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Manuscript Number: YCLNU-D-14-00186R1

Title: Hyperinsulinemia is associated with the loss of appendicular skeletal muscle mass at 4.6 year
follow-up in older men and women

Article Type: Full Length Article

Keywords: Hyperinsulinemia, loss of skeletal muscle, older adults

Corresponding Author: Dr. HELIODORO ALEMAN MATEO, Ph.D

Corresponding Author's Institution: CENTRO DE INVESTIGACION EN ALIMENTACION Y DESARROLLO,
AC

First Author: HELIODORO ALEMAN MATEO, Ph.D

Order of Authors: HELIODORO ALEMAN MATEO, Ph.D; Miriam T Lpez Teros, Master in Science;
Ftima A Ramrez Caballero, Master in Science

Abstract: Background and aim: Homeostasis model assessment as a marker of insulin resistance has
been associated with the pronounced loss of appendicular skeletal muscle mass in older adults. In the
present study, we hypothesized that hyperinsulinemia as an early predictor of insulin resistance may
be associated with the loss of appendicular skeletal muscle mass (ASM). Methods: This is a cohort
study that included 147 well-functioning older men and women subjects who were followed for a
period of 4.6 1.8 years. Lean tissue in arm and legs, or ASM, was derived from dual-energy x-ray
absorptiometry at baseline with follow-up measurements to obtain the relative change.
Hyperinsulinemia was defined empirically at the 75th percentile. Results: The relative change in ASM
was negative and significant throughout the quartiles of fasting insulin levels (p0.05); however, the
loss of ASM was more pronounced in the later quartiles (-0.7 kg) compared with the relative change in
Q1 and Q2 (-0.5 kg and -0.3 kg). The unadjusted analysis indicates a significant association between
hyperinsulinemia and the loss of ASM (= -0.28, 95% CI-0.57-.009, p=0.05), an association that
remained significant after adjusting for several covariates. Conclusion: Hyperinsulinemia as an early
marker of insulin resistance was associated with the loss of ASM in a cohort study of community-
dwelling older men and women subjects without other chronic health conditions. The use of fasting
insulin levels >8.4 U/mL may help clinicians identify individuals in the geriatric population who are at
a high risk of loss of appendicular skeletal muscle mass.





ICMJE Conflict of Interest
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ICMJE Conflict of Interest
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March 21, 2014


N. E. P. Deutz, MD, PhD
Editor of Clinical Nutrition


Dear Editor we are submitting online our Original Article: Hyperinsulinemia is
associated with the loss of appendicular skeletal muscle mass at 4.6 year follow-up in
older men and women by Miriam T. Lpez Teros, Ftima A Ramrez Caballero and
Heliodoro Alemn-Mateo, for review and publication in your prestigious Journal. Dear
Editor, to the best our knowledge this is the first cohort study that demonstrates a
significant association between hyperinsulinemia and the loss of appendicular skeletal
muscle mass in older men and women subjects. We are sure that the clinical importance
of our study rests on the design (cohort study) and the identification of an early marker of
the loss of skeletal muscle mass in the geriatric population.

Thank you for considering our work.

Sincerely,


Heliodoro Alemn-Mateo, PhD.



Centro de Investigacin en Alimentacin y Desarrollo, A.C., Coordinacin de Nutricin. Carretera a la Victoria
Km 0.6, Apartado Postal 1735; Hermosillo, Sonora, Mxico 83000. Tel /FAX + 52 662 280 0094
CENTRO DE INVESTIGACION
EN ALIMENTACION Y DESARROLLO, A.C.
SEP-CONACYT-UNAM-IPN-GOBIERNO DE SONORA-GOBIERNO DE SINALOA-GOBIERNO DE CHIHUAHUA-SEMARNAP
Cover letter
1

Hyperinsulinemia is associated with the loss of appendicular skeletal muscle
mass at 4.6 year follow-up in older men and women

Miriam T. Lpez Teros, Ftima A. Ramrez C, Heliodoro Alemn-Mateo


AFFILIATION: Coordinacin de Nutricin, Centro de Investigacin en Alimentacin
y Desarrollo (CIAD), A.C.

CORRESPONDING AUTHOR: Heliodoro Alemn Mateo, Coordinacin de Nutricin,
Centro de Investigacin en Alimentacin y Desarrollo A.C., Carretera a la Victoria
Km. 0.6, Hermosillo, Sonora, Mxico. Apartado Postal 1735, C.P. 83304. Tel. and
Fax: 52 (662) 280-0094. E-mail: helio@ciad.mx




Running head: Association between hyperinsulinemia and loss of appendicular
skeletal muscle mass


Manuscript
Click here to view linked References
2

ABSTRACT
Background and aim: Homeostasis model assessment as a marker of insulin
resistance has been associated with the pronounced loss of appendicular skeletal
muscle mass in older adults. In the present study, we hypothesized that
hyperinsulinemia as an early predictor of insulin resistance may be associated with
the loss of appendicular skeletal muscle mass (ASM). Methods: This is a cohort
study that included 147 well-functioning older men and women subjects who were
followed for a period of 4.6 1.8 years. Lean tissue in arm and legs, or ASM, was
derived from dual-energy x-ray absorptiometry at baseline with follow-up
measurements to obtain the relative change. Hyperinsulinemia was defined
empirically at the 75
th
percentile. Results: The relative change in ASM was
negative and significant throughout the quartiles of fasting insulin levels (p0.05);
however, the loss of ASM was more pronounced in the later quartiles (-0.7 kg)
compared with the relative change in Q1 and Q2 (-0.5 kg and -0.3 kg). The
unadjusted analysis indicates a significant association between hyperinsulinemia
and the loss of ASM (= -0.28, 95% CI-0.57-.009, p=0.05), an association that
remained significant after adjusting for several covariates. Conclusion:
Hyperinsulinemia as an early marker of insulin resistance was associated with the
loss of ASM in a cohort study of community-dwelling older men and women
subjects without other chronic health conditions. The use of fasting insulin levels
>8.4 U/mL may help clinicians identify individuals in the geriatric population who
are at a high risk of loss of appendicular skeletal muscle mass.
Keywords: hyperinsulinemia, loss of skeletal muscle, older adults
3

1. Introduction
Clinical metabolic studies have demonstrated that the action of insulin declines
progressively with age. In addition to its close association with type 2 diabetes,
which reduces life expectancy in older people, age-related insulin resistance is
implicated in the pathogenesis of several highly prevalent disorders for which aging
is a major risk factor.
1
Age-related loss of skeletal muscle mass below a critical
threshold and sarcopenia are two related disorders often found in the elderly
population.
2-5
Both sarcopenia
6
and insulin resistance
7
(IR) are highly prevalent
disorders in older people around the world. Older subjects who experience muscle
loss and suffer from sarcopenia are exposed to an increased risk of adverse long-
term clinical outcomes such as mobility disorders, disabilities, and poor quality of
life.
8-16

The link between the pancreatic function and skeletal muscle is based on
the role of insulin in regulating body protein mass.
17
The main in vivo effect of
insulin and amino acids on whole-body and skeletal muscle protein metabolism is
to inhibit protein breakdown and stimulate protein synthesis.
18-19
In aged subjects,
the relation between insulin, and the loss of skeletal muscle and sarcopenia is
based on the muscle protein resistance to the anabolic action of insulin,
2,20
a defect
that has been associated with impaired insulin-induced vasodilation and
mammalian target of rapamycin signalling (mTORC1).
20
In fact, this is one of the
potential mechanisms that, it has been suggested, underlies the involuntary loss of
skeletal muscle mass associated with IR in older adults. However, there are no
published studies that allow us to infer a causal association between insulin or
4

hyperinsulinemia as an early predictor of IR and the loss of skeletal muscle mass
in older people.
Hormonal factors that have been implicated in the pathogenesis of the loss
of skeletal muscle mass and sarcopenia include reduced production of
dehydroepiandrosterone sulphate,
21
lower total and free testosterone, and elevated
parathyroid hormone concentrations,
22-23
higher insulin-like growth factor-1 levels,
24

vitamin D deficiency, and high parathyroid hormone levels.
25-27
There is evidence
from studies in humans which suggests that insulin resistance contributes to the
loss of appendicular skeletal muscle mass (ASM). Recently, hyperinsulinemia,
defined by the homeostasis model assessment (HOMA) as a marker of insulin
resistance (HOMA-IR), has been associated with the pronounced or relative loss of
ASM in older non-diabetic men and women subjects.
3-4
In addition,
hyperinsulinemia defined in terms of fasting insulin levels, has been strongly
associated with the risk of developing hypertension,
28-29
and cognitive decline
30
in
older adult populations.
To the best of our knowledge, the literature contains is no longitudinal
evidence of the association between hyperinsulinemia and the loss of ASM. It is
important to recognize that insulin resistance assessed by the homeostatic model
assessment cited above is highly dependent on glucose levels.
3-4
Insulin resistance
occurs in conjunction with a compensatory hyperinsulinemia that initially maintains
plasma glucose levels within normal ranges. In the present study, we hypothesized
that hyperinsulinemia as an early predictor of insulin resistance is associated with
the loss of appendicular skeletal muscle mass in a cohort study of community-
dwelling older men and women subjects.
5


2. Material and methods
2. 1. Study population
This cohort study was designed to assess the association of certain age-related
systemic changes and the loss of ASM in a non-random sample of apparently
healthy, community-living subjects.
4,31
The cohort was derived from a body
composition validation techniques study in 302 subjects over 60 years of age.
32
For
this trial, we report the association between fasting insulin or hyperinsulinemia at
baseline and the loss of appendicular skeletal muscle at 4.6 years of follow-up in a
sample of older men and women subjects. The study was approved by the Ethics
Committee of the Research Centre for Food and Development and written
informed consent was obtained from all participants.
2.2. Subjects
This cohort study was conducted at the Body Composition Laboratory of the
Research Centre for Food and Development in Hermosillo, Sonora, Mexico, and
included 147 older men and women subjects. As reported previously,
4
the original
study was carried out between March 2003 and April 2006, with a second
assessment from March 2008 to May 2011. The total follow-up period was 4.6
1.8 years. At baseline, all participants underwent a medical assessment that
included an oral glucose tolerance test, biochemical analyses, and functionality
and cognitive assessment.
33-34
Body composition, anthropometry (body weight,
6

height, body mass index and waist circumference), physical activity, and
socioeconomic status were also assessed as part of the protocol.
The inclusion and exclusion criteria have been detailed elsewhere.
4
Briefly,
all subjects were over 60 years of age, apparently healthy, physically independent,
and with no pronounced loss of ASM. Subjects with a history of diabetes or plasma
glucose 200 mg/dL at 2 hours after a 75g glucose load, heart attack, cancer,
chronic lung disease, mental disorders, chronic neurological disorders, arthritis and
other musculoskeletal diseases, and liver and kidney disease, were not included.
Other causes of exclusion were a lack of data for any one of the following
parameters: fasting insulin and glucose, DXA measurements, and body mass
index <18.5 kg/m
2
. Volunteers with controlled hypertension, hypothyroidism or
dyslipidemia diagnosed by the medication used or by lipid profile determination
were not excluded. The second assessment included body composition,
anthropometric measurements and a clinical evaluation.
2. 3. Measures of body composition
Body composition was measured by DXA using the DPX-MD+ (GE Lunar Madison,
WI, USA). At baseline and follow-up, evaluations of body composition were carried
out under fasting conditions in accordance with established guidelines. ASM was
determined from the DXA scans following the recommended anatomical
landmarks. The sum of non-fat plus non-bone tissue in both arms and legs was
used to represent ASM.
35

Subjects with low relative ASM or a pronounced loss of ASM at baseline
were not invited for the second assessment; since cohort studies typically focus on
7

new cases of disease that occur during follow-up. To detect subjects with low
relative ASM, the residuals method
36
was applied, and subjects were classified as
having low relative ASM when the values of their residuals fell into the sex-specific
lowest 20% of the baseline survey distribution of residual values. As published
previously,
4
there is no universal criterion for low relative ASM, so the residual
method was used because it is based on measurements of ASM and requires no
reference value from a healthy young population.
36
Using the procedure described
above, 66 subjects proved to have low relative ASM, and so were not included in
the cohort.
2. 4. Response variable
Basal and follow-up measurements of ASM were used to define the response
variable. The absolute relative changes in the response and other variables were
determined as the difference between follow-up and baseline measurements. Also,
the percentage of relative change was calculated as the difference in ASM
between the 4.6-year follow-up examination and baseline, divided by baseline ASM
and multiplied by 100. The absolute relative change in the variable ASM was used
as the continuous variable in the statistical analyses.
2. 5. Biochemical analyses
As reported previously, the following determinations were made at baseline:
plasma glucose, serum insulin, serum interleukin-6 and C-reactive protein, and
serum lipid profiles, especially high-density lipoprotein cholesterol and triglycerides.
Some of these measurements, such as fasting and 2-hour glucose for diagnoses of
8

diabetes, were among the exclusion criteria, while others were measured as
covariates.
4

