Sirt1 Knockout Mice Research Articles

Correction: Corrigendum: SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD+-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

The Full Capacity of AICAR to Reduce Obesity-Induced Inflammation and Insulin Resistance Requires Myeloid SIRT1

Chronic Inflammation is a key link between obesity and insulin resistance. We previously showed that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. AMPK is also a molecular target of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which has been shown to reduce insulin resistance in various animal models. Here we aim to determine whether the therapeutic effects of AICAR against insulin resistance involve its anti-inflammatory function, which requires macrophage SIRT1. Long-term administration of low-dose AICAR significantly suppressed adipose inflammation in established diet-induced obese mice. This was associated with improved glucose homeostasis and insulin sensitivity without changes of body weight. In contrast, SIRT1 deletion in myeloid SIRT1 knockout (MSKO) mice increased infiltration of classically activated M1 macrophages and decreased alternatively activated M2 macrophages in adipose tissue. As a result, MSKO mice on high fat (HF) diets exhibited impaired insulin signaling in skeletal muscle, fat, and liver, and developed systemic insulin resistance in glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamp experiments. Interestingly, the beneficial effects of AICAR on adipose inflammation and insulin sensitivity were absent in MSKO mice fed HF diets, suggesting that the full capacity of AICAR to antagonize obesity-induced inflammation and insulin resistance requires myeloid SIRT1. In summary, AICAR negatively regulates HF diet-induced inflammation, which requires myeloid SIRT1, thereby contributing to the protection against insulin resistance. Myeloid SIRT1 is a therapeutic target of the anti-inflammatory and insulin-sensitizing effects of AICAR.

Abstract 19390: Sirtuin 1 Modulates Cardiac Sodium Channels in vivo

The rapid upstroke in phase 0 of the cardiac action potential is the result of the inward sodium current (I Na ) carried by the cardiac voltage-gated sodium channel Na v 1.5 (SCN5A). We recently reported that the overexpression of Silent Information Regulator 1 (Sirtuin 1, SIRT1), an NAD + dependent protein deacetylase, deacetylated SCN5A, increased its cell surface expression, and increased I Na in HEK-293 cells and rat neonatal cardiac myocytes. Overexpression of a dominant negative SIRT1 construct decreased SCN5A cell surface expression and I Na . The role of acetylation of SCN5A and its regulation by SIRT1 has not been shown in vivo, however. We engineered cardiac specific Sirt1 knockout (cSirt1KO) mice by breeding Sirt1 flox/flox with α-MHC-cre mice on a C57BL-6 background. cSirt1KO compared to littermate Sirt1 flox/flox mice showed much less expression of Sirt1 mRNA (12±6%, n=3) and protein in the heart but not in the kidney, and Scn5a immunoprecipitated from cardiac membrane fractions of the cSirt1KO mice showed an ∼2-fold increase in acetylation (Figure A). At 3 months of age, EKGs from anesthetized cSirt1KO compared to littermate Sirt1 flox/flox mice showed PR prolongation (43±3 ms, n=4, vs. 37±2 ms, n=5; p<0.02) and telemetry from ambulatory cSirt1KO mice showed high degree heart block not seen in the controls (Figure B). Thus, our results show that Scn5a is acetylated in vivo in the mouse, that the degree of acetylation is regulated by cardiac-specific expression of Sirt1, and that decreases in Sirt1 lead to conduction disease similar to those seen in Scn5a +/- mice. These findings raise the possibility that pharmacological manipulation of Sirt1 may provide a means to modulate cardiac I Na and arrhythmias.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Abstract 2315SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures:No relevant conflicts of interest to declare.

Sirtuins as regulators of mammalian aging.

