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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B375-B383 (2001)
© 2001 The Gerontological Society of America

Age-Related Changes in the Autophagic Proteolysis of Rat Isolated Liver Cells

Effects of Antiaging Dietary Restrictions

Alessio Donatia, Gabriella Cavallinia, Cristina Paradisoa, Simona Vittorinia, Maria Polleraa, Zina Goria and Ettore Bergaminia

a Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Italy

Ettore Bergamini, Dipartimento di Patologia Sperimentale, Via Roma 55, 56126 Pisa, Italy E-mail: ebergami{at}ipg.med.unipi.it.

Decision Editor: John Faulkner, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Autophagy is a process that sequesters and degrades organelles and macromolecular constituents of cytoplasm for cellular restructuring and repair and as a source of nutrients for metabolic use in early starvation. The effects of two antiaging dietary regimens (initiated in rats at the age of 2 months), namely, 40% dietary restriction (DR) and every-other-day ad-libitum feeding, that exhibited different effects on metabolism and similar effects on longevity on the age-related changes in the regulation of autophagic proteolysis were studied by monitoring the rate of valine release in the incubation medium from isolated liver cells of male albino Sprague-Dawley rats aged 2, 6, 12, 18, 24, and 27 months. (The liver cells were incubated in vitro with added amino acids and 10-7 M insulin or glucagon.) Age-matched male albino Sprague-Dawley rats fed ad libitum served as a control. Results show that in ad-libitum-fed rats, after a transient increase by age 6 months, autophagic proteolysis and regulation by amino acid exhibit a dramatic age-related decline, and that the age-related changes are prevented by dietary antiaging intervention. A comparison shows that the protective effects of DR and every-other-day ad-libitum feeding are partially different in 24-month-old rats (but the beneficial effects of the two diets on regulation of autophagic proteolysis are always similar). With regard to endocrine regulation, results confirm that the liver cell response to glucagon (but not to insulin) declines with increasing age, and they show that antiaging DRs significantly improve the effects of glucagon (and have no effect on the response to insulin). The interactions of age by diet, glucagon (and in older rats, insulin), and amino acids are significant. It is concluded that DR significantly improves the susceptibility of liver cells to lysosomal degradation, and it prevents decline with increasing age. It is suggested that improved liver autophagy and lysosomal degradation might be part of the antiaging mechanisms of DR.

AUTOPHAGY is a highly conserved, energy dependent, relatively nonselective vacuolar process that sequesters and degrades organelles and the macromolecular constituents of cytoplasm for cellular restructuring and as a source of amino acids for metabolic use in early starvation (1).

In the autophagy process, portions of cytosol and intracellular organelles are first isolated from the cytoplasm into an autophagic vacuole formed from ribosome-free regions of the rough endoplasmic reticulum or other organelles (2), known as autophagosomes, which then fuses with preexisting primary lysosomes to form an autophagolysosome (3). Autophagy is involved in the removal of damaged organelles and membranes during cell injury and remodeling in virtually all eukariotic cells (4).

Most of the studies on autophagy have been carried out in mammalian liver, presumably because of the quantitative importance of this organ (5). Liver autophagy is the main process of intracellular protein degradation during fasting (6)(7), under a moment-to-moment regulation (8) by nine physiological plasma amino acids (Gln, Leu, Tyr, Phe, Pro, Met, His, Trp, Ala) and pancreatic hormones (it is inhibited by insulin and it is stimulated by glucagon; 3,8), involving a receptor-mediated signal transduction pathway (9)(10), the lipid kinase phosphatidylinositol-3-OH kinase, and P70S6 kinase (5)(10). An age-related decline in liver autophagic proteolysis was shown in vitro, using an in situ perfused liver preparation (11) and isolated liver cells (12), and in vivo, with a physiologic model of stimulation of liver autophagy (13). Electron microscopy showed that the formation rate and the elimination of autophagic vacuoles are decreased in hepatocytes of old versus young adult animals (14). Because autophagy is the main cell mechanism for the degradation of altered cell membranes and organelles (15), on a longer time scale, an age-related decline in the function of autophagy could account for the age-dependent abnormal accumulation of the membrane-lipid dolichol (16), and for consequent changes in a number of membrane characteristics, such as fluidity, permeability, fusion capacity, binding of ligands, and modulation of membrane-associated protein activities, including sensitivity to oxidative stress (17).