2. 6. Exposure variable
Clinically, hyperinsulinemia was defined as the exposure variable. Previously,
relevant cut-offs have been reported
37
as fasting insulin 12.2 U/mL; however, it
is unclear precisely what level of insulin resistance is associated with the risk of
developing loss of ASM. Therefore, hyperinsulinemia was defined empirically at the
75
th
percentile based on all subjects in the cohort for whom we had baseline fasting
insulin determinations. In this trial, the 75
th
percentile corresponded to 8.4 U/mL
for fasting insulin.
2. 7. Statistical analyses
Results are presented as means SD, frequencies and percentages. Both mean
absolute change and percentages of relative change in the anthropometric and
body composition characteristics were calculated for each quartile (Q) of the
fasting insulin values. The differences in these variables between quartiles were
assessed by analysis of variance using a Fisher's LSD multiple comparison test. A
significant absolute change in these measurements was determined as p0.05 for
paired t-tests for baseline and 4.6-year follow-up measurements. The association
between fasting insulin values and the loss of ASM was examined using
multivariable linear regression analysis. The association between fasting insulin
values as the dichotomized variable and total appendicular skeletal muscle mass
as continuous variable at baseline was also tested by multivariable linear
9

regression analysis. All statistical analyses were performed using STATA version
11.0 software (StataCorp LP, College Station, Texas).
Covariates were examined in the regression models and included if the
magnitude of association was >10%. Both unadjusted and adjusted coefficients
and 95% confidence intervals (CI) were obtained. The following covariates were
used for this analysis, and were described previously.
4
Briefly, controlled
hypothyroidism, medications, non-steroidal anti-inflammatory drugs, interleukin-6
and C-reactive protein were considered as covariates. Markers of inflammation,
lipid profiles, fasting glucose and insulin, two-hour post-load glucose, age, fat mass
and body weight were all coded as continuous variables; while the categorical
variables included gender (0= female, 1= male); educational level (0 years, 6
years, >6 years of schooling); alcohol use [none, light (7 drinks/wk), moderate-to-
high (>7 drinks/wk)]; smoking (never/former or current); hypertension (0= no
hypertension and 1= hypertension); use of antihypertensive, hypolipidemic agents
and non-steroidal anti-inflammatory drugs (0= no use, 1= use); and physical
activity level by using published predictive equations and the sedentary-
active/moderate-vigorous classification.

3. Results
Two hundred and thirty-six subjects were considered as potential participants;
however, due to various causes, the final cohort consisted of only 147 well-
functioning older men and women subjects who were followed for a period of 4.6
1.8 years. Mean age of the cohort was 68.3 6.0 years at baseline and increased
10

to 73.0 6.3 years at the end of the study. More than fifty percent (56%) of the
total sample were women. The main causes for abandoning the study were: I
prefer not to participate (n= 20); inability to make contact (27); illness and physical
disabilities (n=28); no data for fasting insulin (n=1); undernourishment (n=1); and
death (n=12). Results of the comparative analyses of the baseline measurements
between the cohort and the subjects that did not participate at follow-up show
basically no significant differences between our main response and exposure
variables at baseline, or with respect to any of the other key variables, such as
age, gender, blood pressure, and BMI (Table 1).
Table 2 shows the behaviour of the relative changes in the different
anthropometric and body composition variables according to the quartile
distribution of fasting insulin levels. The relative changes in body weight, body
mass index, total lean tissue, and fat-free mass were negative and significant
throughout the quartiles of fasting insulin levels (p0.05). However, the loss of
ASM was more pronounced in the later quartiles (-0.7 kg) compared to the loss in
the Q1 and Q2 (-0.5 and -0.3 kg). With respect to the fat component total and
truncal body fat the relative changes were negative and only significant in the Q3
and Q4 (p0.05).
It is important to mention that the results of the baseline analysis show that
ASM did not increase according to the quartile distribution of the values for fasting
insulin (Table 2). Also, the unadjusted regression analysis was not significant,
though after adjustment for some baseline covariates such as age, gender, total
fat (kg) and height (cm) a significant and positive association was found. A one-
unit increment in fasting insulin was associated with 120 g increases of ASM
11

(model 1, =0.12, 95% CI -0.03-(-0.20), p=0.00). After additional adjustments
using baseline physical activity as the covariate plus model 1 covariates, the model
remained significant (model 2, =0.11, 95% CI 0.02-0.19, p=0.01), and the same
results were found when model 3 was adjusted for C-reactive protein (mg/L) and
interleukin-6 (pg/mL) plus model 1 ( =0.10, 95% CI 0.01-0.2, p=0.02) (Table 3).
Results of the follow-up analysis of the data on the association between
fasting insulin and the loss of ASM are presented in Table 4 as beta coefficients
and 95% confidence intervals (CI). The unadjusted analysis indicates a significant
negative association between hyperinsulinemia (8.4 U/mL), and the ASM
variable compared to the reference group (<8.4 U/mL). The change of <8.4
U/mL to hyperinsulinemia (8.4 U/mL) decreased the relative change of the
ASM variable in 280 g (= -0.28, 95% CI-0.57-.009, p=0.05). Differences between
groups remained significant after adjustment for baseline variables such as age,
sex, total fat (kg), and height (m) (model 1: =-0.32, 95% CI -0.61-(-0.03), p=0.02).
Also, when adjusted for the baseline physical activity covariate plus model 1
covariates they remained significant (model 2, =-0.33 95% CI -0.62-(-0.04),
p=0.02). This association was also adjusted for C-reactive protein (mg/L) and
interleukin-6 (pg/mL) plus model 1 covariates and continued to be significant,
though to a lesser degree (model 3, =-0.33, 95% CI -0.65-(-0.20), p=0.05).
4. Discussion
In the present cohort study, the results of the baseline survey showed that an
elevated baseline insulin concentration (8.4 U/mL) is associated with increases
of ASM, while at 4.6-year follow-up hyperinsulinemia increased the risk of ASM
12

loss in both elderly men and women. This relationship was maintained after
adjusting for traditional risk factors for ASM loss, including age, gender, smoking,
alcohol use, fat mass, comorbidities such as heart disease and hypertension,
physical activity, use of drugs like anti-hypertension and hypolipidemic agents and
non-steroidal anti-inflammatory, serum lipid profiles, and markers of inflammation.
Thus, this cohort study demonstrates that hyperinsulinemia is a significant risk
marker for the loss of appendicular skeletal muscle mass in an apparently healthy
sample of older non-diabetic people with no pronounced loss of ASM. To the best
of our knowledge, similar findings have not been published before.
The link between insulin and skeletal muscle found in the present cohort
study at baseline could be based on the role of insulin in regulating body protein
mass,
17
since it is a potent anabolic stimulus for skeletal muscle. The importance of
insulin in regulating muscle protein turnover is highlighted by the estimated
contribution of skeletal muscle (30-50%) to whole body protein breakdown.
38-39

Physiological hyperinsulinemia increases skeletal muscle protein synthesis and
anabolism in young healthy subjects, as long as blood flow and amino acid delivery
to the muscles are stimulated by insulin. Both perfusion and nutrients are needed
for the anabolic response of muscle protein synthesis to insulin.
40
The other
proposed mechanism holds that both insulin and amino acids can stimulate the
mTOR signalling pathway. Specifically, insulin promotes the phosphorylation of
Akt, an upstream regulator of mTOR, and enhances mTOR signalling to its
downstream effectors, 4E-binding protein 1 (4EBP1) and ribosomal S6 kinase 1
(S6K1), two key regulators of translation initiation and protein synthesis.
41

13

Human evidence has shown that this latter mechanism is impaired in elderly
people, such that the association between hyperinsulinemia and the loss of ASM at
4.6-year follow-up could be based on the impaired response of muscle protein
synthesis to the anabolic action of insulin in older adults. In fact, Guillet et al.
(2004), showed an impaired anabolic response of muscle protein synthesis
associated with S6K1 disregulation in elderly humans, and this disregulation of
translation factors was proposed as a mechanistic basis of sarcopenia
development during aging.
20

The new evidence from this cohort study on the association between
hyperinsulinemia and the loss of skeletal muscle mass in older people has several
clinical implications. For example, values of fasting insulin 8.4 U/mL could
indicate an early loss of skeletal muscle, and thus allow clinicians to plan timely
interventions designed to decrease the risk of sarcopenia and the loss of
functionality. Importantly, insulin resistance assessed by HOMA has been
associated with a pronounced loss of ASM. Lee et al. reported an association
between HOMA-IR and the loss of ASM, defined as a decrease of 5% or more.
3

Aleman-Mateo et al. also showed a significant association between HOMA-IR and
the loss of ASM defined as the lowest sex-specific 15
th
percentile of the distribution
of the relative change in ASM among the subjects in a cohort study.
4

In order to explore whether hyperinsulinemia could provide timely
information, or similar data to that reported previously, we ran the analysis using
the same procedure described
4
and the results showed no significant association
between hyperinsulinemia and the pronounced or relative loss of ASM.
Interestingly, we did find that hyperinsulinemia (8.4 U/mL) was associated only
14

with the loss of ASM as the continuous variable (= -0.28, 95% CI-0.57-.009,
p=0.05), even after adjusting for several covariates involved in the loss of skeletal
muscle. Clinicians should thus be aware of fasting insulin values in relation to
preventing the loss of ASM. As mentioned in the methodology section, it is unclear
what level of insulin resistance is associated with the risk of developing loss of
ASM, and this cohort study shows clearly that hyperinsulinemia defined empirically
at the 75
th
percentile based on all subjects of the cohort corresponding to 8.4
U/mL is significantly associated with the loss of ASM.
The strength of this study rests on its design, which allowed us to measure
the risk of developing loss of ASM after a follow-up period associated with an
exposure variable, in this case hyperinsulinemia. Also important were the
screening at baseline for loss of ASM, and the care taken in measuring and
analysing the response and exposure variables, as well as all covariates, including
interleukin 6 and C-reactive protein as markers of inflammation. The use of both
DXA for ASM, and fasting insulin as early markers of IR, and the lack of significant
differences between cohort and lost subject strengthened the study. One limitation
of the present cohort study is the use of a non-random sampling method and its
non-population-based nature, both of which limit the ability to generalize results.
Also, we are aware that the reported association could be underestimated because
most of the people were apparently healthy. For these reasons, future randomized
and population-based studies are needed.
5. Conclusion
15

In conclusion, the present cohort study shows that hyperinsulinemia as an early
marker of insulin resistance is associated with the loss of ASM in community-
dwelling older men and women subjects without other chronic health conditions.
The use of fasting insulin levels 8.4 U/mL may help clinicians identify individuals
in the geriatric population who are at a high risk of losing appendicular skeletal
muscle mass. However, new evidence from future cohort studies is required to
infer a possible causal association between hyperinsulinemia and loss of skeletal
muscle. Preventing the loss of skeletal muscle mass and sarcopenia should be a
priority for the geriatric population in order to avoid physical disability and other
comorbidities associated with insulin resistance.
Source of funding
This work derives from Project J37891-M, funded by CONACYT, 2003. CIAD, AC,
also partially supported this research.
Statement of authorship
The authors contributions to the project and the publication process were as
follows: M.T.L.T was involved in data collection, statistical analysis and the writing
of the manuscript; F.A.R.C. designed the study and contributed to data collection;
H.A.M was the project leader and contributed to the design and data collection, the
interpretation of the statistical analyses, the writing and editing of the manuscript,
and financial management.
Conflict of interest
16

All authors state that they have no conflict of interest of any kind.
Acknowledgments
The authors thank the volunteers of the cohort, as well as Ana Luz Blancas Garca,
Bertha I. Pacheco, and Ana Cristina Gallegos for their technical assistance.

