Of note, we recently demonstrated that NMN, an intermediate compound synthesized from nicotinamide in the NAD biosynthetic pathway (Revollo et al., 2007a), circulates systemically in mouse plasma and plays a critical role in the regulation of NAD biosynthesis and GSIS in pancreatic β cells (Revollo et al., 2007b). β cells depend on NMN in blood circulation for the maintenance of normal NAD and GSIS levels. Therefore, we next assessed the levels of NMN in plasma from young and old BESTO mice. Surprisingly, plasma NMN levels were significantly lower in both old male and female BESTO mice compared to young BESTO mice (Fig. 6B), suggesting that in vivo NAD levels must also be significantly reduced in the pancreatic β cells of the aged BESTO mice. In our recent study, we also demonstrated that administration of NMN ameliorates the defects in GSIS observed in mice and islets heterozygous for nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the NAD biosynthetic pathway in mammals (Revollo et al., 2004). We therefore hypothesized that if the reduced plasma NMN levels in the aged BESTO mice contribute to their loss of phenotypes, we might be able to restore the improved glucose tolerance and enhanced GSIS in aged BESTO mice by administration of NMN.
We injected either phosphate-buffered saline (PBS) or NMN (500 mg kg−1 body weight) intraperitoneally to 20-month-old BESTO and control mice 14 h prior to performing IPGTTs. Glucose tolerance and GSIS did not differ significantly between BESTO and control mice following the PBS treatment (Fig. 6C,D, left panels). Interestingly, NMN-treated aged BESTO female mice resumed significantly improved glucose tolerance and enhanced GSIS compared to NMN-treated aged controls (Fig. 6C,D, right panels). While NMN administration slightly impaired glucose tolerance in both the BESTO and control mice compared to PBS-treated mice (Fig. 6C, right panel), it is important to note that NMN administration also augmented GSIS significantly in both BESTO and control mice (compare 30 min results in left and right panels in Fig. 6D). These phenomena appear to be sex specific, as we did not observe similar responses to the same dose of NMN in aged BESTO male mice (Supplementary Fig. S5). Nevertheless, NMN administration restored the positive effect of Sirt1 on glucose tolerance and GSIS, at least in the aged BESTO female mice. Taken together, these findings suggest that an age-associated decline in systemic NAD biosynthesis, and hence Sirt1 activity, accounts for the loss of the phenotypes in aged BESTO mice.

Discussion

In our previous study, we demonstrated that BESTO mice exhibit significantly enhanced GSIS and improved glucose tolerance at both 3 and 8 months of age (Moynihan et al., 2005). In BESTO islets, Sirt1 mediates the repression of Ucp2 and hence increases ATP levels in response to glucose stimulation, resulting in the enhancement of GSIS (Moynihan et al., 2005). Here, we speculated that increasing Sirt1 dosage or activity in pancreatic β cells would provide life-long beneficial effects of enhanced β cell function on glucose homeostasis in the process of aging. In this study, however, we made the unexpected finding that BESTO mice lose their glucose-responsive phenotypes with advanced age, despite maintaining high levels of Sirt1 over-expression in pancreatic β cells. First, both male and female BESTO mice from two independent lines no longer showed glucose-responsive phenotypes at 18–24 months of age (Fig. 1A,B and Supplementary Fig. S1). Second, islets isolated from these aged BESTO mice no longer showed enhanced GSIS in vitro or higher ATP levels compared to controls, consistent with the in vivo results (Fig. 1C,D). Third, aged BESTO islets no longer exhibited the Sirt1-mediated down-regulation of Ucp2 or the up-regulation of Scd1 and Dpp4 that was characteristic of their younger counterparts (Fig. 2). Together, these findings suggest that while Sirt1 overexpression in β cells enhances GSIS and improves glucose tolerance at younger ages, long-term overexpression of Sirt1 does not confer life-long beneficial effects of improved β cell function in mice.