Caloric restriction (CR), which may stimulate liver autophagy by prolonging time of fasting, is the only established intervention that significantly increases the mean and maximum life span in rodents (18) and maintains the physiological processes in a youthful state (19). CR retards many age-related diseases (20) and prevents the age-related accumulation of altered protein (21)(22)(23), nucleic acids (23), and dolichol (16) in cells and tissues. In vitro and in vivo studies have shown that CR may partially counteract the age-related decline in the maximum rate of liver autophagic proteolysis (11)(24). In view of the hypothesis that autophagy may be the extra repair mechanism responsible for the effect of CR on life span in rodents (25)(26), we decided that the age changes in the function of liver autophagy–proteolysis warranted further studies with isolated hepatocytes, related to control by amino acids and pancreatic hormones. We compared the effects of two different types of food restriction, namely 40% dietary restriction (DR) and every-other-day feeding ad libitum (EOD). DR and EOD have different effects on metabolism (27) and similar effects on longevity (28), and it may be assumed that effects in common are more likely to be involved in the antiaging mechanisms.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Random groups of male 2-month-old rats of the Sprague-Dawley strain were maintained on standard laboratory food and water ad libitum or on DR or EOD regimens. As to the mortality rate of the animals, the surviving rats in the ad-libitum fed (AL), EOD, and DR groups were 90%, 95%, and 100% respectively, at 5 months of age; 80%, 92%, and 90%, respectively, at 12 months; 43%, 90%, and 81%, respectively, at 18 months; and 35%, 80%, and 70%, respectively, at 24 months.

Food was withdrawn 16 hours before experimentation. Rats had free access to water. At the given age, rats were anaesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg of body weight).

Isolation of Hepatocytes
Liver parenchymal cells were isolated by the collagenase perfusion method of Seglen (29). Cell viability was tested by Trypan Blue exclusion and was always better than 90%.

Rate of Autophagy
Hepatocytes were incubated in suspension buffer (29) containing 0.3 mM leupeptin, an inhibitor of lysosomal proteases, for 40, 80, or 120 minutes. A mixture of plasma amino acids was added as fraction–multiples of a standard reference mixture of amino acids in rat plasma (30). After incubation, hepatocytes were washed in unbuffered, isotonic (10%) sucrose and electrodisrupted by a single high-voltage pulse (2 kV/cm) as described elsewhere (31). The "cell corpse" containing all lysosomes and other sedimentable components was isolated according to Kopitz and colleagues (32).

Enzyme Assay
Aliquots for enzyme assay were taken from solution of electrodisrupted cells (control) and from a resuspended cell corpse pellet. Lactate dehydrogenase (LDH) was assayed spectrophotometrically by measuring the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with pyruvate as substrate at 340 nm; the assay mixture was 48 mM phosphate buffer, pH 7.5, 0.6 mM sodium pyruvate, and 0.18 mM NADH. The rate of autophagy was expressed as the percent of LDH retained into the sedimentable corpses during 60 minutes of incubation, that is, cell corpse LDH (unit/l)/control LDH (unit/l) x 100. Values were not corrected by the subtraction of the LDH, which may be adsorbed on the cytosolic surface of cell organelles and membranes.

