiscus significantly
suppressed this urea-elevating effect:

Regarding the in vivo study, levels of creatinine and urea in sera, the
gentamicin group displayed significant increases in urea (110.5 ±
17.52 mg/dL) and creatinine (1.62 ± 0.72 mg/dL) compared with the control
group. However, the green tea-treated group demonstrated significant
reduction in urea (43.83 ± 3.45 mg/dL) and creatinine (0.617 ±

IOnderoglu S, Sozer S, Erbil KM, Ortac R, Lermioglu F. The evaluation of long-term effects of cinnamon bark
and olive leaf on toxicity induced by streptozotocin administration to rats. The Journal of Pharmacy &
Pharmacology. 1999; 51(11):1305-12.
IIAnwar Ibrahim D, Noman Albadani R. Evaluation of the Potential Nephroprotective and Antimicrobial

Effect of Camellia sinensis Leaves versus Hibiscus sabdariffa (In Vivo and In Vitro Studies). Advances in
Pharmacological Sciences. 2014

109
0.167 mg/dL) compared with gentamicin group. The hibiscus-treated group
also revealed significant reduction in both nonenzymatic markers of kidney
dysfunctions, urea (43.30 ± 6.47 mg/dL) and creatinine (0.733 ±
0.114 mg/dL) compared with gentamicin group. (Ibidem)

They elaborate on their findings in the Discussion section, indicating how their work accords
with that of others and attributing some of Hibiscus’ efficacy to its antioxidant profile:

Antioxidant compounds are very important for inhibition of oxidative


damage to biological target molecules. They are used for prevention and
treatment of many diseases such as cancer and cardiovascular, autoimmune,
and neurodegenerative diseases as well as inflammatory effect. Gentamicin
was used in this study to induce kidney dysfunction as a model of
nephrotoxicity. Silan et al. 2007 and Soliman et al. 2007 supported our
findings. They showed that gentamicin produced an elevation in the
concentrations of biochemical indicators of kidney function such as blood
urea nitrogen (BUN) and serum creatinine. Both the green tea and hibiscus-
treated group had shown significant nephroprotective effects. They reduced
biochemical indicators or nonenzymatic markers of kidney dysfunction
compared with gentamicin-induced nephrotoxicity. Okoko and Oruambo
(2008)I agree with our outcomes. They found that Hibiscus sabdariffa extract
reduced the levels of serum creatinine, urea, and the elevation of the levels of
kidney GSH and catalase in rats. (Ibidem)

Mind Amenites, this was a medical model of induced disease wherein pathology was
intentionally produced to determine the degree to which promising plant products could
prevent predictable damage. Most of us will not incur kidney damage resulting from
overdosing on gentamicin or another toxic antibiotic. But we assuredly are exposed to toxins
on a daily basis. And it is our kidneys that effectuate the efflux of toxins (as well as
attenuated, modified toxins and waste products) processed by the liver, gastrointestinal
organs and integumentary (skin) system. If the kidneys are compromised, toxins and waste
will accumulate and health will certainly suffer. What is more, the efficiency of the renal and
wider excretory system declines with age (and indulgence). If this decline in the kidney’s
capacity to expunge toxic waste can be decelerated, it is likely that longevity will be increased
accordingly and those diseases which owe their existence most markedly to toxic exposure
will be less likely to afflict us. This general protection of renal function in the face of a
known neprotoxin is in addition to the protein protection provided by Hibiscus’ urea-
reducing property. Add both botanicals to your fraction of Amen Fast fluids and you will be
operating on multiple molecular levels to potentially lengthen your lifespan, heighten health,
and avert disease.

I
Okoko T, Oruambo IF. The effect of Hibiscus sabdariffa calyx extract on cisplatin-induced tissue damage in
rats. Biokemistri. 2008;20(2):47–52.

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Figure II-IB-1. Graphical depiction of the reduction in urea and creatinine, respectively, in rodents
exposed to the antibiotic gentamicin. Excerpted from Anwar et al. 2014.

