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Calorie Restriction Increases Life Span: A Molecular Mechanism

Posted on: Saturday, 25 February 2006, 03:01 CST

By Wolf, George

Calorie restriction increases the life span of many organisms, from yeast to mammals. In yeast, the life span gene affected by calorie restriction is Sir2 (silent information regulator 2). In mammals, Sirt1, an ortholog of Sir2, controls the metabolism of white adipose tissue. Calorie restriction activates Sirt1, and the expressed Sirt1 protein inhibits the action of peroxysome proliferator-activator receptor gamma (PPARγ), the nuclear receptor that promotes adipogenesis. The effect is lipolysis and loss of fat. Lowering of adiposity appears to be one mechanism whereby calorie restriction affects life span.

Key words: adipocytes, adipogenesis, calorie restriction, life span, nicotinamide, NAD^sup +^, NADH, peroxysome proliferator- activator receptor gamma, PPARγ, resveratrol, Sir2 gene, Sirt1 gene, white adipose tissue

2006 International Life Sciences Institute

doi: 10.1301/nr.2006.feb.89-92

Seventy years ago, in a remarkable series of experiments lasting nearly 4 years, McCay et al.1 determined the effect of retarded growth of rats on the length of life span and on the ultimate body size. The rats received a diet providing an excess of "all recognized essentials for rapid growth except sufficient calories." Feed was adjusted to hold body weight constant. A growth of 10 g was permitted every 2 to 3 months "as soon as any member of the group seemed to be failing from the deficiency of calories." The results showed that male rats fed ad libitum averaged a life span of 483 days, whereas calorie-restricted male rats lived an average of 894 days.

More recently, calorie restriction has been found to increase the "life span" of yeast in terms of the number of times mother yeast cells divide to produce daughter cells.2 Calorie restriction lengthens the life of nematodes, insects (Drosophila melanogaster), mice, rats, and probably primates.3

In rodents, calorie restriction (typically 60% to 70%) has been demonstrated not only to increase life span, but also to decrease body temperature, blood glucose and insulin levels, glycogen, fat, and body weight, and to increase insulin sensitivity, organ size (except brain), and reproductive capacity.4

The classical mechanistic explanation of the effect of calorie intake on aging was that reactive oxygen species produced during respiration would cause oxidative damage to DNA, RNA, protein, and lipids. Thus, calorie restriction would decrease the load of reactive oxygen species, resulting in less damage and thus increased life span. Considerable evidence has been adduced for this theory,5 which implies a reduced metabolic rate during calorie restriction. However, an argument against the theory was the observation by McCarter et al.6 that, when normalized to body weight, metabolic rate is not slowed by calorie restriction.

This "passive" theory of aging has recently been upstaged by an "active" theory that depends on a genetic determinant of life span.7 When yeast was grown in a medium with a diluted glucose concentration, the number of times mother yeast cells divided to form offspring cells increased. The gene that determined this increased life span resulting from calorie restriction was found to be silent information regulator 2 (Sir2), which is involved in transcription silencing. Deletion of this gene decreased life span and overexpression increased it.8

The family of proteins coded by the Sir2 gene are conserved in yeast, nematodes, and Drosophila. They function as histone deacetylases, removing the acetyl group from acetyllysine in histones, and require NAD^sup +^ as a cofactor, forming O-acetyl- ADP-ribose and nicotinamide (Figure 1). In calorie-restricted yeast, the NAD^sup +^/NADH ratio is critical for Sir2 activity, particularly since NADH is an inhibitor of Sir2.10 Calorie restriction increases oxidative metabolism in yeast, shifting the ratio NAD^sup +^TNADH in favor of NAD^sup +^ and at the expense of NADH, thus activating Sir2 and resulting in an increased life span.10

Figure 1. The enzyme SIR2 deacetylates the acetyllysine group on histone, with NAD^sup +^ as cofactor, forming the deacetylated histone, nicotinamide and ADP-ribose. (From Landry et al., 2000.9 Used with permission.)

In mammals, white adipose tissue (WAT) in the body increases with aging. Calorie restriction would of course be connected to reducing adiposity. Early work11 suggested that adiposity per se was not a determinant of longevity. Later reports12 showed that insulin was involved in the reversal by calorie restriction of age-related insulin resistance. Blher et al.13 studied transgenic mice made lean by deletion of the gene coding for the insulin receptor specific for adipose tissue. These mice with reduced fat mass lived longer than wild-type (WT) mice. To generate these fat-specific insulin- receptor knock-out (FIRKO) mice, the authors13 crossed mice of two transgenic strains: one that expressed a recombinase under control of the tissue-specific aP2 gene, the gene coding for the adipocyte- specific fatty acid transport protein, and one in which the insulin receptor gene was flanked by two loxP sites, a mutation causing its deletion. The resulting offspring, in which the insulin receptor in WAT had been deleted (FIRKO mice), were normally functional at all sites other than WAT.

