Resveratrol Inhibits Neointimal Growth after Arterial Injury in High-Fat-Fed Rodents: The Roles of SIRT1 and AMPK.

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2.

Unconventional Secretion of Adipocyte Fatty Acid Binding Protein 4 Is Mediated By Autophagic Proteins in a Sirtuin-1-Dependent Manner.

Fatty acid binding protein 4 (FABP4) is a leaderless lipid carrier protein primarily expressed by adipocytes and macrophages that not only functions intracellularly but is also secreted. The secretion is mediated via unconventional mechanism(s), and in a variety of species, metabolic dysfunction is correlated with elevated circulating FABP4 levels. In diabetic animals, neutralizing antibodies targeting serum FABP4 increase insulin sensitivity and attenuate hepatic glucose output, suggesting the functional importance of circulating FABP4. Using animal and cell-based models, we show that FABP4 is secreted from white, but not brown, adipose tissue in response to lipolytic stimulation in a sirtuin-1 (SIRT1)-dependent manner via a mechanism that requires some, but not all, autophagic components. Silencing of early autophagic genes such as Ulk1/2, Fip200, or Beclin-1 or chemical inhibition of ULK1/2 or VPS34 attenuated secretion, while Atg5 knockdown potentiated FABP4 release. Genetic knockout of Sirt1 diminished secretion, and serum FABP4 levels were undetectable in Sirt1 knockout mice. In addition, blocking SIRT1 by EX527 attenuated secretion while activating SIRT1 by resveratrol-potentiated secretion. These studies suggest that FABP4 secretion from adipocytes is regulated by SIRT1 and requires early autophagic components.

Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Modulating SIRT1 activity variously affects thymic lymphoma development in mice.

SIRT1 is a protein deacetylase with a broad range of biological functions, many of which are known to be important in carcinogenesis, however much of the literature regarding the role of SIRT1 in cancer remains conflicting. In this study we assessed the effect of SIRT1 on the initiation and progression of thymic T cell lymphomas. We employed mouse strains in which SIRT1 activity was absent or could be reversibly modulated in conjunction with thymic lymphoma induction using either the N-nitroso-N-methylurea (NMU) carcinogenesis or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) transgene. Decreased SIRT1 activity reduced the development of thymic lymphomas in the NMU-treated mice but was permissive for the formation of lung adenomas. Conversely, in the NPM-ALK transgenic mice, decreased SIRT1 activity had a modest promoting effect in the development of thymic lymphomas. The results of the work presented here add to the growing body of evidence that sirt1 is neither an outright oncogene nor a tumor suppressor. These opposing results in two models of the same disease suggest that the influence of sirt1 on carcinogenesis may lie in a role in tumor surveillance.

Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development.

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.

Ablation of systemic SIRT1 activity promotes nonalcoholic fatty liver disease by affecting liver-mesenteric adipose tissue fatty acid mobilization.

Sirtuin 1 (SIRT1) has been reported to protect against nonalcoholic fatty liver disease (NAFLD) development. The mechanism of how SIRT1 deacetylase activity affects NAFLD has not been well investigated. The current investigation addressed the causal effect of systemic SIRT1 activity on NAFLD development and the underlying mechanism involved in both liver and mesenteric adipose tissue (MAT). Both SIRT1 homozygous mice ablated the catalytic activity (sirt1Y/Y) and their corresponding wild type littermates (WT) were fed a high fat diet (HFD, 60% calories from fat) for 34weeks. Sirt1Y/Y mice showed significantly higher level of hepatic triglyceride which was accompanied with higher levels of SREBP-1 and SCD1and decreased phosphorylation of LKB1 and AMPK in the liver. Compared with WT mice, mRNA expression of lipogenic genes (lxrα, srebp-1c, scd1 and fas) in the MAT increased significantly in sirt1Y/Y mice. Fatty acid oxidation biomarkers (acox1, acox3, cpt, ucp1, sirt3) in both liver and MAT were comparable between groups. Interestingly, we observed that in sirt1Y/Y mice, the mRNA level of hormone sensitive lipase (hsl), adipose triglyceride lipase (atgl) and perilipin-2 (plin-2), all involved in lipolysis, significantly increased in MAT, but not in epididymal adipose tissue. These changes positively correlated with circulating free fatty acid (FFA) concentrations and higher hepatic mRNA expression of cd36 for FFA uptake. The present study has provided novel evidence to suggest that under HFD-induced metabolic surplus, the lack of SIRT1 catalytic activity promotes release of FFA from MAT and escalate NAFLD by interfering with lipid homeostasis in both liver and MAT.

