HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B288-B293 (2001)
© 2001 The Gerontological Society of America

Age-Related Changes in the Regulation of Autophagic Proteolysis in Rat Isolated Hepatocytes

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

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

Decision Editor: John A. Faulkner, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
During intervals between meals, autophagy is a major source of nutrients and may remove damaged organelles and membranes. Age-related changes in the regulation of autophagic proteolysis were studied by monitoring the rate of valine release from liver cells of 2-, 6-, 12-, 18-, and 24-month-old male Sprague–Dawley rats fed ad libitum, and incubated in vitro with added amino acids and 10-7 M of insulin or glucagon. The maximum rate of proteolysis and its maximum inhibition by amino acids were reached at 6 months and declined thereafter. In contrast, the rate of protein degradation in the presence of high concentrations of amino acids was not affected by aging. The inhibitor effect of insulin was additive to that of amino acids and was not altered significantly by age. The conclusion is that altered regulation of autophagic proteolysis decreases susceptibility of older cells to lysosomal degradation, and it may lead to the accumulation of altered organelles and membranes.

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 process of autophagy, 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, that then fuse 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 eukaryotic 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 (6)(7) and the major source of amino acid for metabolic needs when intestinal absorption slows or ceases in the interval between meals. Liver autophagy is under a moment-to-moment regulation by nine physiological plasma amino acids that act as primary regulators (Gln, Leu, Tyr, Phe, Pro, Met, His, Trp) or coregulators (Ala) (8). The hormonally controlled process is inhibited by insulin and stimulated by glucagon (3)(8). Control of autophagy by amino acids and hormones is predominantly exerted at the sequestration step (3)(8). The regulation of autophagic proteolysis by leucine has been shown to depend on the function of a receptor-mediated signal transduction pathway (9)(10), involving the lipid kinase phosphatidylinositol-3-OH kinase and P70S6 kinase (5)(10).

Gerontological interest in this process stems from the observation that autophagy is the main cell mechanism for the degradation of cell membranes and organelles (11). On the longer time scale, an age-related decline in the function of autophagy could account for the age-dependent accumulation of lipids such as dolichol (12). Dolichol exerts a considerable influence on the organization and packing of phospholipids and on a number of membrane characteristics such as fluidity, permeability, fusion capacity, binding of ligands, and modulation of membrane-associated protein activities and sensitivity to oxidative stress (13). Alteration in cellular responsiveness as a result of changes in the membrane and of defective pathways of signal transduction have been suggested to play a key role in the age-related decline of function (14)(15). With this perspective, autophagy may be the extra-repair mechanism accounting for the extension of maximum life span in calorie-restricted rodents (14)(15).

An age-related decline in liver autophagic proteolysis was shown in an in situ perfused liver preparation (16), and in vivo, by the use of a physiologic model of stimulation of liver autophagy (17). Electron microscopy evidence indicated that the formation rate and the elimination of autophagic vacuoles are decreased in hepatocytes of old versus young adult animals (18). These investigations suggested that age changes in the function of liver autophagy–proteolysis warranted further studies with isolated hepatocytes, including the control of the process by amino acids and pancreatic hormones.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male rats of the albino Sprague–Dawley strain were maintained on standard laboratory food and water ad libitum. At 2, 6, 12, 18, 24, and 27 months of age, 18-hour starved rats were anaesthetized by the 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 (16). Cell viability was tested by Trypan Blue exclusion, and it was always better than 90%.

Rate of Autophagy
Hepatocytes were incubated in suspension buffer (19) containing 0.3 mM of leupeptin, an inhibitor of lysosomal proteases, for 40, 80 or 120 minutes. A mixture of plasma amino acids were added as fraction–multiples of a standard reference mixture of amino acids in rat plasma (20). After incubation, hepatocytes were washed in unbuffered, isotonic (10%) sucrose and electrodisrupted by a single high-voltage pulse (2 kV/cm) as described elsewhere (21). The "cell corpse" containing all lysosomes and other sedimentable components were isolated according to Kopitz and colleagues (22).

Enzyme Assay
Aliquots for enzyme assay were taken from the suspension 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 of phosphate buffer, pH 7.5, 0.6 mM of sodium pyruvate, and 0.18 mM of 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 corpses LDH (unit/1) control LDH (unit/1 x 100).

