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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:550-555 (2008)
© 2008 The Gerontological Society of America

Effect of Aging and Anti-Aging Caloric Restriction on the Endocrine Regulation of Rat Liver Autophagy

Alessio Donati, Gianluca Recchia, Gabriella Cavallini and Ettore Bergamini

Centro di Ricerca Biologia e Patologia dell'Invecchiamento, Università di Pisa, Italy.

Address correspondence to Alessio Donati, PhD, Università di Pisa, Centro di Ricerca Biologia e Patologia dell'Invecchiamento, Roma 55, Pisa, Italy 56126. E-mail: a.donati{at}med.unipi.it


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Autophagy is a process that sequesters and degrades altered organelles and macromolecular cytoplasmic constituents for cellular restructuring and repair, and as a source of nutrients for metabolic use in early starvation it may be involved in anti-aging mechanisms of caloric restriction. The effects of 40% daily dietary restriction (DR) and intermittent feeding (EOD) on the age-related changes in the endocrine regulation of autophagic proteolysis were studied by monitoring the rate of valine release from isolated rat liver cells. Results show that in ad libitum-fed rats sensitivity of autophagy to glucagon and insulin declines by one order of magnitude in older rats. Both DR and EOD maintain the sensitivity to glucagon at juvenile levels, whereas only EOD can fully maintain response to insulin. It is concluded that changes in the sensitivity to glucagon may have a role in the aging process.

Key Words: Autophagy • Caloric restriction • Aging • Insulin • Glucagon

AUTOPHAGY is a universal, highly conserved, energy-dependent process, essential for maintenance, restructuring, and nutrition during starvation of eukaryotic cells (1). Autophagy may be active in almost all organs during fasting (2), but most of the studies have been carried out in mammalian liver, presumably because of the quantitative importance of this organ, which is the main site of intracellular protein degradation (3). In the process, portions of organelles are first isolated from the cytoplasm into the autophagic vacuole, known as autophagosome, which then fuses with pre-existing primary lysosomes to form an autophagolysosome (4). Autophagy may recognize and selectively degrade altered cell organelles accumulating in older cells (5–7), and stimulation of autophagy may help to counteract aging (8,9). Activation of autophagy with elimination of unwanted, damaged, or redundant cell organelles and structures is coupled with the time of nutrient shortage, and might account for the well-known anti-aging effect of caloric restriction (CR) (9,10). CR is the only established intervention that significantly increases the mean and maximum life span in rodents (11), maintains the physiological processes in a youthful state (12), and retards many age-related diseases (13).

In vitro, autophagy was shown to be under moment-to-moment primary regulation by nine physiological plasma amino acids (Gln, Leu, Tyr, Phe, Pro, Met, His, Trp, Ala), and regulation by hormones may be secondary (14). In vivo, however, changes in the plasma concentration of regulatory amino acids are very small, and the inhibitory effect of insulin and the stimulatory effect of glucagon may have the major regulatory role (10). Maximum rate of autophagic proteolysis in rat liver peaks at 6 months and then declines during aging (15,16). Regulation of autophagic proteolysis by added amino acids in the perfused liver (15) and isolated liver cells (16) is significantly impaired by aging, starting before the age of 12 months, and alteration is almost fully counteracted by CR (16). Age changes in the regulation of autophagic proteolysis by glucagon and insulin and counteraction by CR were not studied in detail.

The effects of aging on the regulation of autophagic proteolysis was investigated in rat liver cells isolated from male Sprague–Dawley rats either fed ad libitum or on two different types of food restriction, namely 40% dietary restriction (DR) and every-other-day feeding ad libitum (EOD). These two types of CR have different effects on metabolism (17) and similar effects on longevity (18), and it may be assumed that the effects in common could be an essential part in anti-aging mechanisms.


   MATERIALS AND METHODS
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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 EOD or DR regimens. Data on the body weight, the caloric consumption, and the mortality rate of the animals are given in Table 1. Food was withdrawn 16–18 hours before experimentation. Rats had free access to water. At the given age, rats were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg of body weight).


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  		Table 1. Body Weight, Caloric Intake, and Percent Survival of Sprague–Dawley Male Rats Used in This Study.

 
Isolation of Hepatocytes
Liver parenchymal cells were isolated by the collagenase perfusion method of Seglen (19). Cell viability was tested by Trypan Blue exclusion and was always better than 90%.

Rate of Autophagic Proteolysis
Hepatocytes were suspended in Krebs–Ringer bicarbonate buffer (12 mg wet cell weight {cong} 1.5 x 106 cells/mL), and 3 mL of this suspension was 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. Insulin and glucagon were added at the given concentrations. If indicated, amino acids were added as a reference standard 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 hepatocyte suspension were taken at 37 and 47 minutes. The latter were deproteinized in ice-cold perchloric acid (6% final concentration). The rate of proteolysis was assessed by the linear increase in free valine during the 10-minute period following the addition of CHX. Values were corrected by the subtraction of the valine released in the presence of 5 mM 3-methyladenine, an inhibitor of lysosomal proteolysis (21,22).

