The age-related di erences in obese and fatty acid synthase gene expression in white adipose tissue of rat

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1 Biochimica et Biophysica Acta 1533 (2001) 73^80 The age-related di erences in obese and fatty acid synthase gene expression in white adipose tissue of rat Anna Nogalska, Julian Swierczynski * Department of Biochemistry, Medical University of Gdansk, ul. Debinki 1, Gdansk, Poland Received 19 March 2001; received in revised form 6 May 2001; accepted 20 June 2001 Abstract To determine if the age-dependent increase of adiposity is directly related to altered obese (ob) and fatty acid synthase (FAS) gene expression, we assessed an adiposity index, leptin and FAS mrna levels, FAS activity in perirenal adipose tissue and serum leptin concentration in rats aged 1, 2, 3, 6 and 20 months. The results indicate that there are two distinct phases of changes in perirenal white adipose tissue leptin mrna level and serum leptin concentration. The first phase, between 1 and 3 months of the animals' lives, was characterized by a strong positive correlation between adiposity index and leptin mrna level as well as serum leptin concentration. In the second phase (over 3 months) no significant changes of leptin mrna and serum concentration occurred. A close correlation between the age-induced increase of leptin mrna abundance and serum leptin concentration and the age-induced suppression of FAS gene expression in the same tissue was observed. This suggests that the changes of FAS gene expression occur in response to serum leptin concentration and that in mature rats the high level of ob gene expression and consequently the high leptin concentration protect the white adipose tissue cells against fat overload by two independent mechanisms: (a) preventing an increase of food intake through the leptin action on the hypothalamus; (b) inhibiting FAS gene expression and consequently decreasing the rate of lipogenesis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Leptin; Fatty acid synthase; Age-related obesity 1. Introduction It is well known that mammalian aging is usually associated with a relative increase in body weight and adiposity [1]. However, the precise molecular mechanisms responsible for these changes are mostly unknown. The discovery of the obese (ob) gene has o ered new insights into the understanding of the mechanisms that underlie the control of food intake and body weight [2]. Leptin, the protein product of * Corresponding author. Fax: address: juls@amedec.amg.gda.pl (J. Swierczynski). the ob gene [2], is an a erent signal molecule that interacts with appetite and satiety centres in the brain to regulate body weight [3]. Furthermore, leptin appears to be the signal indicating the size of the fat depot in the body [2]. Injected into rodents, leptin reduces food intake and increases energy expenditure, resulting in the loss of body weight [4^7]. Rayner et al. [8] found the presence of leptin mrna in the rat inguinal white adipose tissue taken at the rst day after birth. ob gene expression in rat white adipose tissue markedly increases during the suckling^weaning transition [8,9]. Scarpace et al. [10,11] showed that rat serum leptin concentration and white adipose tissue leptin mrna level in / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 74 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 creased from age 3 to 24 months. Recently Scarpace et al. [12] showed that aged rats demonstrate a reduced responsiveness to leptin. The diminished response to leptin was associated with and may be the result of an impaired suppression of the hypothalamic neuropeptide Y mrna level [12]. It has been shown recently that treatment of mice with fatty acid synthase inhibitors led to the inhibition of feeding and body weight loss [13]. This suggests that fatty acid synthase may also play an important role in the control of feeding behaviour [13]. Furthermore, it has been found that leptin suppresses the fatty acid synthase gene transcription in primary cultured adipocytes [14]. Therefore, one can assume that a chronically elevated leptin mrna level and consequently the increase in leptin concentration in adipocytes should lead to a decrease of fatty acid synthase gene expression in white adipose tissue in vivo. As already mentioned, serum leptin concentration and adipose tissue mrna leptin level increase with age of rats [10,11], while fatty acid synthase activity decreases signi cantly in white adipose tissue with age [15]. To verify this hypothesis, in the present study we used the same animals, maintained under identical conditions, to investigate the relationship between ob and fatty acid synthase gene expression in white adipose tissue from rats aged 1^20 months. Another goal of the present study was to investigate the changes in ob gene expression and serum leptin concentration in rats especially between 30 and 90 days of life, since the previous studies concentrated mainly on ob gene expression in rats between 1 and 30 days [8,9] or 3 and 24 months of age [10,11]. 