Reversible modulation of SIRT1 activity in a mouse strain.

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4-5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2-4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.

Sirt1-deficiency causes defective protein quality control.

Protein quality control is an important mechanism to maintain cellular homeostasis. Damaged proteins have to be restored or eliminated by degradation, which is mainly achieved by molecular chaperones and the ubiquitin-proteasome system. The NAD(+)-dependent deacetylase Sirt1 has been reported to play positive roles in the regulation of cellular homeostasis in response to various stresses. However, its contribution to protein quality control remains unexplored. Here we show that Sirt1 is involved in protein quality control in both an Hsp70-dependent and an Hsp70-independent manner. Loss of Sirt1 led to the accumulation of ubiquitinated proteins in cells and tissues, especially upon heat stress, without affecting proteasome activities. This was partly due to decreased basal expression of Hsp70. However, this accumulation was only partially alleviated by overexpression of Hsp70 or induction of Hsp70 upon heat shock in Sirt1-deficient cells and tissues. These results suggest that Sirt1 mediates both Hsp70-dependent and Hsp70-independent protein quality control. Our findings cast new light on understanding the role of Sirt1 in maintaining cellular homeostasis.

The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance.

Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.

Sirtuin 1 is a negative regulator of parathyroid hormone stimulation of matrix metalloproteinase 13 expression in osteoblastic cells: role of sirtuin 1 in the action of PTH on osteoblasts.

Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.

Sirt1-deficient mice have hypogonadotropic hypogonadism due to defective GnRH neuronal migration.

Hypogonadatropic hypogonadism (HH) can be acquired through energy restriction or may be inherited as congenital hypogonadotropic hypogonadism and its anosmia-associated form, Kallmann's syndrome. Congenital hypogonadotropic hypogonadism is associated with mutations in a group of genes that impact fibroblast growth factor 8 (FGF8) function. The Sirt1 gene encodes a nicotinamide adenine dinucleotide-dependent histone deacetylase that links intracellular metabolic stress to gene expression. Herein Sirt1(-/-) mice are shown to have HH due to failed GnRH neuronal migration. Sirtuin-1 (Sirt1) catalytic function induces GnRH neuronal migration via binding and deacetylating cortactin. Sirt1 colocalized with cortactin in GnRH neurons in vitro. Sirt1 colocalization with cortactin was regulated in an FGF8/fibroblast growth factor receptor-1 dependent manner. The profound effect of Sirt1 on the hormonal status of Sirt1(-/-) mice, mediated via defective GnRH neuronal migration, links energy metabolism directly to the hypogonadal state. Sirt1-cortactin may serve as the distal transducer of neuronal migration mediated by the FGF8 synexpression group of genes that govern HH.

Loss of Sirt1 promotes prostatic intraepithelial neoplasia, reduces mitophagy, and delays PARK2 translocation to mitochondria.

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD(+)-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1(-/-) cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NAD(+)-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.

Modulation of tumorigenesis by dietary intervention is not mediated by SIRT1 catalytic activity.

The protein deacetylase SIRT1 is involved in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. Both SIRT1 activity and tumorigenesis can be influenced by dietary fat and polyphenolics. We set out to determine whether dietary modulations of tumorigenesis are mediated by SIRT1 catalytic functions. We introduced a mammary gland tumor-inducing transgene, MMTV-PyMT, into stocks of mice bearing a H355Y point mutation in the Sirt1 gene that abolishes SIRT1 catalytic activity. Tumor latency was reduced in animals fed a high fat diet but this effect was not dependent on SIRT1 activity. Resveratrol had little effect on tumor formation except in animals heterozygous for the mutant Sirt1 gene. We conclude that the effects of these dietary interventions on tumorigenesis are not mediated by modulation of SIRT1 catalytic activity.

SIRT1-mediated deacetylation of CRABPII regulates cellular retinoic acid signaling and modulates embryonic stem cell differentiation.

Retinoid homeostasis is critical for normal embryonic development. Both the deficiency and excess of these compounds are associated with congenital malformations. Here we demonstrate that SIRT1, the most conserved mammalian NAD⁺-dependent protein deacetylase, contributes to homeostatic retinoic acid (RA) signaling and modulates mouse embryonic stem cell (mESC) differentiation in part through deacetylation of cellular retinoic acid binding protein II (CRABPII). We show that RA-mediated acetylation of CRABPII at K102 is essential for its nuclear accumulation and subsequent activation of RA signaling. SIRT1 interacts with and deacetylates CRABPII, regulating its subcellular localization. Consequently, SIRT1 deficiency induces hyperacetylation and nuclear accumulation of CRABPII, enhancing RA signaling and accelerating mESC differentiation in response to RA. Consistently, SIRT1 deficiency is associated with elevated RA signaling and development defects in mice. Our findings reveal a molecular mechanism that regulates RA signaling and highlight the importance of SIRT1 in regulation of ESC pluripotency and embryogenesis.

SIRT1 protects against cigarette smoke-induced lung oxidative stress via a FOXO3-dependent mechanism.

Oxidative and carbonyl stress is increased in lungs of smokers and patients with chronic obstructive pulmonary disease (COPD), as well as in cigarette smoke (CS)-exposed rodent lungs. We previously showed that sirtuin1 (SIRT1), an antiaging protein, is reduced in lungs of CS-exposed mice and patients with COPD and that SIRT1 attenuates CS-induced lung inflammation and injury. It is not clear whether SIRT1 protects against CS-induced lung oxidative stress. Therefore, we determined the effect of SIRT1 on lung oxidative stress and antioxidants in response to CS exposure using loss- and gain-of-function approaches, as well as a pharmacological SIRT1 activation by SRT1720. We found that CS exposure increased protein oxidation and lipid peroxidation in lungs of wild-type (WT) mice, which was further augmented in SIRT1-deficient mice. Furthermore, both SIRT1 genetic overexpression and SRT1720 treatment significantly decreased oxidative stress induced by CS exposure. FOXO3 deletion augmented lipid peroxidation products but reduced antioxidants in response to CS exposure, which was not affected by SRT1720. Interestingly, SRT1720 treatment exhibited a similar effect on lipid peroxidation and antioxidants (i.e., manganese superoxide dismutase, heme oxygenase-1, and NADPH quinone oxidoreductase-1) in WT and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-deficient mice in response to CS exposure. This indicates that SIRT1 protects against CS-induced oxidative stress, which is mediated by FOXO3, but is independent of Nrf2. Overall, these findings reveal a novel function of SIRT1, which is to reduce CS-induced oxidative stress, and this may contribute to its protective effects against lung inflammation and subsequent development of COPD.

SIRT1 but not its increased expression is essential for lifespan extension in caloric-restricted mice.

The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1(+/-) mice was identical (51%) to that observed in wild-type mice, but SIRT1(+/-) mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.

The SIRT1 deacetylase protects mice against the symptoms of metabolic syndrome.

Type 2 diabetes, hepatic steatosis, and gut dysbiosis are pathophysiological consequences of obesity. Sirtuin (SIRT)-1 is a protein deacetylase implicated in the regulation of metabolic activity. We set out to determine whether the catalytic activity of SIRT1 plays a role in the development of metabolic syndrome, hepatic steatosis, and the distribution of gut microbiota. We challenged with a high-fat diet (HFD) a strain of mice homozygous for a Sirt1 allele carrying a point mutation that ablates the deacetylase activity of SIRT1. When compared to wild-type animals, mice lacking SIRT1 catalytic activity rapidly accumulated excessive hepatic lipid while fed the HFD, an effect evident within 2 wk of HFD feeding. Both white and brown adipose depots became hypertrophic, and the animals developed insulin resistance. The ratio of the major phyla of gut microbiota (Firmicutes and Bacteroidetes) increased rapidly in the SIRT1-deficient mice after HFD challenge. We conclude that the deacetylase activity of SIRT1 plays an important role in regulating glucose and hepatic lipid homeostasis. In addition, the composition of gut microbiota is influenced by both the animals' Sirt1 genotype and diet composition.

SIRT1 catalytic activity has little effect on tumor formation and metastases in a mouse model of breast cancer.

The protein deacetylase SIRT1 has been implicated in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. There are conflicting data that make it unclear whether Sirt1 functions as an oncogene or tumor suppressor. To assess the effect of SIRT1 on the emergence and progression of mammary tumors, we crossed mice that harbor a point mutation that abolishes SIRT1 catalytic activity with mice carrying the polyoma middle T transgene driven by the murine mammary tumor virus promoter (MMTV-PyMT). The absence of SIRT1 catalytic activity neither accelerated nor blocked the formation of tumors and metastases in this model. There was a lag in tumor latency that modestly extended survival in Sirt1 mutant mice that we attribute to a delay in mammary gland development and not to a direct effect of SIRT1 on carcinogenesis. These results are consistent with previous evidence suggesting that Sirt1 is not a tumor promoter or a tumor suppressor.

SIRT1 redresses the imbalance of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9 in the development of mouse emphysema and human COPD.

Sirtuin1 (SIRT1), a protein/histone deacetylase, protects against the development of pulmonary emphysema. However, the molecular mechanisms underlying this observation remain elusive. The imbalance of tissue inhibitor of matrix metalloproteinases (TIMPs)/matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that SIRT1 protects against emphysema by redressing the imbalance between MMPs and TIMPs. To test this hypothesis, SIRT1-deficient and overexpressing/transgenic mice were exposed to cigarette smoke (CS). The protein level and activity of MMP-9 were increased in lungs of SIRT1-deficient mice exposed to CS compared with wild-type (WT) littermates, and these effects were attenuated by SIRT1 overexpression. SIRT1 deficiency decreased the level of TIMP-1, which was augmented in SIRT1 transgenic mice compared with WT littermates by CS. However, the level of MMP-2, MMP-12, TIMP-2, TIMP-3, or TIMP-4 was not altered by SIRT1 in response to CS exposure. SIRT1 reduction was associated with imbalance of TIMP-1 and MMP-9 in lungs of smokers and COPD patients. Mass spectrometry and immunoprecipitation analyses revealed that TIMP-1 acetylation on specific lysine residues was increased, whereas its interaction with SIRT1 and MMP-9 was reduced in mouse lungs with emphysema, as well as in lungs of smokers and COPD patients. SIRT1 deficiency increased CS-induced TIMP-1 acetylation, and these effects were attenuated by SIRT1 overexpression. These results suggest that SIRT1 protects against COPD/emphysema, in part, via redressing the TIMP-1/MMP-9 imbalance involving TIMP-1 deacetylation. Thus redressing the TIMP-1/MMP-9 imbalance by pharmacological activation of SIRT1 is an attractive approach in the intervention of COPD.

SIRT1 is a Highly Networked Protein That Mediates the Adaptation to Chronic Physiological Stress.

SIRT1 is a NAD(+)-dependent protein deacetylase that has a very large number of established protein substrates and an equally impressive list of biological functions thought to be regulated by its activity. Perhaps as notable is the remarkable number of points of conflict concerning the role of SIRT1 in biological processes. For example, evidence exists suggesting that SIRT1 is a tumor suppressor, is an oncogene, or has no effect on oncogenesis. Similarly, SIRT1 is variably reported to induce, inhibit, or have no effect on autophagy. We believe that the resolution of many conflicting results is possible by considering recent reports indicating that SIRT1 is an important hub interacting with a complex network of proteins that collectively regulate a wide variety of biological processes including cancer and autophagy. A number of the interacting proteins are themselves hubs that, like SIRT1, utilize intrinsically disordered regions for their promiscuous interactions. Many studies investigating SIRT1 function have been carried out on cell lines carrying undetermined numbers of alterations to the proteins comprising the SIRT1 network or on inbred mouse strains carrying fixed mutations affecting some of these proteins. Thus, the effects of modulating SIRT1 amount and/or activity are importantly determined by the genetic background of the cell (or the inbred strain of mice), and the effects attributed to SIRT1 are synthetic with the background of mutations and epigenetic differences between cells and organisms. Work on mice carrying alterations to the Sirt1 gene suggests that the network in which SIRT1 functions plays an important role in mediating physiological adaptation to various sources of chronic stress such as calorie restriction and calorie overload. Whether the catalytic activity of SIRT1 and the nuclear concentration of the co-factor, NAD(+), are responsible for modulating this activity remains to be determined. However, the effect of modulating SIRT1 activity must be interpreted in the context of the cell or tissue under investigation. Indeed, for SIRT1, we argue that context is everything.