Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.     

Biochemical characterization,localization, and tissue distribution of the longer form of mouse SIRT3

SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C‐terminus of human SIRT3 but lacks an N‐terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5′ rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full‐length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD⁺. Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.     

trans-(-)-ε-Viniferin increases mitochondrial sirtuin 3 (SIRT3), activates AMP-activated protein kinase (AMPK), and protects cells in models of Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein Huntingtin (Htt). Currently, no cure is available for HD. The mechanisms by which mutant Htt causes neuronal dysfunction and degeneration remain to be fully elucidated. Nevertheless, mitochondrial dysfunction has been suggested as a key event mediating mutant Htt-induced neurotoxicity because neurons are energy-demanding and particularly susceptible to energy deficits and oxidative stress. SIRT3, a member of sirtuin family, is localized to mitochondria and has been implicated in energy metabolism. Notably, we found that cells expressing mutant Htt displayed reduced SIRT3 levels. trans-(-)-ε-Viniferin (viniferin), a natural product among our 22 collected naturally occurring and semisynthetic stilbenic compounds, significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. We demonstrate that viniferin decreases levels of reactive oxygen species and prevents loss of mitochondrial membrane potential in cells expressing mutant Htt. Expression of mutant Htt results in decreased deacetylase activity of SIRT3 and further leads to reduction in cellular NAD(+) levels and mitochondrial biogenesis in cells. Viniferin activates AMP-activated kinase and enhances mitochondrial biogenesis. Knockdown of SIRT3 significantly inhibited viniferin-mediated AMP-activated kinase activation and diminished the neuroprotective effects of viniferin, suggesting that SIRT3 mediates the neuroprotection of viniferin. In conclusion, we establish a novel role for mitochondrial SIRT3 in HD pathogenesis and discovered a natural product that has potent neuroprotection in HD models. Our results suggest that increasing mitochondrial SIRT3 might be considered as a new therapeutic approach to counteract HD, as well as other neurodegenerative diseases with similar mechanisms.     

Emerging role of SIRT3 in mitochondrial dysfunction and cardiovascular diseases

As a nicotinamide adenine dinucleotide (NAD)⁺-dependent protein deacetylase, SIRT3 is highly expressed in tissues with high metabolic turnover and mitochondrial content. It has been demonstrated that SIRT3 plays a critical role in maintaining normal mitochondrial biological function through reversible protein lysine deacetylation. SIRT3 has a variety of substrates that are involved in mitochondrial biological processes such as energy metabolism, reactive oxygen species production and clearance, electron transport chain flux, mitochondrial membrane potential maintenance, and mitochondrial dynamics. In the suppression of SIRT3, functional deficiencies of mitochondria contribute to the development of various cardiovascular disorders. Activation of SIRT3 may represent a promising therapeutic strategy for the improvement of mitochondrial function and the treatment of relevant cardiovascular disorders. In the current review, we discuss the emerging roles of SIRT3 in mitochondrial derangements and subsequent cardiovascular malfunctions, including cardiac hypertrophy and heart failure, ischemia-reperfusion injury, and endothelial dysfunction in hypertension and atherosclerosis.     

Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3‐targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3‐dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3‐targeted sites to chemical acetylation in vitro and fasting‐induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low‐level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.     

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.     

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.     

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.     

Metformin Reduces Hepatic Expression of SIRT3, the Mitochondrial Deacetylase Controlling Energy Metabolism

Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.     

SIRT4 Regulates Fatty Acid Oxidation and Mitochondrial Gene Expression in Liver and Muscle Cells

SIRT4, a member of the sirtuin family, has been implicated in the regulation of insulin secretion by modulation of glutamate dehydrogenase. However, the role of this enzyme in the regulation of metabolism in other tissues is unknown. In this study we investigated whether depletion of SIRT4 would enhance liver and muscle metabolic functions. To do this SIRT4 was knocked down using an adenoviral shRNA in mouse primary hepatocytes and myotubes. We observed a significant increase in gene expression of mitochondrial and fatty acid metabolism enzymes in hepatocytes with reduced SIRT4 levels. SIRT4 knockdown also increased SIRT1 mRNA and protein levels both in vitro and in vivo. In agreement with the increased fatty acid oxidation (FAO) gene expression, we showed a significant increase in FAO in SIRT4 knockdown primary hepatocytes compared with control, and this effect was dependent on SIRT1. In primary myotubes, knockdown of SIRT4 resulted in increased FAO, cellular respiration, and pAMPK levels. When SIRT4 was knocked down in vivo by tail vein injection of a shRNA adenovirus, we observed a significant increase in hepatic mitochondrial and FAO gene expression consistent with the findings in primary hepatocytes. Taken together these findings demonstrate that SIRT4 inhibition increases fat oxidative capacity in liver and mitochondrial function in muscle, which might provide therapeutic benefits for diseases associated with ectopic lipid storage such as type 2 diabetes.     

Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1

SIRT3 (sirtuin 3) modulates respiration via the deacetylation of lysine residues in electron transport chain proteins. Whether mitochondrial protein acetylation is controlled by a counter-regulatory program has remained elusive. In the present study we identify an essential component of this previously undefined mitochondrial acetyltransferase system. We show that GCN5L1 [GCN5 (general control of amino acid synthesis 5)-like 1; also known as Bloc1s1] counters the acetylation and respiratory effects of SIRT3. GCN5L1 is mitochondrial-enriched and displays significant homology with a prokaryotic acetyltransferase. Genetic knockdown of GCN5L1 blunts mitochondrial protein acetylation, and its reconstitution in intact mitochondria restores protein acetylation. GCN5L1 interacts with and promotes acetylation of SIRT3 respiratory chain targets and reverses global SIRT3 effects on mitochondrial protein acetylation, respiration and bioenergetics. The results of the present study identify GCN5L1 as a critical prokaryote-derived component of the mitochondrial acetyltransferase programme.     

Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and Implications for Metabolic Disease

The sirtuins are a family of NAD⁺-dependent protein deacetylases that regulate cell survival, metabolism, and longevity. Three sirtuins, SIRT3–5, localize to mitochondria. Expression of SIRT3 is selectively activated during fasting and calorie restriction. SIRT3 regulates the acetylation level and enzymatic activity of key metabolic enzymes, such as acetyl-CoA synthetase, long-chain acyl-CoA dehydrogenase, and 3-hydroxy-3-methylglutaryl-CoA synthase 2, and enhances fat metabolism during fasting. SIRT5 exhibits demalonylase/desuccinylase activity, and lysine succinylation and malonylation are abundant mitochondrial protein modifications. No convincing enzymatic activity has been reported for SIRT4. Here, we review the emerging role of mitochondrial sirtuins as metabolic sensors that respond to changes in the energy status of the cell and modulate the activities of key metabolic enzymes via protein deacylation.     

SIRT4 is the last puzzle of mitochondrial sirtuins

Sirtuins are recently redefined as a family of nicotinamide adenine dinucleotide (NAD)-dependent deacylases. Sirtuins in mammals including human have seven members, which are SIRT1-7. Compared to other sirtuin members, not much study is focused on mitochondrial sirtuins (SIRT3-5). In mitochondrial sirtuins, SIRT4 was the last of less well-understood mitochondrial sirtuins especially for its robust enzymatic activity. This makes SIRT4 become the last puzzle of mitochondrial sirtuins, and thus brings some obstacles for studying SIRT4 biological functions or developing SIRT4 modulators. In this review, we will summarize and discuss the current findings for substrates, biological functions and possible enzymatic activities of SIRT4. The purpose of this review is to facilitate in discovering the robust enzymatic activity of SIRT4 and eventually finish this last puzzle of mitochondrial sirtuins.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

Icariin attenuates excessive alcohol consumption-induced susceptibility to atrial fibrillation through SIRT3 signaling

Excessive alcohol consumption has long been identified as a risk factor for adverse atrial remodeling and atrial fibrillation (AF). Icariin is a principal active component from traditional Chinese medicine Herba Epimedii and has been demonstrated to exert potential antiarrhythmic effect. The present study was designed to evaluate the effect of icariin against alcohol-induced atrial remodeling and disruption of mitochondrial dynamics and furthermore, to elucidate the underlying mechanisms. Excessive alcohol-treated C57BL/6 J mice were infected with serotype 9 adeno-associated virus (AAV9) carrying mouse SIRT3 gene or negative control virus. Meanwhile, icariin (50 mg/kg/d) was administered to the animals in the presence or absence of AAV9 carrying SIRT3 shRNA. We noted that 8 weeks of icariin treatment effectively attenuated alcohol consumption-induced atrial structural and electrical remodeling as evidenced by reduced AF inducibility and reversed atrial electrical conduction pattern as well as atrial enlargement. Furthermore, icariin-treated group exhibited significantly enhanced atrial SIRT3-AMPK signaling, decreased atrial mitoSOX fluorescence and mitochondrial fission markers, elevated mitochondrial fusion markers (MFN1, MFN2) as well as NRF-1-Tfam-mediated mitochondrial biogenesis. Importantly, these beneficial effects were mimicked by SIRT3 overexpression while abolished by SIRT3 knockdown. These data revealed that targeting atrial SIRT3-AMPK signaling and preserving mitochondrial dynamics might serve as the novel therapeutic strategy against alcohol-induced AF genesis. Additionally, icariin ameliorated atrial remodeling and mitochondrial dysfunction by activating SIRT3-AMPK signaling, highlighting the use of icariin as a promising antiarrhythmic agent in this circumstance.     

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD⁺-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD⁺-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3^−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD⁺-dependent deacetylase, SIRT3.     

The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity

As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.     

The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection

Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. By defining the temporal dynamics of SIRT3-substrate interactions during infection, and overlaying acetylome and proteome information, we find altered SIRT3 associations with the mitochondrial fusion factor OPA1 and acetyl-CoA acyltransferase 2 (ACAA2), concomitant with changes in their acetylation levels. Using mutagenesis, microscopy, and virology assays, we determine OPA1 regulates mitochondrial morphology of infected cells and inhibits HCMV production. OPA1 acetylation status modulates these functions, and we establish K834 as a site regulated by SIRT3. Control of SIRT3 protein levels or enzymatic activity is sufficient for regulating mitochondrial filamentous structure. Lastly, we establish a virus restriction function for ACAA2, an enzyme involved in fatty acid beta-oxidation. Altogether, we highlight SIRT3 activity as a regulatory hub for mitochondrial acetylation and morphology during HCMV infection and point to global acetylation as a reflection of mitochondrial health.