SIRT1 attenuates high glucose-induced insulin resistance via reducing mitochondrial dysfunction in skeletal muscle cells

Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes mellitus (T2DM). Sustained high glucose is an important extracellular environment that induces insulin resistance. Acquired insulin resistance is associated with reduced insulin-stimulated mitochondrial activity as a result of increased mitochondrial dysfunction. Silent information regulator 1 (SIRT1) is one member of the SIRT2 (Sir2)-like family of proteins involved in glucose homeostasis and insulin secretion in mammals. Although SIRT1 has a therapeutic effect on metabolic deterioration in insulin resistance, it is still not clear how SIRT1 is involved in the development of insulin resistance. Here, we demonstrate that pcDNA3.1 vector-mediated overexpression of SIRT1 attenuates insulin resistance in the high glucose-induced insulin-resistant skeleton muscle cells. These beneficial effects were associated with ameliorated mitochondrial dysfunction. Further studies have demonstrated that SIRT1 restores mitochondrial complex I activity leading to decreased oxidative stress and mitochondrial dysfunction. Furthermore, SIRT1 significantly elevated the level of another SIRT which is named SIRT3, and SIRT3 siRNA-suppressed SIRT1-induced mitochondria complex activity increments. Taken together, these results showed that SIRT1 improves insulin sensitivity via the amelioration of mitochondrial dysfunction, and this is achieved through the SIRT1–SIRT3–mitochondrial complex I pathway.     

Overexpression of a dominant-negative mutant of SIRT1 in mouse heart causes cardiomyocyte apoptosis and early-onset heart failure

SIRT1,a mammalian ortholog of yeast silent information regulator 2(Sir2),is an NAD+-dependent protein deacetylase that plays a critical role in the regulation of vascular function.The current study aims to investigate the functional significance of deacetylase activity of SIRT1 in heart.Here we show that the early postnatal hearts expressed the highest level of SIRT1deacetylase activity compared to adult and aged hearts.We generated transgenic mice with cardiac-specific expression of a dominant-negative form of the human SIRT1(SIRT1H363Y),which represses endogenous SIRT1 activity.The transgenic mice displayed dilated atrial and ventricular chambers,and died early in the postnatal period.Pathological,echocardiographic and molecular phenotype confirmed the presence of dilated cardiomyopathy.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed a greater abundance of apoptotic nuclei in the hearts of transgenic mice.Furthermore,we show that cardiomyocyte apoptosis caused by suppression of SIRT1 activity is,at least in part,due to increased p53acetylation and upregulated Bax expression.These results indicate that dominant negative form of SIRT1(SIRT1H363Y)overexpression in mouse hearts causes cardiomyocyte apoptosis and early-onset heart failure,suggesting a critical role of SIRT1 in preserving normal cardiac development during the early postnatal period.     

SIRT1 Regulates Dendritic Development in Hippocampal Neurons

Dendritic arborization is required for proper neuronal connectivity. SIRT1, a NAD+ dependent histone deacetylase, has been associated to ageing and longevity, which in neurons is linked to neuronal differentiation and neuroprotection. In the present study, the role of SIRT1 in dendritic development was evaluated in cultured hippocampal neurons which were transfected at 3 days in vitro with a construct coding for SIRT1 or for the dominant negative SIRT1H363Y, which lacks the catalytic activity. Neurons overexpressing SIRT1 showed an increased dendritic arborization, while neurons overexpressing SIRT1H363Y showed a reduction in dendritic arbor complexity. The effect of SIRT1 was mimicked by treatment with resveratrol, a well known activator of SIRT1, which has no effect in neurons overexpressing SIRT1H363Y indicating that the effect of resveratrol was specifically mediated by SIRT1. Moreover, hippocampal neurons overexpressing SIRT1 were resistant to dendritic dystrophy induced by Aβ aggregates, an effect that was dependent on the deacetylase activity of SIRT1. Our findings indicate that SIRT1 plays a role in the development and maintenance of dendritic branching in hippocampal neurons, and suggest that these effects are mediated by the ROCK signaling pathway.     

Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs as novel SIRT1 activators

SIRT1 is an NAD⁺-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.     