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 41/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 (20). 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). 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 of 3-methyladenine (23)(24).

Analytical Procedure
The acid-soluble supernatants were neutralized with KOH, and then the amino acids were derivatized with dansyl chroride as described by Taphui and colleagues (25). 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 (particle size, 5 µm) 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-water/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 emission).

Statistical Analysis
The analysis of variance (ANOVA) test was used to evaluate differences among multiple conditions. If positive, the Tukey test was used to test for their statistical significance. Values of p > .05 were considered not to be significant.

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


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The rate of autophagy and the rate of protein breakdown were correlated in a linear fashion in both younger and older cells (Fig. 1). Similar correlations were obtained in the presence of the regulatory pancreatic hormones, insulin or glucagon (not shown). Hence both in younger and older cells, (i) autophagy and proteolysis were controlled at the same early step, at the level of sequestration of cytoplasm and organelles, and (ii) the rate of lysosomal breakdown is not a limiting step of proteolysis. In conclusion, the easier and faster assay of the 3-methyladenine sensitive release of valine can be assumed to give a picture of the age changes of autophagically mediated lysosomal proteolysis.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Scatter diagram showing the relation between the rate of autophagy (percent lactate dehydrogenase sequestration) and the rate of proteolysis (nanomoles of valine per minute, per gram of wet cells). Liver cells were isolated from 2- and 24-month-old rats and incubated in the presence of different amino acids concentration: 2 months, y = 0.0463x + 0.4212, r = .698, p < .01; 24 months, y = 0.0364x + 0.8384, r = .454, p < .02.

 
The age of the donor rats affected the rate of valine release by isolated liver cells and the influence of the concentration of amino acids in the incubation medium on the rate of release (Fig. 2). The maximum rate of proteolysis, in the absence of amino acids, peaked by age 6 months, and declined thereafter. The magnitude of the inhibition of proteolysis by the added amino acid mixtures was maximum at 2 and 6 months of age. In liver cells from young rats, the maximum inhibition of proteolysis by amino acids was obtained in the presence of the highest concentration of amino acid mixture and caused an 80% decrease in the rate of valine release. The rate of proteolysis measured in the supplemented medium containing the highest concentration of amino acid mixture was not affected by increasing age.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 2. Effect of increasing age on the autophagically mediated proteolysis (rate is in nanomoles of valine per minute, per gram of wet cells) of isolated liver cells incubated in vitro with different concentrations of amino acids. Means ± SE of at least six cases are given. Results of two-way (age by amino acids concentration) analysis of variance: age main effect, p < .01; Tukey test: 2 and 6 months vs 12, 18, 24, and 27 months; 18 vs 24 months (p < .05); amino acids concentration main effect, p < .01; Tukey test: 0x vs 0.5x, 1x, 2x, and 4x; 0.5x vs 1x, 2x, and 4x; 1x vs 2x and 4x; 2x vs 4x (p < .05); age by amino acids concentration interaction, p < .01.

 
When liver cells were isolated from rats of different ages and incubated in the presence of different concentrations of amino acids, a high concentration (10-7 M) of glucagon in the incubation medium increased the rate of valine release, p < .01 (Fig. 3). The effects of glucagon and of amino acids appeared to be additive because interaction was not significant. An effect of increasing age on the response to glucagon was observed (age by hormone interaction; p < .05), with a higher response at age 6 months and a lower response by age 12 months and above.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Effect of 10-7 M glucagon on rate of proteolysis (nanomoles of valine per minute, per gram of wet cells) of liver cells isolated from rats of different ages in the presence of different concentrations of amino acids: A, Ox; B, 1x; C, 2x; D, 4x. Open circles, no glucagon; closed circles, glucagon 10-7 M. Means ± SE of at least six cases are given. Results of three-way (age by amino acid concentration by glucagon) analysis of variance: age main effect, p < .01; amino acid concentration main effect, p < .01; glucagon main effect, p < .01; age by amino acid concentration interaction, p < 0.01; amino acid concentration by glucagon interaction, not significant; age by glucagon interaction, p < .05; age by amino acid concentration by glucagon, not significant.

