Cellular signaling, AGE accumulation and gene expression in hepatocytes of lean aging rats fed ad libitum or food-restricted

Mechanisms of Ageing and Development 123 (2002) 427– 439 www.elsevier.com/locate/mechagedev

Cellular signaling, AGE accumulation and gene expression in hepatocytes of lean aging rats fed ad libitum or food-restricted L. Teillet a, P. Ribie`re b, S. Gouraud b, H. Bakala c, B. Corman b,* a

b

Hoˆpital Sainte-Pe´rine, Assistance Publique-Hoˆpitaux de Paris (AP-HP), Paris, 75016, France Ser6ice de Biologie Cellulaire, CEA/Centre d’Etudes de Saclay, Gif-sur-Y6ette, 91191 Cedex, France c Faculte´ des Sciences, Uni6ersite´ Paris VII, Paris, 75005, France Received 30 May 2001; received in revised form 3 October 2001; accepted 10 October 2001

Abstract The effects of food restriction on liver glucagon and vasopressin V1a receptors, on AGE accumulation and on gene expression were investigated in 10- and 30-month-old WAG/Rij female rats fed ad libitum or chronically food-restricted by 30%. The age-related increase in glucagon and vasopressin V1a receptor density, as well as the rise in glucagon-induced cAMP generation was prevented by the restriction. AGE accumulation, characteristic of the aging process, was normalized in food-restricted animals. Gene expression determined with rat Atlas™ cDNA Expression Arrays containing 1176 cDNA indicates that a few genes exhibited a greater than twofold change in mRNA ratios with age. Most down-regulated genes were related to oxidative metabolism of lipids, and most of the up-regulated genes were concerned with the cell cycle and transcription factors. Chronic food restriction partially prevents these changes in gene expression and induces up- and down-regulation of several mRNAs which are not modified with age in ad libitum fed rats. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Glucagon receptor; cAMP; AGEs; Gene expression; Liver; WAG/Rij rats

1. Introduction Advanced glycation endproduct (AGEs) accumulation contributes to cross-linking of extracellular proteins and macromolecules, to changes in cell – matrix interactions and to alterations in cellular signaling (Cerami, 1985; Sell et al., 1996; * Corresponding author. Tel.: + 33-1-6908-6399; fax: + 331-6908-3570. E-mail address: [email protected] (B. Corman).

Ulrich and Cerami, 2001; Vlassara, 1996a,b). Such AGE accumulation is determined by local concentration of glucose over time and by macromolecule turnover. In this respect, the impaired glucose tolerance and the reduced turnover of proteins currently reported with age will both contribute to enhanced AGE accumulation. These age-related changes in glucose homeostasis are linked to insulin resistance, and, as recently reported in experiments performed in WAG/Rij rats, to glucagon and vasopressin V1a receptors

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overexpression in liver (Teillet et al., 2000, 2001). The latter was associated with an increased intracellular generation of cAMP by glucagon and a larger production of glucose by hepatocytes. Several studies have shown that, in rodents, chronic food restriction reduced plasma insulin concentration in baseline condition or following a glucose load, and thus reduced the integral of glucose concentration. As a result this would tend to lower plasma glucose concentration over time and the AGE accumulation (Cefalu et al., 1995; Masoro et al., 1989, 1992; Novelli et al., 1998; Reiser, 1994; Sell et al., 1996; Teillet et al., 2000). In contrast, the effect of food restriction on the G-protein-coupled receptors involved in gluconeogenesis is less documented. Although it was found that the increased expression of hepatic adrenergic receptors was mostly prevented by a 40% food restriction in male Fischer 344 rats (Katz, 1988), the effect of diet on age-related increase in glucagon receptor expression is unknown. A reduction in liver glucagon receptor activity would tend to lower circulating glucose and consequently AGE accumulation, while an increase in glucagon receptor expression by food restriction would result in the opposite. To test this hypothesis, cellular signaling and AGE accumulation were studied in the liver of female WAG/Rij rats fed ad libitum or restricted by 30% from 10 to 30 months. Density and cAMP signaling of glucagon receptors were determined in isolated hepatocytes, and compared to the vasopressin and angiotensin II G-protein-coupled receptor expression. The effect of the restriction on AGEs accumulation was assessed by competitive ELISA with anti-AGE antibodies in the liver extracellular matrix. How such food restriction would also be efficient to prevent overall changes in gene expression was determined in parallel with high-density rat Atlas™ cDNA Expression Arrays.

