We and others previously demonstrated that Sirt1 promotes GSIS partly through the repression of Ucp2 expression and the increase in ATP levels in pancreatic β cells (Moynihan et al., 2005; Bordone et al., 2006). In young BESTO islets, Ucp2 expression levels were significantly decreased compared to controls (Moynihan et al., 2005; see Fig. 2). However, despite maintaining high levels of Sirt1 overexpression, islets from aged BESTO mice no longer displayed reduced levels of Ucp2 (Fig. 2A,B), consistent with the lack of a difference in ATP content (Fig. 1D).
In order to determine whether the loss of Sirt1-dependent regulation with age was specific to Ucp2 or whether it was a more general phenomenon, we also examined the mRNA levels of stearoyl-CoA desaturase 1 (Scd1) and dipeptidyl peptidase IV (Dpp4), two genes found to be up-regulated by Sirt1 in microarray analyses comparing young BESTO and control islets (unpublished findings). While Sirt1 overexpression resulted in significant up-regulation of Scd1 (2.7-fold) and Dpp4 (1.5-fold) mRNA levels in young BESTO islets compared to controls, up-regulation of these two genes was reduced and no longer significantly different in old BESTO compared to control islets (Fig. 2C). Together, these results suggest either that Sirt1 activity decreases with advanced age or that the actions of some alternative age-associated factors negate the effect of Sirt1 in aged BESTO mice.
BESTO islets and mice still respond partially to other insulin secretagogues
In addition to being hyperresponsive to glucose, islets isolated from young BESTO mice also elicit a pronounced increase in insulin secretion in response to KCl, a potent insulin secretagogue, compared to controls (Moynihan et al., 2005). Because the enhancement of GSIS and the repression of Ucp2 were completely abolished in the aged BESTO mice and islets, we speculated that they also might have lost their ability to hyper-secrete insulin in response to other secretagogues, such as KCl. We found that islets isolated from BESTO mice at 18–24 months of age still maintained their ability to secrete higher levels of insulin in response to KCl (Fig. 3A). However, the extent of enhancement of insulin secretion in aged BESTO islets was less pronounced than it was in young BESTO islets (Moynihan et al., 2005). To confirm these results in vivo, we intraperitoneally injected arginine, which acts similarly to KCl in that it directly depolarizes the β cell membrane (Newsholme et al., 2005), to 20-month-old BESTO male mice and measured plasma insulin levels at 0, 2, and 5 min after arginine injection. Consistent with the in vitro results, the same aged BESTO mice that no longer showed enhanced GSIS maintained their ability to secrete more insulin in response to arginine than controls (Fig. 3B). Thus, interestingly, while Sirt1-stimulated GSIS was completely blunted as the BESTO mice reached old age, the Sirt1-mediated mechanism that acts downstream of β cell depolarization still remained partially intact in old BESTO islets.