2. Materials and methods 2.1. Animals Male Wistar rats aged 1, 2, 3, 6 and 20 months (n = 5/age) were housed in wire mesh cages at 22³C under a light^dark (12^12 h) cycle with lights on at h. Food (commercial diet composition described in [16]) and tap water were provided ad libitum. Average daily food intake was measured by the di erences in weight between the amount of food provided and the amount remaining over a 1 day period. The rats were killed from to h. Epididymal, inguinal and perirenal white adipose tissues were collected and rapidly frozen in liquid nitrogen. The tissues were stored at 380³C until analysis Determination of adiposity levels Adiposity was determined by the adiposity index expressed as the sum of the weight of epididymal, perirenal and inguinal white adipose divided by body weightu100 [10,17]. Plasma leptin concentration was measured by radioimmunoassay [18] Probe design and labelling Fatty acid synthase (FAS) and malic enzyme mrna levels have been detected using 32-mer oligonucleotides (5P-GAT AGA GGT GCT GAG CCA GCG TGC TGA GCG TG-3P; 5P-CTC ACT CGC CTG TGC CGC AGC CCA ATA TAC AA-3P) [19] complementary to the rat FAS and to the rat malic enzyme coding sequence respectively. The probe to detect leptin mrna was a 33-mer antisense oligonucleotide (5P-GGT CTG AGG CAG GGA GCA GCT CTT GGA GAA GGC-3P) [20]. The 31-mer antisense oligonucleotide (5P-CGC CTG CTG CCT TCC TTG GAT GTG GTA GCC G-3P) was used as a probe for the 18S rrna [20]. The oligonucleotides were synthesized commercially (Genset, Paris, France) with a single digoxigenin ligand at the 3P end Isolation of RNA and Northern blot RNA analysis Total cellular RNA was extracted from frozen tissue by a guanidine isothiocyanate-phenol/chloroform method [21] and nally dissolved in dimethyl pyrocarbonate (DMPC) treated water. The RNA concentration of the extracts was determined from the absorbance at 260 nm and all samples had a 260/280 nm absorbance ratio of about 2.0. RNA samples were applied (10 Wg per lane) to a 1% agarose gel containing 0.41 M formaldehyde, and fractionated by horizontal gel electrophoresis. After electrophoretic fractionation, RNA was transferred overnight to a positively charged nylon membrane by capillary blotting and xed with UV light. Prehybridization

3 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 75 and hybridization were performed as described recently [19,22,23]. Signals were scanned and quanti ed using the Sigma Scan software program (Jandel Scienti c), the levels of mrna for leptin, FAS and malic enzyme were estimated. The values were normalized for the corresponding amount of 18S rrna. Results expressed in arbitrary units are presented as means þ S.D. of samples from ve rats Enzyme activity assay 1 g of perirenal white adipose tissue was placed in 8 ml ice-cold 20 mm Tris^Cl bu er (ph 7.8) containing 0.2% Triton X-100. The tissue was minced nely with scissors, homogenized manually with a Te on pestle homogenizer, and centrifuged at Ug for 20 min. The resulting supernatant was decanted, and the pellet resuspended in 5 ml isolation medium, rehomogenized, and centrifuged as above. The resulting supernatant was combined with this after the rst centrifugation step and used for enzyme assay. The fatty acid synthase (EC ) and malic enzyme (EC ) activities were assayed as described previously [24,25]. Fig. 2. Adiposity index in rats at ages 1, 2, 3, 6 and 20 months. Data represent means þ S.D. of ve animals per age. *P ; **P ; NS, not signi cant Statistics The statistical signi cance of di erences between groups was assessed by one-way analysis of variance (ANOVA) followed by Student's t-test using Systat software (Systat). In addition, regression analysis was performed based on indicated data points. Differences between the groups and correlations were considered as signi cant when P Results Fig. 1. Body weight of rats at ages 1, 2, 3, 6 and 20 months. Data represent means þ S.D. of ve animals per age. *P ; **P ; ***P The rats, under ad libitum feeding conditions, demonstrated a progressive increase in body weight with age between 1 and 20 months (Fig. 1). The most signi cant changes occurred at the rst 3 months of life. The weight of epididymal, perirenal and inguinal adipose tissues in these rats also increased with age (not shown). Consequently the adiposity index increased between 1 month and 20 months. In this case, the most signi cant changes were observed during the rst 6 months of life (Fig. 2). The average daily food intake by 1, 2 and 3 month old rats was 9.1 þ 1.0, 19.7 þ 1.6 and 23.9 þ 1.5 g respectively, but remained unchanged between the third and the 20th month of old animals' lives. The e ect of age on