2. Materials and methods

2.1. Animals WAG/Rij rats are inbred Wistar rats which remain lean even when fed ad libitum, and are free

of chronic progressive nephrosis, hypertension or diabetes in the course of aging (Corman and Michel, 1987). Breeding conditions and the present food restriction protocol have previously been described (Teillet et al., 2000). Female WAG/Rij rats were born and raised in the animal care facilities of the CEA, Centre d’Etudes de Saclay, Gif-sur-Yvette, France. They were maintained on a 12:12 light–dark cycle, 50% humidity and a temperature of 21 °C. The animals were fed a commercial diet (DO3, UAR, Villemoisson, France) containing 2% fish and 15% vegetable proteins, 0.71% phosphate, 0.78% calcium, 0.62% potassium, 0.27% sodium, 0.22% magnesium and a total of 2900 kcal/kg. Daily food intake of animals fed ad libitum was close to 10 g/day. The restricted animals were fed 7 g/day of the same diet, that is a 30% restriction, starting at 10 months till 30 months, the time at which they were sacrificed. During this period the survival curves were similar between restricted and ad libitum fed rats, i.e. 36% of the animals were dead by 30 months in both groups. As previously discussed, similar survival of ad libitum and restricted animals is consistent with previous experiments performed on lean animals which were submitted to food restriction at the age of 10–12 months. It does not exclude that maximal lifespan of the cohort would be increased by such restriction protocol. However, despite similar survival until 30 months, several age-related changes in regulatory systems, including insulin resistance, were prevented by food restriction performed from 10 to 30 months in WAG/Rij rats (Teillet et al., 2000).

2.2. Hepatocytes preparation Unless otherwise stated, all the chemicals were from Sigma–Aldrich Co. Hepatocytes were isolated from the liver according to Berry’s collagenase perfusion method as modified by Seglen (Berry and Friend, 1969; Seglen, 1973). The animals were anaesthetized by an intraperitoneal injection of 10 mg/100 g body weight Inactin (Byk-Gulden, Constance, Germany), the portal vein was cannulated with a catheter (Biotrol no. 7), and the liver was rapidly transferred to a chamber

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thermostated at 37 °C. Solutions used for hepatocyte isolation contained 116 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, 5.5 mM glucose, which were gassed with 95% O2/5% CO2, pH 7.4. The liver was first perfused for 10 min with a calcium-free buffer containing 2 mM EGTA, and thereafter with a second solution containing 4.4 mM calcium and 10 mg/100 ml collagenase A (0.28 U/ mg, Boehringer Mannheim GmbH) for 7 min. The liver was thinly sliced and incubated with the collagenase solution for an additional 7 min, with gentle shaking. The suspension was filtered on surgical gauze to remove undissociated tissues, and the cells were washed three times by centrifugation (40 g) in medium containing 1.8 mM calcium and 2.25 g/l gelatine. Before use, hepatocytes were incubated at room temperature in a conservation medium containing 1.8 mM calcium and 15 g/l gelatine, and supplemented with amino acids and vitamins (BME amino acids and vitamins, Gibco-Brl). Enumeration of hepatocytes was performed with a Malassez haemocytometer, and the viability of the cells was defined from their ability to exclude Trypan Blue (0.4% solution). The diameters of hepatocytes were measured under an inverted microscope with a graduated eyepiece using 200 cells per preparation (Telaval 31, Zeiss).

2.3. Binding experiments Hepatocytes in the conservation medium were washed twice by centrifugation (70g) in a binding buffer solution containing 50 mM Tris– HCl, 3 mM MgSO4, 120 mM NaCl, 5 mM KCl, 1 mM EGTA and 0.1% BSA, pH 7.5. Enumeration was performed to adjust hepatocytes suspension to a concentration of 21×106 cells/ml. Cells were thereafter maintained at 4 °C until the binding experiments. The number of binding sites for glucagon was determined with 125I glucagon (2000 Ci/mmol, Amersham, UK). Hepatocytes (106 cells in 2 ml) were incubated for 1 h at room temperature with gentle rocking in binding buffer containing 0.01% bacitracin and 2×10 − 8 M 125I glucagon.

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Preliminary experiments indicated that this concentration was saturating for glucagon binding sites. Unspecific binding was assessed by incubation of the hepatocytes under the same conditions but with addition of 6× 10 − 6 M glucagon. At the end of the incubation period, the cell suspensions were diluted in 3.5 ml of ice-cold binding buffer free of glucagon and spotted on a Millipore vacuum manifold onto GF/C glass fiber filters which were previously coated in buffer medium with 1% BSA and 0.01% polyethylenimine for at least 1 h. The filters were washed three times with 3.5 ml of binding buffer free of 125I glucagon, and their radioactivity was measured over 4 min in an Automatic Counter Gamma (1272 Clinigamma, LKB Wallac). Each determination was performed in triplicate. The results, calculated from the difference between total and nonspecific binding, were expressed in number of binding sites per cell. Binding of vasopressin and angiotensin II was determined following the same protocol using 2×10 − 8 M 3H vasopressin (59 Ci/mM, NEN, Boston, MA) or 2 × 10 − 8 M 125I (Sar-1, Ile-8) angiotensin II (2000) Ci/mmol, Amersham, UK). These concentrations were found to be saturating for vasopressin and angiotensin II binding sites. Unspecific binding was assessed in the presence of 6× 10 − 6 M vasopressin or Sar-1 angiotensin II. Radioactivity of 3H vasopressin was measured by liquid scintillation counting on dry filters (Liquid Scintillation Analyzer, 1500 Tricarb, Packard).