Rate of Proteolysis
Hepatocytes were suspended (3 ml, 1.5 x 106/ml) in Krebs-Ringer bicarbonate buffer and incubated in four rows of water-jacketed 10-ml conical flasks enclosed within a Lucite box attached to a Dubnoff apparatus with shaking. Flask temperature was maintained at 37°C by means of a constant-temperature recirculating water bath; optimal gas exchange was achieved by continuous flow of humidified 95% O2–5% CO2 at 4 l/min through the box. Mixtures of plasma amino acids were added as fraction–multiples of a standard reference mixture of amino acids in rat plasma (30). After 30 minutes, cycloheximide (CHX; 10 µM) was added to inhibit protein synthesis and 0.5-ml samples of the hepatocytes suspension were taken at 37 and 47 minutes. The latter were deproteinized in ice-cold perchloric acid (PCA; 6% final concentration). The rate of proteolysis was assessed by the linear release of free valine in the 17-minute period following the addition of CHX and was normalized to 106 cells/ml. Values were corrected by the subtraction of the valine released in the presence of an inhibitor of lysosomal proteolysis, 5 mM 3-methyladenine (33)(34).

Analytical Procedure
The acid-soluble supernatants were neutralized with KOH, and then the amino acids were derivatized with dansyl chloride as described by Taphui and colleagues (35). L-Norvaline was added as an internal standard to all samples.

Amino acid separation was carried out on a 4.0 x 250 mm Bio-Sil ODS-5S column with a particle size of 5 (m) in a Beckman (System Gold) high-performance liquid chromatography system (Beckman Instruments, Fullerton and San Ramon, CA, respectively). The column was eluted with a linear gradient of eluants A (88:12 ratio of water to acetonitrile, 0.3% glacial acetic acid, 0.035% triethylamine) and B (100% methanol). Valine was determined by measuring the fluorescence of its dansylated derivative with a Jasco spectrofluorometer (340-nm excitation, 525-nm emission).

Statistical Analysis
The analysis of variance (ANOVA) test was used to evaluate differences among multiple conditions. If they were positive, the Tukey test was used to test for their statistical significance. A least-squares regression analysis was used to determine the correlation between the rate of autophagy and the rate of proteolysis (36). In all analyses, a p < .05 was considered significant.

Materials
Dansyl chloride was obtained from Pierce (Pierce Europe, Beijerland, Netherlands). Amino acids, collagenase (type IV), and CHX were obtained from Sigma (St. Louis, MO). All other reagents were of the highest quality that was commercially obtainable.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Fig. 1 shows the rate of autophagy (given on the ordinate as percent sequestration of the cytosolic enzyme LDH) and the rate of proteolysis (on the abscissa, as the rate of valine release) in liver cells isolated from 24-month-old AL rats (Fig. 1), EOD rats (Fig. 1), or DR rats (Fig. 1); the cells were incubated in the absence or presence of multiples of a physiologic amino acid mixture. Values of the valine released in the presence of 5 mM 3-methyladenine were subtracted, but LDH activy absorbed on the cytosolic surface of cytomembranes was not, and lines intersect the y axis at positive values. Results show that in all three instances, the rate of autophagy and the rate of protein breakdown are correlated in a linear fashion; similar correlations were obtained in the presence of the regulatory pancreatic hormones (insulin or glucagon; not shown). Hence it appears that, both in AL and DR rats, the control of autophagy and proteolysis is produced at the same early step, at the level of sequestration of volumes of cytoplasm and organelles, and the rate of lysosomal breakdown is not the limiting step of proteolysis. In conclusion, the easier, faster, and more precise assay of the 3-methyladenine-sensitive release of valine can be used to get a picture of the age changes of autophagically mediated lysosomal proteolysis.



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  		Figure 1. Scatter diagram showing autophagy rates (i.e., % sequestration of the cytosolic lactate dehydrogenase [LDH] enzyme activity/60 min) as a function of proteolysis rates (i.e., valine release as nmol/min per gram of wet cells). Liver cells isolated from 24-month-old rats fed (A) ad libitum or (B) on the every-other-day (EOD) or (C) on the dietary restriction (DR) antiaging diets were incubated with different amino acid concentrations. AL: y = 0.0364x + 0.8384, r2 = 0.2062, p < .02; EOD: y = 0.0392x + 0.8897, r2 = 0.4472, p < .01; DR: y = 0.0475x6 + 0.5199, r2 = 0.3295, p < .01.

