Journalof Gerontology: BIOLOGICAL SCIENCES Copyright 1999 by The Gerontological Societyof America

1999,Vol.54A,No.8, B318-B323

The Age-Related Accumulation of Protein Carbonyl

in Rat Liver Correlates With the Age-Related Decline
in Liver Proteolytic Activities
Simona Vittorini, CristinaParadiso, AlessioDonati, GabriellaCavallini, Matilde Masini,
Zina Gori, Maria Pollera, and Ettore Bergamini

Istituto di Patologia Generale, University of Pisa,Italy,

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Increases ofprotein carbonylin animal tissues have been associatedwith the agingprocess. So far, the accumulation of
oxidizedproteins,highly susceptibleto proteolysis, has been attributedto age-relatedchangesin proteasomalalkalinepro-
teases.Carbonylin protein wasmonitored in six differenttissues ofmale Sprague-Dawley ratsfed ad libitum up to the age
of27 months, and of24- and 27-month-oldrats subjectedto anti-agingdiet restriction (every-other-day feeding ad libitum).
Alkaline protease activitiesand liver lysosomalproteolysiswerestudied. The levelsofprotein carbonyl were significantly
different in differenttissues,and quite stablethroughoutlife; accumulation wasrestricted to livertissue verylate in life~ be-
tween ages 24 and 27 months; wasfuUypreventedby diet restriction; wasnot accompaniedby any diet-restriction-sensitive
decline ofalkalineproteaseactivity;and wasaccompanied by a dramaticage-relateddeclinein lysosomalproteolysisthat
waspartiallypreventedby anti-agingdiet restriction. No correlation wasfound betweenlevelsofalkaline proteaseactivity
and levelsofproteincarbonylin the different tissuesfrom younger animals. It is concludedthat the processofautophagy, a
well-knownmechanismfor cellmaintenance, may deserve more interestin aging studies.

ELLULAR aging has been definedas a gradually decreas-
C ing ability of the cell to maintain homeostasis (1). The
age-relateddeteriorationof cellularhomeostasis can be associ-
ated with an accumulation of abnormal and oxidativelyaltered
proteinsin cells. An age-dependent increasein protein carbonyl
content was found in cultured fibroblasts from human donors
afterthe age of 60 years (2), in human brain (3), in gerbil cortex
(4), and in rat hepatocytes,over the range of 20-26 months (5).
In mouse brain,heart, and kidney, the age-relatedaccumulation
of protein carbonyls was markedly inhibited by the anti-aging
40% diet restriction(6). In houseflies, the carbonyl content was
associated with the physiological age or life expectancy rather
than the chronologicalage and was proposedto be a biomarker
of aging (7).
It was postulated that the steady-statelevel of oxidized pro-
teins reflectsthe balance between the rates of protein oxidation
and the rates of protein degradation, and the age-related accu-
mulation of altered proteins was thought to be due to an in-
crease of free-radical mediated damage, a loss of protease ac-
tivity, or a combination of both factors (8). Alkaline protease
was supposed to play an important role in this process (5), the
oxidized forms of proteins being degraded more rapidly than
their native counterparts (9,10). The proteasome, the major
componentof alkaline protease (11,12),is the main proteolytic
system involved in the removal of oxidatively damaged pro-
teins (13).Alkalineprotease activity was measured in crude tis-
sue homogenates and in purified proteasomes from old and
young animals (5,8,14-17), but results failed to give a clear an-
swer on the possibility that protease activity is declining with
age (18).
Protein degradation can be accomplished by two primary
pathways: the nonlysosomally mediated pathway mentioned
above, and the lysosomally mediated pathway. In the liver, the
lysosomal pathway has the major function of breaking down
the longer-lived protein by the process of macroautophagy
under plasma aminoacidand hormone control according to the
metabolicneed of the organism (19).
In view of a previous hypothesis that the age-related decrease
in liver autophagy and proteolysis might contributeto the age-
related accumulation of altered protein in this organ (20), we
explored the temporal patterns of the age-related changes in
protein carbonyl, alkaline protease, and autophagy. In this re-
port we show that the protein carbonyl contentsare remarkably
stable during life span in different tissues of Sprague-Dawley
rats fed ad libitum, age-related changes being restricted to the
liver tissue of very old animals; that the level of protein car-
bonyl and the level of alkaline protease activity are not bound
to change together in a reverse relationship;and the accumula-
tion of protein carbonyl in the liver of older rats is not accom-
panied by any significant decrease in the levelsof alkalinepro-
tease activities, but is simultaneouswith a dramatic age-related
decrease in lysosomal proteolysis. In addition, we report that
dietary restriction prevents the age-relatedaccumulation of pro-
tein carbonyl in rat liver tissue,may decreasethe levelsof alka-
line protease activities, and partially and significantly prevents
the decline in lysosomal proteolysis. Part of these results has
been the subjectof a preliminary communication (21).
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats aged 2,8, 16,24, and 27 months
and female rats of the same strain, aged 2 months, either fed ad
libitum (AL) or on a controlleddiet (namely "every other day"
ad libitum feeding, EOD), were used in this study.
As to the mortality rate of the animals, the surviving rats in