2.4. cAMP accumulation measurements Intracellular cAMP accumulation was measured in hepatocyte suspensions (2× 105 cells in 500 ml conservation medium). The samples were preincubated for 5 min in the presence or absence of the phosphodiesterase inhibitor 3isobutyl-1-methyl-xanthine (IBMX, Sigma Chemical Co.) before incubation with the agonist for 4 min. During all preincubation and incubation periods, the cells were continuously bubbled with a 95% O2/5% CO2 gas mixture and maintained at 37 °C. The reaction was

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stopped by cooling the test tubes in an alcohol– solid CO2 mixture for 10 s. The tubes were rapidly centrifuged and the supernatant was removed. To extract cAMP from the cells, the pellets were suspended in 250 ml of a 5% formic acid– ethanol solution. The samples were then evaporated overnight, and resuspended in an appropriate amount of immunoassay buffer for cAMP measurement. Experiments were performed in triplicate. For each sample, cAMP was determined by enzyme immunoassay according to Pradelles et al. (Pradelles et al., 1989). cAMP enzyme immunoassay was from Cayman Chemical. The results were expressed in picomoles per 4 min per 106 cells.

RNase) incubated for 60 days with 0.5 M glucose in PBS pH 7.5, according to the protocol of Makita et al. (1992). The specificity of the resulting antibody has been previously tested with soluble and structural glycoproteins, fibronectin, Type IV collagen, laminin and AGE–BSA (Verbeke et al., 1997). ELISA was set up according to Papanastasiou et al. (1994); the standard curve was established with AGE– BSA dilutions (1– 5000 ng) and AGE –RNase polyclonal antibody (1/500). The reaction was quantified by means of goat antibody directed against rabbit IgG coupled to peroxidase (1/1000, Sigma). The data were expressed as arbitrary units/microgram hydroxyproline (AU/mg OH-Pro), with 1 AU corresponding to the reactivity of 1 ng AGE–BSA.

2.5. Collagen extraction Collagen extracts were prepared from whole liver. Rats were sacrificed by cervical dislocation and livers were rapidly removed, frozen in liquid N2 and stored at − 80 °C. They were further homogenized with a polytron (Ultra-Turrax, IKA labortechnik) and suspended in PBS (pH 7.4). The resulting suspension was centrifuged at 40000g for 30 min, 4 °C. Lipid extraction of the pellet was performed by addition of 5 ml chloroform– methanol (2:1 vol/vol) followed by gentle shaking, and standing overnight at 4 °C. The upper layer was removed and the pellet was washed three times with methanol and distilled water. Then, the pellets were resuspended in 0.5 M acetic acid and 1 mg/ml pepsin, incubated for 18 h at 4 °C to remove noncollagen proteins, and washed three times with 0.1 M CaCl2, 0.02 M Tris – HCl (pH 7.5) and 0.05% toluene. The pellets were digested with 0.1 mg/ml type VII collagenase (Sigma) by gentle shaking at 37 °C for 24 h and centrifuged at 40000g for 30 min at 4 °C. The resulting collagen supernatant was quantified from its hydroxyproline content according to Bergman and Loxley (Bergman and Loxley, 1970).

2.6. Quantification of AGE content by competiti6e ELISA The AGE polyclonal antibodies were raised against bovine pancreatic ribonuclease A (AGE–

2.7. Gene expression Animals were sacrificed by decapitation, their liver was promptly excised, weighed and frozen in liquid nitrogen. Total RNA for each liver was extracted with Trizol (Gibco-Brl, Life Technologies), suspended in water with DEPC, and aliquots were stored at −80 °C. For each sample, 250 mg of total RNA samples were digested by 25 ml of RNase-free DNase I (1 U/ml, GibcoBrl, Life Technologies) to remove possible genomic DNA contamination, and the integrity of the RNA obtained was checked on agarose gel. The efficiency of DNase treatment was assessed by negative control using a 35-cycle PCR amplification with primers for the G3PDH gene. Retrotranscription of 5 mg from the RNA and its labeling with 70 mCi a-32P dATP (6000 Ci/mmol, NEN) was performed according to the supplier’s instructions (Clontech Laboratories Inc., Atlas™ cDNA Expression Arrays, User Manual, Palo Alto, CA). Hybridization of Atlas™ Rat cDNA Expression Array containing 1176 cDNAs with the labeled cDNA was performed overnight at 68 °C. The membranes were then washed five times with SSC solutions and exposed for 3–5 days on a phosphorimager screen for quantification (STORM 840, Molecular Dynamics). The results were analyzed with the Atlas Image software from Clontech.

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2.8. Statistical analysis Results were expressed as means9SEM. Statistical analysis was performed by Student’s t-test for comparison between two groups or with twoway ANOVA and Bonferroni test for multiple comparisons among more than two groups. Significance was set at P B0.05.

3. Results Mean body weight of female WAG/Rij rats significantly increased from 10 to 30 months and was reduced in food-restricted 30-month-old animals (2139 3, n=44, 24197, n =28 and 1609 12 g, n=28, respectively). Liver weight changed in proportion (6.190.3 g, n =6, 7.490.4 g, n = 6 and 4.19 0.1 g, n=6, respectively) and the ratio of liver over body weight was comparable in the three groups (3.09 0.1, 3.090.1, and 2.79 0.1 g/100 g body weight (BW), respectively). The percentage of viable hepatocytes, as measured with blue trypan exclusion, was regularly larger than 90% in the three groups of rats. Mean diameter of the isolated hepatocytes was 22.19 1.3 mm, n=9, in adult and 24.292.5 mm, n = 11, in senescent rats (Teillet et al., 2001). As shown in Fig. 1, this age-related increase in the size of hepatocytes was prevented by chronic food restriction in 30-month-old rats (mean diameter 22.0 90.5 mm, n =9). In contrast, food restriction performed for 3 months in 10-month-old animals did not change the size of hepatocytes (mean diameter 22.390.4 mm, n =4).

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cell was more variable than that of glucagon and vasopressin receptors, and the differences between the three groups were not statistically significant. Since hepatocyte size was affected by age and food-restriction, for comparison, receptor density was expressed by membrane surface area assuming that the cells are spherical (Table 1). The number of glucagon receptors per membrane surface area was larger in senescent than in adult animals, but comparable in 10-month-old and food-restricted 30-month-old rats. The difference in vasopressin receptor expression per membrane surface area was not significant between the three groups as stated from variance analysis.

3.1. Glucagon, 6asopressin and angiotensin II receptor expression The densities of glucagon, vasopressin and angiotensin II receptors in isolated hepatocytes from adult and senescent animals are presented in Table 1. The doubling of glucagon binding sites per cell noted from 10 to 30 months in rats fed ad libitum was prevented by food restriction. Vasopressin V1a density increased by 55% with age, and this was partially reversed by chronic restriction. The number of angiotensin II receptors per

Fig. 1. Hepatocytes sizes. Distribution of hepatocyte diameters in 10- and 30-month-old animals fed ad libitum (upper panel) and in 30-month-old rats food-restricted or fed ad libitum (lower panel). Determinations have been performed on 200 hepatocytes in each experiment. The data represent the mean 9SEM of 9 and 11 experiments in 10- and 30-month-old rats, respectively, and of nine experiments in 30-month-old food-restricted female WAG/Rij rats.

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Table 1 Density of glucagon, vasopressin and angiotensin II binding sites in hepatocytes isolated from 10- and 30-month-old rats fed ad libitum and from 30-month-old rats food-restricted animals by 30% 125

I glucagon

3

125

H vasopressin

I angiotensin II

Sites/cell 10-month-old 30-month-old 30-month-old, restricted

58823 9 8775 (n=6) 10918497574*† (n=6) 471089 6585 (n= 5)

44354 95919 (n = 8) 68700 9 5531* (n =6) 51967 9 4104 (n =5)

286006 9 16681 (n =5) 430215 985892 (n = 7) 203886 922359 (n = 5)

Sites/cell surface (n/vm 2) 10-month-old 30-month-old 30-month-old, restricted

33.8 95.1 (n= 6) 59.59 4.6*† (n= 6) 30.394.2 (n= 5)

25.6 9 3.0 (n =8) 34.6 92.7 (n = 6) 33.4 92.6 (n = 5)

182.4 910.6 (n = 5) 239.7 952.4 (n =7) 131.1 9 14.4 (n =5)

*Significantly different from 10 months (PB0.05). significantly different from 30 months food-restricted (PB0.05).

†

3.2. cAMP accumulation Baseline content of cAMP in hepatocytes of 10and 30-month-old rats fed ad libitum and food-restricted was, respectively, 4.89 0.8 (n =11), 9.69 1.7 (n =8) and 8.090.8 (n =6) pmol/4 min/106 cells. Stimulation of adenylyl cyclase with 5× 10 − 5 M forskolin increased intracellular cAMP content to 97920 (n = 11), 230916 (n = 8), and 101915 (n=6), pmol/4 min /106 cells, in the same groups. This significantly enhanced activity of adenylyl cyclase in aging rats, and normalization by food restriction, were also noted when the cells were stimulated with forskolin in the presence of 10 − 5 M of the phosphodiesterase inhibitor IBMX (mean values 308933, 470942 and 267935 pmol/4 min /106 cells, n =11, n = 8 and n=6, respectively). The effect of chronic food restriction on the concentration –response curve of glucagon-induced cAMP is shown in Fig. 2 a and b. Maximal accumulation of cAMP following glucagon receptor stimulation was larger in 30-month-old than in 10-month-old rats (3259 30 and 241910 pmol/4 min/106 cells, n = 8 and n = 11, respectively), whereas their EC50 were not significantly different (7.891.2 and 9.592.5 nmol/l). Chronic food restriction prevented this age-related increase in glucagon-dependent cAMP accumulation (Fig. 2a). Maximal cAMP accumulation was 2809 31 pmol/4 min/106 cells, n =6, a value significantly lower than that of the 30-month-old rats fed ad

libitum, and very close to that of the 10-monthold animals. Mean EC50 calculated in restricted animals was comparable to that of the 10- or 30-month-old rats fed ad libitum (14.19 4.0 nmol/l, n= 7). The proper effect of food restriction on glucagon-dependent cAMP accumulation was tested in adult animals which were food-restricted by 30% for 3 months between 10 and 13 months. As evidenced in Fig. 2b, food restriction per se modified neither maximal cAMP accumulation induced by glucagon (2409 18 pmol/4 min/ 106 cells, n= 5) nor EC50 (10.09 3.9 nmol/l, n=5). Cross-talk between G protein-coupled receptors was determined in the presence of 10 − 5 M IBMX by simultaneous incubation of hepatocytes with 10 − 7 M glucagon and 10 − 7 M vasopressin or 10 − 7 M angiotensin II. Vasopressin and angiotensin II both inhibited the glucagon-dependent accumulation of cAMP whether the animals were fed ad libitum or food-restricted (Table 2). The glucagon-dependent cAMP accumulation in the presence of vasopressin or angiotensin II was still larger in 30-month-old rats than in 10-monthold fed ad libitum rats, but comparable in 10month-old and food-restricted 30-month-old animals.

3.3. AGE accumulation Immunoenzymatic determinations of AGEs in collagen extracted from the whole liver revealed a

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threefold increase in glycation products between 10 and 30 months (Fig. 3). This rise in AGEs

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accumulation was prevented by chronic food restriction (Fig. 3).

3.4. Gene expression

Fig. 2. Glucagon-induced cAMP accumulation. Concentration– response curve of glucagon-induced cAMP accumulation in hepatocytes isolated from 30-month-old female WAG/Rij rats fed ad libitum or food-restricted for 20 months (a), and isolated from 10-month-old rats fed ad libitum and 13-monthold animals food-restricted for 3 months (b). Hepatocytes were preincubated and incubated in the presence of 10 − 5 M IBMX to inhibit phosphodiesterase activity. Fig. 2a represents mean 9 SEM of eight and six experiments in 30-month-old rats fed ad libitum and food-restricted, respectively. Fig. 2b represents mean 9 SEM of 11 and five experiments in 10month-old rats fed ad libitum and 13-month-old animals food-restricted.

Hybridization signals between the different membranes may be normalized either from housekeeping genes or from the average intensity of all the cDNAs present on the membrane. When expression of housekeeping genes present on the rat Atlas cDNA Expression Arrays was normalized by average intensity of all the cDNAs, their ratios were comparable in liver of 30-month-old and 10-month-old rats (polyubiquitin 1.2, hypoxanthine-guanine phosphoribosyltransferase 1.3, glyceraldehyde 3-phosphate dehydrogenase 1.3, cytoplasmic b-actin 1.1 and 40S ribosomal protein S29 1.1 ). It indicates that housekeeping gene expression is not greatly affected with age in the liver, or that the age-related changes are in the same proportion as that of the whole mRNA. This is consistent with the unchanged content of GAPDH and b-actin mRNAs which has already been demonstrated by Northern blot from 6 to 36 months in liver of WAG/Rij rats (Slagboom et al., 1990). However, because large inter-individual variations in expression of housekeeping genes were found by these authors, gene expression was further normalized by the average intensity of all the cDNA hybridization signals in each membrane. The experimental variability in gene expression using the rat Atlas cDNA Expression Arrays was assessed from the comparison of hybridization profiles obtained with liver RNA from 3 different 10-month-old rats. A gene expression was defined as positive when the hybridization signal was more than twice the background. Under these experimental conditions, 260 to 300 over 1176 genes were positive in the different experiments. Each array from one animal was successively compared to the two others, and the mean values of the ratios of the three comparisons were calculated. As shown in Fig. 4, these ratios were included between 0.6 and 1.7. Comparable control experiments were performed with the arrays from liver RNA isolated from three different 30-monthold rats. The number of positive genes, and distri-

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Table 2 Cross-talk between glucagon, vasopressin and angiotensin II receptors

10-month-old (n =12) 30-month-old (n =9) 30-month-old, restricted (n= 5)

Glucagon

Glucagon and vasopressin

% inhibition

Glucagon and angiotensin II

% inhibition

258.49 13.9 376.99 41.7*† 259.2936.5

200.29 14.7 303.69 37.2*† 205.2 9 18.8

27.3 9 2.7 19.9 9 2.5 17.7 9 7.5

196.8 9 11.4 299.1 933.2*† 181.0 9 12.6

26.6 9 2.9 20.5 9 3.2 20.2 9 7.9

cAMP accumulation was measured in the presence of glucagon 10−7 M9 vasopressin 10−7 M or angiotensin II 10−7 M (pmol/4 min/106 cells). *significantly different from 10-month-old animals. † significantly different from 30-month-old animals food-restricted animals.

bution of the hybridization ratios, were similar to those reported in 10-month-old rats (data not shown). From these experiments, it was considered that changes in liver genes expression would be significant when the ratios between two experimental conditions were lower than 0.5 or larger than 2. The age-related changes in gene expression were assessed comparing liver RNA from three 10month-old rats and three 30-month-old animals. The resulting hybridization of each 30-month-old animal was compared to each array of the 10month-old rats with the ATLAS ARRAY software after normalization. This procedure resulted in 3× 3 ratios the mean values of which were calculated (n=9). Under these conditions, seven genes were considered as down-regulated with a ratio equal to or lower than 0.5, and eight genes were considered as up-regulated with a ratio equal to or larger than 2 (Table 3). Two additional genes were not detected in adult animals but were evidenced in senescent rats, the insulin-like growth factor binding protein 2 (J04486, GenBank accession number) and the multidrug resistance protein MDR1 (M81855, GenBank accession number). Similar comparisons and analysis with liver RNA from three food-restricted 30-month-old rats and from the same 10-month-old rats showed that diet reduced the amplitude of the age-related changes in the expression of seven genes, but amplified the changes of five genes (Table 3). Two genes which were down-regulated in 30-month-old rats were up-regulated in food-restricted 30-month-old animals as compared to adults (Table 3). In addition,

several genes whose expression was not affected by age in the 30-month-old animals fed ad libitum were either up- or down-regulated in food-restricted 30-month-old animals. These are named by their GenBank accession number, followed by the ratio of 30-month-old over 10-month-old rats, the ratio of food-restricted 30-month-old over 10-month-old rats, and the ratio of food-restricted 30-month-old over 30-month-old rats fed ad libitum: J03754, 1.0, 0.1, 0.1 ; S81448, 0.6, 0.1, 0.2 ; J00757, 1.1, 0.2, 0.2 ; J03552, 1.6, 0.3, 0.2 ; J03509, 0.6, 0.2, 0.3 ; M00002, 1.1, 0.4, 0.4 ; D16102, 0.7, 0.2, 0.4 ; L27513, 1.1, 0.4, 0.5 ; L15079, 0.8, 0.4, 0.7 ; M85214, 0.9, 0.4, 0.5 ; J02997, 1.0, 0.4, 0.6 ; D50696, 1.0, 0.5, 1.7 ; M58593, 1.0, 0.5, 1.1 ; M88595, 0.9, 0.5, 0.6 ; M94548, 0.7, 0.5, 1.0 ;

Fig. 3. AGE content of the liver. AGE content in collagen extracted from the whole liver in 10- and 30-month-old female WAG/Rij rats fed ad libitum and 30-month-old animals foodrestricted by 30%. Mean 9SEM of six animals in each group. * significantly different from 10-month-old fed ad libitum and 30-month-old food-restricted female WAG/Rij rats.

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Fig. 4. Gene expression profile. Comparison of expression profile obtained with RNA extracted from liver of three different 10-month-old WAG/Rij female rats. Each array from one animal was successively compared to the two others, and the mean values of the three comparisons were calculated.

M29859, 0.9, 0.5, 0.6 ; M83680, 1.3, 0.5, 0.5 for genes down-regulated in food-restricted 30month-old animals. M58041, 1.5, 2.1, 1.3 ; X91810, 1.1, 2.1, 1.3 ; M29275, 0.9, 2.3, 2.4 ; U88036, 1.0, 2.4, 3.3 ; L46791, 1.4, 2.4, 2.1 ; M95578, 1.3, 2.5, 1.8 ; J02720, 1.2, 2.6, 1.6 ; M91808, 1.5, 2.6, 1.5 ; J05509, 1.1, 2.6, 2.5 ; L27843, 1.3, 3.0, 2.7 ; J02701, 1.0, 4.0, 2.2 ; M12516, 1.3, 7.4, 5.2 for genes up-regulated in food-restricted 30-month-old animals.

4. Discussion Binding experiments performed on hepatocytes showed that the doubling in glucagon receptor density between 10 and 30 months in ad libitum fed rats was prevented by chronic food restriction. This does not result from a change in the size of hepatocytes, since the same conclusion was reached when density was expressed per cell or per surface area. Such reduced glucagon receptor expression in food-restricted animals was consistent with a lower intracellular cAMP accumulation following glucagon stimulation. As the reduced cAMP content of hepatocytes was noted in the absence and presence of the phosphodiesterase inhibitor IBMX, we suggest that food restriction decreases the generation of cAMP,

rather than increase the hydrolysis of nucleotides. A reduction in adenylyl cyclase activity as judged from forskolin experiments will also contribute to lower cAMP accumulation in restricted animals. This was not due to acute food restriction, as demonstrated by similar cAMP accumulation in hepatocytes of adult rats fed ad libitum or foodrestricted for 3 months. Comparable long-term effects of food restriction have been reported for b-adrenergic receptors which contribute to the production of glucose by hepatocytes (Katz, 1988). It is also applied to vasopressin V1a receptor, although in this case the lower number of binding sites was apparently linked to a reduction in the size of hepatocytes rather than a decrease in density per membrane surface area. These effects of food restriction would tend to limit the glucagon-dependent generation of glucose, and in addition to the wellknown effect of diet restriction on insulin resistance, would lower plasma glucose concentration over time and the generation of AGEs. This hypothesis was supported by previous AGE determinations in arteries and kidney, and the present measurements in the liver which demonstrates that food restriction reduces the tissue content of AGEs in rodents (Teillet et al., 2000; Corman et al., 1998; Masoro et al., 1989; Sell et al., 1996; Vlassara, 1996a,b).

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How this protocol of food restriction in lean rats would also prevent the age-related changes in liver gene expression was investigated using highdensity rat Atlas™ cDNA Expression Arrays. Considering hybridization signals with mRNA ratios lower than 0.5 or larger than 2 between 10and 30-month-old animals, 17 genes from the 260– 300 detectable genes exhibited age-related changes in expression. This proportion, close to 5%, is comparable to that found with arrays or DNA chips in mouse muscle, liver or brain (Han et al., 2000; Lee et al., 1999, 2000; Weindruch et al., 2001), suggesting that transcription of a few genes occurs in the course of aging, as far as these arrays are representative of the whole transcriptome. Nine genes were overexpressed in the liver of senescent rats. The apolipoprotein A1 is the major component of the high-density lipoprotein responsible for the transport of cholesterol in liver. Increases in plasma concentration of apolipo-

protein A1 and the corresponding liver mRNA have already been documented in aging rats (Mooradian et al., 1997). Overexpression of protein kinase C zeta has been reported at the end of life in the medfly, and is implicated in growth factor and oncogene signaling (Galve-Roperh et al., 1996; Nakanishi et al., 1993). The corticosteroide 11-b-dehydrogenase is mainly responsible for the metabolism of cortisone (Monder and Lakshmi, 1989). Its increased expression may be related to the high glucocorticoid level reported in aging rats (Sapolsky, 1992). The other overexpressed genes are related to cell cycle and interaction with DNA. Nerve growth factor-induced protein A, or Egr1, is a transcription factor whose mRNA synthesis is stimulated by oxidative stress (Nose and Ohba, 1996). It is known to inhibit growth of tumoral cells and to increase the synthesis of the nuclear factor Id1, an inhibitor of cellular differentiation (Huang et al., 1995). The protein Gax also re-

Table 3 Age-related modifications of gene expression assessed with liver mRNA extracted from 10- and 30-month-old rats fed ad libitum and from 30-month-old rats food-restricted (10 months, 30 months and 30 months R, respectively) GenBank Accession Number

Ratios 30 months/ months

30 months R/ months

30 months R/ months

Proteins down-regulated with age Cytochrome P-450 3A9 Mitochondrial carnitine O-palmitoyltransferase 1 liver isoform Brain fatty acid binding protein Glutamyl aminopeptidase A 2-Arylpropionyl-CoA epimerase c-met proto-oncogene Liver fatty acid binding protein

U60085 L07736

0.4 0.4

0.9 3.0

2.5 6.3

U02096 S73583 Y08172 U65007 M35991

0.5 0.5 0.5 0.5 0.5

1.9 0.6 0.5 0.9 0.3

3.7 0.9 1.7 1.2 0.4

Proteins up-regulated with age LIM domain kinase 2 Growth-arrest-specific protein Gax DNA-binding protein inhibitor ID1 c-jun proto-oncogene Protein kinase C zeta Apolipoprotein A-1 precursor Corticosteroid 11-beta-dehydrogenase isoenzyme 1 Nerve growth factor-induced protein A

D31874 Z17223 D10862 X17163 M18332 M00001 J05107 J04154

2.0 2.1 2.1 2.4 2.6 2.7 3.3 3.6

1.8 1.5 4.0 5.0 1.4 1.7 10.7 6.8

0.9 0.8 2.2 2.0 0.6 0.5 3.7 1.6

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duces cellular proliferation (Perlman et al., 1999). Lim kinase 2 controls the activity of GTPase Rho and of CD42 (Sumi et al., 1999). The proto-oncogene C-jun mRNA is currently increased during hepatocarcinogenesis (Sakai et al., 1989). Insulin-like growth factor binding protein 2 (IGFBP2) and the multidrug resistance protein MDR1 (M81855) were two proteins whose mRNA was undetectable in the liver of 10-monthold animals and widely expressed in aging rats. IGFBP2 in the liver is inversely proportional to tumoral differentiation in the hepatoblastome (Akmal et al., 1995). An increase in insulin-like growth factor binding protein has recently been evidenced by subtractive hybridization in replicative senescence of endothelial cells (Grillari et al., 2000). MEDR1 is a membrane carrier coupled to ATP which is able to extrude several substrates from inside to outside the cell, including drugs. Seven genes were down-regulated with age, considering a ratio lower than 0.5 as compared to adults. These are mostly involved in metabolic pathways with the exception of the protooncogene c-met, a membrane tyrosine kinase receptor associated with cellular proliferation (Cantley and Cantley, 1995). Cytochrome P450 3A9, preferentially expressed in the liver of female rats and down-regulated by ovariectomy, is implicated in the metabolism of different substrates including 17-b-estradiol (Wang and Strobel, 1997). Glutamyl aminopeptidase A is an ectoenzyme anchored in the plasma membrane which hydrolyzes biological peptides such as angiotensin II (Troyanovskaya et al., 2000). Four of the genes whose mRNA content is reduced in aging liver are to some extent linked to the metabolism of lipids. A fatty acid binding protein mRNA, or protein Z, responsible for the transport of fatty acids between the cytosol and other cell compartments is decreased, in agreement with the previous data of Singer et al. in rat liver (Singer et al., 1996). The 2 arylpropionylcoenzyme A epimerase which is implicated in the catabolism of drugs such as ibuprofen, is also involved in lipid metabolism (Reichel et al., 1997). The brain fatty acid binding protein, which is found in the liver but not in other peripheral organs, carries fatty acid in the cytoplasm from

437

the plasma membrane to mitochondria (Glatz et al., 1998). The mitochondrial carnitine O-palmitoyltransferase I liver isoform is responsible for the transport of long-chain fatty acids inside the mitochondria and is considered as a limiting factor for oxidative metabolism of the lipids (Van der Leij et al., 1999). Altogether, these changes in mRNA content of hepatocytes suggest a shift in lipid metabolism with age towards other oxidative pathways. Chronic food restriction which prevent AGE accumulation in this experimental model has various effects on changes in gene expression, depending on the genes. For five of the overexpressed mRNAs and three of the down-regulated mRNAs in aging rats, food restriction tends to lower the differences in comparison to 10-month-old animals. In contrast, it increases the amplitude of these changes for one down-regulated mRNA and five up-regulated mRNAs including the insulinlike growth factor binding protein 1. Moreover, the mitochondrial carnitine O-palmitoyltransferase and the brain fatty acid binding protein whose expression were reduced with age, were overexpressed in food-restricted 30-month-old rats as compared to adults fed ad libitum. This suggests that food restriction is not a single intervention which simply prevents the age-related changes in the expression of certain genes, but rather induces a new and complex metabolic pattern influencing transcription of many proteins. Such an incomplete reversal of age-related changes in gene expression by calorie restriction was also reported by Han et al. in mouse liver for genes which were present on the mice— but not on the rat— Atlas™ cDNA Arrays (Han et al., 2000). In summary, the present experiments indicate that chronic food restriction in lean rats prevents the age-related increase in glucagon receptor expression and glucagon-dependent cAMP accumulation. It also reduces AGE accumulation in liver collagen, probably by a beneficial effect on glucose homeostasis. Data from high-density cDNA expression arrays shows that food restriction does not have a unique effect on genes whose transcription is modified with age, but rather induces new profiles in genes expression, including genes

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related to pathways.

lipid

metabolism

and

energetic

Acknowledgements Laurent Teillet was supported by a grant from the Assistance Publique-Hoˆ pitaux de Paris, direction de la recherche clinique (Paris, France). We are grateful to Patrick Herry and Jean-Charles Robillard for outstanding work in animal care.

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