 
A high concentration of insulin (10-7 M) had a highly significant effect on the release of valine from isolated hepatocytes of rats of different ages (p < .01), as shown in Fig. 4. The effects of amino acids and insulin appear to be additive because the interaction was not significant. Unlike the case of glucagon, response to insulin appeared to be very well preserved with the exception of liver cells of 27-month-old rats incubated with low amino acid concentrations.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Effect of 10-7 M insulin on rate of proteolysis (nanomoles of valine per minute, per gram of wet cells) of liver cells isolated from rats of different ages in the presence of different concentrations of amino acids: A, Ox; B, 0.5x; C, 1x; D, 2x. Open squares, no insulin; closed squares, insulin 10-7. Means ± SE of at least six cases are given. Results of three-way (age by amino acid concentration by insulin) analysis of variance: age main effect, p < .01; amino acid concentration main effect, p < .01; insulin main effect, p < .01; age by amino acid concentration interaction, p < .01; amino acid concentration by insulin interaction, not significant; age by insulin interaction, not significant; age by amino acid concentration by insulin, not significant.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In young rats, autophagic degradation of cytoplasmic protein has been studied extensively, with isolated liver cells and the perfused liver preparation with autophagy measured by either morphometry (26) or by biochemical methods (27). In most instances, proteolysis was measured as the production and release of a long-chain amino acid, valine or leucine in the presence of cycloheximide to prevent simultaneous protein synthesis (28). At any age and both in the absence or in the presence of added pancreatic hormones, the present data and previous work (29) show a linear relationship between the lysosomal pools of degradable protein and the rate of proteolysis. The degradable proteins include internalizable proteins and cytosolic enzymes, such as LDH, and the release of valine is the measure of the rate of breakdown of the liver protein. The quantitative relationship between accelerated protein breakdown and macroautophagy leaves little doubt that the release of valine sensitive to the 3-methyladenine inhibitor is a measure of accelerated macroautophagically mediated proteolysis in the hepatocyte. This conclusion is consistent with the hypotheses that sequestration is the rate-limiting step in protein degradation and that the process of degradation is controlled by amino acids and hormones (30)(31).

From the quantitative point of view, the present data on the 3-methyladenine sensitive release of valine by liver cells isolated from 2-month-old Sprague–Dawley rats are in good agreement with previous work on male Wistar rats of similar age (20)(32). Furthermore, results with isolated liver cells concur with studies on the rate of proteolysis of the perfused liver preparation and may provide a valid representation of autophagically mediated proteolysis in vivo. For example, the total release of valine from the liver of nonfasted male Wistar rats aged 6 weeks, perfused with an unsupplemented medium (i.e., without any added amino acid), may approximate 100 nmol/min1 per gram of liver tissue (33), and approximately 45 nmol of valine/min1 per gram of liver tissue with a supplemented medium containing a physiological amino acid mixture four times higher than plasma amino acid concentration. A 4x normal amino acid concentration represents the lowest concentration that consistently gives maximal or near-maximal inhibition and also corresponds to the upper physiological limit of amino acids in portal vein plasma (34). A lower proteolytic capacity (~30 nmol/min1 per gram of wet liver) was reported with 3-month-old male Fisher 344 rats fed ad libitum (35). The rate of valine production in vivo by the liver was studied in 2-month-old 18-hour starved male Sprague–Dawley rats by the use of short-term single-pass liver perfusion and averaged 50 nmol/min1 of fresh liver, and the values doubled after stimulation of proteolysis by the injection of the antilipolytic agent 3',5'-dimethyl pyrazole (36). In conclusion, although it cannot be claimed that the valine release rates we have measured reflect the rates of proteolysis in vivo, we do believe that they may be related to the peak function and the control of autophagically mediated proteolysis in the liver tissue of the living animal.

We could find no previous report in the literature comparable with our report on the effects of age on autophagically mediated proteolysis in isolated liver cells. Our observations that the maximum proteolytic capacity of liver cells increased from 2 to 6 months of age and then declined in an approximately linear fashion through 27 months are in excellent agreement with a previous report on the maximum proteolytic capacity of the perfused rat liver (35). In contrast, no significant age-related decrease in proteolysis was detected when the medium was supplemented with amino acids at the physiological concentration, or higher. The main effect of aging on rat liver autophagy and proteolysis appears to be a decrease of the reserve capacity of the function. Previous work from this laboratory has shown that the increase in liver carbonyl group content between 24 and 27 months of age correlates with the age-related decline in autophagically mediated proteolytic activity (37). This may suggest that the changes between 24 and 27 months may be related more to pathological effects than aging effects.