B318
 PROTEINCARBONYL, PROTEOLYSIS, AND AGING
the AL and EOD were 90% and 95%, respectively, at 5 months
of age; 80% and 92%, respectively, at 12 months;43 and 90%,
respectively, at 18 months; 35% and 80%, respectively, at 24
months.
At the given age, the extensor digitorum longus (EDL), the
soleus, blood from the inferior vena cava, a fragment from the
median lobe of the liver, the upper pole of the right kidney,the
tail of the pancreas, and a fragment of the free wall of the left
ventricle of the heart were taken under Nembutal anesthesia (50
mglKgbw).
ProteinCarbonylAssay
Fresh tissues(200-300 mg) were finely minced,and the pro-
teins were extracted in 4 ml of buffer(50 mM phosphatebuffer,
pH 7.4, containing 0.1% digitonin, a cocktailof antiproteases-
40 ug/rnl phenylmethylsulphonyl fluoride [PMSF], 5 ug/ml
leupeptin, 7 ug/ml pepstatin, 5 ug/ml aprotonin-and 1 mM
ethylenediaminetetracetic acid [EDTA] for 15 min at room tem-
perature. Maximum extraction was obtained in 10 min, and
yield of protein was constantbetween 10 and 30 min, using up
to 100 mg fresh tissue/ml buffer; yields with liver and kidney
were significantly higher than those with skeletal muscles and
heart. The fluid with the extracted proteins was removed from
the solid tissuesand centrifugedto removethe debris.One per-
cent streptomycin was added to the samplesto remove nucleic
acids.
Carbonyls in proteins were labeled by 2,4-dinitrophenylhy-
drazine [DNPH; (22)], precipitated with 10% trichloroacetic
acid (TCA), washed with TCA and thrice with ethanol-ethyl
acetate, and dissolvedin 6 M guanidinehydrochloride; the dif-
ference spectrumof the sample treatedwith DNPH in HCI was
determinedversus the sample treated with HCI alone.With all
used tissues, similar absorption spectra were obtained at any
tested age both in ad libitum-fedand in food-restricted animals.
A linear relationship was found between levels of carbonyls
and amountof used tissueirrespective of age and diet.Variation
coefficient was 7%. Results are expressed as nmoles of
DNPH incorporated per milligram of protein calculated from
extinction coefficient of 21.0 mM-1cm- 1for aliphatic hydra-
zones (22).
Alkaline Protease Assay
Fresh tissues (200-300 mg) were homogenized in 20 mM
Hepes/potassiumhydroxide(KOH),pH 8.0, containing 1 roM
EDTAand 1 roM 2-mercaptoethanol (49 ml/l g tissue) using a
Potter Elvehijem homogenizer. The soluble protein fraction
was preparedaccording to Cao and Cutler(17),using two steps
of centrifugation (12,000g 10 min and 23,000g 66 min at 4C)
and then used immediately to assay proteaseactivityaccording
to the procedure described by Barrett (23) and Rivett and col-
leagues(23,24).
The reactionmixtures (total volume,200 ul) contained solu-
ble protein fraction and varying concentration of fluorogenic
substrates in 50 roM Hepes bufferpH 8.0.
The chymotrypsin-like and the trypsin-like activities were
assayed with 50 JlMAla-Ala-Phe-7-amido-4-methy1coumarin
(AAP-AMC) and 50 JlM N-t-Boc-Leu-Thr-Ser-Arg-7-amido-
4-methy1coumarin (LSTR-AMC) as a substrate; the peptidyl-
glutamyl-peptide hydrolyzing activity was assayed with 100
J1M N-Z-Leu-Leu-Glu-l3-naphtylamide (LLE-NA). The mix-
B319
tures were incubatedfor 20 min (AAP-AMC and LSTR-AMC)
or 60 min (LLE-NA) at 37C, and reactions were stopped by
the addition of 109 ul of Stop Mix (175 roM aceticacid,74 mM
sodium acetate trihydrate)in theAAP-AMC and LSTR-AMC
assaysor 300 Jll of ethanolin the LLE-NAassay. The releaseof
7-amino-4-methy1coumarin (excitation370 nm, emission 430
nm) and l3-naphtylamine (excitation 323 nm, emission 400 nm)
was determined fluorometrically, and the proteolytic products
were quantitated by using 7-amino-t-methylcoumarin and
l3-naphtylamine as standards.
Sodium dodecyl sulfate(SDS) activation curves were estab-
lished with soluble fractions from liver, kidney, heart, and
soleus muscles as describedin (25) by measuring alkalinepro-
tease activity in the presence of increasing concentrations of
SDS rangingfrom 0% to .06%using .005%increments.
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Rate ofProteolysis
Liver cells were isolated by the collagenase procedure ac-
cordingto Seglen (26).Cell viability was testedby Trypanblue
exclusion and was always better than 90%. Hepatocytes were
suspendedin Krebs-Ringer bicarbonate bufferand incubatedin
conical flasks at 37C: optimal gas exchange was achievedby
continuous flow of humidified 95% OZ/ 5% CO2 at 4 Umin.
Maximum rate of lysosomalproteolysiswas obtainedby incu-
bating the isolated hepatocytes in the absence of any added
aminoacid (27). After 30 min, cycloheximide (10 J,lM) was
added to inhibit protein synthesis;0.2 ml samples of the hepa-
tocyte suspension were taken at 37 and 47 minutes, respec-
tively. The latter were deproteinized in ice-cold perchloricacid
(6% final concentration). The rate of proteolysis in vitro was
assessed by the linearreleaseof free valinein the 17-minutepe-
riod followingthe addition of cycloheximideand was normal-
ized to 1(T6 cells/ml. Valueswere corrected by the subtraction
of the valine released in the presence of a specificinhibitor of
lysosomal proteolysis, 5mM 3-methyladenine (27). Valinein
deproteinized extracts was determined by high performance
liquid chromatographyanalysis after neutralizing the perchlo-
rate supernatants with KOH then derivatizingthe amino acids
with dansylchloride; L-norvaline was added as an internal stan-
dard to all samples(28).
Materials
Digitonin, PMSF, leupeptin, pepstatin, aprotonin, EDTA,
DNPH, AAP-AMC, LSTR-AMC, LLE-NA, l3-naphtylamine,
7-amino-4-methy1coumarin, streptomycin, 2-mercaptoethanol,
collagenase (type IV), 3-methyladenine, and cycloheximide
were obtained from Sigma Chemical Company (St. Louis,
MO). Dansyl chloride was obtained from Pierce (Pierce
Europe, Beijerland, The Netherlands).TCA, ethanol, ethylac-
etate, acetic acid, and sodium acetate trihydrate were obtained
from J.T.Baker Italia (Milan, Italy). Hepes was obtained from
BDH Laboratory Supplies (Poole, England). Guanidine hy-
drochloridewas obtained from Fluka Chemie AG (Buchs, SG,
Switzerland). SDS was obtained from Bio-Rad Laboratories
(Hercules, CA).
StatisticalAnalysis
Data are given as the mean SE. One-wayor two-way anal-
ysis of variance (ANOVA) was used to evaluate differences
among multiple conditions as appropriate. Values of p ~ .05
B320 VI1TORINI EFAL.

were considered significant. If ANOVA for a variable reached
the significancelevel, then the Tukey test was used to determine
the groups of rats having differentmean values of the variable.
REsULTS
Table 1 shows the levels of carbonyl in the crude protein ex-
tracts from several rat tissues. Values are in good agreement
with data on the content in protein carbonyl of plasma and liver
of 2-month-old rats obtained by the same technique (22). The
levels of carbonyl are significantly different in the different tis-
sues; the highest values are observed in the heart and then in the
soleus, kidney,EDL, and liver; the lowest levels are observed in
liver and pancreas. Age-related changes in carbonyl are re-
stricted to the liver: in this organ, a significant age-dependent
increase in carbonyl content is observed between ages 24 and
activities are likely to include significant levels of cytosolic
peptidase, in addition to proteasomal peptidase activities.)In all
tissues, the highest level alkaline protease activity is seen with
AAP-AMC as substrate, and lower levels of peptidase activity
are found with LLE-NA and LSTR-AMC. Levels of peptidase
activities are significantlydifferent in the various tissues: higher
values are seen in the liver and kidney, and much lower values
are found in the heart and the skeletal muscles. AAP-AMC and
LLE-NA activities are significantly higher in the heart than in
the skeletal muscle; these differences do not appear to be at-
tributable to tissue differences in protease latency (unpub-
lished).All activities are similar in fast and slow muscles.
In view of the tissue specificity of the age-related changes in
protein carbonyl, age-relatedchanges of alkaline protease activ-
ity were studied in the liver tissue only (Table4). Between ages

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27 months.
Table 2 shows the effects of the anti-aging EOD regimen on
the levels of protein carbonyl in the same tissues. Differences
among the varioustissues are confirmed. EOD has no significant
effect on carbonyl levels in EDL, soleus, heart, kidney, and pan-
creas tissues but completely preventsthe age-dependentincrease
of protein carbonyl observed in the liver between ages 24 and 27
months. A similar preventive effect was observed in 4 rats sub-
mitted to 40% caloricrestriction(1.53 .201 unpublished).
It is assumed that alkaline protease is a major determinant of
the levels of carbonyl in rodent tissues. Table 3 shows the levels
of alkaline protease activities assayed by using three different
peptide substrates in the crude tissue extracts of young rats.
(Having been used as a soluble cell fraction, alkaline protease
2 and 27 months, AAP-AMC and LSTR-AMC activitiesdo not
change, whereas LLE-NA activity decreases significantly. Anti-
aging diet restriction causes a minor and significantdecrease in
AAP-AMC and LSTR-AMC activities (-30% in both cases)
and does not affect the age-dependent LLE-NA decrease.
Table 5 shows the rate of valine release (a measure of the
rate of proteolysis) from isolated rat liver cells incubated in
vitro. In control (AL) rats, rate of valine release is decreased
significantly by age 24 months, and by age 27 months it is al-
most fully suppressed. EOD feeding enhances the rate of valine
release at any tested age and partially prevents the age-related
decrease in the old rats. In 27-month-old EOD rats, valine re-
lease is smaller than in younger rats, but it is much larger than
in ad libitum-fed controls.

Table1.Levelsof CarbonylPer Unit of Protein in Crude Extractsof Different Tissues

From Rats FedAd Libitum

Age EDL Soleus Heart Liver Kidney Pancreas Serum

2Mo(1O) 1.99 .078 3.03 .220 3.03 .172 1.63 .112 2.16 .144 1.02 .059 0.47 .064
8-9Mo (6) 2.43 .121 2.86 .446 3.39 .273 1.40 .157 1.90 .241 0.96 .112 1.03 .223
16-18 Mo (7) 1.86 .176 2.49 .207 3.34 .272 1.73 .132 2.05 .204 0.78 .085 0.78 .254
24Mo(6) 1.99 .145 2.71 .31O 3.58 .658 1.68 .103 2.24 .223 1.32 .227 1.06 .191
27 Mo (5) 2.29 .189 2.93 .163 3.28 .343 3.05 .544 2.34 .449 1.05 .329 0.33 .015
Notes: Results are given as Means SE for nmolescarbonyl/mgprotein.The number of cases are in parentheses. Results of two-way (Age X Organ)ANOVA:
organ main effect,p<.Ol; Tukeytest: EDL vs Soleus,Heart, Pancreas,Serum;Heart vs Liver,Kidney,Pancreas,Serum;Pancreas vs Heart; Serum vs EDL, Soleus,
Heart,p's < .05.Age main effect and Age X Organ interaction not significant. One-way(age)ANOVA withineach organ gives the followingresults:Liverp<.OI;
Tukeytest:24 mo. vs 27 mo.p <.05. Serum p<.05; Thkeytest: 24 mo. vs 27 mo. p <.05.EDL, Soleus,Heart,Kidney, and Panceasnot significant.

Table2. Levelsof Carbonyl Per Unit of Proteinin CrudeExtractsof Different Tissues FromYoung(2 months)
and Old (24 and 27 months)Rats Fed Ad Libitum(AL) or Every-Other-Day Ad Libitum(EOD)

Age EDL Soleus Heart Liver Kidney Pancreas Serum

2Mo (10) 1.99 .078 3.03 .220 3.03 .172 1.63 .112 2.16 .144 1.02 .059 0.47 .064
24 MoAL (6) 1.99 .145 2.71 .310 3.58 .658 1.68 .103 2.24 .223 1.32 .227 1.06 .191
24 Mo EOD (6) 1.83 .238 2.86 .407 2.91 .469 1.62 .138 2.00 .507 1.01 .127 0.88 .111
27 MoAL (5) 2.29 .189 2.93 .163 3.28 .343 3.05 .544 2.34 .449 1.05 .329 0.33 .015
27 MoEOD (9) 2.02.151 2.68 .281 3.11 .272 1.72 .129* 2.78 .324 1.32.313 0.33 .020

Notes: Restrictionwas started by age 2 months.In this table and in Tables4 and 5, 2 months EOD was pooled with 2 monthsAL, given the absenceof a differ-
ence at this time.TheANOVAs were done beforethe 2-monthsdata were pooled.Resultsare given as Means SE for nmoles carbonyl/mgprotein.The numberof
cases are in parentheses. Results of two-way (Age X Diet) ANOVAwithin each organ: Liver: age main effect, diet main effect, and Age X Diet interaction, all
p's<.Ol;Tukeytest: 2 mo. and 24 mo. vs 27 mO.,p's<.05; Serum: age main effect,p < .01; Tukeytest: 2 mo. vs 24 mo. and 24 mo. vs 27 mo.,p < .05.Diet main ef-
fect andAge X Diet interaction not significant. Pancreas, Heart, Kidney, BDL,and Soleusnot significant.
*p < .05, as comparedto the age-matched controlsfed ad libitum(Tukey test).
 PROTEIN CARBONYL, PROTEOLYSIS, AND AGING B321

Table3. Levelsof AlkalineProteaseActivities Assayed DISCUSSION

With Three DifferentPeptides,in Extractsof DifferentTissues Many authors have shown that protein carbonyl derivatives
of Young (2-month-old) Rats are formed in vitro as a result of metal-catalyzed oxidations and
Substrate AAP-AMC LSTR-AMC LLE-NA accumulate during aging in disparate model systems [reviewed
in (29)]. It has been postulated that this molecular oxidative
EDL(7) 37 1.4 8.4 1.25 25 1.8
damage to cellular proteins is a major contributory factor in the
Soleus (7) 47 4.5 1O 1.1 48 1.4
Heart (7) 137 8.1
loss of various homeostatic functions associated with the pro-
14 1.6 55 2.3
Liver (7) 423 12.9 80 5.2 1037.4
cess of aging (30-32). Our results on the rat liver tissue of 27-
Kidney (7) 394 18.2 138 14.5 904.6 month-old rats and previous observations on hepatocytes iso-
lated from rats of similar age (5) show that the carbonyl content
Notes: Results are given as Means SE for nmoles AMC or NNminlmg of rat liver proteins increases significantly after age 24 months.
tissue. The number of cases are given in parentheses. Result of one-way (sub-
strate) ANOVA: p < .01; Tukey test: AAP-AMC vs LSTR-AMC and LLE-NA, No age-related accumulation of carbonyl in liver protein was
p's < .05. Results of one-way (organ) ANOVA within each substrate: AAP- found in 24-month-old rats, in accord with (33). In this respect,
AMC: p < .01; Tukey test: Liver and Kidney vs Heart, EDL, Soleus; Heart vs it should be mentioned that no evidence for an accumulation of
EDL and Soleus, p's < .05. LSTR-AMC: p < .01; Tukey test: Liver vs Kidney, oxidatively damaged nuclear DNA was found in the liver tissue

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Heart, EDL, Soleus; Kidney vs Heart, EDL, Soleus,p's < .05. LLE-NA: p <
of male Fischer 344 rats by age 24 months (34). The age-
.01; Tukey test: Liver vs Kidney, Heart, EDL, Soleus; Kidney vs Heart, EDL,
Soleus; Heart vs EDL, Soleus, p's < .05. dependent accumulation of protein carbonyls was restricted to
the liver (no significant accumulation was found in extra-hep-
atic tissues) and was fully prevented by anti-aging dietary re-
striction. An age-dependent accumulation of protein carbonyl
fully prevented by dietary restriction was observed in the pro-
tein of male Fischer 344XBNF splenic lymphocytes at an older
Table4. Levelsof AlkalineProteaseActivity on Three Peptides,
in the Liverof Younger (2 months) and Older (24 and 27 months) age [by age 31 months: (35)]. In mice, accumulation of car-
Rats Fed Ad Libitum (AL) or on a RestrictedDiet (EOD) bonyls in protein (6) and of 8-hydroxy-desoxyguanosine in
DNA was found both in the liver and extrahepatic tissues at a
Substrate younger age (6,36), and dietary restriction resulted in a remark-
Age AAP-AMC LSTR-AMC LLE-NA
able protection (36).
The age-related accumulation of altered protein was sup-
2Mo(4) 351 59.4 80 5.2 95 3.3 posed to be the consequence of an increase of free-radical me-
24Mo diated damage, of a decrease in protease activity, or of a combi-
AL(4) 322 23.5 83 11.0 95 19.9 nation of both factors (8). It is widely recognized that oxidative
EOD(6) 231 9.5 62 4.0 104 13.3 stress is a contributory factor in the aging process and that the
27Mo
balance between generation of oxidants and antioxidants (the
AL(7) 341 22.3 98 9.1 60 3.4
redox status) shifts to become more oxidizing in aging (37,38).
EOD(9) 273 22.7 70 7.7 63 3.1
However, in mice quite dramatic differences in the balance be-
Notes: Restriction was started by age 2 months. Results are given as Means tween rates of prooxidant generation and antioxidant levels
SE for nmoles AMC or NA/rninlmg tissue. The number of cases are given in may generate rather small changes in the steady-state level of
parentheses. Results of two-way (Age X Diet) ANOVA within each substrate:
AAP-AMC: age main effect not significant, diet main effect p <.01, Age X
protein carbonyl in tissues (39). To our knowledge, no dramatic
Diet interaction not significant. LSTR-AMC: age main effect not significant, changes in oxidative stress, restricted to the rat liver tissue, have
diet main effect p < .01, Age X Diet interaction not significant. LLE-NA: age been reported to suddenly occur between ages 24 and 27 months.
main effect, p < .01; Tukey test: 2 mo. and 24 mo. vs 27 mo. p's < .05; diet With regard to the activity of proteases, age changes in alkaline
main effect and Age X Diet interaction not significant.
proteases were explored extensively, and reports suggesting that
liver alkaline protease activity may decline by 70% in 26-month-
old rats (5) have been questioned recently (17). A comparison of
3-, 13-, and 23-month-old Sprague-Dawley rats indicated a 50%
Table5. Valine Release From LiverCells Isolated decline in alkaline protease in the liver, no age-related decline
FromYoung or Older Rats FedAd Libitum (AL) in brain, and a 20% decline in the heart during 13 to 22 months
or on a RestrictedDiet (EOD) (14); no changes in the liver alkaline protease activities were
found in 24-month-old Fischer 344 rats (17); our data show that
Age AL EOD
the peptidyl-glutamyl peptide hydrolase activity only exhibits a
2Mo 0.476 .0266 (12) significant (30-40%) age-related decrease both in ad libitum-
6Mo 0.551 .0224 (8) 0.764 .0379 (6) fed and in diet-restricted animals; recent studies with the puri-
18Mo 0.408 .0277 (8) 0.572 .0336 (5) fied proteasome revealed that the age-related decrease in activ-
24Mo 0.365 .0144 (10) 0.558 .0360 (10)
ity is likely to be due to a very subtle modification of the
27Mo 0.080 .0087 (5) 0.170 .0045 (8)
proteasome (16,18), and that the anti-aging diet restriction does
Notes: Restriction was started by age 2 months. Results are given as Means not prevent the change (16). In conclusion, there is no support
SE for nmo1es val/rnin/1O mg of cell weight. The number of cases are in paren- to the hypothesis that age-related changes in free radical medi-
theses. Results of two-way (Age X Diet) ANOVA: age main effect,p < .01;
Tukey test: 2 mo. vs 6 mo. and 27 mo.; 6 mo. vs 18 mo., 24 mo., and 27 mo.; 18
ated damage and/or in the levels of alkaline protease activity
mo. vs 24 mo. and 27 mo.; 24 mo. vs 27 mo. (P's< .05). Diet main effect, are responsible for the age-related accumulation of protein car-
p < .01; Age X Diet interaction not significant. bonyl in the liver tissue. Data, by exclusion, suggest that
B322 VI1TORINI ETAL
macroautophagy [reviewed in (19)] declines in the rat liver be-
tween ages 24 and 27 months, and that anti-aging diet restric-
tion partially prevents this decline. In the rat liver, lysosomal
proteolytic pathways (including macroautophagy, microau-
tophagy, crinophagy, and selective chaperonin-mediated, direct
uptake of proteins) have a major role in the degradation of in-
tracellular protein, and macro autophagy is the most important
process by far, from the quantitative point of view (40-42).
Hence, there is support to the hypothesis that the dramatic age-
related impairment of the amino acid and hormone-regulated
process of liver autophagy and proteolysis might lead to an ear-
lier age-dependent overloading of the extralysosomal prote-
olytic machinery and to accumulation of altered proteins in
liver cells (20). In view of the fact that autophagy is the process
responsible for the disposal of the cell organelles, impairment
of the process might account for selective accumulation of mi-
tochondrial DNA damage in liver cells also, and for the protec-
tion by dietary restriction (35). In conclusion, the process of au-
tophagy is a well-known mechanism for cell maintenance that
may deserve more investigation in aging studies.
With regard to extrahepatic tissues, differences in the levels
of altered protein carbonyl in various tissues of rodents have
been reported (e.g., 21,37). Different levels of 8-hydroxy-
guanosine are also seen in different mouse tissues (6). Higher
levels of protein carbonyls are seen in metabolically active tis-
sues (heart and the slow-twitch muscle soleus> kidney> fast-
twitch muscle EDL > liver). The difference between fast-twitch
and slow-twitch muscles appears by the time of differentiation
and does not disappear after a 12-hour denervation period (un-
published results). Higher levels of protein carbonyl are seen in
the tissues with lower levels of alkaline protease activity (AAP-
AMC: liver> kidney> heart> soleus> EDL; LSTR-AMC:
kidney> liver> heart> soleus> EDL; LLE-NA: liver> kidney
> heart> soleus> EDL). Perhaps levels of altered proteins are
under a very tight multifactorial control throughout the life
span, similar to the case of essential homeostatic parameters
and functions.
ACKNOWLEDGMENfS
This research was supported in part by funds of Ministero dell'Universita e
della Ricerca Scientifica e Tecnologica (MURST, 40%) and funds from the
University of Pisa (60%).
Address correspondence to Prof. Ettore Bergamini, Istituto di Patologia
Generale, Via Roma 55,56126 Pisa, Italy. E-mail: e.bergamini@ipg.med.unipi.it
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ReceivedJuly 21, 1998
AcceptedMarch 23, 1999
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