 
Fig. 2 shows that the age of the donor rats and the antiaging dietary regimens significantly affect the rate of valine release by isolated liver cells and the sensitivity to the control by amino acids. Both in AL and EOD rats the maximum rate of proteolysis, in the absence of added amino acids, peaks by age 6 months (Fig. 2) and declines thereafter; values are always higher in DR rats at any tested age. In AL rats, the size of the regulatory effect of the added amino acid mixtures is maximum in 2- and 6-month-old rats, and then it declines progressively with increasing age. A minimum is reached when the rats are 27 months old. In EOD rats a transient maximum is seen by age 6 months, and then the amplitude of regulation goes back to the 2-month value and does not show any further decrease with increasing age. The minimum rate of proteolysis is observed in the presence of a 4x amino acid mixture, which causes an 80% decrease in the rate of valine release by the younger liver cells. In both diet groups, the rate of proteolysis measured in the supplemented medium containing a 4x amino acid mixture is not significantly affected by increasing age.



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  		Figure 2. Effect of increasing age on the autophagic proteolysis of liver cells isolated from rats fed ad libitum (AL) and every other day (EOD) incubated in vitro with different amino acid concentrations. A, 2; B, 6; C, 12; D, 18; E, 24; F, 27 months. Results are given as nmol val/min per gram of wet cells. Means ± SE of at least 5 cases are given. Results of a three-way analysis of variance are as follows: Main effect of age, amino acid concentration and diet, p < .01. Interactions: age by amino acid concentration, age by diet, amino acid concentration by diet, ps < .01; amino acid concentration by age by diet, p < .05.

 
The inhibitory effect of the amino acid mixture on the 3-methyladenine-sensitive release of valine declines significantly with increasing age in AL rats only, from 30 (nmol/min)g in 2- and 6-month-old rats down to less than 7 (nmol/min)g in 27-month-old rats (p < .01). At any tested age the largest decline in function is seen in AL rats at 27 months of age, and maximum protection by EOD is seen at 27 months of age.

Fig. 3 and Fig. 3 show the effects of 40% DR and of EOD on maximum and minimum rates of autophagic proteolysis and on the size of the regulatory effects of the amino acid mixtures in rat liver cells isolated from 12-month-old (Fig. 3) and 24-month-old (Fig. 3) rats. Both antiaging dietary regimens preserve a higher maximum rate of proteolysis and a larger regulatory effect of amino acids. In 12-month-old rats, the protective effects of DR and EOD on both parameters are almost identical. At the older age, the effects on the maximum rate of autophagic proteolysis of DR and EOD are different, with the beneficial effect of DR being significantly smaller; in contrast, at lower amino acid concentrations in the medium, protection by DR and that by EOD from the age-related decline in regulation are almost identical.



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  		Figure 3. Effect of different types of caloric restriction on the autophagically mediated proteolysis of liver cells isolated from 12-month-old (A) and 24-month-old (B) rats. Results are given as nmol val/min per gram of wet cells. Means ± SEM of at least 5 cases are given. Results of two-way analysis of variance are as follows: A: Amino acid concentration main effect, p < .01; Tukey test, 0x vs 0.5x, 1x vs 2x vs 4x (ps < .05); diet main effect, p < .01; Tukey test, AL vs DR, EOD (ps < .05); amino acid concentration by diet interaction, p < .01. B: Amino acid concentration main effect, p < .01; Tukey test, 0x vs 0.5x vs 1x vs 2x vs 4x (ps < .05); diet main effect, p < .01; Tukey test, AL vs DR vs EOD (ps < .05); amino acid concentration by diet interaction, p < .01. AL = ad libitum; DR = dietary restriction; EOD = every other day.

