SUMMARY Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD+-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.

During early fasting, increases in skeletal muscle proteolysis liberate free amino acids for hepatic gluconeogenesis in response to pancreatic glucagon. Hepatic glucose output diminishes during the late protein-sparing phase of fasting, when ketone body production by the liver supplies compensatory fuel for glucose-dependent tissues 1–4. Glucagon stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of the CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2), while parallel decreases in insulin signaling augment gluconeogenic gene expression through the de-phosphorylation and nuclear shuttling of Forkhead Box O1 (FOXO1) 5–7. Here we show that a fasting-inducible switch, consisting of the histone acetyl-transferase (HAT) P300 and the nutrient-sensing deacetylase Sirtuin 1 (SIRT1), maintains energy balance through the sequential induction of CRTC2 and FOXO1. Following glucagon induction, CRTC2 stimulated gluconeogenic gene expression through an association with P300, which we show here is also activated by de-phosphorylation at Ser89 during fasting. In turn, P300 increased hepatic CRTC2 activity by acetylating it at Lys628, a site that also targets CRTC2 for degradation following its ubiquitination by the E3 ligase Constitutive Photomorphogenic Protein (COP1) 8. Glucagon effects were attenuated during late fasting, when CRTC2 was down-regulated due to SIRT1-mediated deacetylation and when FOXO1 supported expression of the gluconeogenic program. Disrupting SIRT1 activity, by liver-specific knockout of the SIRT1 gene or by administration of SIRT1 antagonist, increased CRTC2 activity and glucose output, while exposure to SIRT1 agonists reduced them. In view of the reciprocal activation of FOXO1 and its coactivator peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α) by SIRT1 activators 9–12, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.

Calorie restriction (CR) has been reported to increase SIRT1 protein levels in mice, rats, and humans, and elevated activity of SIRT1 orthologs extends life span in yeast, worms, and flies. In this study, we challenge the paradigm that CR induces SIRT1 activity in all tissues by showing that activity of this sirtuin in the liver is, in fact, reduced by CR and activated by a high-caloric diet. We demonstrate this change both by assaying levels of SIRT1 and its small molecule regulators, NAD and NADH, as well as assessing phenotypes of a liver-specific SIRT1 knockout mouse on various diets. Our findings suggest that designing CR mimetics that target SIRT1 to provide uniform systemic benefits may be more complex than currently imagined.Supplemental material is available at http://www.genesdev.org.Received January 11, 2008; revised version accepted April 29, 2008. Caloric intake influences life span, and the incidence of diseases in animals (Koubova and Guarente 2003). Food excess accounts for the recent historic increase in metabolic disorders in humans. Conversely, calorie restriction (CR) promotes metabolic fitness, long life, and disease protection in rodent models (Weindruch 1988). Several genetic pathways have been identified that govern diet, metabolism, and life span (Van Remmen et al. 2001;Koubova and Guarente 2003;Kenyon 2005;Sinclair 2005).Genes related to yeast SIR2, called sirtuins, encode NAD-dependent deacetylases, and promote longevity in yeast, worms, and flies (Chen and Guarente 2007). In model systems ranging from yeast to mice, sirtuins have also been associated with the salutary effects of CR. The mammalian Sir2 ortholog SIRT1 targets numerous regulatory factors affecting stress management and metabolism (Sinclair 2005;Chen and Guarente 2007). The levels of SIRT1 have been reported to increase in rodent and human tissues in response to CR (Cohen et al. 2004;Nisoli et al. 2005;Civitarese et al. 2007), and this increase is proposed to cause favorable changes in metabolism and stress tolerance triggered by this diet. The polyphenol resveratrol has also been proposed to partially mimic CR by activating SIRT1 to induce beneficial effects on health (Baur et al. 2006;Lagouge et al. 2006). Results and DiscussionTo address the relationship between SIRT1 activity and the diet, we first analyzed SIRT1 protein levels in the liver, white adipose tissue (WAT), and skeletal muscle in mice fed ad libitum (AL) or CR. While SIRT1 was induced in the WAT and muscle, as previously reported (Cohen et al. 2004;Nisoli et al. 2005;Civitarese et al. 2007), levels surprisingly were lower in CR liver (Fig. 1A). It is not clear whether the increased SIRT1 expression in CR liver reported by Cohen et al. (2004) is specific to rat or to the time point when the tissue samples are removed from animals after daily feeding. Because SIRT1 activity is also regulated by the NAD/NADH ratio (Lin et al. 2004), we determined NAD and NADH levels in these tissues. Whereas the NAD/NADH ratio increased significantly in the muscle during...

Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. How stem cells maintain metabolic homeostasis remains elusive. Here, we identified a regulatory branch of the mitochondrial unfolded protein response (UPRmt), which is mediated by the interplay of SIRT7 and NRF1 and is coupled to cellular energy metabolism and proliferation. SIRT7 inactivation caused reduced quiescence, increased mitochondrial protein folding stress (PFSmt), and compromised regenerative capacity of hematopoietic stem cells (HSCs). SIRT7 expression was reduced in aged HSCs, and SIRT7 up-regulation improved the regenerative capacity of aged HSCs. These findings define the deregulation of a UPRmt-mediated metabolic checkpoint as a reversible contributing factor for HSC aging.