4 76 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 Fig. 3. Representative Northern blot analysis of ob, fatty acid synthase (FAS), malic enzyme (ME) and 18S rrna in perirenal white adipose tissue of rats at ages 1, 2, 3, 6 and 20 months. (The blot was sequentially probed for leptin mrna, FAS mrna, ME mrna and 18S rrna.) leptin, fatty acid synthase and malic enzyme mrna levels is presented in Fig. 3, which exhibits a representative Northern blot analysis. The lms were quanti ed by densitometry, and the levels of leptin, fatty acid synthase and malic enzyme mrnas in aging rats were compared to the corresponding 18S rrna level in these animals (Fig. 4). The results indicate that the leptin mrna level was very low in perirenal white adipose tissue of 1 month old animals and then signi cantly increased, reaching its maximal value at the age of 3 months (Figs. 3 and 4). The amount of perirenal white adipose tissue leptin mrna level was correlated with both the body weight (r = 0.9, P ) and the adiposity index; however, in the latter instance the correlation was much weaker (r = 0.4, P ) over the period tested (between 1 and 20 months). The correlation between the amount of adipose tissue and leptin mrna level was much stronger in 1^3 month old rats (r = 0.77, P ). In 3^20 month old rats no correlation between leptin mrna level and the amount of adipose tissue was observed (r = 0.14). Unlike leptin, fatty acid synthase and malic enzyme mrna levels were high in perirenal white adipose tissue of 1 and 2 month old rats and then gradually decreased, reaching a low level in 20 month old ani- Fig. 4. Quanti cation of the e ect of age (1, 2, 3, 6 and 20 months) on leptin (a), fatty acid synthase (b) and malic enzyme (E) mrna in rat perirenal white adipose tissue. Blots obtained as described in Section 2 and in the legend to Fig. 3 were scanned and quanti ed by the Sigma Scan software program.

5 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 77 Fig. 5. Serum leptin concentration in rats aged 1, 2, 3, 6 and 20 months. Data represent means þ S.D. of ve animals per age. *P = 0.01, NS, not signi cant. mals (Figs. 3 and 4). For a better representation of the e ect of age on whole body leptin synthesis and secretion, serum leptin concentration was estimated. Serum leptin concentration increased signi cantly from age 1 to 3 months, followed by a non-signi cant decline at the age of 20 months (Fig. 5). The pattern of the age-related changes in serum leptin concentration qualitatively resembled that of the adiposity index and abundance of leptin mrna level in perirenal white adipose tissue (Fig. 4). Additionally, a strong correlation between serum leptin concentration and body weight (r = 0.73, P ), and a weaker correlation between serum leptin concentration and adiposity index (r = 0.56, P ) in rats between 1 and 20 months of age were found. However, in 1^3 month old rats a strong correlation between adiposity index and serum leptin concentration was found (r = 0.88, P ). There was no correlation between adiposity index and serum leptin concentration in 3^20 month old rats. As expected (based on mrna measurements), perirenal white adipose tissue fatty acid synthase (Fig. 6) and malic enzyme activities (not shown) decreased signi cantly with age. The pattern of changes in these enzyme activities resembled that of changes in abundance of fatty acid synthase and malic enzyme mrnas. The data presented in Fig. 6 clearly indicate a strong inverse correlation (r = 0.75, P ) between serum leptin concentration and fatty acid synthase activity in perirenal white adipose tissue of aging rats. Essentially similar results were obtained when the correlation between leptin mrna and malic enzyme activity was tested (not shown). The e ect of aging on leptin and fatty acid syn- Fig. 6. Relationship between fatty acid synthase activity (b) in perirenal white adipose tissue and serum leptin concentration (a) in rats aged 1, 2, 3, 6 and 20 months.