 
Fig. 4 shows that a very high concentration of insulin (10-7 M) has a highly significant inhibitory effect on the release of valine (p < .01). The effect of the hormone does not change with the concentration of the added amino acid (the insulin by amino acid interaction is not significant) and varies according to the diet and age of the cell-donor animal (p < .01 for both). The effects of diet, age, and amino acid on the maximum rate and control of autophagic proteolysis are confirmed (p < .01; all). In addition, data in Fig. 6 below confirm that the regulation of liver cell proteolysis by amino acids is significantly impaired by the older age of the donor rat (amino acid by age interaction, p < .01); that the sensitivities of AL and EOD rat cells to stimulation by lack of amino acids are different (p < .01); and that EOD effectively counteracts the age-dependent impairment of regulation (p < .05). In general, sensitivity to insulin appears to be much better preserved than that to glucagon in the aging cells.



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  		Figure 4. Effect of 10-7 M insulin on proteolysis rate of liver cells isolated from rats of different ages fed ad libitum (AL) or on an every-other-day (EOD) dietary regimen in the presence of different amino acid concentrations. A, 2; B, 6; C, 12; D, 18; E, 24; F, 27 months. Results are given as nmol val/min per gram of wet cells. Means ± SE of at least 6 cases are given. Results of a four-way analysis of variance are as follows: main effect of age, diet, amino acid concentration and insulin, ps < .01. Interactions: age by diet, age by amino acid concentration, age by insulin, diet by amino acid concentration, ps < .01; diet by insulin, p < .05; amino acid concentration by insulin, NS; age by diet by amino acid concentration, p < .05; age by amino acid concentration by insulin, age by diet by insulin, diet by amino acid concentration by insulin, age by diet by amino acid concentration by insulin, NS.

 


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  		Figure 6. Effect of 10-7 M glucagon and 10-7 M insulin on proteolysis rate of hepatocytes isolated from (A) 12-month-old and (B) 24-month-old rats submitted to different types of caloric restriction. Results are expressed as the increase or the decrease of proteolysis rate in the presence of hormones and are given as nmol val/min per gram of wet cells. Values are the mean ± SEM of at least 5 cases. Results of a two-way analysis of variance are as follows: 10-7M glucagon: 12 months: diet main effect, p < .01; Tukey test, AL vs DR; amino acid concentration main effect, p < .01; Tukey test, 0x vs 1x vs 2x, 4x; diet by amino acids concentration interaction, p < .01. 24 months: diet main effect, p < .01; Tukey test, AL vs DR and EOD; amino acid concentration main effect, p < .01; Tukey test, 0x vs 1x, 2x, 4x; diet by amino acid concentration interaction, p < .01. 10-7M insulin: 12 months: diet main effect, NS; amino acid concentration main effect, p < .01; Tukey test, 0x vs 0.5 and 1x; diet by amino acid concentration interaction, NS. 24 months: diet main effect, p < .01; Tukey test, AL vs DR and EOD; amino acid concentration main effect, NS; diet by amino acid concentration interaction, p < .01. AL = ad libitum; DR = dietary restriction; EOD = every other day.

 
Fig. 5 show that a very high concentration (10-7 M) of glucagon in the incubation medium significantly increases the rate of valine release from liver cells (p < .01) and that the effect of the hormone is significantly different according to the age and diet of the rats and to the concentration of amino acids (p < .01; all). It should be mentioned that the rate of proteolysis itself is significantly affected by the diet and the age of the rats and by the added amino acids (p < .01; all). With regard to the interaction among treatments, an ANOVA shows that age and diets interact with the effects of glucagon as a function of the added amino acid concentration (p < .01; both). Glucagon does not change the size of the protective effect of antiaging DR on the age changes in regulation (the glucagon by diet by age by amino acid interaction is not significant). In addition, Fig. 5 confirms that age and diet affect the proteolytic response to added amino acids, and that the effects of diet on rate and control of autophagic proteolysis change with age (they are bigger by age 6 months). The effect of diet's preventing the age-dependent decline in autophagic proteolysis is highly significant (p < .01).