The FIRKO mice had a 15% to 25% reduction in body weight and a 50% to 70% reduction in fat mass compared with WT mice. However, their food intake per gram body weight was 55% greater than that of the WT mice. They were healthy and did not suffer the age-related reduction in glucose tolerance found in the aged WT mice. Of the control group of WT mice, only 45% to 50% lived 30 months, whereas 80% of the FIRKO mice lived that long. Mean life span of the lean mice was 18% greater than that of the WT mice. The authors concluded that "leanness, not food restriction, is a key contributor to extended longevity."13

Clearly, adiposity is closely connected to longevity. A recent report by Picard et al.14 explored the question of how the Sirt1 gene, an ortholog of the yeast Sir2 gene that promotes longevity, affects adipogenesis, insulin, and fat mobilization.

Picard et al.14 used cultured 3T3-L1 cells that can be made to differentiate into adipocytes. The nuclear receptor PPARγ promotes adipogenesis in these cells.15 During differentiation, the Sirt1 protein appeared in the differentiating adipocytes. When the cells were caused to overexpress Sirtl 10-fold by transfection with the virus-derived retroviral construct pBABE-Sirt1, intracellular triglyceride decreased by 50% compared with control cells treated with the vector alone. When Sirt1 was down-regulated 7-fold by transfection with the Sirt1 interfering-RNA vector SUPER-Sirt1-RNAi construct, a considerable increase in triglyceride level was observed. Thus, Sirt1 appears to be a negative regulator of adipogenesis.

Since insulin regulates adipogenesis, the authors14 next determined at which point in the insulin signaling cascade Sirt1 exerts its action. They found that in the absence of insulin, by addition of the PPARγ-stimulating and thereby adipogenesis- stimulating drug rosiglitazone, neither Sirt1 overexpression nor Sirt1 down-regulation had any effect on adipogenesis. Therefore, Sirt signals downstream of insulin in the adipogenesis signaling cascade. Sirt1 overexpression reduced the expression of the genes driving WAT differentiation: PPARγ, c/EBP-d, and c/EBP-α, as well as that for the fatty acid-binding protein aP2.

Applying the Sirt1 activator resveratrol, a small molecule found in red grapes, to fully differentiated 3T3-L1 adipocytes greatly reduced the triglyceride content of the cells. Therefore, Sirt1 activation, even in fully differentiated adipocytes, mobilizes fat. Even primary cultures of rat WAT cells treated with adrenalin (to mobilize fat) respond to resveratrol by greatly enhancing free fatty acid release. Conversely, nicotinamide, the inhibitor of Sirt1 activity (Figure 1), significantly reduced adrenalin-stimulated free fatty acid release (Figure 2).

The authors14 tested their hypothesis that Sirt1 may function by inhibiting the action of PPARγ, the transcription factor essential in stimulating adipocyte differentiation, using the chromatin immunoprecipitation assay.17 For this assay, the proteins were cross-linked by formaldehyde to DNA in primary human pre- adipocytes. After sonication, the resulting protein-DNA complexes were each separately immunoprecipitated with the respective antibodies to Sirt1 and PPARγ, and the DNA was extracted and amplified by the polymerase chain reaction (PCR). The authors found that in 3T3-L1 adipocytes, Sirt1 and PPARγ both bound to the same promoter region on the PPARγ gene as well as the aP2 gene (the target gene of PPARγ). In those adipocytes overexpressing Sirt1, binding was much stronger; in Sirt1-down-regulated cells, there was no binding. Luciferase reporter assays showed that Sirt1 repressed transactivation by PPARγ. The authors concluded that: 1) Sirt1 and PPARγ bound to the same promoter sequences, and 2) that Sirt1 is a repressor of PPARγ (Figure 2).

Figure 2. Scheme representing SRT1 activation upon fasting and/ or calorie restriction. In WAT, SRT1 binds to PPARγ and its co- repressor NCoR, repressin\g PPARγ transcriptional activity, thus reducing adipogenesis and promoting lipolysis. (From Picard et al., 2005.16 Used with permission.)

When PPARγ was immunoprecipitated, Sirt1 precipitated with it. Further, the PPARγ co-repressor NCoR immunoprecipitated with the anti-Sirt1 antiserum. Using the glutathione-S-transferase pull-down method, the authors14 observed that two NCoRs bound to one Sirt1 repressor domain.

Chromatin immunoprecipitation assays revealed that NCoR bound to the PPARγ already bound to the promoter on the adipogenic genes. As already described above, 3T3-L1 cells, when made to overexpress the Sirt1 gene, reduced fat accumulation. When the Sirt1- overexpressing cells were then infected with an interfering RNA virus targeted to NCoR to inhibit the repressor action of NCoR, the reduction of fat accumulation was prevented. The authors14 concluded that Sirt1 exerts its action of fat accumulation through NCoR (Figure 2).

The authors14 extended their studies to fat mobilization in mice in vivo, and showed that the Sirt1 protein was present in all WAT tested. Using the chromatin immunoprecipitation assay, Sirt1 was found not to be bound to the promoters of the PPARγ gene of normally fed WT mice. When fasted overnight, Sirt1 became bound to the promoter. Therefore, Sirt1 is "recruited"14 to the PPARγ gene of adipocytes upon calorie restriction (fasting).

Picard et al.14 then generated a strain of transgenic mice in which the Sirt1 gene had been deleted. This mutation (Sirt1^sup +/- ^) was postnatally lethal. Heterozygotes (Sirt1^sup +/-^) were phenotypically normal: their fat deposits were the same as those of WT mice. In vivo assays of circulating free fatty acids showed that Sirt1^sup +/-^ mice had 40% to 45% less free fatty acids in blood after overnight fasting compared with fasted WT mice. A similar result was obtained when primary WAT cells from Sirt1^sup +/-^ mice were cultured and free fatty acid release was stimulated by adrenalin treatment.

The initial finding that set off the search for a genetic determinant of calorie restriction on life span was made in yeast. In yeast, as well as in Drosophila and the flatworm, one gene, Sir2, was found to be responsible for lengthening the reproductive life span. The discovery of an ortholog of the Sir2 gene in mammals, Sirt1, led to the hypothesis that activation of Sirt1, similar to Sir2 in yeast, could be the mechanism whereby calorie restriction in mammals lengthens life span. The Sirt1 gene in mammals controls the expression of a multitude of metabolic and neuroendocrine systems.18 Clearly, a likely candidate for a target of calorie restriction would be fat tissue, since fat storage is most readily affected by calorie intake. A stimulus to this line of thinking was the finding that decreased adiposity leads to increased life span.13 Picard et al.14 have now shown that Sirt1 can act as a sensor for decreased calorie intake by being activated. This activation results in fat mobilization by inhibition by Sirt1 of the nuclear receptor PPARγ necessary for adipogenesis. This mechanism could be one way, but certainly not the only way, that calorie restriction extends life span in mammals.

REFERENCES

1. McCay CM, Cromwell MF, Maynard LA. The effect of retarded growth upon the length of life span and ultimate body size. J Nutr. 1935;10:63-79.

2. Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science. 2000;289:2126-2128.

3. Ingram DK, Anson RM, de Cabo R et al. Development of calorie restriction mimetics as a prolongevity strategy. Ann N Y Acad Sci. 2004;1019:412-423.

4. Weindruch R, Walford RL. The Retardation of Aging and Diseases by Dietary Restriction. Springfield, IL: Charles C. Thomas Pub.; 1988.

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6. McCarter RJ, Masoro EJ, Yu BP. Does food restriction retard aging by reducing the metabolic rate? Am J Physiol. 1985;248:E488- E890.

7. Guarente L. Diverse and dynamic functions of the Sir silencing complex. Nature. 1999;23:281-285.

8. Kaeberlein M, McVey M, Guarente L. The SIR2/3/4 complex and Sir2 alone promote longevity in Saccharomyces cerevisiae by 2 different mechanisms. Gen Dev. 1999;13:2570-2580.

9. Landry J, Slama JT, Sternglanz R. Role of NAD+ in the deacetylase activity of the SIR2-like proteins. Biochem Biophys Res Comm. 2000;278:685-690.

10. Lin SJ, Ford E, Haigis M, Liszt G, Guarente L. Calorie restriction extends yeast life span by lowering level of NADH. Gen Dev. 2004;18:12-16.

11. Harrison DE, Archer JR, Astle CM. Effects of food restriction on aging: separaton of food intake and adiposity. Proc Natl Acad Sci USA. 1984;81:18351838.

12. Gabriely I, Ma XH, Yang XM, et al. Removal of visceral fat prevents insulin resistance and glucose intolerance of aging: an adipokine-mediated process. Diabetes. 2002;51:2951-2958.

13. Bluher M, Kahn BB, Kahn CR. Extended longevity in mice lacking the insulin receptor in adipose tissue. Science. 2003;299:572-574.

14. Picard F, Kurtev M, Chung N, et al. Sirt1 promotes fat mobilization in white adipocytes by repressing PPARγ. Nature 2004;429:771-6.

15. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor. Cell. 1994;79:1147-1156.

16. Picard F, Guarente L. Molecular links between aging and adipose tissue, Int J Obes. 2005;29:536-539.

17. Nakae J, Kitamura T, Kitamura Y, Briggs WH, Arden KC, Accili D. The forkhead transcription factor Foxol regulates adipocyte transcription. Dev Cell. 2003;4:119-129.

18. Guarente L, Picard P. Calorie restriction-the SIR2 connection. Cell. 2005;120:473-482.

George Wolf, DPhil

Dr. Wolf is with the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California, USA.

Please address all correspondence to: Dr. George Wolf, c/o Nutrition Reviews, One Thomas Circle NW, 9th Floor, Washington, DC 20005; Phone: 202-6590074; Fax: 202-659-3859; E-mail: nutritionreviews@ ilsi.org.

Copyright International Life Sciences Institute Feb 2006

Source: Nutrition Reviews

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