Carbamylation is a condition that increases with age. It is a consequence of urea


binding to bodily proteins. To the extent that urea can be reduced or proteins can be
protected from forming adducts therewith, a deleterious age-related process can be
attenuated. Several substances consumed during the Amen Fast exhibit urea-reducing ability
and can thuswise curtail carbamylation. The capacity of the Amen Fast/Apothecary to
combinatorially mitigate the ‘proteo-senescent’ state of carbamylation reinforces our regard
for our Regimen as an unparalleled Anti-Aging System.

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AMEN-RA’S
AGE INHIBITION REGIMEN (AIR)
AN EXPOSITION ON THE EXPERIMENTAL, THEORETICAL &
PRACTICAL UNDERPINNINGS OF LIFESPAN EXTENTION
& OPTIMAL HEALTH PROMOTION,
A DISCOURSE ON THE METHOD OF GEROSTASIS

VOLUME II
Tractatus I-C of X (Carbonylation & AOEs)

ESSENTIALS OF THE AMEN FAST


AVERTING THE EFFECTS OF ELEVATED OXIDANTS

Dr. Nūn Sava-Siva Amen-Ra, Dr.PH, MA, MSW

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The preservation of protein integrity is the principal pathway to lifespan prolongation. The
truth of this proclamation should be evident to the Adherent merely by considering the
triune topics of this Tractate. Glycation compromises protein integrity and its inhibition
attenuates aging; Carbamylation compromises protein integrity and its diminution mitigates
mortality. Additionally, it is evident that extended exposure of proteins to oxidative
conditions compromises their structure and function and correlates with curtailment of
longevity in multiple animal models. Thus we have a third chemical combatant with which to
contend and we shall see how the Amen Regimen generally and the Amen Fast more
specifically can protect our proteins from ‘Advanced Oxidative Endproducts’ (AOEs) and
enable us to attain our ideal of lengthier, healthier lives.

Carbonylation Increases with Age and Aggravates Protein Aggregation

Among the molecular manifestations of protein damage is the aberrant aggregation thereof.
Such clustering of proteins prevents them from fulfilling their functional roles in the body.
As has been underscored before by the Author, the collective integrity of our individual
proteins fundamentally defines our effective, actual age. Indeed, autophagy is so imperative to
ensuring optimal health and longevity precisely because it degrades damaged proteins,
curtails their clustering, and keeps them from cluttering of our somatic systems. We are
aware that glycation and carbamylation contribute to protein pathomorphosis. We shall now
see that pervasive oxidative damage to proteins prompts them to aggregate and that this
condition increases with age in animals. As summarized in Scientific Reports:

Protein aggregation is a common biological phenomenon, observed in


different physiological and pathological conditions. Decreased protein
solubility and a tendency to aggregate is also observed during physiological
aging but the causes are currently unknown. Herein we performed a
biophysical separation of aging-related high molecular weight aggregates,
isolated from the bone marrow and splenic cells of aging mice and followed
by biochemical and mass spectrometric analysis. The analysis indicated that
compared to younger mice an increase in protein post-translational
carbonylation was observed. The causative role of these modifications in
inducing protein misfolding and aggregation was determined by inducing
carbonyl stress in young mice, which recapitulated the increased protein
aggregation observed in old mice. Altogether our analysis indicates that
oxidative stress-related post-translational modifications accumulate in the
aging proteome and are responsible for increased protein aggregation and
altered cell proteostasis.I

Clearly, carbonylation, oxidation, autophagy and aging are intimately intertwined. Consider
what the researchers did. They compared the quantity of oxidized proteins in young and old

ITanase M, Urbanska AM, Zolla V, Clement CC, Huang L, Morozova K, Follo C, Goldberg M, Roda B,
Reschiglian P, Santambrogio L. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation.
Scientific Reports. 2016; 6:19311.

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mice and confirmed that the latter had significantly higher concentrations of such
carbonylated proteins. They then sought to ascertain its cause by simulating molecular
conditions that they conjectured would conduce to such observed derangements. The
substances selected for the simulation were carbonyl compounds; such compounds,
Amenites will recall, include the oxidative by-products of advanced glycation reactions—so-
called reactive carbonyl compounds. Dr. Tanase’s team succeeded in simulating in silico
what was seen in the aging animals—extensive aggregation of proteins.