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  		Figure 5. Effect of 10-7 M glucagon on proteolysis rate of liver cells isolated from rats of different ages fed ad libitum (AL) or on an every-other-day (EOD) dietary regimen in the presence of different amino acid concentrations. A, 2; B, 6; C, 12; D, 18; E, 24; F, 27 months. Results are given as nmol val/min per gram of wet cells. Means ± SE of at least 6 cases are given. Results of a four-way analysis of variance are as follows: main effect of age, diet, amino acid concentration and glucagon, ps < .01. Interactions: age by diet, age by amino acid concentration, age by glucagon, diet by amino acid concentration, diet by glucagon, amino acid concentration by glucagon, age by diet by amino acid concentration, ps < .01; age by amino acid concentration by glucagon, age by diet by glucagon, NS; diet by amino acid concentration by glucagon, p < .01; age by diet by amino acid concentration by glucagon, NS.

 
Fig. 6 shows that the two antiaging diets DR and EOD may differ in their effects on the proteolytic responses to glucagon and insulin of liver cells from 12-month-old (Fig. 6) and 24-month-old (Fig. 6) rats. No effect of glucagon on valine release can be seen at 0x amino acid concentration in all 12-month age groups and in 24-month-old DR rats. By age 12 months, cells of DR rats exhibit the biggest response to glucagon (p < .01). By age 24 months, AL cells may not respond to glucagon at low amino acid concentrations, and both DR and EOD prevent an age-related decline in autophagic proteolysis. At both ages, in the presence of glucagon, liver cells from rats on different antiaging diets exhibit different sensitivities to the added amino acid concentration (diet by amino acid interaction, p < .01). With regard to insulin, interactions with diet and with amino acid concentrations are not significant by the age of 12 months; they are significant by the age of 24 months. At the latter age, the inhibitory effect of insulin on the proteolytic response of DR cells declines when the amino acid concentration in the medium is increased (p < .01), unlike the case of cells from the EOD rats.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The autophagic degradation of cytoplasmic protein was studied extensively in younger and older ad-libitum fed rats, with isolated liver cells and a perfused liver preparation made by using morphometrical (37) or biochemical methods (3), that is, by measuring the density volume of autophagic vacuoles or by quantitating the cytosolic enzyme activities and the electroinjected radiolabeled sugar sequestered into the autophagic vacuoles, or by measuring the production and release of a branched-chain amino acid (valine or leucine) in the presence of CHX to prevent simultaneous protein synthesis (38). The linear relationship between the lysosomal pools of degradable proteins and the rate of proteolysis in older AL and DR rats leaves little doubt that valine-release sensitivity to a 3-methyladenine inhibitor is a measure of accelerated macroautophagically mediated proteolysis in the hepatocytes and is consistent with the hypothesis that sequestration is the rate-limiting step of protein degradation, which is sensitive to control by amino acids and hormones (12)(39)(40). From the quantitative point of view, the present data agree with previous work from different laboratories on male younger Sprague-Dawley and Wistar rats (30)(41) and on older Sprague-Dawley rats fed ad libitum (12)(13), and these data may provide a valid representation of autophagically mediated proteolysis in vivo (12).

This paper deals with the age-dependent changes in the autophagically mediated proteolysis of liver cells isolated from AL and DR rats. The effects of aging on the control of autophagic proteolysis in rat liver cells have been reported recently by this laboratory (12). We could find no previous report in the literature comparable with our report on autophagic proteolysis in liver cells from DR rats. Our observations that in AL-fed rats the maximum proteolytic capacity of liver increased from 2 to 6 months of age and then declined in an approximatively linear fashion through 27 months are in excellent agreement with previous reports (11)(12). Antiaging dietary restrictions magnified the increase in maximum proteolytic capacity from 2 to 6 months of age and minimized the aging-related decline beyond age 12 months. In contrast, no significant effects of aging and of the antiaging treatments were detected when the medium was supplemented with amino acids at the physiological concentration or higher. The main effect of the antiaging dietary intervention on rat liver autophagy and proteolysis appears to be a protection from the aging-related decrease of the reserve capacity of the function. Previous work from this laboratory has shown that the increase of the liver content in protein carbonyl between ages 24 and 27 months correlates with the age-related decline in autophagically mediated proteolytic activity, and that antiaging dietary restriction fully prevents the change (22). This may suggest that the antiaging dietary intervention can also retard changes that may be more related to pathology effects than aging effects.