These unexpected results raised an interesting question: Why do BESTO mice lose their glucose-responsive phenotypes with advanced age? Because Sirt1 expression remained high in the aged BESTO islets, two possible explanations were considered to account for the loss of the BESTO phenotypes: (i) Sirt1 activity decreases with advanced age or (ii) the actions of some alternative age-associated factors negate the Sirt1-mediated enhancement of GSIS. We first suspected that the increased body weight and circulating lipids might contribute to the loss of the BESTO phenotypes with age because these parameters were all significantly elevated in the old compared to young male mice (Supplementary Figs S2 and S3). However, following a Western-style HFD regimen for up to 30 weeks, the BESTO mice still maintained significantly improved glucose tolerance and enhanced GSIS compared to controls, and Ucp2 expression was still suppressed in islets from HFD-fed BESTO mice, as it was in islets from regular chow-fed young BESTO mice (Figs 4 and and5,5, and Supplementary Fig. S4). These results suggest that Sirt1 can still function to enhance GSIS and improve glucose tolerance even in the face of diabetogenic dietary conditions. Considering that Sirt1 mediates the repression of Ucp2 expression in β cells (Moynihan et al., 2005; Bordone et al., 2006), it is interesting to note that the results from HFD-fed BESTO mice are similar to those from HFD-fed Ucp2-deficient mice that also exhibit enhanced β cell glucose sensitivity compared to controls after HFD (Joseph et al., 2002). Therefore, our findings on HFD-fed BESTO mice reemphasize the notion that increasing Sirt1 dosage or activity in pancreatic β cells provides beneficial effects of enhanced β cell function on glucose homeostasis (Moynihan et al., 2005). These findings also suggest that common physiological changes observed in both aged and HFD-fed mice alone are not enough to abolish the positive effects of Sirt1 in β cells.
Because Sirt1 requires NAD for its enzymatic activity (Imai et al., 2000), it is also possible that a decline in NAD biosynthesis with age would result in a significant reduction of Sirt1 activity and subsequent loss of the glucose-responsive phenotypes. Although no difference was detected in the ability of young and old BESTO islets to synthesize NAD in culture conditions, we found that plasma levels of NMN, a critical component for β cells to maintain normal NAD biosynthesis and GSIS (Revollo et al., 2007b), were significantly reduced in old compared to young BESTO mice (Fig. 6A,B), indicating that in vivo NAD levels must also be reduced in old BESTO islets. Consistent with these findings, administration of NMN was able to restore improved glucose tolerance and enhanced GSIS compared to controls in aged BESTO female mice (Fig. 6C,D). While NMN leads to a slight impairment of glucose tolerance in both BESTO and control female mice (Fig. 6C), the levels of insulin secretion post-glucose injection in NMN-treated aged BESTO female mice were remarkably augmented compared to those in the PBS-treated counterparts (compare 30 min results in left and right panels, Fig. 6D), resulting in a pronounced difference in insulin secretion between aged BESTO and control mice (right panel, Fig. 6D). The restoration of increased GSIS and hence the improvement of glucose tolerance by NMN administration have also been observed in female Nampt-heterozygous mice, another mouse model in which plasma NMN levels are also reduced (Revollo et al., 2007b). Given that Nampt-mediated NAD biosynthesis has been shown to play an important role in the regulation of Sirt1 activity (Revollo et al., 2004), it is very likely that NMN administration restored the activity of over-expressed Sirt1 in β cells and thereby enhanced GSIS and improved glucose tolerance in aged BESTO female mice compared to old controls. Currently, why the aged BESTO male mice did not respond similarly to NMN administration remains unclear, and it should be noted that a similar gender disparity has also been observed in the case of the Nampt-heterozygous mice (Revollo et al., 2007b). Further investigation will be necessary to clarify this sex-dependent difference. Nonetheless, these findings strongly suggest that an age-dependent decline in plasma NMN levels contributes to decreased systemic NAD biosynthesis and reduced Sirt1 activity in aged animals (Fig. 7).