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 (23). L-Norvaline was added as an internal standard to all samples. Amino acid separation was carried out on a 4.6 x 250 mm Bio-Sil ODS-5S column with a particle size of 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 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. In all analyses, p <.05 was considered significant.

Materials
Dansyl chloride was obtained from Pierce (Pierce Europe, Beijerland, The Netherlands). Amino acids, insulin, collagenase (type IV), and CHX were obtained from Sigma (St. Louis, MO). Glucagon was obtained from Novo Nordisk A/S (Bagsvaerd, Denmark). All other reagents were of the highest quality that was commercially obtainable.


   RESULTS
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Effects of Insulin and Glucagon on Liver Autophagy at Different Ages
High insulin and glucagon concentrations (e.g., 10–7/10–6 M) have been used to investigate the endocrine control of sugar and protein metabolism. Figure 1 shows the effects of age and diet on modulation by 10–7 M insulin and glucagon of the amino acid regulation of autophagic proteolysis in liver cells isolated from 16- to 18-hour-fasted rats. Cell suspension (106 cells/mL) was incubated in vitro with a physiological concentration of added amino acids. In the ad libitum–fed rats a significant decline in the autophagic proteolysis of liver cells was seen between 2 and 12 months of age, when modulation by hormones was preserved. In cells from older ad libitum–fed rats, no further decrease in basal autophagic proteolysis was detected, but a significant decrease in the stimulatory effect of 10–7 M glucagon was observed. In older cells, modulation by 10–7 M insulin was preserved. Both EOD and DR fully counteracted both the decline in autophagy in middle age and the age-related alteration in the response of older cells to 10–7 M glucagon.


Figure 01
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  		Figure 1. Effect of dietary regimen on the modulation of autophagy 100 nm insulin (INS) and 100 nm glucagon (GLU) in liver cells isolated from young (2 months), middle-age (12 months), and old rats (24 months). Cells were incubated in the presence of a physiological amino acid concentration. Results are expressed as nanomoles of released valine per minute per gram of wet tissue and represent the mean ± standard error of the mean of at least six cases. The inhibitory and stimulatory effects (in percentages) of insulin and glucagon are given at the right of the bars. #p <.05 with respect to the age-matched control (CTRL); *p <.01 with respect to the age-matched control

 
Age-Associated Changes in the Autophagic Response of Isolated Liver Cells to Physiological Concentrations of Insulin and Glucagon
The physiological plasma levels of insulin in rat portal vein were shown to be much lower than 10–7 M and to vary during the day (24), ranging from 0.1–0.2 x 10–9 (25) up to 5 x 10–9 M (26). In humans, a prandial-like hyperinsulinemia may attain a 0.5 nM level (27). Figure 2 shows that age significantly affects the sensitivity of autophagic proteolysis of liver cells isolated from 16- to 18-hour-fasted rats to the inhibitory effect of insulin within the physiological concentration range. With cells from 2-month-old fasted rats, the release of valine in the medium was significantly decreased by a 0.5 nM insulin concentration, and maximum inhibition was caused by a 1 nM concentration (the apparent Ki of the insulin effect is 2.8 x 10–10). With cells isolated from 24-month-old fasted rats, the dose–response curve was displaced to the right by one order of magnitude. Cells from fasted rats on an EOD or a DR feeding regimen exhibited different behaviors: EOD fully counteracted the age-related displacement to the right of the response to added insulin; cells from DR rats showed a highly significant response to insulin only at a very high hormone concentration.


Figure 02
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  		Figure 2. Effect of age and diet on the modulation of autophagy by insulin, at different concentrations, within and over the physiological range. Hepatocytes were incubated in the presence of physiological amino acid concentration. Results are expressed as nanomoles released of valine per minute per gram of wet tissue and represent the mean ± standard error of the mean of at least five cases. #p <.05 with respect to the age-matched control (ctrl); *p <.01 with respect to the age-matched control. AL = ad libitum-fed; EOD = fed intermittently (every other day); DR = 40% daily dietary restriction

 
Glucagon plasma levels too may vary during the day (28) and rise above a 0.025 nM concentration in portal vein plasma during fasting (26,29). Figure 3 shows that liver cells from young, fasted rats exhibited a significant increase in the rate of autophagic proteolysis in the 0.01–0.05 nM glucagon concentration range (the apparent KM of the glucagon effect is 5 x 10–11). The response to glucagon of the liver cells from older ad libitum-fed 16- to 18-hour-fasted rats is displaced to the right by one order of magnitude. Cells from CR (both EOD and DR) fasted rats exhibited a nearly juvenile response.


