Original Publication: Ramsey KM, Mills KF, Satoh A, Imai S. Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice. Aging cell. 2008;7(1):78-88. doi:10.1111/j.1474-9726.2007.00355.x.

Summary

The Sir2 (silent information regulator 2) family of NAD-dependent deacetylases regulates aging and longevity across a wide variety of organisms, including yeast, worms, and flies. In mammals, the Sir2 ortholog Sirt1 promotes fat mobilization, fatty acid oxidation, glucose production, and insulin secretion in response to nutrient availability. We previously reported that an increased dosage of Sirt1 in pancreatic β cells enhances glucose-stimulated insulin secretion (GSIS) and improves glucose tolerance in beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice at 3 and 8 months of age. Here, we report that as this same cohort of BESTO mice reaches 18–24 months of age, the GSIS regulated by Sirt1 through repression of Ucp2 is blunted. Increased body weight and hyperlipidemia alone, which are observed in aged males and also induced by a Western-style high-fat diet, are not enough to abolish the positive effects of Sirt1 on β cell function. Interestingly, plasma levels of nicotinamide mononucleotide (NMN), an important metabolite for the maintenance of normal NAD biosynthesis and GSIS in β cells, are significantly reduced in aged BESTO mice. Furthermore, NMN administration restores enhanced GSIS and improved glucose tolerance in the aged BESTO females, suggesting that Sirt1 activity decreases with advanced age due to a decline in systemic NAD biosynthesis. These findings provide insight into the age-dependent regulation of Sirt1 activity and suggest that enhancement of systemic NAD biosynthesis and Sirt1 activity in tissues such as β cells may be an effective therapeutic intervention for age-associated metabolic disorders such as type 2 diabetes.
Keywords: aging, BESTO mice, insulin secretion, pancreatic β cells, Sirt1, systemic NAD biosynthesis