Amen Adherents are interested in the integrity of their skeletal muscle for both vain
and valid, superficial and substantive reasons. On the loftier level, we know that the
preservation of lean muscle is linked to longevity and lessens levels of circulating sugar
owing to the repletion of insulin receptors on myocytes. On the lower, less lofty (but by no
means base) level we perceive that the preservation of lean muscle is aesthetically appealing
and enables one to maintain a desirable level of strength, stamina and general vigor with
increasing age. Central to the maintenance of skeletal muscle are satellite cells whose
replicative expansion replaces damaged myocytes. Satellite cells are subject to oxidative
assault and the more extensive these insults are the less capable we are of repairing and
replacing damaged muscle tissue. Of course this compromised capacity to replace deranged
muscle increases with age and is called sarcopenia. So, sarcopenia and carbonylation are closely
linked. As indicated in the journal Free Radical Biology & Medicine....

Accumulation of damaged macromolecules, including irreversibly oxidized


proteins, is a hallmark of cellular and organismal ageing. Failure of protein
homeostasis is a major contributor to the age-related accumulation of
damaged proteins. In skeletal muscle, tissue maintenance and regeneration is
assured by resident adult stem cells known as satellite cells. During
senescence their replication and differentiation is compromised contributing
to sarcopenia. In this study we have addressed the impact of oxidatively
modified proteins in the impaired metabolism of senescent human satellite
cells. By using a targeted proteomics analysis we have found that proteins
involved in protein quality control and glycolytic enzymes are the main
targets of oxidation (carbonylation) and modification with advanced
glycation/lipid peroxidation end products during replicative senescence of
satellite cells. Inactivation of the proteasome in aged cells appeared as a key
contributor to the accumulation of such damaged proteins. Untargeted
metabolomic profiling and functional analyses indicated glucose metabolism
impairment in senescent cells, although mitochondrial respiration remained
unaffected. A metabolic shift leading to increased mobilization of non-
carbohydrate substrates as branched chain amino acids or long chain fatty
acids was observed in senescent cells. In addition, phospho-and glycerolipids
metabolism was altered. Increased levels of acyl-carnitines indicated
augmented turnover of storage and membrane lipids for energy production.
Such changes reflect alterations in membrane composition and dysregulation
of sphingolipids signaling during senescence. This study establishes a new
concept connecting oxidative protein modifications with the altered cellular
metabolism associated with the senescent phenotype. In addition, these
findings highlight the molecular mechanisms implicated in satellite cells
dysfunction during ageing, paving the road for future therapeutic

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interventions aimed at preventing oxidative modifications of proteins and/or
stimulating their elimination.I

Contributing further to the catabolism of skeletal muscle, carbonylation caused those amino
acids most concentrated in the contractile fibers of muscle—the branched chain amino acids
(BCAAs)—to be deconstructed so as to serve as fuel. Concomitantly, damage to glycolytic
enzymes rendered them less able to provide the glycogen-generated glucose that makes the
possession of copious skeletal muscle especially salubrious in a glycemic sense. To be clear,
when one is relatively replete with muscle, one metabolizes sugar more readily and its
concentration is consequently lowered in the serum as compared to a corpulent state; this
condition of marked muscularity mitigates glycation. Now we see, on the strength of this
superb study that excessive oxidation of proteins alters the gross morphology and
biochemical physiology of muscle in a manner that is synergistically sinister.
There are, of course, two distinct types of striated muscle—skeletal and cardiac. It
has been established that carbonylation compromises cardiac function by disabling enzymes
involved in autophagy; the impairment of autophagy promotes the accretion of
dysfunctional proteins and this the defining hallmark of aging. The ensuing analysis in
Oncotarget is complex but the basics have already been bestowed upon the Reader. The
researchers relate that...