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21. Valenti G, Denti L, Maggio M, Ceda G, Volpato S, Bandinelli S, et al. Effect of
DHEAS on skeletal muscle over the life span: the InCHIANTI study. J Gerontol
A Biol Sci Med Sci 2004;59:466-472.
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28. Xun P, Liu K, Cao W, Sidney S, Williams OD, He K. Fasting insulin level is
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evidence on the association between interleukin-6 and C-reactive protein with
the loss of total appendicular skeletal muscle in free-living older men and
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Valencia ME. Body composition by the four-compartment model: validity of the
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Appendicular skeletal muscle mass: measurement by dual-photon
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Sarcopenia: alternative definitions and associations with lower extremity
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et al. Diagnosing insulin resistance in the general population. Diabetes Care
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Measurement of L-[114C] leucine kinetics in splanchnic and leg tissues in
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Pharmacological vasodilation improves insulin-stimulated muscle protein
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2771.
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Biochem 2002;269:53385349.





22

Table 1. Social and demographic characteristics, anthropometry, body
composition, biochemical variables, and health status: cohort subjects vs. lost
subjects.
a

Variables Cohort
(Men and women
n=147)
Lost subjects
(Men and women
n=89)
P-Values

Age, years 68.3 0.5 67.9 0.7 0.57
Sex, %
Male
Female

43.9
56.1

54.5
45.5


0.11
Educational level, %
0 years 7.4 12.6
6-9 years 54.7 46
>10 years 37.8 41.4 0.27
Body weight, kg 72.5 1.0 73.1 1.3 0.44
ASM, kg 17.2 0.3 19.2 0.5 0.20
BMI, kg/m
2
27.8 0.3 27.6 0.4 0.31
Waist circumference, cm 99.5 0.9 98.6 1.3 0.33
Total body fat, kg 26.8 0.7 25.4 0.9 0.00
Truncal fat, kg 16.3 0.4 15.1 0.5 0.15
SBP, mmHg 137.1 1.8 136.9 2.4 0.09
DBP, mmHg 79.4 0.8 82.4 1.1 0.54
HDL-C, mg/dL 45.7 1.2 45.7 1.5 0.62
Triglycerides, mg/dL 148.5 6.7 144.8 6.9 0.77
Fasting glucose, mg/dL 96.3 0.8 96.3 1.2 0.75
Fasting insulin, IU/mL 8.6 (8.1-9.1)
b
8.6 (6.8-10.4)
b
0.62
HOMA-IR 2.1 (1.9-2.2)
b
2.1 (1.6-2.5)
b
0.07
NSAIDs, % 43.2 17.0 0.00
Antihypertensives, % 29.7 4.5 0.00
Hypolipidemic agents, % 2.0 2.3 0.89
Note: ASM = Appendicular skeletal muscle mass; BMI = Body mass index; SBP = Systolic blood
pressure; DBP =Diastolic blood pressure; HDL-C = High-density lipoprotein cholesterol; HOMA-IR =
Homeostatic model assessment of insulin resistance; NSAIDs = Non-steroidal anti-inflammatory
drugs.
a
Results are presented as means standard error, unless otherwise indicated.
b
Geometric mean (95% CI) due to skewed distribution.
P-values are presented for chi-square tests for categorical variables and GLM ANOVA for
continuous variables adjusted for sex between the cohort and the lost subjects sub-sample.






23

Table 2. Relative changes in the anthropometry and body composition variables at 4.6-years follow-up
according to quartile (Q) of fasting insulin values (U/mL)
Variables Q1 (n=37) Q2 (n=37) Q3 (n=37) Q4 (n=36)
(7) (>7-8.1) (>8.1-9.4) (>9.4)
Body Weight (kg)
Baseline 67.5 11.3 71.5 9.5 74.9 14.5 75.7 12.4
e,f

Follow-up 66.5 12.3 70.8 10.8 72.7 14.1
h
73.6 10.7
a, e

Absolute change, (CI) -1.0 (-0.4-2.5) -0.6 (-0.6-1.8) -2.0 (1.1-3.0) -2.4 (1.1-3.8)
% Relative change, (CI) -1.9 (-4.1-0.4) -0.8 (-2.6-1.1) -2.7 (-3.5-1.9) -2.9 (-3.8-1.9)
BMI (kg/m
2
)
Baseline 25.4 3.5 28.5 2.9 27.9 3.6 29.4 4.2
e,f

Follow-up 25.1 4.6 28.5 2.9 27.1 3.7
h
28.8 3.7
a,e,f,g

Absolute change, (CI)
% Relative change, (CI)
-0.1 (-0.4-.87)
-0.9 (-3.3-1.6)
-0.01 (-0.6-0.5)
-0.1 (-1.8-1.9)
-0.8 (0.4-1.2)
-2.0 (-3.4-0.7)
-0.6 (0.2-1.0)
-2.5 (-4.1-0.9)
Waist Circumference (cm)
Baseline 94.2 9.9 101.6 8.7 99.2 11.1 102.7 11.9
e

Follow-up 91.4 13.0
h
98.6 10.3 96.9 9.6
h
100.0 10.0
a,e

Absolute change, (CI)
% Relative change, (CI)
-3.8 (1.3-7.5)
-4.5 (-7.7-1.3)
-0.1 (-2.6-2.4)
-0.2 (-2.4-2.9)
-3.2 (0.04-6.3)
-2.6 (-5.3-0.05)
-3.3 (0.5-6.2)
-2.6 (-5.3-0.05)
ASM (kg)
Baseline 16.9 3.4 16.5 3.5 18.3 4.9 17.1 3.5
Follow-up 15.9 3.6
h
16.3 3.1
h
17.2 4.2
h
17.2 4.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.5 (0.2-0.8)
-2.9 (-4.4-1.1)
-0.3 (0.3-0.5)
-1.4 (-2.9-01)
-0.71 (0.4-0.9)
-0.4 (-5.5-2.3)
-0.7 (0.3-1.1)
-3.9 (-5.9-1.8)
TLT (kg)
Baseline 41.77.1 39.7 7.9 44.2 11.5 41.8 8.2
Follow-up 40.86.9 39.4 8.1 43.0 11.1
h
40.7 7.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.9(.4-1.4)
-1.9(-3.1 -0.6)
-0.3 (-0.2-0.7)
-0.9 (-1.9-0.10)
-1.2 (0.7-1.6)
-2.9 (-4.1-1.7)
-1.0 (0.4-1.7)
-2.2 (-3.7-0.7)
FFM (kg)
Baseline 44.1 7.5 42.1 8.4 46.8 12.1 44.1 8.6
Follow-up 43.1 7.3
h
41.8 8.5
h
45.6 11.7
h
43.1 7.8
h

Absolute change, (CI)
% Relative change, (CI)
-0.9 (.5-1.5)
-1.8 (-3.0 to -0.7)
-0.3(-0.2-0.7)
-0.9(-1.8-0.1)
-1.2 (0.8-1.6)
-2.6 (-3.7-2)
-0.9 (0.3-1.6)
-1.8 (-3.3-0.3)
Total body fat, kg
Baseline 22.0 8.7 27.8 7.1 26.6 6.9 30.3 10.0
e

Follow-up 21.6 10 27.3 7.4 25.4 6.5
h
28.9 8.5
h,e

Absolute change, (CI)
% Relative change, (CI)
-0.4 (-0.5-1.7)
-2.4 (-0.8-3.4)
-0.5(-0.9-1.2)
-0.2(-4.9-4.4)
-1.2 (0.02-2.5)
-3.2 (-7.7-1.3)
-1.4 (0.3-2.6)
-1.8 (-3.3-0.3)
Truncal fat, kg
Baseline 14.0 5.0 16.7 3.8 16.3 4.2 17.9 4.8
e

Follow-up 13.8 5.7 16.68 3.8 15.7 3.9
h
17.6 4.3
h,e

Absolute change (CI)
% Relative change,(CI)
-0.2 (-0.5-1.0)
-1.6 (-7.9-4.6)
-0.02 (-0.6-0.7)
0.6 (-4.3-5.5)
-0.6 (-0.005-1.3)
-1.3 (-5.8-3.1)
-0.3 (-0.3-0.9)
-1.4 (-5.3-2.6)
Notes: ASM = Appendicular skeletal muscle mass; TLT = Total lean tissue; BMI = Body mass index; FFM = Fat-free
mass (TLT plus total bone mineral content); CI= 95% CI.
a
Difference for trend across categories of fasting insulin values, p 0.05.
b
Difference between Q1 and Q2 of fasting insulin values, p 0.05.
c
Difference between Q2 and Q3 of fasting insulin values, p 0.05.
d
Difference between Q1 and Q3 of fasting insulin values, p 0.05.
e
Difference between Q1 and Q4 of fasting insulin values, p 0.05.
f
Difference between Q2 and Q4 of fasting insulin values, p 0.05.
g
Difference between Q3 and Q4 of fasting insulin values, p 0.05.
h
Difference between baseline and 5-year follow-up measurements, p 0.05

24

Table 3. Association between fasting insulin levels and ASM at baseline survey

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/ml


0.004
(-0.21-.21)


0.97


0.12
(-0.03- -0.20)


0.00


0.11
(0.02-0.20)


0.01


0.10
(.01- .19)


0.02

Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6(pg/mL).

































25

Table 4. Association between hyperinsulinemia and the risk of developing loss of
ASM at 4.6 years of follow-up

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/mL
8.4
> 8.4




Reference
-0.28
(-0.57-.009)




0.05



Reference
-0.32
(-0.61- (-0.03))




0.02



Reference
-0.33
(-0.62-(-0.04))




0.02



Reference
-0.33
(-0.65-(-0.20))




0.05


Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6 (pg/mL).













ICMJE Conflict of Interest
Click here to download ICMJE Conflict of Interest: YCLNU_Conflict_of_InterestHELIODORO.pdf
1

Hyperinsulinemia is associated with the loss of appendicular skeletal muscle
mass at 4.6 year follow-up in older men and women adults

Miriam T. Lpez Teros, Ftima A. Ramrez C, Heliodoro Alemn-Mateo


AFFILIATION: Coordinacin de Nutricin, Centro de Investigacin en Alimentacin
y Desarrollo (CIAD), A.C.

CORRESPONDING AUTHOR: Heliodoro Alemn Mateo, Coordinacin de Nutricin,
Centro de Investigacin en Alimentacin y Desarrollo A.C., Carretera a la Victoria
Km. 0.6, Hermosillo, Sonora, Mxico. Apartado Postal 1735, C.P. 83304. Tel. and
Fax: 52 (662) 280-0094. E-mail: helio@ciad.mx




Running head: Association between hyperinsulinemia and loss of appendicular
skeletal muscle mass


Field Code Changed
*Manuscript (including track changes)
2

ABSTRACT
Background and aim: Homeostasis model assessment as a marker of insulin
resistance has been associated with the pronounced loss of appendicular skeletal
muscle mass in older adults. In the present study, we hypothesized that
hyperinsulinemia as an early predictor of insulin resistance may be associated with
the loss of appendicular skeletal muscle mass (ASM). Methods: This is a cohort
study that included 147 well-functioning older men and women subjects who were
followed for a period of 4.6 1.8 years. Lean tissue in arm and legs, or ASM, was
derived from dual-energy x-ray absorptiometry at baseline with follow-up
measurements to obtain the relative change. Hyperinsulinemia was defined
empirically at the 75
th
percentile. Results: The relative change in ASM was
negative and significant throughout the quartiles of fasting insulin levels (p0.05);
however, the loss of ASM was more pronounced in the later quartiles (-0.7 kg)
compared with the relative change in Q1 and Q2 (-0.5 kg and -0.3 kg). The
unadjusted analysis indicates a significant association between hyperinsulinemia
and the loss of ASM (= -0.28, 95% CI-0.57-.009, p=0.05), an association that
remained significant after adjusting for several covariates. Conclusion:
Hyperinsulinemia as an early marker of insulin resistance was associated with the
loss of ASM in a cohort study of community-dwelling older men and women
subjects without other chronic health conditions. The use of fasting insulin levels
>8.4 U/mL may help clinicians identify individuals in the geriatric population who
are at a high risk of loss of appendicular skeletal muscle mass.
Keywords: hyperinsulinemia, loss of skeletal muscle, older adults
3

