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Insulin resistance is a major risk factor for type 2 diabetes mellitus. The protein encoded by the sirtuin 1 (Sirt1) gene, which is a mouse homolog of yeast Sir2, is implicated in the regulation of glucose metabolism and insulin sensitivity; however, the underlying mechanism remains elusive. Here, using mice with a liver-specific null mutation of Sirt1, we have identified a signaling pathway involving Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and Foxo1 that regulates gluconeogenesis. We found that Sirt1 positively regulates transcription of the gene encoding Rictor, triggering a cascade of phosphorylation of Akt at S473 and Foxo1 at S253 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck). Liver-specific Sirt1 deficiency caused hepatic glucose overproduction, chronic hyperglycemia, and increased ROS production. This oxidative stress disrupted mTorc2 and impaired mTorc2/Akt signaling in other insulin-sensitive organs, leading to insulin resistance that could be largely reversed with antioxidant treatment. These data delineate a pathway through which Sirt1 maintains insulin sensitivity and suggest that treatment with antioxidants might provide protection against progressive insulin resistance in older human populations. IntroductionThe 7 members of the mammalian sirtuin family (SIRT1-SIRT7) have NAD + -dependent deacetylase activity and/or mono-ADP ribosylase activity (1-4). Their substrates include histones and nonhistone proteins that are involved in numerous biological functions, including cell growth, apoptosis, senescence, neuronal protection, adaptation to calorie restriction, organ metabolism and diseases, DNA damage response and repair, and tumorigenesis (1-3, 5-11).Functions of sirtuin 1 (SIRT1), a founding member of the sirtuin family, in glucose metabolism have been extensively studied at the whole-organism level. SIRT1 expression is induced upon nutritional stress (12, 13). Activation of SIRT1 protein by fasting induces gluconeogenic genes and hepatic glucose production, whereas acute knockdown of SIRT1 in the mouse liver with the adenovirus system reduces glucose output (14,15). It was also reported that SIRT1 knockdown in the liver by using an antisense oligonucleotide decreases basal gluconeogenesis and increases hepatic insulin responsiveness in diabetic rats (16). In contrast, transgenic mice with moderate overexpression of SIRT1 displayed improved glucose tolerance due to decreased hepatic glucose production, suggesting a negative role of SIRT1 in gluconeogenesis (17). Moreover, a recent report using mice carrying a liver-specific deletion of exon 4 of the Sirt1 gene mediated by albumin-Cre (Sirt1 exon4/exon4 ;Alb-Cre mice) revealed normal levels of fasting and fed blood glucose (18). These observations may reflect a complex role of SIRT1 in the regulation of glucose metabolism at different

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1alpha mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1alpha in vivo using three different antibodies. PP1alpha was phosphorylated at Thr-320 during M-phase and again in late G(1)- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1alpha phosphorylation. In vitro, PP1alpha was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1alpha occurred in M-phase but not at G(1)/S. G(1)/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1alpha phosphorylated at Thr-320 co-precipitated with pRB during G(1)/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1alpha may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1alpha regulate the G(1)/S and G(2)/M transitions and that PP1alpha complexed to pRB requires inhibitory phosphorylation by G(1)-specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G(1)- to S-phase and/or complete S-phase.