Cardiac aging is characterized by accumulation of damaged proteins and


decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated
inactivation in aged heart, we determined the benefits of activation of
aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the
ALDH2 KO mice progressively developed age-related heart dysfunction and
showed reduction in the lifespan, which strongly suggests that ALDH2
ablation leads to cardiac aging. What's more, aged hearts displayed a
significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-
protein adducts and protein carbonyls, impairment in the autophagy flux,
and, consequently, deteriorated cardiac function after starvation. Sustained
Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2
activity and abrogated these effects. Using SIRT1 deficient heterozygous
(Sirt1+/-) mice, we found that SIRT1 was necessary for ALDH2 activation-
induced autophagy. We further demonstrated that ALDH2 activation
attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby
increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially,
ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1
increased Rab7 expression, which were both necessary and sufficient for
restoring autophagy flux. These results highlight that both accumulation of
proteotoxic carbonyl stress linkage with autophagy decline contribute to
heart senescence. ALDH2 activation is adequate to improve the autophagy

IBaraibarM, Hyzewicz J, Rogowska-Wrzesinska A, Bulteau AL, Prip-Buus C, Butler-Browne G, Friguet B.


Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic
enzymes. Free Radical Biology & Medicine. 2014; 75 Suppl 1:S23.

115
flux by reducing the carbonyl modification on SIRT1, which in turn plays an
important role in maintaining cardiac health during aging.I

To elaborate, aldehyde dehydrogenase is an enzyme that rectifies glycation by removing


glyoxal, a reactive carbonyl compound. Concomitantly, it also actuates autophagy. Thus, the
inactivation of this enzyme with age exacerbates and correlates with senescence. It is the
carbonylation of SIRT, so the study established, that suppressed aldehyde dehydrogenase
and led to discernible cardiac damage. Astute Amenites will recognize SIRT as a molecule
that mediates genome silencing and is implicated in multiple models of lifespan extension.
Indeed, one reason that our Regimen includes resveratrol in the Amen Apothecary is due to
its SIRT/sirtuin-stimulating efficacy. Allow the Author to add to this intricate lattice linking
longevity to multiple molecules by mentioning that the compounds created by glycation and
glycolysis (e.g. glyoxal and methylglyoxal) are agents that, in turn, oxidize proteins; in effect,
glycation leads to carbonylation. This is explained in an Abstract in the aptly named journal,
Chemico-Biological Interactions:

Chronic hyperglycemia in diabetic patients often leads to chronic side effects


associated with protein glycation and the formation of reactive carbonyl
species, such as methylglyoxal (MGO) and glyoxal (GO). We have shown
that both MGO and GO carbonylated bovine serum albumin (BSA) in vitro
to the same degree and stability. The carbonylated BSA formed initially could
be a reversible Schiff base as the UV absorbance formed after the addition of
2,4-dinitrophenylhydrazine was decreased when sodium borohydride was
added. MGO and GO also carbonylated hepatocyte protein rapidly with
similar dose and time dependence. In contrast to BSA carbonylation, the
amount of carbonylated proteins in hepatocytes decreased over time, much
more rapidly for hepatocytes treated with MGO than with GO. This could
be attributed to the rapid hepatocyte metabolism of MGO with glyoxalase I,
the predominant detoxification enzyme for MGO. Protein carbonylation and
the associated toxicity caused by GO and MGO were studied in the
following hepatocyte models: (1) control hepatocytes, (2) glutathione (GSH)-
depleted hepatocytes, (3) mitochondrial aldehyde dehydrogenase (ALDH2)-
inhibited hepatocytes, (4) hepatocyte inflammation model, and (5) catalase-
inhibited hepatocyte model. Carbonylation and cytotoxicity caused by MGO
or GO was markedly increased in GSH-depleted hepatocytes as compared to
control hepatocytes. Hepatocytes exposed to non-toxic concentrations of
H2O2 or hepatocytes treated with catalase inhibitors also showed a marked
increase in GO-caused cytotoxicity and protein carbonylation, whereas there
were only minor increases with MGO. The GO effect was attributed to
potential radical formation and the inhibition effect of H2O2on aldehyde
dehydrogenase, a major GO metabolising enzyme. GO-caused cytotoxicity
and protein carbonylation were also increased with ALDH2-inhibited

IWu B, Yu L, Wang Y, Wang H, Li C, Yin Y, Yang J, Wang Z, Zheng Q, Ma H. Aldehyde dehydrogenase 2


activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1. Oncotarget.
2016; 7(3):2175-88.