1. Introduction
Clinical metabolic studies have demonstrated that the action of insulin declines
progressively with age. In addition to its close association with type 2 diabetes,
which reduces life expectancy in older people, age-related insulin resistance is
implicated in the pathogenesis of several highly prevalent disorders for which aging
is a major risk factor.
1
Age-related loss of skeletal muscle mass below a critical
threshold and sarcopenia are two related disorders often found in the elderly
population.
2-5
Both sarcopenia
6
and insulin resistance
7
(IR) are highly
prevalentprevalent disorders in older people around the world. Older sSubjects
whit o experience muscle loss and suffer from sarcopenia are exposed to an
increased risk of adverse long-term clinical outcomes such as mobility disorders,
physical disabilities, and poor quality of life.
8-16

The link between the pancreastic function and skeletal muscle is based on
the role of insulin in regulating body protein mass.
17
The main in vivo effect of
insulin and amino acids on whole-body and skeletal muscle protein metabolism is
to Particularly, inhibits protein breakdown and stimulates e protein synthesis.
18-19
In
aged subjects, the relation between insulin, and the loss of skeletal muscle and
sarcopenia is based on the muscle protein resistance to the anabolic action of
insulin,
2,20
a defect that has been associated with impaired insulin-induced
vasodilation and mammalian target of rapamycin signalling (mTORC1).
20
In fact,
this is one of the potential mechanisms that, it has been suggested, underlies the
involuntary loss of skeletal muscle mass associated with IR in older adults.
However, there are no published studies that allow us to infer a causal association
4

between insulin or hyperinsulinemia as an early predictor of IR and the loss of
skeletal muscle mass in older people.
Several Hhormonesal factors that have been implicated in the pathogenesis
of the loss of skeletal muscle mass and sarcopenia include reduced production of
dehydroepiandrosterone sulphate,
21
lower total and free testosterone, and elevated
parathyroid hormone concentrations,
22-23
higher insulin-like growth factor-1 levels,
24

vitamin D deficiency, and high parathyroid hormone levels.
25-27
There is evidence
from studies in humans which suggests that insulin resistance IR contributes to
the loss of appendicular skeletal muscle mass (ASM). Recently, hyperinsulinemia,
defined by the homeostasis model assessment (HOMA) as a marker of insulin
resistance (HOMA-IR), has been associated with the pronounced or relative loss of
ASM in older non-diabetic men and women subjects.
3-4
In addition,
hyperinsulinemia defined in terms of fasting insulin levels, has been strongly
associated with the risk of developing hypertension,
28-29
and cognitive decline
30
in
older adult populations.
To the best of our knowledge, the literature contains is no longitudinal
evidence of the association between hyperinsulinemia and the loss of ASM. It is
important to recognize that insulin resistance IR assessed by the homeostatic
model assessment cited above is highly dependent on glucose levels.
3-4
Insulin
resistance occurs in conjunction with a compensatory hyperinsulinemia that initially
maintains plasma glucose levels within normal ranges. In the present study, we
hypothesized that hyperinsulinemia as an early predictor of insulin resistance is
associated with the loss of appendicular skeletal muscle mass. Therefore, the main
5

objective was to investigate whether hyperinsulinemia is associated with the loss of
ASM in a cohort study with community-dwelling older adults.


2. Materials and methods
2. 1. Study population
This cohort study was designed to assess the association of certain age-related
systemic changes and the loss of ASM in a non-random sample of apparently
healthy, community-living subjects.
4,31
The cohort was derived from a body
composition validation techniques study in 302 subjects over 60 years of age.
32
For
this trial, we report the association between fasting insulin or hyperinsulinemia at
baseline and the loss of appendicular skeletal muscle at 4.6 years of follow-up in a
sample of older men and women subjects. The study was approved by the Ethics
Committee of the Research Centrer for Food and Development and written
informed consent was obtained from all participants.
2.2. Subjects
This cohort study was conducted at the Body Composition Laboratory of the
Research Centrer for Food and Development in Hermosillo, Sonora, Mexico, and
included 147 older men and women subjects. As reported previously,
4
the original
study was carried out between March 2003 and April 2006, with a second
assessment from March 2008 to May 2011. The total follow-up period was 4.6
1.8 years. At baseline, all participants underwent a medical assessment that
Formatted: Font: (Default) Arial, 12
pt
6

included an oral glucose tolerance test, biochemical analyses, and functionality
and cognitive assessment.
33-34
Body composition, anthropometry (body weight,
height, body mass index and waist circumference), physical activity, and
socioeconomic status were also assessed as part of the protocol.
The inclusion and exclusion criteria have been detailed elsewhere.
4
Briefly,
all subjects were over 60 years of age, apparently healthy, physically independent,
and with no pronounced loss of ASM. Subjects with a history of diabetes or plasma
glucose 200 mg/dL at 2 hours after a 75g glucose load, heart attack, cancer,
chronic lung disease, mental disorders, chronic neurological disorders, arthritis and
other musculoskeletal diseases, and liver and kidney disease, were not included.
Other causes of exclusion were a lack of data for any one of the following
parameters: fasting insulin and glucose, DXA measurements, and body mass
index <18.5 kg/m
2
. Volunteers with controlled hypertension, hypothyroidism or
dyslipidemia diagnosed by the medication used or by lipid profile determination
were not excluded. The second assessment included body composition,
anthropometric measurements and a clinical evaluation.
2. 3. Measures of body composition
Body composition was measured by DXA using the DPX-MD+ (GE Lunar Madison,
WI, USA). At baseline and follow-up, evaluations of body composition were carried
out under fasting conditions in accordance with established guidelines. ASM was
determined from the DXA scans following the recommended anatomical
landmarks. The sum of non-fat plus non-bone tissue in both arms and legs was
used to represent ASM.
35

7

Subjects with low relative ASM or a pronounced loss of ASM at baseline
were not invited for the second assessment; since cohort studies typically focus on
new cases of disease that occur during follow-up. To detect subjects with low
relative ASM, the residuals method
36
was applied, and subjects were classified as
having low relative ASM when the values of their residuals fell into the sex-specific
lowest 20% of the baseline survey distribution of residual values. As published
previously,
4
there is no universal criterion for low relative ASM, so the residual
method was used because it is based on measurements of ASM and requires no
reference value from a healthy young population.
36
Using the procedure described
above, 66 subjects proved to have low relative ASM, and so were not included in
the cohort.
2. 4. Response variable
Basal and follow-up measurements of ASM were used to define the response
variable. The absolute relative changes in the response and other variables were
determined as the difference between follow-up and baseline measurements. Also,
the percentage of relative change was calculated as the difference in ASM
between the 4.6-year follow-up examination and baseline, divided by baseline ASM
and multiplied by 100. The absolute relative change in the variable ASM was used
as the continuous variable in the statistical analyses.
2. 5. Biochemical analyses
As reported previously, the following determinations were made at baseline:
plasma glucose, serum insulin, serum interleukin-6 and C-reactive protein, and
8

serum lipid profiles, especially high-density lipoprotein cholesterol and triglycerides.
Some of these measurements, such as fasting and 2-hour glucose for diagnoses of
diabetes, were among the exclusion criteria, while others were measured as
covariates.
4

2. 6. Exposure variable
Clinically, hyperinsulinemia was defined as the exposure variable. Previously,
relevant cut-offs have been reported
37
as fasting insulin 12.2 U/mL; however, it
is unclear precisely what level of insulin resistance is associated with the risk of
developing loss of ASM. Therefore, hyperinsulinemia was defined empirically at the
75
th
percentile based on all subjects in the cohort for whom we had baseline fasting
insulin determinations. In this trial, the 75
th
percentile corresponded to 8.4 U/mL
for fasting insulin.
2. 7. Statistical analyses
Results are presented as means SD, frequencies and percentages. Both mean
absolute change and percentages of relative change in the anthropometric and
body composition characteristics were calculated for each quartile (Q) of the
fasting insulin values. The differences in these variables between quartiles were
assessed by analysis of variance using a Fisher's LSD multiple comparison test. A
significant absolute change in these measurements was determined as p0.05 for
paired t-tests for baseline and 4.6-year follow-up measurements. The association
between fasting insulin values and the loss of ASM was examined using
multivariable linear regression analysis. The association between fasting insulin
9

values as the dichotomized variable and total appendicular skeletal muscle mass
as continuous variable at baseline was also tested by multivariable linear
regression analysis. The assumptions of linear regression and collinearity of the
final models were tested. The interactions of the independent variables in the
model were tested with p 0.1. All statistical analyses were performed using
STATA version 11.0 software (StataCorp LP, College Station, Texas).
Covariates were examined in the regression models and included if the
magnitude of association was >10%. Both unadjusted and adjusted coefficients
and 95% confidence intervals (CI) were obtained. The following covariates were
used for this analysis, and were described previously.
4
Briefly, controlled
hypothyroidism, medications, non-steroidal anti-inflammatory drugs, interleukin-6
and C-reactive protein were considered as covariates. Markers of inflammation,
lipid profiles, fasting glucose and insulin, two-hour post-load glucose, age, fat mass
and body weight were all coded as continuous variables; while the categorical
variables included gender (0= female, 1= male); educational level (0 years, 6
years, >6 years of schooling); alcohol use [none, light (7 drinks/wk), moderate-to-
high (>7 drinks/wk)]; smoking (never/former or current); hypertension (0= no
hypertension and 1= hypertension); use of antihypertensive, hypolipidemic agents
and non-steroidal anti-inflammatory drugs (0= no use, 1= use); and physical
activity level by using published predictive equations and the sedentary-
active/moderate-vigorous classification.

3. Results
10

Two hundred and thirty-six subjects were considered as potential
participants; however, due to various causes, the final cohort consisted of only 147
well-functioning older men and women subjects who were followed for a period of
4.6 1.8 years. Mean age of the cohort was 68.3 6.0 years at baseline and
increased to 73.0 6.3 years at the end of the study. More than fifty percent (56%)
of the total sample were women. The main causes for abandoning the study were:
I prefer not to participate (n= 20); inability to make contact (27); illness and physical
disabilities (n=28); no data for fasting insulin (n=1); undernourishment (n=1); and
death (n=12). Results of the comparative analyses of the baseline measurements
between the cohort and the excluded subjects for the present analysis showed
basically no significant differences in some social and demographic characteristics
such as age and sex; anthropometric variables such as BMI and body composition,
biochemical profile and health status (blood pressure). Importantly, the main
response and exposure variables at baseline were also no significant (Table 1).
Table 2 shows the behaviour of the relative changes in the different
anthropometric and body composition variables according to the quartile
distribution of fasting insulin levels. The relative changes in body weight, body
mass index, total lean tissue, and fat-free mass were negative and significant
throughout the quartiles of fasting insulin levels (p0.05). However, the loss of
ASM was more pronounced in the later quartiles (-0.7 kg) compared to the loss in
the Q1 and Q2 (-0.5 and -0.3 kg). With respect to the fat component total and
truncal body fat the relative changes were negative and only significant in the Q3
and Q4 (p0.05).
11

It is important to mention that the results of the baseline analysis show that
ASM did not increase according to the quartile distribution of the values for fasting
insulin (Table 2). Also, the unadjusted regression analysis was not significant,
though after adjustment for some baseline covariates such as age, gender, total
fat (kg) and height (cm) a significant and positive association was found. A one-
unit increment in fasting insulin was associated with 120 g increases of ASM
(model 1, =0.12, 95% CI -0.03-(-0.20), p=0.00). After additional adjustments
using baseline physical activity as the covariate plus model 1 covariates, the model
remained significant (model 2, =0.11, 95% CI 0.02-0.19, p=0.01), and the same
results were found when model 3 was adjusted for C-reactive protein (mg/L) and
interleukin-6 (pg/mL) plus model 1 ( =0.10, 95% CI 0.01-0.2, p=0.02) (Table 3).
Results of the follow-up analysis of the data on the association between
fasting insulin and the loss of ASM are presented in Figure 1 and in Table 4 as
beta coefficients and 95% confidence intervals (CI). The unadjusted analysis
indicates a significant negative association between hyperinsulinemia (8.4
U/mL), and the ASM variable compared to the reference group (<8.4 U/mL). The
change of <8.4 U/mL to hyperinsulinemia (8.4 U/mL) decreased the relative
change of the ASM variable in 280 g (= -0.28, 95% CI-0.57-.009, p=0.05).
Differences between groups remained significant after adjustment for baseline
variables such as age, sex, total fat (kg), and height (m) (model 1: =-0.32, 95%
CI -0.61-(-0.03), p=0.02). Also, when adjusted for the baseline physical activity
covariate plus model 1 covariates they remained significant (model 2, =-0.33
95% CI -0.62-(-0.04), p=0.02). This association was also adjusted for C-reactive
protein (mg/L) and interleukin-6 (pg/mL) plus model 1 covariates and continued to
12

be significant, though to a lesser degree (model 3, =-0.33, 95% CI -0.65-(-0.20),
p=0.05). There were no problems of collinearity and any interaction between the
independent variables of the final models.
4. Discussion
In the present cohort study, the results of the baseline survey showed that an
elevated baseline insulin concentration (8.4 U/mL) is associated with increases
of ASM, while at 4.6-year follow-up hyperinsulinemia increased the risk of ASM
loss in both elderly men and women. This relationship was maintained after
adjusting for traditional risk factors for ASM loss, including age, gender, smoking,
alcohol use, fat mass, comorbidities such as heart disease and hypertension,
physical activity, use of drugs like anti-hypertension and hypolipidemic agents and
non-steroidal anti-inflammatory, serum lipid profiles, and markers of inflammation.
Thus, this cohort study demonstrates that hyperinsulinemia is a significant risk
marker for the loss of appendicular skeletal muscle mass in an apparently healthy
sample of older non-diabetic people with no pronounced loss of ASM. To the best
of our knowledge, similar findings have not been published before.
The link between insulin and skeletal muscle found in the present cohort
study at baseline could be based on the role of insulin in regulating body protein
mass,
17
since it is a potent anabolic stimulus for skeletal muscle. The importance of
insulin in regulating muscle protein turnover is highlighted by the estimated
contribution of skeletal muscle (30-50%) to whole body protein breakdown.
38-39

Physiological hyperinsulinemia increases skeletal muscle protein synthesis and
anabolism in young healthy subjects, as long as blood flow and amino acid delivery
13

to the muscles are stimulated by insulin. Both perfusion and nutrients are needed
for the anabolic response of muscle protein synthesis to insulin.
40
The other
proposed mechanism holds that both insulin and amino acids can stimulate the
mTOR signalling pathway. Specifically, insulin promotes the phosphorylation of
Akt, an upstream regulator of mTOR, and enhances mTOR signalling to its
downstream effectors, 4E-binding protein 1 (4EBP1) and ribosomal S6 kinase 1
(S6K1), two key regulators of translation initiation and protein synthesis.
41

Human evidence has shown that this latter mechanism is impaired in elderly
people, such that the association between hyperinsulinemia and the loss of ASM at
4.6-year follow-up could be based on the impaired response of muscle protein
synthesis to the anabolic action of insulin in older adults. In fact, Guillet et al.
(2004), showed an impaired anabolic response of muscle protein synthesis
associated with S6K1 disregulation in elderly humans, and this disregulation of
translation factors was proposed as a mechanistic basis of sarcopenia
development during aging.
20

The new evidence from this cohort study on the association between
hyperinsulinemia and the loss of skeletal muscle mass in older people has several
clinical implications. For example, values of fasting insulin 8.4 U/mL could
indicate an early loss of skeletal muscle, and thus allow clinicians to plan timely
interventions designed to decrease the risk of sarcopenia and the loss of
functionality. Importantly, insulin resistance assessed by HOMA has been
associated with a pronounced loss of ASM. Lee et al. reported an association
between HOMA-IR and the loss of ASM, defined as a decrease of 5% or more.
3

Aleman-Mateo et al. also showed a significant association between HOMA-IR and
14

the loss of ASM defined as the lowest sex-specific 15
th
percentile of the distribution
of the relative change in ASM among the subjects in a cohort study.
4

In order to explore whether hyperinsulinemia could provide timely
information, or similar data to that reported previously, we ran the analysis using
the same procedure described
4
and the results showed no significant association
between hyperinsulinemia and the pronounced or relative loss of ASM.
Interestingly, we did find that hyperinsulinemia (8.4 U/mL) was associated only
with the loss of ASM as the continuous variable (= -0.28, 95% CI-0.57-.009,
p=0.05), even after adjusting for several covariates involved in the loss of skeletal
muscle. Clinicians should thus be aware of fasting insulin values in relation to
preventing the loss of ASM. As mentioned in the methodology section, it is unclear
what level of insulin resistance is associated with the risk of developing loss of
ASM, and this cohort study shows clearly that hyperinsulinemia defined empirically
at the 75
th
percentile based on all subjects of the cohort corresponding to 8.4
U/mL is significantly associated with the loss of ASM.
The strength of this study rests on its design, which allowed us to measure
the risk of developing loss of ASM after a follow-up period associated with an
exposure variable, in this case hyperinsulinemia. Also important were the
screening at baseline for loss of ASM, and the care taken in measuring and
analysing the response and exposure variables, as well as all covariates, including
interleukin 6 and C-reactive protein as markers of inflammation. The use of both
DXA for ASM, and fasting insulin as early markers of IR, and the lack of significant
differences between cohort and lost subject strengthened the study. One limitation
of the present cohort study is the use of a non-random sampling method and its
15

non-population-based nature, both of which limit the ability to generalize results.
Also, we are aware that the reported association could be underestimated because
most of the people were apparently healthy. For these reasons, future randomized
and population-based studies are needed.
5. Conclusion
In conclusion, the present cohort study shows that hyperinsulinemia as an early
marker of insulin resistance is associated with the loss of ASM in community-
dwelling older men and women subjects without other chronic health conditions.
The use of fasting insulin levels 8.4 U/mL may help clinicians identify individuals
in the geriatric population who are at a high risk of losing appendicular skeletal
muscle mass. However, new evidence from future cohort studies is required to
infer a possible causal association between hyperinsulinemia and loss of skeletal
muscle. Preventing the loss of skeletal muscle mass and sarcopenia should be a
priority for the geriatric population in order to avoid physical disability and other
comorbidities associated with insulin resistance.
Source of funding
This work derives from Project J37891-M, funded by CONACYT, 2003. CIAD, AC,
also partially supported this research.
Statement of authorship
The authors contributions to the project and the publication process were as
follows: M.T.L.T was involved in data collection, statistical analysis and the writing
16

of the manuscript; F.A.R.C. designed the study and contributed to data collection;
H.A.M was the project leader and contributed to the design and data collection, the
interpretation of the statistical analyses, the writing and editing of the manuscript,
and financial management.
Conflict of interest
All authors state that they have no conflict of interest of any kind.
Acknowledgments
The authors thank the volunteers of the cohort, as well as Ana Luz Blancas Garca,
Bertha I. Pacheco, and Ana Cristina Gallegos for their technical assistance.

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22

Table 1. Social and demographic characteristics, anthropometry, body
composition, biochemical variables, and health status: cohort subjects vs. lost
subjects.
a

Variables Cohort
(Men and women
n=147)
Lost subjects
(Men and women
n=89)
P-Values

Age, years 68.3 0.5 67.9 0.7 0.57
Sex, %
Male
Female

43.9
56.1

54.5
45.5


0.11
Educational level, %
0 years 7.4 12.6
6-9 years 54.7 46
>10 years 37.8 41.4 0.27
Body weight, kg 72.5 1.0 73.1 1.3 0.44
ASM, kg 17.2 0.3 19.2 0.5 0.20
BMI, kg/m
2
27.8 0.3 27.6 0.4 0.31
Waist circumference, cm 99.5 0.9 98.6 1.3 0.33
Total body fat, kg 26.8 0.7 25.4 0.9 0.00
Truncal fat, kg 16.3 0.4 15.1 0.5 0.15
SBP, mmHg 137.1 1.8 136.9 2.4 0.09
DBP, mmHg 79.4 0.8 82.4 1.1 0.54
HDL-C, mg/dL 45.7 1.2 45.7 1.5 0.62
Triglycerides, mg/dL 148.5 6.7 144.8 6.9 0.77
Fasting glucose, mg/dL 96.3 0.8 96.3 1.2 0.75
Fasting insulin, IU/mL 8.6 (8.1-9.1)
b
8.6 (6.8-10.4)
b
0.62
HOMA-IR 2.1 (1.9-2.2)
b
2.1 (1.6-2.5)
b
0.07
NSAIDs, % 43.2 17.0 0.00
Antihypertensives, % 29.7 4.5 0.00
Hypolipidemic agents, % 2.0 2.3 0.89
Note: ASM = Appendicular skeletal muscle mass; BMI = Body mass index; SBP = Systolic blood
pressure; DBP =Diastolic blood pressure; HDL-C = High-density lipoprotein cholesterol; HOMA-IR =
Homeostatic model assessment of insulin resistance; NSAIDs = Non-steroidal anti-inflammatory
drugs.
a
Results are presented as means standard error, unless otherwise indicated.
b
Geometric mean (95% CI) due to skewed distribution.
P-values are presented for chi-square tests for categorical variables and GLM ANOVA for
continuous variables adjusted for sex between the cohort and the lost subjects sub-sample.






23

Table 2. Relative changes in the anthropometry and body composition variables at 4.6-years
follow-up according to quartile (Q) of fasting insulin values (U/mL)
Variables Q1 (n=37) Q2 (n=37) Q3 (n=37) Q4 (n=36)
(7) (>7-8.1) (>8.1-9.4) (>9.4)
Body Weight (kg)
Baseline 67.5 11.3 71.5 9.5 74.9 14.5 75.7 12.4
e,f

Follow-up 66.5 12.3 70.8 10.8 72.7 14.1
h
73.6 10.7
a, e

Absolute change, (CI) -1.0 (-0.4-2.5) -0.6 (-0.6-1.8) -2.0 (1.1-3.0) -2.4 (1.1-3.8)
% Relative change, (CI) -1.9 (-4.1-0.4) -0.8 (-2.6-1.1) -2.7 (-3.5-1.9) -2.9 (-3.8-1.9)
BMI (kg/m
2
)
Baseline 25.4 3.5 28.5 2.9 27.9 3.6 29.4 4.2
e,f

Follow-up 25.1 4.6 28.5 2.9 27.1 3.7
h
28.8 3.7
a,e,f,g

Absolute change, (CI)
% Relative change, (CI)
-0.1 (-0.4-.87)
-0.9 (-3.3-1.6)
-0.01 (-0.6-0.5)
-0.1 (-1.8-1.9)
-0.8 (0.4-1.2)
-2.0 (-3.4-0.7)
-0.6 (0.2-1.0)
-2.5 (-4.1-0.9)
Waist Circumference (cm)
Baseline 94.2 9.9 101.6 8.7 99.2 11.1 102.7 11.9
e

Follow-up 91.4 13.0
h
98.6 10.3 96.9 9.6
h
100.0 10.0
a,e

Absolute change, (CI)
% Relative change, (CI)
-3.8 (1.3-7.5)
-4.5 (-7.7-1.3)
-0.1 (-2.6-2.4)
-0.2 (-2.4-2.9)
-3.2 (0.04-6.3)
-2.6 (-5.3-0.05)
-3.3 (0.5-6.2)
-2.6 (-5.3-0.05)
ASM (kg)
Baseline 16.9 3.4 16.5 3.5 18.3 4.9 17.1 3.5
Follow-up 15.9 3.6
h
16.3 3.1
h
17.2 4.2
h
17.2 4.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.5 (0.2-0.8)
-2.9 (-4.4-1.1)
-0.3 (0.3-0.5)
-1.4 (-2.9-01)
-0.71 (0.4-0.9)
-0.4 (-5.5-2.3)
-0.7 (0.3-1.1)
-3.9 (-5.9-1.8)
TLT (kg)
Baseline 41.77.1 39.7 7.9 44.2 11.5 41.8 8.2
Follow-up 40.86.9 39.4 8.1 43.0 11.1
h
40.7 7.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.9(.4-1.4)
-1.9(-3.1 -0.6)
-0.3 (-0.2-0.7)
-0.9 (-1.9-0.10)
-1.2 (0.7-1.6)
-2.9 (-4.1-1.7)
-1.0 (0.4-1.7)
-2.2 (-3.7-0.7)
FFM (kg)
Baseline 44.1 7.5 42.1 8.4 46.8 12.1 44.1 8.6
Follow-up 43.1 7.3
h
41.8 8.5
h
45.6 11.7
h
43.1 7.8
h

Absolute change, (CI)
% Relative change, (CI)
-0.9 (.5-1.5)
-1.8 (-3.0 to -0.7)
-0.3(-0.2-0.7)
-0.9(-1.8-0.1)
-1.2 (0.8-1.6)
-2.6 (-3.7-2)
-0.9 (0.3-1.6)
-1.8 (-3.3-0.3)
Total body fat, kg
Baseline 22.0 8.7 27.8 7.1 26.6 6.9 30.3 10.0
e

Follow-up 21.6 10 27.3 7.4 25.4 6.5
h
28.9 8.5
h,e

Absolute change, (CI)
% Relative change, (CI)
-0.4 (-0.5-1.7)
-2.4 (-0.8-3.4)
-0.5(-0.9-1.2)
-0.2(-4.9-4.4)
-1.2 (0.02-2.5)
-3.2 (-7.7-1.3)
-1.4 (0.3-2.6)
-1.8 (-3.3-0.3)
Truncal fat, kg
Baseline 14.0 5.0 16.7 3.8 16.3 4.2 17.9 4.8
e

Follow-up 13.8 5.7 16.68 3.8 15.7 3.9
h
17.6 4.3
h,e

Absolute change (CI)
% Relative change,(CI)
-0.2 (-0.5-1.0)
-1.6 (-7.9-4.6)
-0.02 (-0.6-0.7)
0.6 (-4.3-5.5)
-0.6 (-0.005-1.3)
-1.3 (-5.8-3.1)
-0.3 (-0.3-0.9)
-1.4 (-5.3-2.6)
Notes: ASM = Appendicular skeletal muscle mass; TLT = Total lean tissue; BMI = Body mass index; FFM = Fat-free
mass (TLT plus total bone mineral content); CI= 95% CI.
a
Difference for trend across categories of fasting insulin values, p 0.05.
b
Difference between Q1 and Q2 of fasting insulin values, p 0.05.
c
Difference between Q2 and Q3 of fasting insulin values, p 0.05.
d
Difference between Q1 and Q3 of fasting insulin values, p 0.05.
e
Difference between Q1 and Q4 of fasting insulin values, p 0.05.
f
Difference between Q2 and Q4 of fasting insulin values, p 0.05.
g
Difference between Q3 and Q4 of fasting insulin values, p 0.05.
h
Difference between baseline and 5-year follow-up measurements, p 0.05

24

Table 3. Association between fasting insulin levels and ASM at baseline survey

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/ml


0.004
(-0.21-.21)


0.97


0.12
(-0.03- -0.20)


0.00


0.11
(0.02-0.20)


0.01


0.10
(.01- .19)


0.02

Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6(pg/mL).

































25

Table 4. Association between hyperinsulinemia and the risk of developing loss of
ASM at 4.6 years of follow-up

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/mL
8.4
> 8.4




Reference
-0.28
(-0.57-.009)




0.05



Reference
-0.32
(-0.61- (-0.03))




0.02



Reference
-0.33
(-0.62-(-0.04))




0.02



Reference
-0.33
(-0.65-(-0.20))




0.05


Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6 (pg/mL).







































26













Figure 1. Association between the loss of appendicular skeletal muscle mass at
follow-up period and basal levels of fasting insulin in older men and women aged
subjects.












1

Hyperinsulinemia is associated with the loss of appendicular skeletal muscle
mass in older adults

Miriam T. Lpez Teros, Ftima A. Ramrez C, Heliodoro Alemn-Mateo


AFFILIATION: Coordinacin de Nutricin, Centro de Investigacin en Alimentacin
y Desarrollo (CIAD), A.C.

CORRESPONDING AUTHOR: Heliodoro Alemn Mateo, Coordinacin de Nutricin,
Centro de Investigacin en Alimentacin y Desarrollo A.C., Carretera a la Victoria
Km. 0.6, Hermosillo, Sonora, Mxico. Apartado Postal 1735, C.P. 83304. Tel. and
Fax: 52 (662) 280-0094. E-mail: helio@ciad.mx




Running head: Association between hyperinsulinemia and loss of skeletal muscle


*Manuscript with changes
Click here to view linked References
2

ABSTRACT
Background and aim: Homeostasis model assessment as a marker of insulin
resistance has been associated with the pronounced loss of appendicular skeletal
muscle mass in older adults. In the present study, we hypothesized that
hyperinsulinemia as an early predictor of insulin resistance may be associated with
the loss of appendicular skeletal muscle mass (ASM). Methods: This is a cohort
study that included 147 well-functioning older men and women subjects who were
followed for a period of 4.6 1.8 years. Lean tissue in arm and legs, or ASM, was
derived from dual-energy x-ray absorptiometry at baseline with follow-up
measurements to obtain the relative change. Hyperinsulinemia was defined
empirically at the 75
th
percentile. Results: The relative change in ASM was
negative and significant throughout the quartiles of fasting insulin levels (p0.05);
however, the loss of ASM was more pronounced in the later quartiles (-0.7 kg)
compared with the relative change in Q1 and Q2 (-0.5 kg and -0.3 kg). The
unadjusted analysis indicates a significant association between hyperinsulinemia
and the loss of ASM (= -0.28, 95% CI-0.57-.009, p=0.05), an association that
remained significant after adjusting for several covariates. Conclusion:
Hyperinsulinemia as an early marker of insulin resistance was associated with the
loss of ASM in a cohort study of community-dwelling older men and women
subjects without other chronic health conditions. The use of fasting insulin levels
>8.4 U/mL may help clinicians identify individuals in the geriatric population who
are at a high risk of loss of appendicular skeletal muscle mass.
Keywords: hyperinsulinemia, loss of skeletal muscle, older adults
3

1. Introduction
Clinical metabolic studies have demonstrated that the action of insulin declines
progressively with age. In addition to its close association with type 2 diabetes,
age-related insulin resistance is implicated in the pathogenesis of several highly
prevalent disorders for which aging is a major risk factor.
1
Age-related loss of
skeletal muscle mass below a critical threshold and sarcopenia are two related
disorders in the elderly population.
2-5
Both sarcopenia and insulin resistance (IR)
are highly prevalent in older people around the world.
6-7
Subjects with muscle loss
and sarcopenia are exposed to an increased risk of adverse long-term clinical
outcomes such as mobility disorders, physical disabilities, and poor quality of life.
8-
16

The link between the pancreas and skeletal muscle is based on the role of
insulin in regulating body protein mass.
17
Particularly, inhibits protein breakdown
and stimulates protein synthesis.
18-19
In aged subjects, the relation between insulin,
and the loss of skeletal muscle is based on the muscle protein resistance to the
anabolic action of insulin,
2,20
a defect that has been associated with impaired
insulin-induced vasodilation and mammalian target of rapamycin signalling
(mTORC1).
20
In fact, this is one of the potential mechanisms that, it has been
suggested, underlies the involuntary loss of skeletal muscle mass associated with
IR in older adults. However, there are no published studies that allow us to infer a
causal association between insulin or hyperinsulinemia as an early predictor of IR
and the loss of skeletal muscle mass in older people.
4

Several hormones have been implicated in the pathogenesis of the loss of
skeletal muscle mass and sarcopenia.
21-27
There is evidence from studies in
humans which suggests that IR contributes to the loss of appendicular skeletal
muscle mass (ASM). Recently, hyperinsulinemia, defined by the homeostasis
model assessment (HOMA) as a marker of insulin resistance (HOMA-IR), has
been associated with the relative loss of ASM in older non-diabetic men and
women subjects.
3-4
In addition, hyperinsulinemia defined in terms of fasting insulin
levels, has been strongly associated with hypertension,
28-29
and cognitive decline
30

in older adult populations.
To the best of our knowledge, the literature contains no longitudinal
evidence of the association between hyperinsulinemia and the loss of ASM. It is
important to recognize that IR assessed by the homeostatic model assessment
cited above is highly dependent on glucose levels.
3-4
Insulin resistance occurs in
conjunction with a compensatory hyperinsulinemia that initially maintains plasma
glucose levels within normal ranges. In the present study, we hypothesized that
hyperinsulinemia as an early predictor of insulin resistance is associated with the
loss of skeletal muscle. Therefore, the main objective was to investigate whether
hyperinsulinemia is associated with the loss of ASM in a cohort study with
community-dwelling older adults.

2. Materials and methods
2. 1. Study population
5

This cohort study was designed to assess the association of certain age-related
systemic changes and the loss of ASM in a non-random sample of apparently
healthy, community-living subjects.
4,31
The cohort was derived from a body
composition validation techniques study in 302 subjects over 60 years of age.
32
For
this trial, we report the association between fasting insulin or hyperinsulinemia at
baseline and the loss of appendicular skeletal muscle at 4.6 years of follow-up in a
sample of older men and women subjects. The study was approved by the Ethics
Committee of the Research Center for Food and Development and written
informed consent was obtained from all participants.
2.2. Subjects
This cohort study was conducted at the Body Composition Laboratory of the
Research Center for Food and Development in Hermosillo, Sonora, Mexico, and
included 147 older men and women subjects. As reported previously,
4
the original
study was carried out between March 2003 and April 2006, with a second
assessment from March 2008 to May 2011. The total follow-up period was 4.6
1.8 years. At baseline, all participants underwent a medical assessment that
included an oral glucose tolerance test, biochemical analyses, and functionality
and cognitive assessment.
33-34
Body composition, anthropometry (body weight,
height, body mass index and waist circumference), physical activity, and
socioeconomic status were also assessed as part of the protocol.
The inclusion and exclusion criteria have been detailed elsewhere.
4
Briefly,
all subjects were over 60 years of age, apparently healthy, physically independent,
and with no pronounced loss of ASM. Subjects with a history of diabetes or plasma
6

glucose 200 mg/dL at 2 hours after a 75g glucose load, heart attack, cancer,
chronic lung disease, mental disorders, chronic neurological disorders, arthritis and
other musculoskeletal diseases, and liver and kidney disease, were not included.
Other causes of exclusion were a lack of data for any one of the following
parameters: fasting insulin and glucose, DXA measurements, and body mass
index <18.5 kg/m
2
. Volunteers with controlled hypertension, hypothyroidism or
dyslipidemia diagnosed by the medication used or by lipid profile determination
were not excluded. The second assessment included body composition,
anthropometric measurements and a clinical evaluation.
2. 3. Measures of body composition
Body composition was measured by DXA using the DPX-MD+ (GE Lunar Madison,
WI, USA). At baseline and follow-up, evaluations of body composition were carried
out under fasting conditions in accordance with established guidelines. ASM was
determined from the DXA scans following the recommended anatomical
landmarks. The sum of non-fat plus non-bone tissue in both arms and legs was
used to represent ASM.
35

Subjects with low relative ASM or a pronounced loss of ASM at baseline
were not invited for the second assessment; since cohort studies typically focus on
new cases of disease that occur during follow-up. To detect subjects with low
relative ASM, the residuals method
36
was applied, and subjects were classified as
having low relative ASM when the values of their residuals fell into the sex-specific
lowest 20% of the baseline survey distribution of residual values. As published
previously,
4
there is no universal criterion for low relative ASM, so the residual
7

method was used because it is based on measurements of ASM and requires no
reference value from a healthy young population.
36
Using the procedure described
above, 66 subjects proved to have low relative ASM, and so were not included in
the cohort.
2. 4. Response variable
Basal and follow-up measurements of ASM were used to define the response
variable. The absolute relative changes in the response and other variables were
determined as the difference between follow-up and baseline measurements. Also,
the percentage of relative change was calculated as the difference in ASM
between the 4.6-year follow-up examination and baseline, divided by baseline ASM
and multiplied by 100. The absolute relative change in the variable ASM was used
as the continuous variable in the statistical analyses.
2. 5. Biochemical analyses
As reported previously, the following determinations were made at baseline:
plasma glucose, serum insulin, serum interleukin-6 and C-reactive protein, and
serum lipid profiles, especially high-density lipoprotein cholesterol and triglycerides.
Some of these measurements, such as fasting and 2-hour glucose for diagnoses of
diabetes, were among the exclusion criteria, while others were measured as
covariates.
4

2. 6. Exposure variable
8

Clinically, hyperinsulinemia was defined as the exposure variable. Previously,
relevant cut-offs have been reported
37
as fasting insulin 12.2 U/mL; however, it
is unclear precisely what level of insulin resistance is associated with the risk of
developing loss of ASM. Therefore, hyperinsulinemia was defined empirically at the
75
th
percentile based on all subjects in the cohort for whom we had baseline fasting
insulin determinations. In this trial, the 75
th
percentile corresponded to 8.4 U/mL
for fasting insulin.
2. 7. Statistical analyses
Results are presented as means SD, frequencies and percentages. Both mean
absolute change and percentages of relative change in the anthropometric and
body composition characteristics were calculated for each quartile (Q) of the
fasting insulin values. The differences in these variables between quartiles were
assessed by analysis of variance using a Fisher's LSD multiple comparison test. A
significant absolute change in these measurements was determined as p0.05 for
paired t-tests for baseline and 4.6-year follow-up measurements. The association
between fasting insulin values and the loss of ASM was examined using
multivariable linear regression analysis. The association between fasting insulin
values as the dichotomized variable and total appendicular skeletal muscle mass
as continuous variable at baseline was also tested by multivariable linear
regression analysis. The assumptions of linear regression and collinearity of the
final models were tested. The interactions of the independent variables in the
model were tested with p 0.1. All statistical analyses were performed using
STATA version 11.0 software (StataCorp LP, College Station, Texas).
9

Covariates were examined in the regression models and included if the
magnitude of association was >10%. Both unadjusted and adjusted coefficients
and 95% confidence intervals (CI) were obtained. The following covariates were
used for this analysis, and were described previously.
4
Briefly, controlled
hypothyroidism, medications, non-steroidal anti-inflammatory drugs, interleukin-6
and C-reactive protein were considered as covariates. Markers of inflammation,
lipid profiles, fasting glucose and insulin, two-hour post-load glucose, age, fat mass
and body weight were all coded as continuous variables; while the categorical
variables included gender (0= female, 1= male); educational level (0 years, 6
years, >6 years of schooling); alcohol use [none, light (7 drinks/wk), moderate-to-
high (>7 drinks/wk)]; smoking (never/former or current); hypertension (0= no
hypertension and 1= hypertension); use of antihypertensive, hypolipidemic agents
and non-steroidal anti-inflammatory drugs (0= no use, 1= use); and physical
activity level by using published predictive equations and the sedentary-
active/moderate-vigorous classification.

3. Results
Two hundred and thirty-six subjects were considered as potential participants;
however, due to various causes, the final cohort consisted of only 147 well-
functioning older men and women subjects who were followed for a period of 4.6
1.8 years. Mean age of the cohort was 68.3 6.0 years at baseline and increased
to 73.0 6.3 years at the end of the study. More than fifty percent (56%) of the
total sample were women. The main causes for abandoning the study were: I
10

prefer not to participate (n= 20); inability to make contact (27); illness and physical
disabilities (n=28); no data for fasting insulin (n=1); undernourishment (n=1); and
death (n=12). Results of the comparative analyses of the baseline measurements
between the cohort and the excluded subjects for the present analysis showed
basically no significant differences in some social and demographic characteristics
such as age and sex; anthropometric variables such as BMI and body composition,
biochemical profile and health status (blood pressure). Importantly, the main
response and exposure variables at baseline were also no significant (Table 1).
Table 2 shows the behaviour of the relative changes in the different
anthropometric and body composition variables according to the quartile
distribution of fasting insulin levels. The relative changes in body weight, body
mass index, total lean tissue, and fat-free mass were negative and significant
throughout the quartiles of fasting insulin levels (p0.05). However, the loss of
ASM was more pronounced in the later quartiles (-0.7 kg) compared to the loss in
the Q1 and Q2 (-0.5 and -0.3 kg). With respect to the fat component total and
truncal body fat the relative changes were negative and only significant in the Q3
and Q4 (p0.05).
It is important to mention that the results of the baseline analysis show that
ASM did not increase according to the quartile distribution of the values for fasting
insulin (Table 2). Also, the unadjusted regression analysis was not significant,
though after adjustment for some baseline covariates such as age, gender, total
fat (kg) and height (cm) a significant and positive association was found. A one-
unit increment in fasting insulin was associated with 120 g increases of ASM
11

(model 1, =0.12, 95% CI -0.03-(-0.20), p=0.00). After additional adjustments
using baseline physical activity as the covariate plus model 1 covariates, the model
remained significant (model 2, =0.11, 95% CI 0.02-0.19, p=0.01), and the same
results were found when model 3 was adjusted for C-reactive protein (mg/L) and
interleukin-6 (pg/mL) plus model 1 ( =0.10, 95% CI 0.01-0.2, p=0.02) (Table 3).
Results of the follow-up analysis of the data on the association between
fasting insulin and the loss of ASM are presented in Figure 1 and in Table 4 as
beta coefficients and 95% confidence intervals (CI). The unadjusted analysis
indicates a significant negative association between hyperinsulinemia (8.4
U/mL), and the ASM variable compared to the reference group (<8.4 U/mL). The
change of <8.4 U/mL to hyperinsulinemia (8.4 U/mL) decreased the relative
change of the ASM variable in 280 g (= -0.28, 95% CI-0.57-.009, p=0.05).
Differences between groups remained significant after adjustment for baseline
variables such as age, sex, total fat (kg), and height (m) (model 1: =-0.32, 95%
CI -0.61-(-0.03), p=0.02). Also, when adjusted for the baseline physical activity
covariate plus model 1 covariates they remained significant (model 2, =-0.33
95% CI -0.62-(-0.04), p=0.02). This association was also adjusted for C-reactive
protein (mg/L) and interleukin-6 (pg/mL) plus model 1 covariates and continued to
be significant, though to a lesser degree (model 3, =-0.33, 95% CI -0.65-(-0.20),
p=0.05). There were no problems of collinearity and any interaction between the
independent variables of the final models.
4. Discussion
12

In the present cohort study, the results of the baseline survey showed that an
elevated baseline insulin concentration (8.4 U/mL) is associated with increases
of ASM, while at 4.6-year follow-up hyperinsulinemia increased the risk of ASM
loss in both elderly men and women. This relationship was maintained after
adjusting for traditional risk factors for ASM loss, including age, gender, smoking,
alcohol use, fat mass, comorbidities such as heart disease and hypertension,
physical activity, use of drugs like anti-hypertension and hypolipidemic agents and
non-steroidal anti-inflammatory, serum lipid profiles, and markers of inflammation.
Thus, this cohort study demonstrates that hyperinsulinemia is a significant risk
marker for the loss of appendicular skeletal muscle mass in an apparently healthy
sample of older non-diabetic people with no pronounced loss of ASM. To the best
of our knowledge, similar findings have not been published before.
The link between insulin and skeletal muscle found in the present cohort
study at baseline could be based on the role of insulin in regulating body protein
mass,
17
since it is a potent anabolic stimulus for skeletal muscle. The importance of
insulin in regulating muscle protein turnover is highlighted by the estimated
contribution of skeletal muscle (30-50%) to whole body protein breakdown.
38-39

Physiological hyperinsulinemia increases skeletal muscle protein synthesis and
anabolism in young healthy subjects, as long as blood flow and amino acid delivery
to the muscles are stimulated by insulin. Both perfusion and nutrients are needed
for the anabolic response of muscle protein synthesis to insulin.
40
The other
proposed mechanism holds that both insulin and amino acids can stimulate the
mTOR signalling pathway. Specifically, insulin promotes the phosphorylation of
Akt, an upstream regulator of mTOR, and enhances mTOR signalling to its
13

downstream effectors, 4E-binding protein 1 (4EBP1) and ribosomal S6 kinase 1
(S6K1), two key regulators of translation initiation and protein synthesis.
41

Human evidence has shown that this latter mechanism is impaired in elderly
people, such that the association between hyperinsulinemia and the loss of ASM at
4.6-year follow-up could be based on the impaired response of muscle protein
synthesis to the anabolic action of insulin in older adults. In fact, Guillet et al.
(2004), showed an impaired anabolic response of muscle protein synthesis
associated with S6K1 disregulation in elderly humans, and this disregulation of
translation factors was proposed as a mechanistic basis of sarcopenia
development during aging.
20

The new evidence from this cohort study on the association between
hyperinsulinemia and the loss of skeletal muscle mass in older people has several
clinical implications. For example, values of fasting insulin 8.4 U/mL could
indicate an early loss of skeletal muscle, and thus allow clinicians to plan timely
interventions designed to decrease the risk of sarcopenia and the loss of
functionality. Importantly, insulin resistance assessed by HOMA has been
associated with a pronounced loss of ASM. Lee et al. reported an association
between HOMA-IR and the loss of ASM, defined as a decrease of 5% or more.
3

Aleman-Mateo et al. also showed a significant association between HOMA-IR and
the loss of ASM defined as the lowest sex-specific 15
th
percentile of the distribution
of the relative change in ASM among the subjects in a cohort study.
4

In order to explore whether hyperinsulinemia could provide timely
information, or similar data to that reported previously, we ran the analysis using
the same procedure described
4
and the results showed no significant association
14

between hyperinsulinemia and the pronounced or relative loss of ASM.
Interestingly, we did find that hyperinsulinemia (8.4 U/mL) was associated only
with the loss of ASM as the continuous variable (= -0.28, 95% CI-0.57-.009,
p=0.05), even after adjusting for several covariates involved in the loss of skeletal
muscle. Clinicians should thus be aware of fasting insulin values in relation to
preventing the loss of ASM. As mentioned in the methodology section, it is unclear
what level of insulin resistance is associated with the risk of developing loss of
ASM, and this cohort study shows clearly that hyperinsulinemia defined empirically
at the 75
th
percentile based on all subjects of the cohort corresponding to 8.4
U/mL is significantly associated with the loss of ASM.
The strength of this study rests on its design, which allowed us to measure
the risk of developing loss of ASM after a follow-up period associated with an
exposure variable, in this case hyperinsulinemia. Also important were the
screening at baseline for loss of ASM, and the care taken in measuring and
analysing the response and exposure variables, as well as all covariates, including
interleukin 6 and C-reactive protein as markers of inflammation. The use of both
DXA for ASM, and fasting insulin as early markers of IR, and the lack of significant
differences between cohort and lost subject strengthened the study. One limitation
of the present cohort study is the use of a non-random sampling method and its
non-population-based nature, both of which limit the ability to generalize results.
Also, we are aware that the reported association could be underestimated because
most of the people were apparently healthy. For these reasons, future randomized
and population-based studies are needed.
15

5. Conclusion
In conclusion, the present cohort study shows that hyperinsulinemia as an early
marker of insulin resistance is associated with the loss of ASM in community-
dwelling older men and women subjects without other chronic health conditions.
The use of fasting insulin levels 8.4 U/mL may help clinicians identify individuals
in the geriatric population who are at a high risk of losing appendicular skeletal
muscle mass. However, new evidence from future cohort studies is required to
infer a possible causal association between hyperinsulinemia and loss of skeletal
muscle. Preventing the loss of skeletal muscle mass and sarcopenia should be a
priority for the geriatric population in order to avoid physical disability and other
comorbidities associated with insulin resistance.
Source of funding
This work derives from Project J37891-M, funded by CONACYT, 2003. CIAD, AC,
also partially supported this research.
Statement of authorship
The authors contributions to the project and the publication process were as
follows: M.T.L.T was involved in data collection, statistical analysis and the writing
of the manuscript; F.A.R.C. designed the study and contributed to data collection;
H.A.M was the project leader and contributed to the design and data collection, the
interpretation of the statistical analyses, the writing and editing of the manuscript,
and financial management.
16

Conflict of interest
All authors state that they have no conflict of interest of any kind.
Acknowledgments
The authors thank the volunteers of the cohort, as well as Ana Luz Blancas Garca,
Bertha I. Pacheco, and Ana Cristina Gallegos for their technical assistance.