SIRT2 negatively regulates insulin resistance in C2C12 skeletal muscle cells

SIRT2 is primarily a cytoplasmic protein deacetylase and is abundantly expressed in metabolically active tissues like adipocytes and brain. However, its role, if any, in regulating insulin signaling in skeletal muscle cells, is not known. We have examined the role of SIRT2 in insulin-mediated glucose disposal in normal and insulin resistant C2C12 skeletal muscle cells in vitro. SIRT2 was over expressed in insulin resistant skeletal muscle cells. Pharmacological inhibition of SIRT2 increased insulin-stimulated glucose uptake and improved phosphorylation of Akt and GSK3β in insulin resistant cells. Knockdown of endogenous SIRT2 and over expression of catalytically-inactive SIRT2 mutant under insulin-resistant condition showed similar amelioration of insulin sensitivity. Our results suggest that down-regulation of SIRT2 improved insulin sensitivity in skeletal muscle cells under insulin-resistant condition. Previously it has been reported that down-regulation of SIRT1 and SIRT3 in C2C12 cells results in impairment of insulin signaling and induces insulin resistance. However, we have observed an altogether different role of SIRT2 in skeletal muscle. This implicates a differential regulation of insulin resistance by sirtuins which otherwise share a conserved catalytic domain. The study significantly directs towards future approaches in targeting inhibition of SIRT2 for therapeutic treatment of insulin resistance which is the major risk factor in Type 2 diabetes.     

Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha

Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. We tested whether resveratrol (RSV), which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis. Treatment of mice with RSV significantly increased their aerobic capacity, as evidenced by their increased running time and consumption of oxygen in muscle fibers. RSV's effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis and were largely explained by an RSV-mediated decrease in PGC-1alpha acetylation and an increase in PGC-1alpha activity. This mechanism is consistent with RSV being a known activator of the protein deacetylase, SIRT1, and by the lack of effect of RSV in SIRT1(-/-) MEFs. Importantly, RSV treatment protected mice against diet-induced-obesity and insulin resistance. These pharmacological effects of RSV combined with the association of three Sirt1 SNPs and energy homeostasis in Finnish subjects implicates SIRT1 as a key regulator of energy and metabolic homeostasis.     

Sirtuin 1 (SIRT1) deacetylase activity is not required for mitochondrial biogenesis or peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation following endurance exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Paradoxical increase in TAG and DAG content parallel the insulin sensitizing effect of unilateral DGAT1 overexpression in rat skeletal muscle

Background

The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity.

Methodology/Principal Findings

The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle.

Conclusions/Significance

We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling.     

Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK

Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation.The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities.Primary rat podocytes cultured with standard or high glucose concentrations for 5 days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning.Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.     

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.     

Metabolic syndrome exacerbates amyloid pathology in a comorbid Alzheimer's mouse model

Alzheimer's disease (AD) often coexists with other aging-associated diseases including obesity, diabetes, hypertension, and cardiovascular diseases. The early stage of these comorbidities is known as metabolic syndrome (MetS) which is highly prevalent in mid-life. An important cause of MetS is the deficiency of SIRT3, a mitochondrial deacetylase which enhances the functions of critical mitochondrial proteins, including metabolic enzymes, by deacetylation. Deletion of Sirt3 gene has been reported to result in the acceleration of MetS. In a recently published study, we demonstrated in the brain of Sirt3^−/− mice, downregulation of metabolic enzymes, insulin resistance and elevation of inflammatory markers including microglial proliferation. These findings suggested a novel pathway that could link SIRT3 deficiency to neuroinflammation, an important cause of Alzheimer's pathogenesis. Therefore, we hypothesized that MetS and amyloid pathology may interact through converging pathways of insulin resistance and neuroinflammation in comorbid AD. To investigate these interactions, we crossed Sirt3^−/− mice with APP/PS1 mice and successfully generated APP/PS1/Sirt3^−/− mice with amyloid pathology and MetS. In these comorbid AD mice, we observed exacerbation of insulin resistance, glucose intolerance, amyloid plaque deposition, markers of neuroinflammation, including elevated expression of IL-1β, TNF-α and Cox-2 at 8 months of age. There was also increased microglial proliferation and activation. Our observations suggest a novel mechanism by which MetS may interact with amyloid pathology during the cellular phase of AD. Therapeutic targeting of SIRT3 in AD with comorbidities may produce beneficial effects.     

SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated beta-galactosidase (SA-beta-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan. Western blot results showed that SIRT1 reduced the expression of p16(INK4A) and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16(INK4A) and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.     

SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.     

Lack of skeletal muscle liver kinase B1 alters gene expression,mitochondrial content,inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice.KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling.KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.