Since the discovery that overexpression of yeast Sir2 deacetylase extends lifespan by as much as 30% over a decade ago [1], much effort has been invested in researching whether this effect is conserved in higher organisms as well. Indeed, in wormsand flies, two separate groups found that SIR2 extended lifespan as well, by 50% and 18%, respectively [1]. In parallel to the work in worms and flies, researchers were trying to make headway in the role of sirtuins in higher organisms. There are seven mammalian homologs to the yeast Sir2, SIRT1-SIRT7. SIRT1 is the most well-researched and has been shown to regulate metabolism and age-related diseases. However, SIRT1 overexpression did not increase lifespan [2], although this was said to be due to the relatively weak expression of the transgene. Therefore, a role for sirtuins in regulating lifespan of mammals looked bleak. Despite the controversy surrounding sirtuins and longevity, there has never been any doubt that mammalian sirtuins are important regulators of health and disease [3]. Previous results from our lab have shown SIRT6 to be involved in the calorie restriction response [4], and demonstrate that SIRT6 overexpression in mice protects against diet-induced obesity and its metabolic consequences [4]. These results, along with data that SIRT6 knockout mice display a premature aging-like phenotype[5], prompted us to turn towards SIRT6 as a potential regulator of mammalian aging. Over the course of three years, we measured the lifespan of mice overexpressing exogenous SIRT6 (MOSES). This study was performed in two separate lines from distinct founders, to ensure that the random integration of the transgene into the genome did not influence the results. We found that the gene insertion in both lines did not disrupt any neighboring genes, and results were similar in both lines. In this way we overcame the issue of site-specific integration, which was previously shown to be a problem in sirtuin studies [6]. Additionally, we chose to work with a mixed background, to ensure no strain-specific effects. Strikingly, both male MOSES lines had significant mean and median lifespan extension, of 14.5% and 9.9% [7]. Even more interesting, there was no lifespan extension in either female lines examined, attesting to a gender-specific role for SIRT6. Additionally, male MOSES displayed improved glucose tolerance in comparison to their wild type littermates. We therefore went on to perform whole-gene microarray and pathway analysis, to elucidate which genes and pathways were altered. Significantly, the IGF-1 signaling pathway was decreased in males only. A reduction in IGF-1 is an integral factor in increasing lifespan in worms, flies, and mice [8]. We then tested the major metabolic tissues to see whether SIRT6 decreased IGF-1 signaling. Surprisingly, we found significantly reduced IGF-1 signaling in the white adipose tissue (WAT) of male mice. These tissue-specific effects were not seen in the muscle or liver, nor were they seen in female mice. Altogether, we found SIRT6 has an integral role in IGF-1 signaling in the fat tissues of male mice, which could potentially explain the longevity seen in male mice overexpressing SIRT6. For the first time, we have shown evidence for a sirtuin to extend mammalian lifespan. Notably, these results were male-specific, and caused a partial “feminized” effect on the IGF-1 signaling in males. Indeed, very few genes were differentially regulated in females, while over 80 were significantly changed in males. Interestingly, calorie restriction and starvation have previously been shown to have an overall feminized effect, and 30% of differentially expressed genes in these treatments also changed in male SIRT6 transgenic mice [9]. While the exact cause of this feminization shift is unknown, these results may well lead us to the deeper mystery of why females live longer than males in this background. Additionally, of interest is the fact that alterations in the IGF-1 pathway were found in the WAT tissue. This tissue has garnered attention in recent years, as mice with insulin receptor knockout in adipose tissue displayed increased lifespan [10]. We are currently in the process of researching the exact mechanism of this tissue-specific regulation by creating tissue-specific SIRT6 overexpression. These exciting results in the sirtuin field have great potential to lead to new medical discoveries, to combat the increasing occurrence of the metabolic syndrome and other age-related diseases. In other words, with SIRT6 around to counteract the effects of high-fat diet and aging, we can have our cake, and eat it too.

Sirt7 promotes adipogenesis by binding to and inhibiting Sirt1

Background Members of the mammalian sirtuin family, Sirt1 – Sirt7, are known to regulate metabolic processes especially carbohydrate and fat metabolism [1,2]. Sirt1 and Sirt2 inhibit adipocyte differentiation [3,4] while Sirt1 and Sirt6 prevent liver steatosis [5]. These examples illustrate a synergistic action of different sirtuins in promoting lean, “healthy” phenotypes. We have previously shown that Sirt7 knockout mice display signs of premature aging, suffer from progressive cardiomyopathy and have a reduced lifespan [6]. Here, we investigate the biological function of Sirt7 in the regulation of metabolism in white adipose tissue (WAT) and liver.

Muscle or liver-specific Sirt3 deficiency induces hyperacetylation of mitochondrial proteins without affecting global metabolic homeostasis

Sirt3 is a mitochondrial sirtuin, predominantly expressed in highly metabolic tissues. Germline ablation of Sirt3 has major metabolic consequences, including increased susceptibility to metabolic damage and oxidative stress after high fat feeding. In order to determine the contribution of liver and skeletal muscle to these phenotypes, we generated muscle-specific Sirt3 (Sirt3skm−/−) and liver-specific Sirt3 (Sirt3hep−/−) knock-out mice. Despite a marked global hyperacetylation of mitochondrial proteins, Sirt3skm−/− and Sirt3hep−/− mice did not manifest any overt metabolic phenotype under either chow or high fat diet conditions. Similarly, there was no evidence for increased oxidative stress in muscle or liver when Sirt3 was ablated in a tissue-specific manner. These observations suggest that the mitochondrial hyperacetylation induced by Sirt3-deletion in a tissue specific manner is not necessarily linked to mitochondrial dysfunction and does not recapitulate the metabolic abnormalities observed in the germline Sirt3 knock-out mice.

Fortifying the Link between SIRT1, Resveratrol, and Mitochondrial Function

The molecular mechanisms behind the health benefits of resveratrol remain enigmatic and controversial. In this issue of Cell Metabolism, Price et al. establish a clear chemical-genetic connection between SIRT1 and resveratrol, providing strong evidence that SIRT1 is critical for resveratrol to stimulate mitochondrial biogenesis and a switch toward oxidative muscle fibers (Price et al., 2012).

SIRT1 acutely modulates glucose sensing of POMC neurons in the hypothalamus

POMC neurons in the hypothalamus are glucose‐excited neurons to sense glucose via AMPK and KATP channel. NAD+‐dependent SIRT1 deacetylase in POMC neurons is essential for maintaining energy balance. However, it is unknown if SIRT1 modulates glucose sensing by POMC neurons. Our aims were to determine if SIRT1 in POMC neurons is essential for homeostatic control of blood glucose in addition to body weight; and if SIRT1 directly modulates glucose sensing by POMC neurons. [1] Using the Cre‐LoxP system, we generated POMC‐specific SIRT1 knockout (KO) mice. Using hyperinsulinemic euglycemic clamp, we demonstrated that the KO mice had increased hepatic glucose production and decreased peripheral glucose removal during the clamp. [2] Using the whole‐cell patch clamp, we showed that firing rate of POMC neurons was significantly lower (50%) in the KO brain slice at 5 mM glucose than that in the WT brain slice, indicating that POMC neurons with SIRT1 deletion require a higher level of glucose to be excited. Firing rate of POMC neurons in the WT slice decreased by 57% when glucose decreased from 5 mM to 3 mM; however, it was not altered in the KO slice. Moreover, 80% of POMC neurons in the WT slice responded to changes in glucose concentration, while only about 10% of POMC neurons in the KO slice did. These data suggest that POMC neurons with SIRT1 deletion are not sensitive to glucose levels. [3] 60% of POMC neurons in the WT slice were sensitive to genipin (UCP2 blocker, 20 μM) by increasing (+120%) firing rate at 3 mM glucose. However, POMC neurons with SIRT1 deletion were not responsive to genipin anymore. We conclude that SIRT1 in POMC neurons is essential for glucose homeostasis; and SIRT1 deletion in POMC neurons impairs glucose sensing in a UCP2‐dependent manner. We propose that SIRT1‐mediated glucose sensing in POMC neurons may play an important role in the maintenance of glucose homeostasis.

Sirt1 protects against thrombomodulin down-regulation and lung coagulation after particulate matter exposure

AbstractExposure to ambient particulate matter (PM) air pollution has been reported to trigger inflammation and thrombosis. However, molecular mechanisms underlying the modulation of coagulation pathways in PM-induced thrombosis remain largely unknown. We report here that Sirt1, a member of class III histone deacetylase, controls lung inflammation and coagulation after PM exposure. Sirt1 knock-out mice exhibited aggravated lung vascular leakage and inflammation after PM exposure, which was correlated with increased NF-κB acetylation and activation. Furthermore, Sirt1 knock-out mice were highly susceptible to PM-induced lung coagulation as demonstrated by increased fibrin formation. The increased fibrin formation was associated with reduced tissue factor pathway inhibitor (TFPI) expression and increased plasminogen activator inhibitor-1 (PAI-1) activity in the lungs, thus favoring elevated coagulation and disrupted fibrinolysis responses. Thrombomodulin (TM), a central player of the anticoagulant protein C system, is regulated by Kruppel-like factor 2 (KLF2) at the transcriptional level. Our data show that PM exposure led to decreased lung KLF2 and TM expression in wild-type mice, and lung KLF2 and TM protein levels were further decreased in Sirt1 knock-out mice. Importantly, Sirt1 gene delivery inhibited TM and KLF2 down-regulation and reduced lung coagulation after PM exposure. Collectively, our studies indicate that Sirt1 functions as a suppressor of coagulation after particulate matter exposure.

Mitochondrial Acetylome Analysis in a Mouse Model of Alcohol-Induced Liver Injury Utilizing SIRT3 Knockout Mice

Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD.

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-KithighSca-1+Lineage− (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Abstract 18251: Sirt1 Mediates Pressure Overload-Induced Cardiac Hypertrophy and Failure in vivo

Mitochondrial dysfunction is a hallmark of the failing heart. Sirt1, a class III histone deacetylase, plays an important role in mediating transcriptional regulation of metabolic genes in response to stress. The function of endogenous Sirt1 during heart failure is poorly understood. Here we investigated the role of Sirt1 in pressure overload-induced cardiac hypertrophy and failure using genetically altered mouse models, namely cardiac specific Sirt1 heterozygous (Sirt1 +/- ) and homozygous (Sirt1 -/- ) knockout (KO) (C57BL/6) and Sirt1 overexpression (Tg-Sirt1) mice (FVB). These mice were subjected to pressure overload with transverse aortic constriction (TAC). Notably, C57BL/6 mice are more susceptible to TAC-induced cardiac hypertrophy and failure than FVB mice. Cardiac hypertrophy induced by 4 weeks of TAC was exacerbated in Tg-Sirt1 (heart weight/body weight (HW/BW, mg/g), Wild type (WT) 6.0, Tg-Sirt1 8.4*, p<0.05 vs WT), whereas it was ameliorated in Sirt1 KO mice (HW/BW, (mg/g), WT 7.9, Sirt1 +/- 6.9*, Sirt1 -/- 6.0*, * p<0.05 vs WT). TAC-induced systolic dysfunction was also exacerbated in Tg-Sirt1 (ejection fraction (EF) (%), WT 71, Tg-Sirt1 48*, p<0.05 vs WT), whereas it was ameliorated in Sirt1 KO mice (EF (%), WT 55, Sirt1 +/- 65*, Sirt1 -/- 63*, * p<0.05 vs WT). The expression of nuclear encoded mitochondrial genes, such as Cpt2, Sdha, Fh1 and Ndufv1, was downregulated by TAC in WT mice, an effect that was normalized in Sirt1 -/- mice (relative mRNA levels vs WT sham, Sdha: WT TAC 0.56, Sirt1 -/- sham 1.2, Sirt1 -/- TAC 0.81*, Ndufv1: WT TAC 0.59, Sirt1 -/- sham 0.84, Sirt1 -/- TAC 1.1*, * p<0.05 vs WT TAC). On the other hand, ATP content was decreased in Tg-Sirt1 mice after TAC (45% reduction vs WT). Chromatin immunoprecipitation assays revealed that pressure overload induces recruitment of Sirt1 to the promoter region of these mitochondrial genes (relative promoter occupancy after 4 weeks of TAC, Cpt2 2.6*, Sdha 7.4*, Fh1 5.4*, Ndufv1 5.6*, * p<0.05 vs sham), indicating that endogenous Sirt1 interacts with mitochondrial genes, thereby negatively regulating their expression during TAC. These results suggest that Sirt1 mediates pressure overload-induced cardiac hypertrophy and failure, possibly through attenuation of mitochondrial function.

Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production

Sirt3 is a member of the sirtuin family of protein deacetylases that is localized in mitochondria and regulates mitochondrial function. Sirt3 expression in skeletal muscle is decreased in models of type 1 and type 2 diabetes and regulated by feeding, fasting, and caloric restriction. Sirt3 knockout mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling. This effect is mimicked by knockdown of Sirt3 in cultured myoblasts, which exhibit reduced mitochondrial oxidation, increased reactive oxygen species, activation of JNK, increased serine and decreased tyrosine phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 plays an important role in diabetes through regulation of mitochondrial oxidation, reactive oxygen species production, and insulin resistance in skeletal muscle.

SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD+-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

Overexpression of SIRT1 in mouse forebrain impairs lipid/glucose metabolism and motor function.

SIRT1 plays crucial roles in glucose and lipid metabolism, and has various functions in different tissues including brain. The brain-specific SIRT1 knockout mice display defects in somatotropic signaling, memory and synaptic plasticity. And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter. These mice, especially females, exhibited increased fat accumulation accompanied by significant upregulation of adipogenic genes in white adipose tissue. Glucose tolerance of the mice was also impaired with decreased Glut4 mRNA levels in muscle. Moreover, the SIRT1 overexpressing mice showed decreased energy expenditure, and concomitantly mitochondria-related genes were decreased in muscle. In addition, these mice showed unusual spontaneous physical activity pattern, decreased activity in open field and rotarod performance. Further studies demonstrated that SIRT1 deacetylated IRS-2, and upregulated phosphorylation level of IRS-2 and ERK1/2 in striatum. Meanwhile, the neurotransmitter signaling in striatum and the expression of endocrine hormones in hypothalamus and serum T3, T4 levels were altered. Taken together, our findings demonstrate that SIRT1 in forebrain regulates lipid/glucose metabolism and motor function.

Reduced mitochondrial function in obesity-associated fatty liver: SIRT3 takes on the fat

Aging is associated with various metabolic disorders that may have their origin in the liver, including non-alcoholic fatty liver disease, obesity, type 2 diabetes mellitus, and atherosclerosis. Although well-characterized in models of caloric restriction, relatively little is known about the role of sirtuins and acetylation under conditions of caloric excess. Sirtuins are NAD (+)-dependent protein deacetylases that mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized within the mitochondrial matrix, where it regulates acetylation levels of a diverse set of metabolic enzymes. When normal mice are fed a high fat diet they demonstrate reduced SIRT3 activity, impaired mitochondrial function, and hyperacetylation of a diverse set of proteins in their livers. Furthermore, SIRT3 knockout mice have signs of accelerated aging and cancer. Understanding SIRT3?s biochemical function and regulation in the liver under conditions of caloric excess may potentially increase our understanding of the normal aging process and diseases associated with aging, such as diabetes, fatty liver disease, or cancer.

Regulation of the mitochondrial transition pore: impact on mammalian aging

During the last few years, data from different biological systems have revealed evidence for an age-related increase in the abundance of CypD, a peptidyl prolylcis, trans-isomerase located in the mitochondrial matrix. Such an increase was reported in the short-lived aging model organism Podospora anserina [1,2] but also in rat gastrocnemius muscle and in brain tissue of mice and humans [2-3]. The enzyme is known to be involved in the regulation of the mitochondrial transition pore (mPTP) [4], a macromolecular mitochondrial protein complex the structure of which is still unsolved and controversial. The activity of CypD can be repressed by the cyclosporine A, a cyclic peptide used in human transplantation medicine or in treatment of Ullrich congenital dystrophy. In a recent aging study, a strong effect of CypD overexpression on mitochondrial ultrastucture structure and on aging was reported in P. anserina [5]. Overexpression strains were characterized by mitochondrial dysfunction and accelerated aging as the result of the induction of apoptosis. Treatment of CypD overexpressors was found to be effective in a reversion of the accelerated aging phenotype and it was suggested that cyclosporine A or other molecules affecting the function of CypD may be promising compounds for the development of anti-aging interventions by interfering into mitochondrial apoptotis pathways. In P. anserina apoptosis is the final pathway leading to death of this simple microbial aging model [6-9]. In contrast, in mammalian systems, it is rather represents a pathway relevant for development and protection against cancer. However, more and more data accumulate which also point to an important role of apoptosis in the final life stage of mammals. For exemple, transgenic mice expressing a mutated gene of the mitochondrial DNA polymerase gamma, which, due to loss of the proof reading activity of this enzyme, express an accelerated aging phenotype with sarcopenia and an induction of apoptosis markers [10,11]. In the last 2010 issue of AGING [12] Hafner et al. report the regulation of the mPTP of mice via deacetylation of CypD at lysine 166 by the NAD+-dependent SIRT3 deacetylase. Mice lacking SIRT3 display an accelerated aging phenotype correlated with increased mitochondrial swelling as the result of mPTP opening. Most remarkably, this phenotype is related to heart impairments and can be reverted by treatment with cyclosporine A. Sirt3 knockout mice are hypersensitive to heart stress, display cardiac hypertrophy and fibrosis. From their work the authors suggest that the phenotype of the mice is the result of an initial reduction of SIRT3 activity leading to increases in susceptibility of mPTP formation. Subsequently this leads to impairments in NAD+ generation by mitochondria and, via a positive feedback cycle, to accelerated mitochondrial depolarization and destruction. In mammals, CypD thus appears to of relevance not only for skeletal muscle function but also in the heart connecting this pathway to one of the most important age-related death in humans, cardiac failure. Overall the data are an important piece of work towards the elucidation of the mechanistic details of mPTP regulation. Moreover, the work provides additional and very promising perspectives for the development of medical strategies to intervene into the part of the molecular network governing aging via mitochondrial pathways involved in the control of apoptosis as they have been put forward earlier [7,13].

Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy

Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues.

Calorie Restriction Reduces Oxidative Stress by SIRT3-Mediated SOD2 Activation

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.