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22

Table 1. Social and demographic characteristics, anthropometry, body
composition, biochemical variables, and health status: cohort subjects vs. lost
subjects.
a

Variables Cohort
(Men and women
n=147)
Lost subjects
(Men and women
n=89)
P-Values

Age, years 68.3 0.5 67.9 0.7 0.57
Sex, %
Male
Female

43.9
56.1

54.5
45.5


0.11
Educational level, %
0 years 7.4 12.6
6-9 years 54.7 46
>10 years 37.8 41.4 0.27
Body weight, kg 72.5 1.0 73.1 1.3 0.44
ASM, kg 17.2 0.3 19.2 0.5 0.20
BMI, kg/m
2
27.8 0.3 27.6 0.4 0.31
Waist circumference, cm 99.5 0.9 98.6 1.3 0.33
Total body fat, kg 26.8 0.7 25.4 0.9 0.00
Truncal fat, kg 16.3 0.4 15.1 0.5 0.15
SBP, mmHg 137.1 1.8 136.9 2.4 0.09
DBP, mmHg 79.4 0.8 82.4 1.1 0.54
HDL-C, mg/dL 45.7 1.2 45.7 1.5 0.62
Triglycerides, mg/dL 148.5 6.7 144.8 6.9 0.77
Fasting glucose, mg/dL 96.3 0.8 96.3 1.2 0.75
Fasting insulin, IU/mL 8.6 (8.1-9.1)
b
8.6 (6.8-10.4)
b
0.62
HOMA-IR 2.1 (1.9-2.2)
b
2.1 (1.6-2.5)
b
0.07
NSAIDs, % 43.2 17.0 0.00
Antihypertensives, % 29.7 4.5 0.00
Hypolipidemic agents, % 2.0 2.3 0.89
Note: ASM = Appendicular skeletal muscle mass; BMI = Body mass index; SBP = Systolic blood
pressure; DBP =Diastolic blood pressure; HDL-C = High-density lipoprotein cholesterol; HOMA-IR =
Homeostatic model assessment of insulin resistance; NSAIDs = Non-steroidal anti-inflammatory
drugs.
a
Results are presented as means standard error, unless otherwise indicated.
b
Geometric mean (95% CI) due to skewed distribution.
P-values are presented for chi-square tests for categorical variables and GLM ANOVA for
continuous variables adjusted for sex between the cohort and the lost subjects sub-sample.






23

Table 2. Relative changes in the anthropometry and body composition variables at 4.6-years
follow-up according to quartile (Q) of fasting insulin values (U/mL)
Variables Q1 (n=37) Q2 (n=37) Q3 (n=37) Q4 (n=36)
(7) (>7-8.1) (>8.1-9.4) (>9.4)
Body Weight (kg)
Baseline 67.5 11.3 71.5 9.5 74.9 14.5 75.7 12.4
e,f

Follow-up 66.5 12.3 70.8 10.8 72.7 14.1
h
73.6 10.7
a, e

Absolute change, (CI) -1.0 (-0.4-2.5) -0.6 (-0.6-1.8) -2.0 (1.1-3.0) -2.4 (1.1-3.8)
% Relative change, (CI) -1.9 (-4.1-0.4) -0.8 (-2.6-1.1) -2.7 (-3.5-1.9) -2.9 (-3.8-1.9)
BMI (kg/m
2
)
Baseline 25.4 3.5 28.5 2.9 27.9 3.6 29.4 4.2
e,f

Follow-up 25.1 4.6 28.5 2.9 27.1 3.7
h
28.8 3.7
a,e,f,g

Absolute change, (CI)
% Relative change, (CI)
-0.1 (-0.4-.87)
-0.9 (-3.3-1.6)
-0.01 (-0.6-0.5)
-0.1 (-1.8-1.9)
-0.8 (0.4-1.2)
-2.0 (-3.4-0.7)
-0.6 (0.2-1.0)
-2.5 (-4.1-0.9)
Waist Circumference (cm)
Baseline 94.2 9.9 101.6 8.7 99.2 11.1 102.7 11.9
e

Follow-up 91.4 13.0
h
98.6 10.3 96.9 9.6
h
100.0 10.0
a,e

Absolute change, (CI)
% Relative change, (CI)
-3.8 (1.3-7.5)
-4.5 (-7.7-1.3)
-0.1 (-2.6-2.4)
-0.2 (-2.4-2.9)
-3.2 (0.04-6.3)
-2.6 (-5.3-0.05)
-3.3 (0.5-6.2)
-2.6 (-5.3-0.05)
ASM (kg)
Baseline 16.9 3.4 16.5 3.5 18.3 4.9 17.1 3.5
Follow-up 15.9 3.6
h
16.3 3.1
h
17.2 4.2
h
17.2 4.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.5 (0.2-0.8)
-2.9 (-4.4-1.1)
-0.3 (0.3-0.5)
-1.4 (-2.9-01)
-0.71 (0.4-0.9)
-0.4 (-5.5-2.3)
-0.7 (0.3-1.1)
-3.9 (-5.9-1.8)
TLT (kg)
Baseline 41.77.1 39.7 7.9 44.2 11.5 41.8 8.2
Follow-up 40.86.9 39.4 8.1 43.0 11.1
h
40.7 7.4
h

Absolute change, (CI)
% Relative change, (CI)
-0.9(.4-1.4)
-1.9(-3.1 -0.6)
-0.3 (-0.2-0.7)
-0.9 (-1.9-0.10)
-1.2 (0.7-1.6)
-2.9 (-4.1-1.7)
-1.0 (0.4-1.7)
-2.2 (-3.7-0.7)
FFM (kg)
Baseline 44.1 7.5 42.1 8.4 46.8 12.1 44.1 8.6
Follow-up 43.1 7.3
h
41.8 8.5
h
45.6 11.7
h
43.1 7.8
h

Absolute change, (CI)
% Relative change, (CI)
-0.9 (.5-1.5)
-1.8 (-3.0 to -0.7)
-0.3(-0.2-0.7)
-0.9(-1.8-0.1)
-1.2 (0.8-1.6)
-2.6 (-3.7-2)
-0.9 (0.3-1.6)
-1.8 (-3.3-0.3)
Total body fat, kg
Baseline 22.0 8.7 27.8 7.1 26.6 6.9 30.3 10.0
e

Follow-up 21.6 10 27.3 7.4 25.4 6.5
h
28.9 8.5
h,e

Absolute change, (CI)
% Relative change, (CI)
-0.4 (-0.5-1.7)
-2.4 (-0.8-3.4)
-0.5(-0.9-1.2)
-0.2(-4.9-4.4)
-1.2 (0.02-2.5)
-3.2 (-7.7-1.3)
-1.4 (0.3-2.6)
-1.8 (-3.3-0.3)
Truncal fat, kg
Baseline 14.0 5.0 16.7 3.8 16.3 4.2 17.9 4.8
e

Follow-up 13.8 5.7 16.68 3.8 15.7 3.9
h
17.6 4.3
h,e

Absolute change (CI)
% Relative change,(CI)
-0.2 (-0.5-1.0)
-1.6 (-7.9-4.6)
-0.02 (-0.6-0.7)
0.6 (-4.3-5.5)
-0.6 (-0.005-1.3)
-1.3 (-5.8-3.1)
-0.3 (-0.3-0.9)
-1.4 (-5.3-2.6)
Notes: ASM = Appendicular skeletal muscle mass; TLT = Total lean tissue; BMI = Body mass index; FFM = Fat-free
mass (TLT plus total bone mineral content); CI= 95% CI.
a
Difference for trend across categories of fasting insulin values, p 0.05.
b
Difference between Q1 and Q2 of fasting insulin values, p 0.05.
c
Difference between Q2 and Q3 of fasting insulin values, p 0.05.
d
Difference between Q1 and Q3 of fasting insulin values, p 0.05.
e
Difference between Q1 and Q4 of fasting insulin values, p 0.05.
f
Difference between Q2 and Q4 of fasting insulin values, p 0.05.
g
Difference between Q3 and Q4 of fasting insulin values, p 0.05.
h
Difference between baseline and 5-year follow-up measurements, p 0.05

24

Table 3. Association between fasting insulin levels and ASM at baseline survey

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/ml


0.004
(-0.21-.21)


0.97


0.12
(-0.03- -0.20)


0.00


0.11
(0.02-0.20)


0.01


0.10
(.01- .19)


0.02

Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6(pg/mL).

































25

Table 4. Association between hyperinsulinemia and the risk of developing loss of
ASM at 4.6 years of follow-up

Unadjusted

(95% CI)

P
Model 1

(95% CI)

P
Model 2

(95% CI)

P
Model 3

(95% CI)

P
Fasting
insulin
U/mL
8.4
> 8.4




Reference
-0.28
(-0.57-.009)




0.05



Reference
-0.32
(-0.61- (-0.03))




0.02



Reference
-0.33
(-0.62-(-0.04))




0.02



Reference
-0.33
(-0.65-(-0.20))




0.05


Notes: = Beta coefficient; CI = Confidence Interval.
Model 1 = Adjusted for age, gender, baseline total fat (kg) and height (m).
Model 2 = Adjusted for Model 1 covariates plus baseline physical activity.
Model 3 = Adjusted for Model 1 covariates plus C-reactive protein (mg/L) and interleukin-6 (pg/mL).







































26













Figure 1. Association between the loss of appendicular skeletal muscle mass at
follow-up period and basal levels of fasting insulin in older men and women aged
subjects.












Responses to the reviewers
And
Thanks for all your valuable comments for our work: "Hyperinsulinemia is associated with
the loss of appendicular skeletal muscle mass at 4.6 year follow-up in older men and
women" MS. Ref. No.: YCLNU-D-14-00186

All changes made in the original text are highlighted in red and blue color

Comments from the Editors and Reviewers:

Reviewer #1: Manuscript #: YCLNU-D-14-00186

Title: Hyperinsulinemia is associated with the loss of appendicular skeletal muscle mass at
4.6 year follow-up older men and women.

Comment: The title can be shortened.

Thanks for this comment, and here the new title: Hyperinsulinemia is associated with the
loss of skeletal muscle in older adults
Also the introduction section was also shortened

General: This manuscript examined risk whether hyperinsulinemia a risk factor for insulin
resistance is associated with loss of appendicular skeletal mass (ASM) among older adults
aged 60 years and older.

Abstract: Concise and specific.

Introduction: The literature used is pertinent to the study. Please provide the study
objective. Study hypothesis is clearly stated.

This was attended at the end of the last paragraph of the introduction section.

Methods: Study design, sample, and all measures are clearly described. Statistical analyses
used are appropriate.

Comments:
-Please provide a brief description of excluded participants from the analysis.

-Were non-linear relationship between hyperinsulinemia and ASM investigated?
Effectively, this relationship was explored; we did not find a significant association using
the ASM as dichotomous variable with hyperinsulinemia. This was stated in the paragraph
5 of the discussion section. Possibly, the clinical implication of this result is that the use of
fasting insulin levels 8.4 U/mL may help clinicians identify individuals in the geriatric
population who are at a high risk of losing appendicular skeletal muscle mass and not the
pronounced loss of ASM, and maybe sarcopenia (as stated in conclusion section).

*Revision Note response latter
-Insulin levels at baseline were analyzed as continuous and dichotomy variable in the
multivariable regression analysis, but as quartiles for the descriptive analysis. Was the
relationship between quartiles of insulin level and ASM investigated in the multivariable
regression analysis?
Definitively, we explored this analysis; however for the small subjects of the cohort, we
decided to run the analysis using the exposition variable as dichotomy. We are sure, that
increasing the number of subjects of the cohort and as reported in other published papers,
probably we could find a significant association between higher quartiles of the exposition
variables with the response variable. As you can see in table 2, there is a more pronounced
loss of skeletal muscle mass at quartiles 3 and 4 (p<0.05).

-Were interaction terms for age and gender investigated?
We tested this and other interaction terms including age and gender, and we did not find an
interaction between independents variables. Thanks for this comment, now it was
incorporated in methodology and results section.

-A Figure presenting the findings of the relationship between insulin levels and ASM will
be helpful.
Thanks for this suggestion; we added. Figure 1 shows the correlation between the loss of
appendicular skeletal muscle mass and fasting insulin levels.

-Did any of the participants developed diabetes or insulin resistance during follow-up or
other medical condition that could have an effect on ASM?
Yes, some older men and women subjects developed type 2 diabetes and other
comorbidities such as heart diseases, EPOC, dementia, and physical disabilities. These
volunteers were excluded because it is well known that a diabetic patient has an
accelerated loss of skeletal muscle.

Results: Three tables well presented.

Comments:
I don't see any p-value for relative change in ASM in Table 2 as it states in the text and in
the abstract. Also, those values in the Table correspond to absolute change. Please clarify.
Literal and p values are in the legend in table 2 and the values in the same table are both
absolute and percentage.

Discussion:
-Previous pertinent literature was compared with author's findings.
-Study strengthens, limitations and implications were identified.

Minor comment: Delete "is" on page 4, first line of the last paragraph.

Thanks it was delete

References: There were 41 and all are appropriate.