6 78 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 thase gene expression in epididymal white adipose tissue is essentially similar to that in the perirenal fat pad (not shown). 4. Discussion The e ect of aging on white adipose tissue ob gene expression and serum leptin concentration has been reported recently [8^11]. However, these studies concentrated mainly on the changes occurring in 1^30 day old rats [8,9] or in mature animals (over the age of 3 months) [10,11]. The results presented in this paper indicate that there are two distinct phases of changes in perirenal white adipose tissue leptin mrna level and serum leptin concentration. The rst phase, between 1 and 3 months, is characterized by a strong positive correlation between adiposity index and perirenal white adipose tissue leptin mrna level as well as between adiposity index and serum leptin concentration. The second phase is characterized by no signi cant change in perirenal white adipose tissue ob gene expression. The increase in ob gene expression at the rst phase (between 1 and 3 month old rats) could be related, at least in part, to the increase in food intake by the rats. However, these changes might also be related to the development or simply be secondary to di erences in the amount of fat pads appearing in rats at di erent ages, since the size of fat cells and fat content of adipocytes are important determinants of lipid metabolism [26]. Taken together, these results suggest that the responsiveness of perirenal white adipose tissue ob gene expression undergoes rapid changes during the development of animals. In mature rats this responsiveness was substantially blunted and the level of adipose tissues leptin mrna abundance and serum leptin concentration were maintained on a relatively high, but constant level. It seems that in old rats (over 3 months) the high level of leptin mrna and consequently the high serum leptin concentration prevent a further increase in daily food intake and the overaccumulation of fat in adipocytes. This is consistent with the prediction that under a low serum leptin level, adipocytes are able to accumulate fat at a relatively high rate (as can be observed in young animals), but once the leptin concentration reaches a relatively high, stable level (as was found in mature animals) it protects the white adipose tissue cells against fat accumulation at the rate observed in young rats. Thus, these data support the hypothesis that serum leptin is a signal molecule that indicates the size of the fat depots in the body [2]. The present data also indicate a close correspondence between the age-induced increase of leptin mrna abundance in white adipose tissue (and serum leptin concentration) and age-induced suppression of fatty acid synthase gene expression in white adipose tissue. An essentially similar correspondence between the age-induced increase of leptin mrna level and the age-induced suppression of malic enzyme, which is involved in the de novo synthesis of long chain fatty acids by providing NADPH, was also observed. This close correspondence and the inhibitory e ect of leptin on fatty acid synthase gene transcription in primary cultured adipocytes [14] suggest that the changes in fatty acid synthase and malic enzyme gene expression occur in response to serum leptin concentration. Taken together, these results appear to support the assumption that leptin exerts an autocrine e ect as an anti-obesity hormone controlling the expression of key lipogenic enzymes and consequently the rate of lipogenesis. This is consistent with the suggestion that leptin shifts the adipocyte metabolism, reducing the synthesis of lipids from glucose, and increasing the oxidation rate of glucose that could otherwise be stored as fat in adipose tissue [27]. The importance of leptin as an inhibitor of fatty acid synthase gene expression in vivo is also disclosed by: (a) ob/ob mice, which are bearing a mutation in the leptin gene and consequently do not synthesize leptin [2]; (b) fa/fa (Zucker) rats, which are bearing the mutation in the leptin receptor gene and consequently lose leptin action [28]. Accordingly these animals are characterized by an overexpression of lipogenic enzymes and a high rate of lipogenesis [29^31]. However, the increase in serum leptin concentration between the rst and the second month of age is associated with no signi cant change in fatty acid synthase and malic enzyme gene expression. These data suggest that: (a) some other factors may be involved in overriding the inhibitory action of leptin at this period of time; (b) between the rst 2 months of the rat's life the leptin concentration in white adipose tissue is too low to inhibit the expression of