The supplementation of the incubation medium with very high concentrations of pancreatic hormones affected the rate of protein breakdown both in younger and older cells. The stimulation by glucagon at a very high concentration significantly increased protein degradation at any age and diet; at lower amino acid concentrations the effect was bigger in EOD than in AL rats. Inhibition by insulin was additive to that of amino acid and did not decline significantly with increasing age, except perhaps the case of the liver cells isolated from 27-month-old rats incubated at lower amino acid concentrations. This effect of insulin may be surprising: with the use of different models, it was shown that older liver cells are resistant to stimulation by the hormone, and that resistance is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (42)(43), with higher membrane viscosity compared with young controls and with additional nonmembrane factors (42). Antiaging dietary intervention had significant effects on glucagon and (at the older age) on insulin action. The effects of EOD and DR are partially different: EOD was more effective than DR on the maximum rate of autophagic proteolysis; in contrast, EOD and DR had similar beneficial effects on the control of the function. In view of the similarity of the beneficial effects of EOD and DR on life span (28), it appears that the deterioration of the regulation of autophagy by amino acid and glucagon levels may be more relevant to longevity than the decline in the maximum capacity of the function. Perhaps experimentation with lower concentrations of hormones and shorter incubation times might give a better picture of the susceptibility of older liver cells to lysosomal degradation and of the in vivo recruitment of the function. Such experimentation may also lead to a better understanding of differences between data of in vitro and in vivo studies.

The age-related changes in regulation of autophagic proteolysis in older hepatocytes of AL rats, and the preventive effect of dietary restriction, may deserve discussion. The activation of liver autophagy by a lack of amino acids depends upon the function of a receptor-mediated signal transduction pathway (5)(9)(10) involving the lipid kinase phosphatidylinositol-3-OH kinase and P70S6 kinase (44)(45)(46). An impairment in both P70S6 kinase and extracellular signal regulated kinase signalling pathway was reported in aged rat hepatocytes (42). Changes in membrane lipids affecting signal transduction were detected in older liver cells (43) together with a remarkable, age-dependent accumulation of the long-chain polyisoprenoid dolichol (16). It has been mentioned that dolichol exerts a considerable influence on the organization and packing of phospholipids in model membranes and can affect a number of membrane characteristics, including binding of ligands by elevating Kd values without affecting the number of the binding sites (47). We could find no reports on the effects of antiaging dietary restriction on the phosphatidylinositol-3-OH kinase and P70S6 kinase-mediated signal transduction pathway. It is conceivable that dietary restriction may enhance liver autophagy during the longer time period of fasting, and that increased degradation by autophagy may stimulate turnover, and improve maintenance, of cell membranes and organelles. Decreased susceptibility to degradation in older cells may bear on the rate of progression of aging itself, because of the vicious circle, and perhaps a continuing stimulation of lysosomal degradation by dietary restriction might break the circle. The data on the age-related changes in proteolysis may be an example of an age-related alteration in transmembrane signalling, possibly related to the age-related changes in membrane composition and structure and to the general mechanism of aging and antiaging dietary intervention (16).


   Acknowledgments
 
This research was supported in part by funds from the Ministero dell'Universitá e della Ricerca Scientifica e tecnologica (MURST, confinanziamento) and funds from the University of Pisa.

Received July 19, 2000

Accepted April 6, 2001


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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