116
hepatocytes whereas such an increase was only observed with MGO in GSH-
depleted hepatocytes.I

It was intelligent for the investigators to observe and recount that the glycation compounds
caused less damage to whole hepatocytes than to the purified proteins comprising BSA. This
difference was due to the presence of glutathione resident within the hepatic tissue. The
Reader will recall from Tractate I-A of this Volume that agents in our Apothecary stimulate
the glutathione-containing glyoxalase system and that glutathione itself is included as a
supplement in the Amen Apothecary. Inhibiting the antioxidant enzyme catalase was also
found to facilitate carbonylation. Recall that the catalytic activity of catalase or its
concentration is increased by agents included in the Amen Apothecary and Amen Meal alike.
Thus we see that some of the strategies we employ against advanced glycation end-products
are effective against ‘advanced oxidation end-products’.
We shall end our enumeration of the inimical nature of protein oxidation with a
study chronicling the damaging effects of carbonylation on fibrocartilage. The insidious
stiffening of cartilage with increasing age is an effect that is doubly dooming for it forces
sufferers to assume a more stooped, sedentary state which, in turn, exacerbates the condition
owing to the increased inflammation, glycation, and autophagic insufficiency that follows
weight gain and inadequate exercise. The study is summarized in Chemistry & Biology thusly:

Aging-related oxidative stress has been linked to degenerative modifications


in different organs and tissues. Using redox proteomic analysis and
illustrative tandem mass spectrometry mapping, we demonstrate oxidative
posttranslational modifications in structural proteins of intervertebral discs
(IVDs) isolated from aging mice. Increased protein carbonylation was
associated with protein fragmentation and aggregation. Complementing these
findings, a significant loss of elasticity and increased stiffness was measured
in fibrocartilage from aging mice. Studies using circular dichroism and
intrinsic tryptophan fluorescence revealed a significant loss of secondary
and tertiary structures of purified collagens following oxidation. Collagen
unfolding and oxidation promoted both nonenzymatic and enzymatic
degradation. Importantly, induction of oxidative modification in healthy
fibrocartilage recapitulated the biochemical and biophysical modifications
observed in the aging IVD. Together, these results suggest that protein
carbonylation, glycation, and lipoxidation could be early events in promoting
IVD degenerative changes.II

Attenuate glycation and carbonylation and we can considerably curtail the skeletal
degeneration that commonly characterizes aging and cruelly confines seniors to a premature
state of immobile impotence.

IYang K, Qiang D, Delaney S, Mehta R, Bruce WR, O'Brien PJ. Differences in glyoxal and methylglyoxal
metabolism determine cellular susceptibility to protein carbonylation and cytotoxicity. Chemico-Biological
Interactions. 2011; 191(1-3):322-9.

IIScharf B, Clement CC, Yodmuang S, Urbanska AM, Suadicani SO, Aphkhazava D, Thi MM, Perino G,
Hardin JA, Cobelli N, Vunjak-Novakovic G, Santambrogio L. Age-related carbonylation of fibrocartilage
structural proteins drives tissue degenerative modification. Chemistry & Biology. 2013; 20(7):922-34.

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Now enter the Agents. Alas Amenites, our task concerning carbonylation is yet
incomplete until we confirm that there is a practical way of rectifying or precluding pervasive
protein oxidation. The Author has already ascertained this and it should come as little
surprise to the Amenite that many of the agents in our Apothecary that are effective against
glycation are analogously inhibitory toward carbonylation. The active compounds in Camellia
sinensis are the first substances that we shall consider. The following in Abstract in Food &
Function informs us that...