Activation of liver autophagy–proteolysis by lack of amino acids depends upon the function of a receptor-mediated signal transduction pathway (9)(10)(38) involving the lipid kinase phosphatidylinositol-3-OH-kinase and P70S6 kinase (10)(38)(39). An impairment in both kinase and extracellular signal regulated kinase signaling pathway was reported in aged rat hepatocytes (40). Changes in membrane lipids affecting signal transduction were detected in older liver cells (41) together with a remarkable, age-dependent accumulation of the long-chain polyisoprenoid dolichol (12). Dolichol has been reported to exert a considerable influence on the organization and packing of phospholipids in model membranes and can affect a number of membrane characteristics. Enrichment of membranes with dolichol affects binding of ligands by elevating Kd values without affecting the number of the binding sites (42).

Degradation by autophagy may stimulate turnover and improve maintenance of cell membranes and organelles, and a decreased susceptibility to degradation in cells of old animals may have some bearing on the rate of progression of aging itself. The incubation medium in a very high concentration of pancreatic hormones can affect the rate of protein breakdown in cells from young and old animals. At all ages, the addition of glucagon-enhanced protein degradation at any amino acid concentration was greater than zero. Furthermore, in liver cells from 24-month-old rats, glucagon restored the stimulatory effect of amino acid deprivation. Except in the case of the isolated liver cells from 27-month-old rats incubated at a low amino acid concentration, the inhibitory effect of insulin did not decline with increasing age; the effect of insulin was additive to that of amino acids.

The effect of age on liver protein degradation has been reviewed by Ward and Richardson (43). Indirect evidence suggested that protein degradation decreased with age, because protein synthesis decreased while the protein content of a cell–tissue remained relatively constant. Several laboratories have studied the degradation–turnover in vivo of mixed long- and short-lived protein populations in liver tissue from ad-libitum fed laboratory rodents (e.g., (44),(45)). Various ages were studied by measuring the release of radioactively labeled amino acids from protein, or the rate of disappearance of radioactivity from the liver protein of prelabeled animals. The anticipated age-related decline in protein degradation was not observed. The present data indicate that this observation may be misleading because of technical issues. For example, the rate of liver protein degradation is influenced greatly by nutrition, is stimulated during fasting, and is low in ad-libitum fed animals, irrespective of age.