7 A. Nogalska, J. Swierczynski / Biochimica et Biophysica Acta 1533 (2001) 73^80 79 lipogenic enzyme genes, but once it reaches a relatively high level it inhibits the expression of these genes. A key question concerns the possible mechanism(s) by which leptin suppresses fatty acid gene expression in vivo. A previous study described a leptin-mediated inhibition of insulin action in adipocytes [32]. In recent reports, leptin was shown to decrease in vitro the insulin stimulation of fatty acid synthase gene expression by reducing the binding capacity of receptors [14,33]. Thus, one possible mechanism is through the decrease by leptin of the insulin receptor binding capacity. Another possible mechanism is that leptin inhibits insulin secretion in pancreatic islets [34]. Irrespective of the underlying mechanism, our results suggest that leptin can also play an important role in lipogenic enzyme activity regulation in vivo. If this is true, the amount of fat in rats can be a ected not only by the leptin action on the hypothalamus, but also by a direct action on white adipose tissue lipogenesis. In this context, it should be noted that leptin suppresses transcription of the ATP-citrate lyase gene in adipocytes [14] and acetyl-coa carboxylase in preadipocytes [33]. Furthermore, Bai et al. [33] demonstrated that leptin inhibits the insulin and dexamethasone stimulated synthesis of fatty acid in 30A5 preadipocytes. Our data also indicate that the ATP-citrate lyase and acetyl-coa carboxylase activities were much higher in white adipose tissue of young rats (below 3 months) than in old rats (not shown). Taking together, the above results suggest that the physiological role of the hyperleptinaemia found in mature rats is to protect adipocytes from fat overload in part by preventing the upregulation of lipogenesis in these cells during caloric excess. Acknowledgements We are indebted to Prof. Mariusz M. Zydowo for criticism and discussion of the manuscript, and Mrs Elzbieta Goyke for her technical assistance. This work was supported by a grant from the Committee for Scienti c Research (within project No. 6P05A 04021) and from the Medical University of Gdansk (Badania Statutowe St-41, Badania Wlasne W-934). References [1] E.J. Masoro, Adipose tissue, in: Handbook of Physiology in Aging, CRC Press, Boca Raton, FL, 1981, pp. 417^421. [2] Y. Zhang, R. Proenca, M. Ma ei, M. Barone, L. Leopold, J. Friedman, Positional cloning of the mouse obese gene and its human homologue, Nature 372 (1994) 425^432. [3] C.A. Meler, Advances in the understanding of the molecular basis of obesity, Eur. J. Endocrinol. 133 (1995) 761^763. [4] L.A. Camp eld, F.J. Smoth, Y. Guisez, R. Devos, P. Burn, Recombinant mouse ob protein: evidence for a peripheral signal linking adiposity and central neural networks, Science 269 (1995) 546^549. [5] J.L. Halaas, K.S. Gaiwala, M. Ma ei, S.L. Cohen, B.T. Chait, D. Rabinowitz, R.L. Lallone, S.K. Burley, J.M. Friedman, Weight-reducing e ects of the plasma protein encoded by the obese gene, Science 269 (1995) 543^546. [6] M. Pelleymounter, M.J. Cullen, M.B. Baker, R. Hecht, D. Winters, T. Boone, F. Collins, E ects of the obese gene product on body weight regulation in ob/ob mice, Science 269 (1995) 540^543. [7] P.J. Scarpace, M. Matheny, Leptin increases uncoupling protein expression and energy expenditure, Am. J. Physiol. 273 (1997) E226^E230. [8] D.V. Rayner, G.D. Dalgliesh, J.S. Duncan, L.J. Hardie, N. Hoggard, P. Trayhurn, Postnatal development of the ob gene system: elevated leptin levels in suckling fa/fa rats, Am. J. Physiol. 273 (1997) R446^R450. [9] V. Rousseau, D.J. Becker, L.N. Ongemba, J. Rahier, J.C. Henquin, S.M. Brichard, Developmental and nutritional changes of ob and PPARQ 2 gene expression in rat white adipose tissue, Biochem. J. 321 (1997) 451^456. [10] H. Li, M. Matheny, M. Nicolson, N. Tumer, P.J. Scarpace, Leptin gene expression increases with age independent of increasing adiposity in rats, Diabetes 46 (1997) 2035^ [11] H. Li, M. Matheny, N. Tumer, P.J. Scarpace, Aging and fasting regulation of leptin and hypothalamic neuropeptide Y gene expression, Am. J. Physiol. 275 (1998) E405^ E401. [12] P.J. Scarpace, M. Matheny, R.L. Moore, N. Tumer, Impaired leptin responsiveness in aged rats, Diabetes 40 (2000) 431^435. [13] T.M. Loftus, D.E. Jaworsky, G.L. Frehywot, C.A. Townsend, G.V. Ronnet, M.D. Lane, F.P. Kuhajda, Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors, Science 288 (2000) 2379^2381. [14] H. Fukuda, N. Iritani, T. Sugimoto, H. Ikeda, Transcriptional regulation of fatty acid synthase gene by insulin/glucose, polyunsaturated fatty acids and leptin in hepatocytes and adipocytes in normal and genetically obese rats, Eur. J. Biochem. 260 (1999) 505^511. [15] A.D. Mooradian, S.G. Albert, The age-related changes in lipogenic enzymes: the role of dietary factors and thyroid hormone responsiveness, Mech. Ageing Dev. 108 (1999) 139^149.

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