SIRT6 Deficiency Results in Severe Hypoglycemia by Enhancing Both Basal and Insulin-stimulated Glucose Uptake in Mice

Glucose homeostasis in mammals is mainly regulated by insulin signaling. It was previously shown that SIRT6 mutant mice die before 4 weeks of age, displaying profound abnormalities, including low insulin, hypoglycemia, and premature aging. To investigate mechanisms underlying the pleiotropic phenotypes associated with SIRT6 deficiency, we generated mice carrying targeted disruption of SIRT6. We found that 60% of SIRT6^−/− animals had very low levels of blood glucose and died shortly after weaning. The remaining animals, which have relatively higher concentrations of glucose, survived the early post-weaning lethality, but most died within one year of age. Significantly, feeding the mice with glucose-containing water increased blood glucose and rescued 83% of mutant mice, suggesting that the hypoglycemia is a major cause for the lethality. We showed that SIRT6 deficiency results in more abundant membrane association of glucose transporters 1 and 4, which enhances glucose uptake. We further demonstrated that SIRT6 negatively regulates AKT phosphorylation at Ser-473 and Thr-308 through inhibition of multiple upstream molecules, including insulin receptor, IRS1, and IRS2. The absence of SIRT6, consequently, enhances insulin signaling and activation of AKT, leading to hypoglycemia. These data uncover an essential role of SIRT6 in modulating glucose metabolism through mediating insulin sensitivity.     

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.     

SIRT6 protects against pathological damage caused by diet‐induced obesity

The NAD+‐dependent SIRT6 deacetylase is a therapeutic candidate against the emerging metabolic syndrome epidemic. SIRT6, whose deficiency in mice results in premature aging phenotypes and metabolic defects, was implicated in a calorie restriction response that showed an opposite set of phenotypes from the metabolic syndrome. To explore the role of SIRT6 in metabolic stress, wild type and transgenic (TG) mice overexpressing SIRT6 were fed a high fat diet. In comparison to their wild‐type littermates, SIRT6 TG mice accumulated significantly less visceral fat, LDL‐cholesterol, and triglycerides. TG mice displayed enhanced glucose tolerance along with increased glucose‐stimulated insulin secretion. Gene expression analysis of adipose tissue revealed that the positive effect of SIRT6 overexpression is associated with down regulation of a selective set of peroxisome proliferator‐activated receptor‐responsive genes, and genes associated with lipid storage, such as angiopoietin‐like protein 4, adipocyte fatty acid‐binding protein, and diacylglycerol acyltransferase 1, which were suggested as potential targets for drugs to control metabolic syndrome. These results demonstrate a protective role for SIRT6 against the metabolic consequences of diet‐induced obesity and suggest a potentially beneficial effect of SIRT6 activation on age‐related metabolic diseases.     

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5′- and 3′-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.     

Muscle-Specific SIRT1 Gain-of-Function Increases Slow-Twitch Fibers and Ameliorates Pathophysiology in a Mouse Model of Duchenne Muscular Dystrophy

SIRT1 is a metabolic sensor and regulator in various mammalian tissues and functions to counteract metabolic and age-related diseases. Here we generated and analyzed mice that express SIRT1 at high levels specifically in skeletal muscle. We show that SIRT1 transgenic muscle exhibits a fiber shift from fast-to-slow twitch, increased levels of PGC-1α, markers of oxidative metabolism and mitochondrial biogenesis, and decreased expression of the atrophy gene program. To examine whether increased activity of SIRT1 protects from muscular dystrophy, a muscle degenerative disease, we crossed SIRT1 muscle transgenic mice to mdx mice, a genetic model of Duchenne muscular dystrophy. SIRT1 overexpression in muscle reverses the phenotype of mdx mice, as determined by histology, creatine kinase release into the blood, and endurance in treadmill exercise. In addition, SIRT1 overexpression also results in increased levels of utrophin, a functional analogue of dystrophin, as well as increased expression of PGC-1α targets and neuromuscular junction genes. Based on these findings, we suggest that pharmacological interventions that activate SIRT1 in skeletal muscle might offer a new approach for treating muscle diseases.     

SIRT2 transgenic over-expression does not impact lifespan in mice

The NAD⁺-dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late-life health and overall lifespan, though of these members, a role for sirtuin-2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under-expressed. Here, we sought to test whether SIRT2 over-expression would impact the overall health and lifespan of mice on a nonprogeroid, wild-type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high-fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2-Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.     

ATP6v0d2 deficiency increases bone mass, but does not influence ovariectomy-induced bone loss

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1.     