Introduction

The silent information regulator 2 (Sir2) family of NAD-dependent deacetylases has been implicated in the regulation of aging and longevity in a wide variety of organisms, including yeast, worms, and flies (Blander & Guarente, 2004; Bordone & Guarente, 2005). An increased dosage of Sir2 proteins extends life span in each of these organisms, while deletion or mutation of Sir2 shortens life span (Kaeberlein et al., 1999; Tissenbaum & Guarente, 2001; Astrom et al., 2003; Howitz et al., 2003; Rogina & Helfand, 2004; Wood et al., 2004). In budding yeast, Sir2-mediated silencing at the rDNA loci is crucial for its ability to regulate life span because Sir2 suppresses homologous recombination and hence the formation and accumulation of toxic extrachromosomal rDNA circles, one of the primary causes of yeast aging (Gottlieb & Esposito, 1989; Sinclair & Guarente, 1997; Kaeberlein et al., 1999). In Caenorhabditis elegans, the Sir2 ortholog SIR-2.1 requires the forkhead transcription factor DAF-16 to promote life span extension (Tissenbaum & Guarente, 2001) and acts in a stress response pathway parallel to the insulin/IGF-1 signaling pathway that converges directly upon DAF-16 (Berdichevsky et al., 2006; Wang & Tissenbaum, 2006; Wang et al., 2006). In Drosophila, whole body and neuronal-specific overexpression of the Sir2 ortholog dSir2 extends life span (Rogina & Helfand, 2004). Finally, caloric restriction (CR), the single most consistent regimen to extend life span across a wide range of species, requires the Sir2 genes for CR-mediated life span extension in certain genetic backgrounds and conditions in these organisms (Lin et al., 2000, 2002, 2004; Anderson et al., 2003; Rogina & Helfand, 2004; Wang & Tissenbaum, 2006).
While it is not yet known whether the mammalian Sir2 ortholog Sirt1 similarly regulates aging and longevity in mammals, Sirt1 has been shown to regulate metabolic responses to changes in nutrient availability in multiple tissues (Bordone & Guarente, 2005; Moynihan & Imai, 2006). Sirt1 levels are increased in tissues such as muscle, brain, liver, and fat in response to fasting and CR in rodents (Al-Regaiey et al., 2004; Cohen et al., 2004; Nemoto et al., 2004; Rodgers et al., 2005). Up-regulation of Sirt1 in adipocytes in response to fasting promotes lipolysis and free fatty acid mobilization through repression of PPARγ, a nuclear hormone receptor that promotes adipogenesis (Picard et al., 2004). Sirt1 also promotes gluconeogenesis and represses glycolysis in hepatocytes in response to nutrient deprivation by interacting with and deacetylating PGC-1α, a key transcriptional regulator of glucose production in the liver (Rodgers et al., 2005). In skeletal muscle, Sirt1-mediated PGC-1α deacetylation is required to induce mitochondrial fatty acid oxidation genes in states of nutrient deprivation (Gerhart-Hines et al., 2007). Finally, we and others demonstrated that Sirt1 promotes insulin secretion in pancreatic β cells in response to glucose partly through repression of uncoupling protein 2 (Ucp2), a mitochondrial inner membrane protein that uncouples respiration from ATP production (Moynihan et al., 2005; Bordone et al., 2006).
Aging is one of the greatest risk factors for metabolic complications, such as obesity, glucose intolerance, and type 2 diabetes (Chang & Halter, 2003; Moller et al., 2003). It has been shown that a progressive decline in β cell function in the elderly is a major contributing factor to the pathophysiology of type 2 diabetes (Iozzo et al., 1999; Basu et al., 2003). A similar age-associated impairment of β cell function has also been demonstrated in rodents (Muzumdar et al., 2004). In our previous study, we reported that pancreatic beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice exhibited enhanced glucose-stimulated insulin secretion (GSIS) and improved glucose tolerance compared to controls at both 3 and 8 months of age (Moynihan et al., 2005). BESTO islets had reduced levels of Ucp2 and correspondingly increased levels of ATP. Furthermore, BESTO pancreata and islets secreted more insulin compared to controls in response to the potent insulin secretagogue potassium chloride (KCl), which directly depolarizes β cells. Based on these findings, we hypothesized that increased Sirt1 dosage or activity in pancreatic β cells would provide life-long beneficial effects of enhanced β cell function on glucose homeostasis and prevent or delay the development of metabolic complications associated with aging.
To address this hypothesis, we followed the same cohorts of BESTO mice that we used in our previous study (Moynihan et al., 2005) and analyzed their β cell function at 18–24 months of age. Unexpectedly, we found that the improved glucose tolerance and enhanced GSIS were completely abolished as the BESTO mice reached these old ages. Furthermore, islets isolated from the aged BESTO mice also no longer showed Sirt1-mediated repression of Ucp2 expression or increased ATP levels compared to controls. Because Sirt1 expression remained high in the aged BESTO islets, our results suggest that either Sirt1 activity decreases with age, perhaps because of a decline in NAD biosynthesis, or the actions of some alternative age-associated factors, such as increased obesity and/or blood lipid levels, negate the Sirt1-mediated enhancement of GSIS. Recently, we have demonstrated that nicotinamide mononucleotide (NMN), a novel NAD biosynthetic metabolite circulating in plasma, plays a critical role in the regulation of NAD biosynthesis and GSIS in pancreatic β cells (Revollo et al., 2007b). Interestingly, we found that old BESTO mice have significantly reduced NMN levels in their plasma and that NMN administration restored the improved glucose tolerance and enhanced GSIS in the aged female BESTO mice. These findings strongly suggest that an age-associated decline in systemic NAD biosynthesis indeed accounts for the reduced activity of Sirt1 in aged pancreatic β cells. These surprising findings provide insight into the age-dependent regulation of Sirt1 activity and may open up new avenues for the treatment of age-associated metabolic complications such as impaired glucose tolerance and type 2 diabetes.

Results

Aged BESTO mice no longer show improved glucose tolerance or enhanced GSIS

We previously reported that an increased dosage of Sirt1 in pancreatic β cells results in enhanced GSIS and improved glucose tolerance in BESTO transgenic mice at 3 and 8 months of age (Moynihan et al., 2005). Based on these previous results, we were interested in examining whether these beneficial effects of improved β cell function would persist into old age in the BESTO mice. Surprisingly, the same cohort of BESTO male and female mice in two independent transgenic lines no longer exhibited such an improvement at 18–24 months of age (Fig. 1A and Supplementary Fig. S1A). Likewise, the enhancement of GSIS observed after glucose injection in the young BESTO mice (Moynihan et al., 2005) was also abolished in the aged BESTO male and female mice (Fig. 1B and Supplementary Fig. S1B). Consistent with these in vivo results, islets isolated from the aged BESTO mice and cultured in RPMI media for 18–20 h also no longer displayed the enhanced GSIS or increased ATP levels compared to controls (Fig. 1C,D) that was characteristic of their younger counterparts (Moynihan et al., 2005). Thus, contrary to our expectations, long-term overexpression of Sirt1 in pancreatic β cells does not result in life-long beneficial effects of improved β cell function in BESTO mice.