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

Received May 15, 2000

Accepted February 7, 2001


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Seglen PO, Gordon PB, Holen I, 1990. Non-selective autophagy. Seminars in Cell Biol. 1:441-448.
  2. Dunn WA, 1990. Studies on the mechanism of autophagy: formation of the autophagic vacuole. J Cell Biol. 110:1923-1933. [Abstract/Free Full Text]
  3. Seglen PO, Bohley P, 1992. Autophagy and other vacuolar protein degradation. Experientia. 48:158-169. [Medline]
  4. Glaumann H, Ericson JLE, Marzella L, 1981. Mechanisms of intralysosomal degradation with special reference to autophagocytosis and heterophagocytosis of cell organelles. Intl Rev Cytol. 73:149-182.
  5. Blommaart EFC, Luiken JJFP, Meijer AJ, 1997. Autophagic proteolysis: control and specificity. Histochem J. 29:365-385. [Medline]
  6. Mortimore GE, 1992. Role of autophagy in hepatic macromolecular turnover. Adv Mol Cell Biol. 4:201-228.
  7. Mortimore GE, Miotto G, Venerando R, Kadowaki M, 1996. Autophagy. Subcell Biochem. 27:93-135. [Medline]
  8. Mortimore GE, Pösö AR, Lardeux B, 1989. Mechanism and regulation of protein degradation in liver. Diabetes Metab Rev. 5:49-70. [Medline]
  9. Miotto G, Venerando R, Marin O, Siliprandi N, Mortimore GE, 1994. Inhibition of macroautophagy and proteolysis in the isolated rat hepatocytes by a non-transportable derivative of the multiple antigen peptide Leu8-Sys4-Lys2-Lys-ßAla. J Biol Chem. 269:25,348-25,353. [Abstract/Free Full Text]
  10. Codogno P, Ogier-Denis E, Houri JJ, 1997. Signal transduction pathways in macroautophagy. Cell Signal. 9:125-130. [Medline]
  11. Hare JF, 1990. Mechanisms of membrane protein turnover. Biochim Biophys Acta. 1031:71-90. [Medline]
  12. Marino M, Dolfi C, Paradiso C, et al. 1998. Age-dependent accumulation of dolichol in rat liver: is tissue dolichol a biomarker of aging?. J Gerontol Biol Sci. 53A:B87-B93. [Abstract]
  13. Van Dessel G, De Wolf M, Hilderson HJ, Lagrois A, Derick W, 1990. Intracellular and extracellular flow of dolichol. Subcell Biochem. 16:227-278. [Medline]
  14. Bergamini E, Gori Z, 1995. Towards an understanding of the antiaging mechanisms of dietary restriction: a signal transduction theory of aging. Aging Clin Exp Res. 7:473-475.
  15. Bergamini E, Cavallini G, Cecchi L, Donati A, Dolfi C, 1998. A proposed mechanism of the antiaging action of diet restriction. Aging Clin Exp Res. 10:174
  16. Ward WF, 1988. Food restriction enhances the proteolytic capacity of the aging rat liver. J Gerontol. 43:B121-B124. [Abstract]
  17. Bergamini E, Kovacs J, 1989. Exploring the age-related changes in hormone-regulated protein breakdown by the use of a physiologic model of stimulation of liver autophagy. Segal HL, Rothstein M, Bergamini E, , ed.Protein Metabolism in Aging 361-370. Wiley–Liss, New York.
  18. Terman A, 1995. The effect of age on formation and elimination of autophagic vacuoles in mouse hepatocytes. Gerontology. 41: (suppl 2) 319-326.
  19. Seglen PO, 1976. Preparation of isolated liver cells. Prescott DM, , ed.Methods in Cell Biology 29-83. Academic Press, New York.
  20. Venerando R, Miotto G, Kadowaki M, Siliprandi N, Mortimore GE, 1994. Multiphasic control of proteolysis by leucine and alanine in the isolated rat hepatocyte. Am J Physiol. 266:C455-C461. [Abstract/Free Full Text]
  21. Gordon PB, Seglen PO, 1982. Autophagic sequestration of [14C]sucrose, introduced into isolated rat hepatocytes by electropermeabilization. Exp Cell Res. 142:1-14. [Medline]
  22. Kopitz J, Kisen GO, Gordon PB, Bohley P, Seglen PO, 1990. Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol. 111:941-953. [Abstract/Free Full Text]
  23. Seglen PO, Gordon PB, 1982. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA. 79:1889-1892. [Abstract/Free Full Text]
  24. Blommaart EFC, Krause U, Schellens JP, Vreeling-Sinderova H, Meijer AJ, 1997. The phosphatidyl inositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem. 243:240-246. [Medline]
  25. Taphui Y, Schmidt DE, Lindner W, Karger BL, 1981. Dansylation of amino acids for high performance liquid chromatography analysis. Anal Biochem. 115:123-139. [Medline]
  26. Pfeifer U, 1978. Inhibition by insulin of the formation of autophagic vacuoles in rat liver. A morphometric approach to the kinetics of intracellular degradation by autophagy. J Cell Biol. 78:152-167. [Abstract/Free Full Text]