SIRT1 reduction causes renal and retinal injury in diabetes through endothelin 1 and transforming growth factor β1

In diabetes, hyperglycaemia causes up‐regulation of endothelin 1 (ET‐1) and transforming growth factor beta 1 (TGF‐β1). Previously we showed glucose reduces sirtuin1 (SIRT1), a class III histone deacetylase. Here, we investigated the regulatory role of SIRT1 on ET‐1 and TGF‐β1 expression. Human microvascular endothelial cells were examined following incubation with 25 mmol/l glucose (HG) and 5 mmol/l glucose (NG) with or without SIRT1 or histone acetylase p300 overexpression or knockdown. mRNA expressions of ET‐1, TGF‐β1, SIRT1, p300 and collagen 1α(I) were examined. SIRT1 enzyme activity, ET‐1 and TGF‐β1 protein levels were measured. Histone acetylation and endothelial permeability were further investigated. Similar analyses were performed in the kidneys and retinas of SIRT1 overexpressing transgenic mice with or without streptozotocin induced diabetes. Renal functions were evaluated. In the endothelial cells (ECs), HG caused increased permeability and escalated production of ET‐1, TGF‐β1, collagen Iα(I). These cells also showed increased p300 expression, histone acetylation and reduced SIRT1 levels. These changes were rectified in the ECs following p300 silencing or by SIRT1 overexpression, whereas SIRT1 knockdown or p300 overexpression in NG mimicked the effects of HG. High ET‐1 and TGF‐β1 levels were seen in the kidneys and retinas of diabetic mice along with micro‐albuminuria and increased fibronectin protein (marker of glucose‐induced cell injury) levels. Interestingly, these detrimental changes were blunted in SIRT1 overexpressing transgenic mice with diabetes. This study showed a novel SIRT1 mediated protection against renal and retinal injury in diabetes, regulated through p300, ET‐1 and TGF‐β1.     

Posttranslational Processing of Brain Actin

Abstract: Two short-lived isoforms of actin, named δ- and ε-actin, have been detected in brain extracts from rats labeled in vivo with [³⁵S]methionine. These two molecular species have PI values slightly more basic than β- and γ-actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ- and ε-actin, synthesized in vivo , can be converted in vitro into β- and γ-actin. This posttranslational processing of δ- and ε-actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH₂-terminal end, is involved in the transformation of these proteins into β- and γ-actin.     

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.     

The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by α-synuclein or amyloid-β (1-42) peptide

Human sirtuins are a family of seven conserved proteins (SIRT1-7). The most investigated is the silent mating type information regulation-2 homolog (SIRT1, NM_012238 ), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer's disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down-regulated by siRNA approach. RES was also able to protect SK-N-BE from the toxicity arising from two aggregation-prone proteins, the AD-involved amyloid-β (1-42) peptide (Aβ42) and the familiar Parkinson's disease linked α-synuclein(A30P) [α-syn(A30P)]. Alpha-syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Aβ42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Aβ42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or α-syn(A30P) aggregation, while RES displayed a SIRT1-independent protective action against Aβ42.     

Deleted in Breast Cancer 1 (DBC1) Protein Regulates Hepatic Gluconeogenesis

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.     

Regulation of SIRT1 protein levels by nutrient availability

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.     

SIRT4 Represses Peroxisome Proliferator-Activated Receptor α Activity To Suppress Hepatic Fat Oxidation

Sirtuins are a family of protein deacetylases, deacylases, and ADP-ribosyltransferases that regulate life span, control the onset of numerous age-associated diseases, and mediate metabolic homeostasis. We have uncovered a novel role for the mitochondrial sirtuin SIRT4 in the regulation of hepatic lipid metabolism during changes in nutrient availability. We show that SIRT4 levels decrease in the liver during fasting and that SIRT4 null mice display increased expression of hepatic peroxisome proliferator-activated receptor α (PPARα) target genes associated with fatty acid catabolism. Accordingly, primary hepatocytes from SIRT4 knockout (KO) mice exhibit higher rates of fatty acid oxidation than wild-type hepatocytes, and SIRT4 overexpression decreases fatty acid oxidation rates. The enhanced fatty acid oxidation observed in SIRT4 KO hepatocytes requires functional SIRT1, demonstrating a clear cross talk between mitochondrial and nuclear sirtuins. Thus, SIRT4 is a new component of mitochondrial signaling in the liver and functions as an important regulator of lipid metabolism.     

Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice

The Sir2 (silent i nformation r egulator 2) family of NAD-dependent deacetylases regulates aging and longevity across a wide variety of organisms, including yeast, worms, and flies. In mammals, the Sir2 ortholog Sirt1 promotes fat mobilization, fatty acid oxidation, glucose production, and insulin secretion in response to nutrient availability. We previously reported that an increased dosage of Sirt1 in pancreatic β cells enhances glucose-stimulated insulin secretion (GSIS) and improves glucose tolerance in be ta cell-specific S ir t 1- o verexpressing (BESTO) transgenic mice at 3 and 8 months of age. Here, we report that as this same cohort of BESTO mice reaches 18–24 months of age, the GSIS regulated by Sirt1 through repression of Ucp2 is blunted. Increased body weight and hyperlipidemia alone, which are observed in aged males and also induced by a Western-style high-fat diet, are not enough to abolish the positive effects of Sirt1 on β cell function. Interestingly, plasma levels of nicotinamide mononucleotide (NMN), an important metabolite for the maintenance of normal NAD biosynthesis and GSIS in β cells, are significantly reduced in aged BESTO mice. Furthermore, NMN administration restores enhanced GSIS and improved glucose tolerance in the aged BESTO females, suggesting that Sirt1 activity decreases with advanced age due to a decline in systemic NAD biosynthesis. These findings provide insight into the age-dependent regulation of Sirt1 activity and suggest that enhancement of systemic NAD biosynthesis and Sirt1 activity in tissues such as β cells may be an effective therapeutic intervention for age-associated metabolic disorders such as type 2 diabetes.