Figure 03
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  		Figure 3. Effect of age and diet on modulation of liver cell autophagy by glucagons, at different concentrations, within and over the physiological range. Hepatocytes were incubated in the presence of physiological amino acid concentration. Results are expressed as nanomoles released of valine per minute per gram of wet tissue and represent the mean ± standard error of the mean of at least five cases. #p <.05 with respect to the age-matched control (ctrl); *p <.01 with respect to the age-matched control. AL = ad libitum-fed; EOD = fed intermittently (every other day); DR = 40% daily dietary restriction

 

   DISCUSSION
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Besides the effects on glucose metabolism, a main action of insulin and glucagon is on the whole-body protein metabolism [protein breakdown is inhibited by insulin (30,31) and accelerated by glucagon (32)], in which autophagy plays a pivotal role (14). The sensitivity of mammalian cells to hormones may decline with increasing age in ad libitum-fed rodents, and CR may prevent these changes (33).

In this article we studied the effects of aging on the regulation of autophagic protein degradation by insulin and glucagon and showed that pancreatic hormones are likely to be a major physiologic regulator in liver cells of ad libitum-fed animals. This juvenile regulation may be lost by older cells, which are much less responsive to hormones added in the physiological concentration range. Interestingly, both types of CR (DR and EOD) were able to counteract the age-dependent loss in the regulation of autophagy by glucagon, whereas EOD only fully preserved regulation by insulin.

Rat liver cells freshly isolated from 2-month-old donors are a good model for studies on the endocrine control of autophagic protein breakdown. A dose–response curve for the autophagic proteolysis was obtained with both hormones. The apparent Ki and KM of insulin and glucagon in cells from young rats were within the physiological range of the plasma levels of the two hormones. Incidentally, the Ki of insulin was very close to the reported value of the insulin KM for the in vivo glucose uptake by young human tissues (34). Maximum inhibition by insulin and stimulation by glucagon were similar in younger and older cells, but an age-dependent impairment of the endocrine regulation of autophagic proteolysis was seen with a rightward shift in the dose–response curve of both insulin and glucagon action. The apparent Ki for insulin and KM for glucagon of the cells isolated from 24-month-old ad libitum-fed, overnight-fasted rats were far above the peak levels that could be attained by the two hormones during alternation between feeding and fasting. Because autophagy may be an essential part in the anti-aging mechanism of CR (10), data might help us to understand the well known but still unexplained finding that CR has smaller or no anti-aging effect if started by middle-age or later (35,36).

It is well known that the effect of insulin declines with increasing age (37), but the mechanism was studied on sugar metabolism only. Interestingly, both in older humans (38) and older Sprague–Dawley rats (39), the insulin dose–response curve for glucose uptake progressively shifted to the right. Most studies agreed that insulin resistance is associated with a postbinding defect (34,40,41), and a juvenile response may be restored in the rat by the administration of vanadate (42). Activation of the insulin receptor might be impaired in older age because of the accumulation of posttranslational modifications of the protein, affecting serine phosphorylation or binding to inhibiting proteins such as PC-1 or members of the SOCS or Grb protein families (see 43).

The dependency of the autophagic/proteolytic response of liver cells to glucagon on the age of the animal had not been studied. Data on the effects of glucagon on sugar metabolism are conflicting and might not be properly applied to this discussion (44–48). The Kd of glucagon binding to isolated membranes did not change, and the maximum binding capacity per unit of protein decreased between young and middle age (49), but the number of glucagon binding sites per cell increased from 60 x 103 to 110 x 103 from maturity to old age (50). Effects of glucagon on the adenyl cyclase activity (49) and cyclic adenosine monophosphate (cAMP) accumulation in older cells were seen at a very high (10–8 to 10–6 M) concentration range (46). It should be mentioned that a selective deprivation of intracellular amino acids may be involved in glucagon-induced autophagy and proteolysis (51), and an age-dependent decline in the oxidation of branched amino acids was reported recently (52).

CR was shown to counteract the age-related alteration in the control of autophagic proteolysis by amino acids and to modulate insulin receptor signalling in liver and skeletal muscle (53,54) and glucagon cellular signalling in hepatocytes (50). Hence, a protective effect of CR from the age-related changes in the response of liver cells to glucagon and insulin could be expected. However, the surprising finding is that the two anti-aging CR regimens, EOD and DR, almost fully prevented the age-related decrease in the apparent KM and maximal activity of glucagon, but EOD only improved the sensitivity of older liver cells to insulin. Because both types of CR have similar effects on life span, it can be proposed that a lifelong maintenance of the physiological response to glucagon might have a role in the anti-aging mechanism of CR. The effectiveness of the glucagon response to a low level of nutrients has been proposed recently to have a key role in the anti-aging effect of CR (55).


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received September 11, 2007

Accepted January 25, 2008


   References
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