Carbonylation is an irreversible modification in oxidized proteins that has


been directly related to a number of health disorders including Type 2
diabetes. Dietary antioxidants have been proposed to counteract the
oxidative stress occurring under hyperglycemic conditions. An understanding
of the nature and consequences of the molecular interactions between
phytochemicals and human plasma proteins is of utmost scientific interest.
Three tea catechins, namely epicatechin (EC), epigallocatechin (EGC) and
epigallocatechin-3-gallate (EGCG) were tested for (i) their affinity to bind to
human serum albumin (HSA) and human hemoglobin (HH) and (ii) their
ability to inhibit tryptophan (Trp) depletion and for the formation of specific
protein carbonyls and pentosidine in the aforementioned proteins. Both
proteins (20 mg mL(-1)) were allowed to react with postprandial plasmatic
concentrations of the catechins (EC: 0.7 µM, EGC: 1.8 µM, and EGCG: 0.7
µM) under simulated hyperglycemic conditions (12 mM glucose/0.2 mM
Fe(3+)/37 °C/10 days). The three catechins were able to inhibit Trp
oxidation and protein carbonylation in both plasma proteins. Some anti-
glycation properties were linked to their binding affinities. The molecular
interactions reported in the present study may explain the alleged beneficial
effects of tea catechins against the redox impairment linked to hyperglycemic
conditions.I

The combination of catechins in Camellia formed complexes with human plasma proteins
and chemically protected them from glycative and oxidative attack. This offers insight into a
mechanism of action with important, practical consequences for Amen Practitioners.
Because our Protocol prescribes continual consumption of tea throughout the course of our
Fast, the compounds contained therein fleetingly affix themselves to our proteins, providing
protection against glycation and subsequent oxidation.

Now Neophytes, consider the anti-carbonylation capacity of coffee along with grape
seed extract, pyridoxamine (B6), carnosine, and the gold-standard anti-glycating agent
aminoguanidine. In the Journal of Pharmacology & Biomedical Analytics, the study is summarized
thusly:

An in vitro high resolution mass spectrometry (MS) method was set-up to


test the ability of compounds, mixtures and extracts to inhibit protein
carbonylation induced by reactive carbonyl species (RCS). The method

IÖzyurtH, Luna C, Estévez M. Redox chemistry of the molecular interactions between tea catechins and
human serum proteins under simulated hyperglycemic conditions. Food & Function. 2016 Mar 16;7(3):1390-400.

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consists of incubating the protein target (ubiquitin) with 4-hydroxy-trans-2-
nonenal (HNE) in the presence and absence of the tested compound. After
24h of incubation, the reaction is stopped and the protein is analyzed by
high-resolution MS. The extent of protein carbonylation is determined by
measuring the area of the +11 multicharged peak of the HNE adduct in
respect to the native form. The method was validated by measuring the effect
of well-known RCS sequestering agents, namely aminoguanidine,
pyridoxamine, hydralazine and carnosine, yielding a good reproducibility and
the possibility to be automatable. All the compounds were found to dose-
dependently inhibit the protein carbonylation with the following order of
potency carnosine≈hydralazine≫aminoguanidine>pyridoxamine, as
determined by calculating the UC50 values, that is the concentration required
to inhibit ubiquitin carbonylation by 50%. A good correlation was found
with the results obtained by measuring HNE consumption using an HPLC
method optimized by a mobile phase set at pH 7.4, in order to stabilize the
eluted adducts. The MS approach was then applied to test the effect of two
selected natural extracts on protein carbonylation, i.e. green coffee bean
extract and procyanidins from Vitis vinifera. In summary, this paper reports a
validated and highly reproducible MS method to test the ability of pure
compounds as well as natural extracts to act as protein carbonylation
inhibitors.I

As we shall see in Tractate I of Volume III, carnosine and pyridoxamine are components of
the Amen Amino Aliksir. Grape seed extract is a constituent of the Amen Apothecary
contained in a capsule in combination with resveratrol. Coffee is consumed several times
throughout the fasting day, especially as a pre-exercise stimulant and fat-oxidizing agent.
Thus do we combat carbonylation on several fronts and at several phases of our Circadian
Cycle, especially during our Fast.
We shall end our exposition on carbonylation with a study of substantial significance
for those Adherents who are most desirous of maintaining adequate mental function for the
duration of their duly-lengthened lives—this should assuredly encompass all Amenites. We
have seen and shall see the ensuing biomedical model again. It is a mouse manufactured to
age rapidly and to exhibit signs and symptoms of Alzheimer’s disease. The scientists that
conducted this study demonstrated that carbonylation contributes to the cognitive
impairment of these organisms and is manifested molecularly by more oxidized proteins
being prevalent in the brain’s hippocampal region. As importantly, the investigators
identified a compound that can avert this oxidative alteration in the age-accelerated animals.
This compound is a constituent of citrus peels and is aptly called nobiletin. Dr. Nakajima
discusses the team’s findings in the pages of Behavioural Brain Research thusly:

Senescence-accelerated mouse prone 8 (SAMP8) is a model of aging


characterized by the early onset of learning and memory impairment and
various pathological features of Alzheimer's disease (AD). Our recent studies

IColzani M, Criscuolo A, De Maddis D, Garzon D, Yeum KJ, Vistoli G, Carini M, Aldini G. A novel high
resolution MS approach for the screening of 4-hydroxy-trans-2-nonenal sequestering agents. J Pharm Biomed
Anal. 2014 Mar;91:108-18.

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have demonstrated that nobiletin, a polymethoxylated flavone from citrus
peels, ameliorates learning and memory impairment in olfactory-
bulbectomized mice, amyloid precursor protein transgenic mice, and NMDA
receptor antagonist-treated mice. Here, we present evidence that this natural
compound improves age-related cognitive impairment and reduces oxidative
stress and tau phosphorylation in SAMP8 mice. Treatment with nobiletin (10
or 50mg/kg) reversed the impairment of recognition memory and context-
dependent fear memory in SAMP8 mice. Treatment with nobiletin also
restored the decrease in the GSH/GSSG ratio in the brain of SAMP8 mice.
In addition, increases in glutathione peroxidase and manganese-superoxide
dismutase activities, as well as a decrease in protein carbonyl level, were
observed in the brain of nobiletin-treated SAMP8 mice. Furthermore,
nobiletin reduced tau phosphorylation in the hippocampus of SAMP8 mice.
Together, the markedly beneficial effects of nobiletin represent a potentially
useful treatment for ameliorating the learning and memory deficits, oxidative
stress, and hyperphosphorylation of tau in aging as well as age-related
neurodegenerative diseases such as AD.I

When it is so simple to suppress a scourge as unrelenting as Alzheimer’s it is incumbent


upon the intelligent to do so. There is a potent preventative product in the peelings of a
pervasive fruit for which we have so little use that we commonly discard it (and its
indwelling nobiletin, naringenin, and numerous citrus flavonoids) without notice. Now
Amenites know better. Neophytes need not steep the skins of citrus fruits and laboriously
extract their essence. Supplements can serve this purpose and, indeed, citrus flavonoids are
included in the micronutrient complex added to our Amen Amino Aliksir [see AIR III:I].
These compounds can also be ingested during the Amen Fast in either the Autophagic or
Anti-Glycative/Antioxidant Aliksir partaken prior to or after the Persistence Exercise
Session(s). Further, far lesser amounts of such citrus bioflavonoid compounds can be
consumed in tea, especially those varieties of black tea infused with spices and essence of
orange zest. In this last line there is a lesson for those Amenites and other Readers reluctant
to embrace the apparent “artificiality” of supplementation. Though we shall have reason to
revisit this issue more meaningfully in AIR III:I, it is fitting to reflect on some pertinent
points. If one is convinced of the soundness and significance of the study above and seeks to
integrate it into a practicable regimen such as ours, could one acquire the quantity of the
active compound from food? Clearly not. Happily, most of us are in the position of
preventing pathologies such as Alzheimer’s from occurring rather than that of curing it or
reducing its severity once we, or those for whom we care, are afflicted therewith. As such, an
animal model wherein the organism exhibits the disease may seem less applicable to us. But
let us not forget that many maladies have long latencies and that we may harbor the
precursors of pathologies without being privy to their presence. The Author prefers more
prudence and prescribes the adoption of all otherwise innocuous, actionable interventions
that demonstrably diminish dementia and mortality. This impetus explains the expansive,
integrative essence of AIR.

INakajimaA, Aoyama Y, Nguyen TT, Shin EJ, Kim HC, Yamada S, Nakai T, Nagai T, Yokosuka A, Mimaki Y,
Ohizumi Y, Yamada K. Nobiletin, a citrus flavonoid, ameliorates cognitive impairment, oxidative burden, and
hyperphosphorylation of tau in senescence-accelerated mouse. Behavioral Brain Research. 2013; 250:351-60

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