  27. Seglen PO, Bohley P, 1992. Autophagy and other vacuolar protein degradation mechanisms. Experientia. 48:158-171.
  28. Khairallah EA, Mortimore GE, 1976. Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA bound valine. J Biol Chem. 251:1375-1384. [Abstract/Free Full Text]
  29. Hutson NJ, Mortimore GE, 1982. Suppression of cytoplasmic protein uptake by lysosomes as the mechanism of protein regain in livers of starved-refed mice. J Biol Chem. 257:9548-9554. [Abstract/Free Full Text]
  30. Ward WF, Cox JR, Mortimore GE, 1977. Lysosomal sequestration of intracellular protein as a regulatory step in hepatic proteolysis. J Biol Chem. 252:6955-6961. [Free Full Text]
  31. Seglen PO, Gordon PB, Holen I, Hoyvick H, 1991. Hepatocytic autophagy. Biomed Biochem Acta. 50:373-381. [Medline]
  32. Blommaart EFC, Luiken JJFP, Meijer AJ, 1997. Regulation of hepatic protein degradation. Contrib Nephrol. 121:101-108. [Medline]
  33. Poso AR, Mortimore GE, 1984. Requirement for alanine in the amino acid control of deprivation-induced protein degradation in the liver. Proc Natl Acad Sci USA. 81:4270-4274. [Abstract/Free Full Text]
  34. Mortimore GE, Khurana KK, 1990. Regulation of protein degradation in the liver. Int J Biochem 22:1075-1080. [Medline]
  35. Ward WF, 1988. Food restriction enhancement of the proteolytic capacity of aging rat liver. J Gerontol. 43:B121-B123.
  36. Bergamini E, Del Roso A, Gori Z, Masiello P, Masini M, Pollera M, 1994. Endocrine and amino acid regulation of liver macroautophagy and proteolytic function. Am J Physiol. 266:G118-G122. [Abstract/Free Full Text]
  37. Vittorini S, Paradiso C, Donati A, et al. 1999. The age-related accumulation of protein carbonyl in rat liver correlates with the age-related decline in liver proteolytic activities. J Gerontol Biol Sci. 54A:B318-B323. [Abstract]
  38. Blommaart EFC, Luiken JJFP, Blommaart PJE, van Woerkom GM, Meijer AJ, 1995. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 270: (5) 2320-2326. [Abstract/Free Full Text]
  39. Shigemitsu K, Tsujishita Y, Hara K, Nanahoshi M, Avruch J, Yonezawa K, 1999. Regulation of translational effectors by amino acid and mammalian target of rapamycin signaling pathways. Possible involvement of autophagy in cultured hepatoma cells. J Biol Chem. 274: (2) 1058-1065. [Abstract/Free Full Text]
  40. Liu Y, Gorospe M, Kokkonen GC, et al. 1998. Impairment in both P70S6 kinase and extracellular signal-regulated kinase signalling pathways contribute to the decline in proliferative capacity of aged hepatocytes. Exp Cell Res. 240: (1) 40-48. [Medline]
  41. Nadiv O, Shinitzky M, Mann H, et al. 1994. Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats. Biochem J. 298:433-450.
  42. Nadiv O, Cohen O, Zyck Y, 1992. Defects of insulin's signal transduction in old rat livers. Endocrinology. 130:1515-1524. [Abstract/Free Full Text]
  43. Ward W, Richardson A, 1991. Effect of age on liver protein synthesis and degradation. Hepatology. 14:935-948. [Medline]
  44. Ove P, Obenrader M, Lansing AI, 1972. Synthesis and degradation of liver proteins in young and old rats. Biochem Biophys Acta. 277:211-221. [Medline]
  45. Goldspink DF, Lewis SEM, Kelly FJ, 1984. Protein turnover and growth in the whole body, liver and kidney of the rat from the fetus to senility. Biochem J. 217:507-516. [Medline]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
R. Kiffin, S. Kaushik, M. Zeng, U. Bandyopadhyay, C. Zhang, A. C. Massey, M. Martinez-Vicente, and A. M. Cuervo
Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age
J. Cell Sci., March 1, 2007; 120(5): 782 - 791.
[Abstract] [Full Text] [PDF]

Home page
 Sci Aging Knowl EnvironHome page
A. M. Cuervo
Autophagy and Aging--When "All You Can Eat" Is Yourself
Sci. Aging Knowl. Environ., September 10, 2003; 2003(36): pe25 - 25.
[Abstract] [Full Text]

Home page
 Journals of Gerontology Series A: Biological Sciences and Medical SciencesHome page
A. Donati, G. Cavallini, C. Paradiso, S. Vittorini, M. Pollera, Z. Gori, and E. Bergamini
Age-Related Changes in the Autophagic Proteolysis of Rat Isolated Liver Cells: Effects of Antiaging Dietary Restrictions
J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2001; 56(9): B375 - 383.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS