Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

Enxuan Jing; Brian T. O’Neill; Matthew J. Rardin; André Kleinridders; Olga R. Ilkeyeva; Siegfried Ussar; James R. Bain; Kevin Y. Lee; Eric M. Verdin; Christopher B. Newgard; Bradford W. Gibson; C. Ronald Kahn

doi:10.2337/db12-1650

Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

Jing, Enxuan; O’Neill, Brian T.; Rardin, Matthew J.; Kleinridders, André; Ilkeyeva, Olga R.; Ussar, Siegfried; Bain, James R.; Lee, Kevin Y.; Verdin, Eric M.; Newgard, Christopher B.; Gibson, Bradford W.; Kahn, C. Ronald 2013-09-17 00:00:00 ORIGINAL ARTICLE Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation 1 1 2 1 3 Enxuan Jing, Brian T. O’Neill, Matthew J. Rardin, André Kleinridders, Olga R. Ilkeyeva, 1 3 1 4,5 3 Siegfried Ussar, James R. Bain, Kevin Y. Lee, Eric M. Verdin, Christopher B. Newgard, 2 1 Bradford W. Gibson, and C. Ronald Kahn Recent reports have shown that mitochondrial dys- Sirt3 is an NAD -dependent deacetylase that regulates mitochon- function is a major contributor to the development of in- drial function by targeting metabolic enzymes and proteins. In sulin resistance and diabetes (6,7). Transcription factors fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion regulating mitochondrial function and biogenesis, such of Sirt3 led to impaired glucose oxidation in muscle, which was as peroxisome proliferator–activated receptor (PPAR)g associated with decreased pyruvate dehydrogenase (PDH) activ- coactivator-1a, nuclear respiratory factor-1, and PPARa ity, accumulation of pyruvate and lactate metabolites, and an in- play critical roles in insulin sensitivity, glucose metabo- ability of insulin to suppress fatty acid oxidation. Antibody-based lism, and lipid metabolism in muscle (8–11). Mutations of acetyl-peptide enrichment and mass spectrometry of mitochon- key metabolic enzymes and subunits of the electron trans- drial lysates from WT and Sirt3 KO skeletal muscle revealed that porter chain can also lead to mitochondrial dysfunction and a major target of Sirt3 deacetylation is the E1a subunit of PDH various degrees of myopathy and neuropathology. Among (PDH E1a). Sirt3 knockout in vivo and Sirt3 knockdown in myo- these, pyruvate dehydrogenase (PDH) complex deﬁciency blasts in vitro induced hyperacetylation of the PDH E1a subunit, altering its phosphorylation leading to suppressed PDH enzy- due to mutations of the E1a subunit gene (PDHA1) that matic activity. The inhibition of PDH activity resulting from reduced encodes the catalytic subunit of PDH is a genetic cause of levels of Sirt3 induces a switch of skeletal muscle substrate uti- mitochondrial dysfunction and inherited neurodegenerative lization from carbohydrate oxidation toward lactate production disease in humans, implicating this subunit’scritical rolein and fatty acid utilization even in the fed state, contributing to metabolism (12,13). a loss of metabolic ﬂexibility. Thus, Sirt3 plays an important role The PDH complex catalyzes the rate-limiting step in in skeletal muscle mitochondrial substrate choice and metabolic aerobic carbohydrate metabolism and mediates the efﬁcient ﬂexibility in part by regulating PDH function through deacetyla- conversion of pyruvate from glycolysis to energy in cells. tion. Diabetes 62:3404–3417, 2013 The activity of this multienzyme complex is regulated, at least in part, by reversible phosphorylation of serine resi- dues of the E1a subunit through PDH kinases (PDHKs) and PDH phosphatases whose enzymatic functions are keletal muscle is the major oxidative tissue in mammals. Metabolic ﬂexibility, i.e., the ability to regulated by cellular nutrient cues (14). Phosphorylation switch between glucose and lipid oxidation, in by PDHKs inhibits the E1a subunit, decreasing PDH ac- Smuscle is essential to maintain normal energy tivity; accordingly, inhibition of PDHKs is a potential metabolism and physiology. In the fed state, the main fuel therapeutic target for diabetes (15). Nutrient deprivation, source in muscle is insulin-induced glucose metabolism such as starvation or diabetes, leads to increased NAD -to- (1,2); during fasting, muscle switches its fuel utilization NADH ratio and increases PDHK expression and activity, from glucose to lipid oxidation (3). Insulin resistance, type thereby inhibiting PDH in muscle; this is reversible with 2 diabetes, and obesity are strongly associated with im- refeeding or insulin treatment (16). Besides phosphor- paired skeletal muscle substrate metabolism including ylation, recent studies suggest that reversible acetylation/ decreased fasting lipid oxidation, impaired postprandial deacetylation may also regulate PDH catalytic subunit E1a glucose oxidation, and reduced capacity for lipid oxidation (PDH E1a) function (17–19), although the pathways con- during exercise (4,5). Thus, the ﬂexibility and capacity of trolling this process have not been fully elucidated. In recent years, NAD -dependent deacetylases called substrate metabolism are compromised in muscle in these states. sirtuins (Sirt) have been shown to play important roles in metabolism (20,21). Among seven members of this protein family, Sirt3 is identiﬁed as the major mitochondrial From the Section on Integrative Physiology and Metabolism, Joslin Diabetes deacetylase (22,23). Several recent studies have shown Center, Harvard Medical School, Boston, Massachusetts; the Buck Institute 3 that Sirt3 regulates lipid metabolism, energy production, and for Research on Aging, Novato, California; the Department of Medicine, stress response in different tissues through its deacetylase Duke University Medical Center, Durham, North Carolina; the Gladstone Institute of Virology and Immunology, San Francisco, California; and the activity (24–26). In muscle, Sirt3 expression is regulated Department of Medicine, University of California, San Francisco, San Fran- by nutrient signals and contractile activity and impacts cisco, California. downstream signaling events through AMP-activated pro- Corresponding author: C. Ronald Kahn, [email protected]. Received 27 November 2012 and accepted 26 June 2013. tein kinase activation and PPARg coactivator-1a expres- DOI: 10.2337/db12-1650 sion (27,28). Sirt3 was implicated in the development of This article contains Supplementary Data online at http://diabetes metabolic disease in humans when a commonly identiﬁed .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1650/-/DC1. E.J. and B.T.O. contributed equally to this study. polymorphism that decreases Sirt3 activity was found to 2013 by the American Diabetes Association. Readers may use this article as be associated with the development of metabolic syndrome long as the work is properly cited, the use is educational and not for proﬁt, (29). We previously demonstrated that skeletal muscle Sirt3 and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. expression is downregulated in rodent models of diabetes 3404 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES reversed-phase nano-high-performance liquid chromatography electrospray and upregulated by caloric restriction and that decreased tandem mass spectrometry (HPLC-ESI-MS/MS) on a QSTAR Elite mass spec- Sirt3 expression induces oxidative stress and impairs trometer. The resulting MS/MS datasets were analyzed using Mascot 2.3.2 insulin signaling in muscle (30). Sirt3 also regulates levels (Matrix Sciences) and Protein Pilot 4.1 (AB Sciex, Foster City, CA) searched of reactive oxygen species (ROS) through deacetylation of against the mouse SwissProt database (SwissProt 2011_04). Peptide scores SOD2 (26,31). In the current study, using a combination of outside of the 5% local false discovery rate were excluded. Skyline MS1 Fil- proteomic, metabolomic, and functional approaches, we tering (34) was used to quantify changes in the MS1 ion abundances among acetylated peptides between WT and KO mice. demonstrate that skeletal muscle Sirt3 regulates substrate PDH activity assay. PDH activity was measured using two methods. The ﬁrst metabolism by targeting mitochondrial PDH E1a subunit used the Mitosciences Pyruvate Dehydrogenase Enzyme Activity Microplate and PDH enzyme activity and thus optimizes the complex Assay Kit (Abcam) in which PDH complexes from isolated mitochondria or and intricate switch of substrate utilization between glu- cellular lysates are immunocaptured on a microplate. A reaction medium cose and lipid oxidation and substrate ﬂexibility. containing pyruvate and NAD is then added. The readout is the rate of pro- duction of NADH, which is coupled to a dye whose formation is monitored on a spectrophotometric plate reader, and activity is calculated from the rate of RESEARCH DESIGN AND METHODS change in optical density at 410 nm. For in vivo experiments, muscle mito- Animal studies were performed according to protocols approved by the In- chondria were isolated as previously described (35), and equal amounts of 2/2 stitutional Animal Care and Use Committee. Male C57Bl/6 mice or Sirt3 and mitochondrial protein were loaded into the immunocapture plate. For in vitro wild-type (WT) littermate controls backcrossed onto a C57Bl/6 background experiments, conﬂuent myoblasts were treated as speciﬁed in the manu- and maintained on a standard chow diet were used. Fed mice were allowed ad facturer’s protocol. For speciﬁed experiments, PI cocktails 2 and 3 (Sigma) or libitum access to food and killed at 9:00 A.M. For fasting studies, mice were 20 mmol/L dichloroacetate (DCA) was added to detergent-soluble fraction and transferred to a new cage without food for 24 h and then killed or refed for buffer 1 prior to microplate incubation. Activity was normalized to protein 4 or 16 h prior to sacriﬁce. content of the detergent-soluble fraction. For Western blots of extracts from Insulin signaling in muscle strips. Extensor digitorum longus (EDL) or PDH activity experiments, an aliquot of detergent soluble extracts was in- hemidiaphragms were dissected and incubated for 30 min in Krebs-Henseleit cubated at room temperature for 3 h to mimic microplate conditions and buffer (KHB) at 37°C and then transferred to KHB with or without 1 or 10 mU/mL blotted using phospho-speciﬁc PDH E1a antibodies. insulin for 10 min. Muscle strips were blotted and snap frozen in liquid PDH activity was also measured in whole muscle homogenates based on 14 14 the evolution of CO from C-labeled pyruvate (36). Brieﬂy, gastrocnemius nitrogen. muscle from randomly fed mice was snap frozen until use. Muscle homoge- Glycolysis, glycogen synthesis, glucose oxidation, and palmitate nates (50 mg wet weight/mL) were incubated for 15 min at 37°C with either 1) oxidation in muscle strips. Glycolysis, glycogen synthesis, glucose oxida- “inactivation” buffer containing 50 mmol/L NaF and 8 mmol/L ATP to prevent tion, and palmitate oxidation were measured in isolated muscle strips as dephosphorylation of the PDH complex with or without the addition of previously described with the following modiﬁcations (32). Brieﬂy, hemi- deacetylase inhibitors (1 mmol/L nicotinamide and 1 mmol/L trichostatin A) diaphragms and EDL muscles were dissected and incubated for 30 min in KHB or 2) “activation” buffer containing 1 mg recombinant PDH phosphatase 1 gassed with 95% O and 5% CO . Muscle strips were transferred to gassed KHB 2 2 (Abcam), 5 mmol/L DCA, 9 mmol/L MgCl , and 0.1 mmol/L CaCl with or containing 5 mmol/L glucose and 0.2 mmol/L palmitate bound to 3% fatty acid– 2 2 free BSA, with or without 1 mU/mL insulin, and with radiolabeled tracers. For without deacetylase inhibitors. Activity was then measured in 20 mL homo- glycolysis, glycogen synthesis, and glucose oxidation, incubation buffer con- genates diluted in reaction buffer for 10 min and terminated with 20% tri- 14 3 tained 20 mCi/mmol [U- C]glucose and 80 mCi/mmol [5- H]glucose, and chloroacetate and 30 mmol/L pyruvate as described in the protocol. Evolved muscles were incubated for 1 h with shaking at 37°C. Reactions were termi- CO was collected in suspended center wells containing 1 mol/L hyamine nated by removal of the tissue from the incubation medium, followed by in- hydroxide in methanol and counted in 4 mL Cytoscint scintillation ﬂuid. Ac- jection of 0.2 mL hyamine hydroxide into the center wells and 0.1 mL 70% tivity of homogenates treated with inactivating solution was termed “PDHa,” (w/v) HClO into 1 mL of the contents of the ﬂask. The rate of glucose oxi- as this likely represents the native activation of PDH in muscle. Activity of dation was determined from the production of CO . Glycogen was puriﬁed homogenates treated with activating solution was termed “PDHt,” represent- by digestion of tissue in 1 mol/L NaOH and then precipitation in 66% ethanol ing total activity of PDH present in muscle. with 100 mg unlabeled glycogen to determine the amount of glycogen syn- ATP and glycogen measurement. ATP levels were measured by an enzymatic 14 3 3 thesized from [U- C]glucose and [5- H]glucose incorporation. H Oformation coupled assay as previously described (37). Brieﬂy, metabolites were isolated was measured by separating H O from 0.5 mL incubation buffer containing from pulverized muscle in 6% perchloric acid and neutralized with KOH [5- H]glucose on Poly-Prep Columns AG 1-X8-731-6212 (Bio-Rad) pretreated with and imidazole, and then ATP was measured as the change in optical 1 mol/L NaOH and then 0.3 mol/L Boric acid. The rate of glycolysis was de- density at 340 nm after addition of glucose, NADP , glucose-6-phosphate termined from thedifferencebetween therateof H O formed and the rate of dehydrogenase, and hexokinase. Glycogen content was measured as substrate recycling determined from the difference between the rates of previously described (38). 14 3 glycogen synthesis from [U- C]glucose and [5- H]glucose. Immunoprecipitation and Western analysis. Powdered muscle tissue, Cell culture maintenance. C2C12 cells (American Type Culture Collection, isolated muscle mitochondria, or conﬂuent myoblasts were homogenized in Manassas, VA) were maintained in high-glucose Dulbecco’s modiﬁed Eagle’s radioimmunoprecipitation assay buffer (Millipore) with protease and PIs medium (DMEM) (Invitrogen) containing 10% FBS (Gemini Bioproducts) un- (Sigma) and deacetylase inhibitors (10 mmol/L nicotinamide/1 mmol/L tri- less otherwise indicated. Sirt3 short hairpin RNA (shRNA) and short hairpin chostatin A). Lysates were subjected to SDS-PAGE and blotted using PDH green ﬂuorescent protein (shGFP) control lentiviral constructs were pur- E1a, PDHK4, LCAD, glyceraldehyde-3-phosphate dehydrogenase, insulin re- chased from Open Biosystems (Huntsville, AL). Stable Sirt3 knockdown and ceptor b (Santa Cruz), phospho-IR/IGFR, phospho-Akt, phospho-ERK, Akt, shGFP control cell lines were generated by viral transduction of C2C12 extracellular signal–related kinase (ERK), VDAC, Sirt3, (Cell Signaling), or myoblasts and selection with puromycin. Wild-type PDH E1a cDNA lentiviral acetyl-lysine (Immunechem or Cell Signaling) antibodies. For immunoprecip- construct (Genecopeia) was used for site-directed mutagenesis (Stratagene) itation assays, mitochondria were isolated in the presence of protease and to generate K336Q and K336R mutations and further sequenced. WT, K336Q, deacetylase inhibitors as previously described (35), resuspended in IP lysis and K336R overexpressing stable C2C12 cell lines were generated. buffer (Pierce), and immunoprecipitated with anti–acetyl-lysine (AcK) agarose Proteomic analysis of acetylated skeletal muscle mitochondrial beads (Immunechem) with streptavidin beads (Pierce) as a control. peptides. Mitochondria were isolated from hindlimb muscle of WT and Oxygen consumption rate and extracellular acidiﬁcation rate assays. SIRT3 KO mice as previously described (33) with the following adaptations: Cellular oxygen consumption rate (OCR) was measured using a Seahorse Muscle was homogenized with teﬂon-pestle in Medium 1 with deacetylase Bioscience XF24 ﬂux analyzer. Cells were seeded at 30,000 cells/well 24 h prior inhibitors (250 mmol/L sucrose, 1 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH to the analysis in low-glucose (100 mg/dL) DMEM containing 10% FBS. Each 7.4; protease and phosphatase inhibitors [PIs] [Sigma]; 10 mmol/L nicotinamide; experimental condition was analyzed using four to six biological replicates. For and 1 mmol/L trichostatin A). Mitochondria were enriched by differential cen- OCR experiments using palmitate, KHB buffer (pH 7.4) was added to each well trifugation and puriﬁed by centrifugation at 59,800g on 5–25% (w/v) linear Ficoll and measurements were performed every 3 min with 2 min intermeasurement (Sigma) gradients. Puriﬁed mitochondria were lysed in radioimmunoprecipitation mixing. BSA-conjugated palmitate (ﬁnal concentration 200 mmol/L) and assay buffer containing protease, phosphatase, and deacetylase inhibitors. etomoxir (ﬁnal concentration 50 mmol/L) were injected sequentially. For ex- Approximately 1 mg total mitochondrial protein was digested with trypsin, and tracellular acidiﬁcation rate (ECAR) experiments with glucose as a substrate, an acetylated peptide fraction was prepared using a combination of anti-AcK sodium carbonate and glucose/pyruvate-free DMEM was used. Glucose and antibodies from ImmuneChem (cat. no. ICP0380-100) and Cell Signaling 2-deoxyglucose were injected sequentially to give ﬁnal concentrations of (cat. no. 9441) as previously described (34). Peptides were analyzed by 25 mmol/L. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3405 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY Metabolomic assays for skeletal muscle amino acids, organic acids, and KO mice to fully characterize muscle substrate metabolism acylcarnitines. Amino acids, acylcarnitines, and organic acids were analyzed in the absence of Sirt3. Glucose oxidation in EDL muscles using stable isotope dilution techniques. Amino acids and acylcarnitine (composed primarily of glycolytic ﬁbers) in the presence measurements were made by ﬂow injection MS/MS using sample preparation of 5 mmol/L glucose, 0.2 mmol/L palmitate, and 1 mU/mL methods described previously (39,40). The data were acquired using a Micro- insulin was signiﬁcantly decreased in Sirt3 KO mice com- mass Quattro MicroTM system equipped with a model 2777 autosampler, a model 1525 HPLC solvent delivery system, and a data system controlled by pared with controls (Fig. 2A). However, glycogen synthe- MassLynx 4.1 operating system (Waters, Millford, MA). Organic acids were sis rates responded normally to insulin, and glycolytic quantiﬁed using methods described previously with Trace Ultra GC coupled to rates were unchanged in Sirt3 KO, suggesting that glucose a Trace DSQ MS operating under Xcalibur 1.4 (Thermo Fisher Scientiﬁc, uptake was normal but that the end products of glycolysis Austin, TX) (41). were not fully oxidized in Sirt3 KO muscle generating Statistical analyses. All data are means 6 SEM. Student t test was performed lactate. Palmitate oxidation rate was quite low in EDL for comparison of two groups or ANOVA was performed for comparison of three or more groups to determine signiﬁcance. muscle and was unchanged between KO and WT. To de- termine whether these changes were due to insulin re- sistance, as we have observed in older 24-week animals RESULTS (30), we measured insulin signaling in muscle strips from Sirt3 expression and mitochondrial acetylation is 8- to 16-week-old Sirt3 KO mice and controls. Interestingly, regulated by fasting. Quantitative real-time PCR using in this ex vivo experiment in EDL strips from younger mice in the fed or fasted state, we found no difference in insulin quadriceps (Quad), EDL, and soleus muscles from 8-week- old male WT C57Bl/6 mice revealed that 24 h of fasting signaling between Sirt3 KO mice and controls (Fig. 2B). In hemidiaphragms (composed of more oxidative ﬁbers) suppressed Sirt3 mRNA expression in hindlimb muscles (Fig. 1A). All of these reductions returned to close to fed palmitate oxidation was increased in the presence of in- levels by 16 h of refeeding and the EDL muscle showed sulin, and glucose oxidation was not signiﬁcantly changed some recovery as early as 4 h after refeeding (Supplemen- in Sirt3 KO but tended to decrease (Fig. 2C). Again, there tary Fig. 1A). Western blotting analysis conﬁrmed a parallel was no change in insulin signaling, glycolysis, or glycogen ;50% decrease of Sirt3 protein level in soleus and gas- synthesis rates in hemidiaphragms from Sirt3 KO com- trocnemius muscle from fasted mice, which increased after pared with WT controls (Supplementary Fig. 2A and B). refeeding to near-normal levels, as we have previously de- These changes in substrate metabolism did not affect total scribed in quadriceps (30) (Fig. 1B). There was some vari- glycogen content or ATP levels in tissues isolated in the fed ability in the recovery of Sirt3 levels from experiment to or fasted states, and AMP-activated protein kinase phos- experiment, but a decrease in Sirt3 levels in the fasted phorylation was decreased in Sirt3 KO muscle upon fasting state was observed in all muscle groups tested (Supple- as previously described (Supplementary Fig. 2D and E)(27). mentary Fig. 1A and C). In the fasted state, levels of ex- The decreased glucose oxidation rate in the insulin- stimulated state in muscle samples from Sirt3 KO mice pression also varied, with muscle ﬁber type being highest in red soleus and lower in white EDL muscle. indicated a potential regulatory role for Sirt3 on mito- Western analysis of isolated mitochondria from skeletal chondrial glucose oxidation. Since PDH E1a function can muscle using an antibody against AcK revealed a general be regulated by phosphorylation on three serine residues increase of mitochondrial protein acetylation after 24 h of (42), we assessed phosphorylation of PDH E1a in WT and fasting, coinciding with decreased Sirt3 levels. We ob- Sirt3 KO skeletal muscle to determine whether deletion of served a speciﬁc increase in acetylation of bands at 97, 72, Sirt3 mimics the effect of fasting on posttranslational and 47 kDa (Fig. 1C). The lower hyperacetylated band was modiﬁcation of PDH E1a. Knockout of Sirt3 resulted in an in the migration position of PDH catalytic subunit E1a.We increase in the phosphorylation of PDH E1a on S300 and immunoprecipitated muscle mitochondrial lysates isolated tended to increase S232 (but not S293) in muscle of fed from either fed or fasted mice using anti-AcK antibody and mice (Fig. 2D and Supplementary Fig. 2F), mimicking the subjected the immunoprecipitates to Western blotting increased phospho–PDH E1a observed in fasted WT mice. analysis with anti–PDH E1a antibody. The abundance of the As observed in fasting, increased PDH E1a acetylation and PDH E1a protein from the immunoprecipitates represents phosphorylation in Sirt3 KO occurred without changes in its acetylation status. This analysis conﬁrmed hyperacet- the total level of PDH E1a or PDHK4 in either the fed or ylation of PDH E1a in fasted skeletal muscle (Fig. 1D). the fasted state (Fig. 2D and F). Posttranslational modiﬁcations known to suppress PDH Posttranslational modiﬁcations like phosphorylation activity include PDH kinase–mediated phosphorylation of PDH E1a are known to have effects on PDH enzymatic targeting three serine residues of PDH E1a (S232, S293, activity (14,43); thus, we investigated whether the in- and S300) (42). We tested serine phosphorylation of PDH creased acetylation and phosphorylation of PDH E1a re- E1a in muscle mitochondria using phospho-speciﬁc anti- lated to Sirt3 deletion would change catalytic activity of bodies and found that S232 and S300 phosphorylation was the PDH enzyme complex. Indeed, PDH activity measured increased during fasting coincident with the PDH E1a by the immunocapture assay (see RESEARCH DESIGN AND hyperacetylation (Fig. 1D). The changes in PDH E1a acet- METHODS) was signiﬁcantly reduced in Sirt3 KO mitochon- drial lysates compared with WT. Quantiﬁcation of the ylation/phosphorylation after fasting were not explained by changes in PDH E1a total protein levels (Fig. 1D). slopes of the linear regressions demonstrated a .70% in- Sirt3 deletion induces a shift in substrate utilization hibition of PDH activity in KO mice (Fig. 2E). To further away from glucose oxidation and increases PDH E1a elucidate the possible regulatory role of acetylation on phosphorylation suppressing PDH activity. We hy- PDH activity, we determined active and total PDH activity pothesized that Sirt3 has its most prominent role in met- in muscle homogenates from fed Sirt3 KO and WT mice as abolic regulation in the fed state, when it is present at well as fasted C57Bl/6 mice by measurement of CO highest levels. To test this hypothesis, we assessed glucose production from C-labeled pyruvate in the presence or and palmitate oxidation, as well as glycolysis and glycogen absence of deacetylase inhibitors (see RESEARCH DESIGN AND synthesis, in isolated muscle strips from fed WT and Sirt3 METHODS). As in the mitochondrial isolates, PDHa activity 3406 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 1. Skeletal muscle Sirt3 expression and mitochondrial protein acetylation are regulated by fasting. Wild-type 8-week-old male C57Bl/6 mice were fed ad libitum, fasted for 24 h, or fasted and then refed for 16 h. After each treatment, RNA and protein were extracted and analyzed by real- time quantitative PCR (A) or Western blotting (B) for assessment of Sirt3 expression in quadriceps (Quad), EDL, soleus (Sol), and gastrocnemius (Gast) muscle groups (n =3–5, #P < 0.05 vs. fed, ANOVA). C: Mixed hindlimb muscles (gastrocnemius and soleus) were collected from mice in the fed or fasted state. Muscle mitochondria were isolated in the presence of protease and deacetylase inhibitors as described in RESEARCH DESIGN AND METHODS. Mitochondrial protein lysates from each animal were subjected to SDS-PAGE and Western blotting using an antibody against AcK. The intensity of speciﬁed bands was quantiﬁed with ImageJ software (n =3,*P < 0.05, Student t test). D: PDH E1a acetylation in muscle of fed or fasted mice was measured by immunoprecipitation (IP) of mitochondrial lysates using anti-AcK antibody. Immunoprecipitates were subjected to Western blotting analysis (IB) using anti–PDH E1a antibody. The same muscle mitochondrial lysates were directly subjected to SDS-PAGE electrophoresis and Western blotting using antibodies against phosphorylated serine sites p-232, p-293, and p-300 of the PDH E1a subunit and total protein of PDH E1a. Autoradiography of Western blots was quantiﬁed with ImageJ software (n =3,*P < 0.05, Student t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. was signiﬁcantly decreased in Sirt3 KO muscle to the level act on PDH, partially restored PDH activity from Sirt3 KO of fasted C57Bl/6 mice (Fig. 2G) without any changes in mice. Since inhibition of PDH activity in fasting muscle has total PDH (Supplementary Fig. 2C). Remarkably, incubation profound effects on substrate metabolism, the observed of the muscle homogenates in the absence of deacetylase reduction of PDH activity induced by Sirt3 deletion inhibitors, which likely allows other deacetylases outside demonstrates that Sirt3 is an important regulator of the the mitochondria but present in the tissue homogenate to metabolic changes in fed skeletal muscle. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3407 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 2. Sirt3 deletion induces synergistic switch from glucose oxidation toward fatty acid oxidation and suppresses PDH activity by inducing PDH E1a hyperacetylation and hyperphosphorylation in fed Sirt3 KO skeletal muscle. A: Basal and insulin-stimulated (1 mU/mL) glucose oxidation, glycogen synthesis, glycolysis, and palmitate oxidation were measured in EDL muscles isolated from randomly fed 8- to 16-week-old WT and Sirt3 KO mice (n =9;§P < 0.05 vs. basal, #P < 0.05 vs. WT, ANOVA). B: Basal and insulin-stimulated (10 mU/mL) phosphorylation of insulin/IGF-1 receptor, Akt, and ERK in tissue lysates of EDL muscles from fed and fasted 8- to 16-week-old WT and Sirt3 KO mice. C: Insulin-stimulated (1 mU/mL) glucose and palmitate oxidation were measured as described in RESEARCH DESIGN AND METHODS in diaphragms isolated from randomly fed 8- to 16- week-old WT and Sirt3 KO mice (n =9; *P < 0.05, Student t test). D: Mitochondrial lysates were subjected to Western blot analysis using anti- bodies against p-232, p-293, and p-300 serine phosphorylation sites on PDH E1a, total PDH E1a, and Sirt3 (densitometry in Supplementary Fig. 2F). E: PDH activity was measured in 20 mg mitochondrial lysate from hindlimb of fed WT and Sirt3 KO mice using a PDH activity microplate kit as described in RESEARCH DESIGN AND METHODS. The PDH activities were calculated by linear regression of the steady-state kinetics and quantiﬁed (n =4–5, 3408 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES PDH E1a subunit is a Sirt3 substrate. Using anti-AcK Knockdown of Sirt3 in C2C12 myoblasts decreases antibody to detect protein acetylation, we observed that PDH activity. To further elucidate the role of Sirt3 in PDH Sirt3 KO muscle mitochondria displayed an elevation in function, we determined PDH E1a acetylation and enzyme acetylation similar to that seen in fasted WT mice (Fig. activity in C2C12 myoblasts in which Sirt3 was knocked 3A), supporting the idea that Sirt3 is the primary mito- down using shRNA (shSirt3). This resulted in a .90% de- chondrial deacetylase and consistent with our previous crease in Sirt3 protein by Western analysis (Fig. 5A, C, and report in Sirt3 KO fed mice (23,30). To identify the po- D). Immunoprecipitation of mitochondrial lysates from tential Sirt3 targets in skeletal muscle during fasting (when shGFP control and shSirt3 myoblasts using anti-AcK anti- Sirt3 levels are low) and in Sirt3 KO muscle, we performed body revealed signiﬁcantly increased acetylated PDH E1a a global acetyl-proteomic analysis (34). To this end, acet- in knockdown cells (Fig. 5A). The increased acetylation of ylated peptides were enriched from trypsinized mito- PDH E1a in vitro was associated with a 27% decrease in chondrial protein lysates of WT and Sirt3 KO muscle using PDH activity in shSirt3 myoblasts measured by microplate AcK antibody immunoprecipitation and subjected to mass immunocapture of PDH (Fig. 5B). However, in contrast to spectrometry analysis. A total of 549 acetylated peptides results in skeletal muscle in vivo from Sirt3 KO mice, shSirt3 were identiﬁed in the combined WT and KO muscle mi- myoblasts showed decreased phosphorylation at both S232 tochondria. Among these, 299 acetylated peptides were and S300 sites (Fig. 5C and D), suggesting that PDH E1a present in both WT and KO samples, only 49 of which were acetylation may inhibit PDH activity independent of detected exclusively in WT, whereas 201 acetylated pep- phosphorylation. tides were exclusive to the Sirt3 KO, indicating increased Previous bioenergetic proﬁling using Seahorse XF ﬂux acetylation induced by Sirt3 deletion (Fig. 3B). These 549 analyzer with glucose and pyruvate as substrates showed acetylated peptides corresponded to a total of 147 acety- that shSirt3 myoblasts had signiﬁcantly lower uncoupled lated proteins, indicating that there were multiple acety- respiration (30). Since changes in the metabolomic proﬁle lation sites for each protein (Fig. 3B). Our analysis revealed suggested a switch in substrate utilization in Sirt3 KO mus- several lysine acetylation sites on PDH E1a (Fig. 3C). cles, we measured glucose and fatty acid oxidation occurred Among the peptides showing signiﬁcantly increased acet- in a cell autonomous manner using shSirt3 knockdown ylation in Sirt3 KO muscle mitochondria was one peptide myoblasts. Basal rates of lactate production, as indicated representing acetylated lysine 336 (K336) of PDH E1a by ECAR, were similar in control and shSirt3 myoblasts. (Supplementary Fig. 3A). Quantitation of the MS1 pre- When given glucose as substrate, shSirt3 cells displayed cursor signals of this peptide between the four WT and a greater increase in ECAR compared with control (Fig. 5E). four KO mice showed a threefold increase in acetylation at The area under the curve (AUC) quantiﬁcation of ECAR K336 in the Sirt3 KO animals (Fig. 3D), indicating that was signiﬁcantly higher in shSirt3 after glucose stimulation K336 is a substrate of Sirt3. (Fig. 5F). As previously reported, OCRs were lower in To conﬁrm that Sirt3 directly acetylates PDH, we immu- shSirt3 myoblasts (Supplementary Fig. 4A and B) (30). noprecipitated WT and Sirt3 KO muscle mitochondrial OCR in the presence of 200 mmol/L palmitate was signiﬁ- lysates from both fed and fasted mice using anti-AcK anti- cantly higher in shSirt3 cells, and this was inhibited by body and subjected the immunoprecipitates to Western addition of etomoxir, indicating higher rates of fatty acid blotting analysis with anti-PDH E1a antibody. This revealed b-oxidation in shSirt3 compared with control cells. AUC that PDH E1a acetylation was increased signiﬁcantly in calculation after palmitate showed a ;35% increase in OCR Sirt3 KO muscle mitochondria (Fig. 3E), and this was in shSirt3 (Fig. 5G and H). Together, these data demon- speciﬁc, as no enrichment was observed using streptavidin strate that decreased Sirt3 expression induces a switch in beads as a control (Supplementary Fig. 3). muscle substrate utilization from glucose to fatty acid oxi- Sirt3 deletion in skeletal muscle induces an altered dation to compensate for decreased carbohydrate oxidation metabolic proﬁle. To fully assess the metabolic effects caused by inhibition of PDH activity. of PDH E1a inhibition in Sirt3 KO skeletal muscle, we Overexpression of K336Q or K336R mutants of PDH performed targeted gas chromatography/mass spectrome- E1a decreases phosphorylation at S232 and S300 but try and MS/MS metabolomic analysis on samples of skel- does not affect PDH activity or substrate metabolism. etal muscle from fed WT and Sirt3 KO mice. We found To determine whether acetylation at lysine 336 of PDH E1a signiﬁcantly elevated levels of lactate, pyruvate, and a- was sufﬁcient to affect PDH activity, we created C2C12 ketoglutarate (a-KG) in Sirt3 KO skeletal muscle (Fig. 4A). myoblast cell lines with stable overexpression of WT PDH The increase in lactate and pyruvate in Sirt3 KO muscles is E1a or with either a K336Q or a K336R mutation. In some consistent with suppression of PDH activity and decreased molecules, the lysine (K) to glutamine (Q) mutation can carbohydrate oxidation. We also observed a signiﬁcant mimic the acetylated state, while the K to arginine (R) decrease in levels of acylcarnitines measured in Sirt3 KO prevents acetylation and mimics the deacetylated state. muscle, suggestive of increased fatty acid use (Fig. 4B). Both K336Q and the K336R mutations caused a reduction in Additionally, amino acids levels were decreased in Sirt3 phosphorylation of S232 and S300 compared with over- KO muscle (Fig. 4C). Taken together, these metabolomic expression of WT PDH E1a (Fig. 6A and B), indicating an data indicate that Sirt3 deletion induces a metabolic de- important role of lysine 336 in control of serine phosphor- rangement in skeletal muscle in which impaired PDH ac- ylation. To dissect the effect of phosphorylation status tivity suppresses glucose oxidation and enhances lipid and versus K336 mutation on PDH activity, we determined PDH amino acid catabolism. activity in the presence of PIs to promote phosphorylation *P < 0.05, Student t test). F: Western blot analysis was performed on tissue lysates from gastrocnemius muscle from fed and fasted WT and Sirt3 KO for PDHK4, Sirt3, and glyceraldehyde-3-phosphate dehydrogenase. G: Native PDH activity (PDHa) was measured in gastrocnemius muscle 14 14 homogenate from WT fed, Sirt3 KO fed, and fasted C57Bl/6 mice by collection of CO release from C-pyruvate. Prior to PDH assay, aliquots of homogenates were incubated in the presence of 50 mmol/L NaF with or without deacetylase inhibitors (1 mmol/L nicotinamide and 1 mol/L tri- chostatin A) as described in RESEARCH DESIGN AND METHODS (n =6–7; #P < 0.05 vs. WT plus deacetylase inhibitors, ANOVA). OD, optical density. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3409 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 3. Discovery of PDH E1a subunit as a target of Sirt3 in skeletal muscle. A: Muscle mitochondrial protein lysates from WT and Sirt3 KO mice in either the fed or fasted state were subjected to Western blotting analysis using anti-AcK antibody. B: Proteomic analysis using anti-AcK antibody– based acetylated peptide enrichment and mass spectrometry discovered a total of 549 acetylated peptides present in WT and Sirt3 KO skeletal muscle mitochondria. These peptides represented a total of 147 proteins. Venn diagrams show overlapping and distinctive patterns of distribution of acetylated peptides and proteins between WT and Sirt3 KO skeletal muscle. C: Schematic diagram of lysine acetylation sites and previously 3+ reported serine phosphorylation sites on PDH E1a. D: A representative MS1 chromatogram of the triply charged precursor peak at m/z 772.7303 (324-MVNSNLASVEELacKEIDVEVR-343) of PDH E1a was highly increased in Sirt3 KO skeletal muscle mitochondria compared with WT (n =4; P < 0.05, Student t test). E: Skeletal muscle mitochondrial lysates from WT and Sirt3 KO mice in both fed and fasted states were immunoprecipitated (IP) with AcK antibody–bound beads. Immunoprecipitates of the mitochondrial lysate were subjected to Western blotting (IB) using an anti–PDH E1a antibody. Densitometry of either fed or fasted animals was calculated (n =4;*P < 0.05, Student t test). 3410 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 4. Sirt3 deletion induces a substrate switch and derangement of metabolites in skeletal muscle. Quadriceps muscles from fed male WT and Sirt3 KO mice were collected and subjected to metabolomic analysis as described in RESEARCH DESIGN AND METHODS. Relative levels of organic acids and Kreb cycle intermediates are shown in A, levels of acylcarnitines in B, and amino acid levels in C (n =4–5; *P < 0.05, Student t test). Asx, asparagine and aspartic acid; Cit, citrulline; Glx, glutamine and glutamic acid; Orn, ornithine. or DCA, a PDH kinase speciﬁc inhibitor, to decrease phos- recapitulate the effects of decreased Sirt3 expression on phorylation of PDH E1a. PDH activity was unchanged in PDH complex activity and substrate metabolism. myoblasts harboring either of the K336Q or K336R mutations compared with WT controls (Fig. 6C) with or without ad- DISCUSSION dition of PI to preserve S293 and S300 phosphorylation or addition of DCA to maximally dephosphorylate PDH E1a Mitochondrial sirtuins are uniquely positioned to regulate (Fig. 6D). energy metabolism via protein deacetylation. Although To assess whether K336Q or K336R mutations could Sirt3, Sirt4, and Sirt5 have all been localized to mito- recapitulate the substrate switch observed in shSirt3 chondria, previous reports have shown that only Sirt3 cells, we analyzed glucose-induced ECAR in WT, K336Q, deletion induces mitochondrial protein hyperacetylation, or K336R myoblasts. No signiﬁcant differences in glucose- suggesting that Sirt3 is the major mitochondrial protein stimulated ECAR were observed in the K336Q or K336R deacetylase (23). Indeed, recent studies have shown that mutant cells compared with control (Fig. 6E and F). Thus, altered Sirt3 expression can have effects on lipid metab- altering the ability of K336 to undergo acetylation did olism, ROS production, oxidative stress response, and cell modify phosphorylation of PDH E1a but could not fully survival (25,26,30,44,45). diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3411 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 5. Sirt3 knockdown in C2C12 myoblasts impairs PDH activity despite decreased phosphorylation of PDH E1a and leads to a substrate switch toward fatty acid utilization. A: Total mitochondrial protein lysates from shGFP control and shSirt3 myoblasts were immunoprecipitated (IP) with AcK (AcLys) antibody and subjected to Western blot analysis (IB) using an anti–PDH E1a antibody. The same mitochondrial lysates were directly subjected to Western blot analysis using antibodies against PDH E1a, Sirt3, and voltage-dependent anion channel (VDAC) as a mitochondrial loading control. Densitometry of PDH E1a from AcK immunoprecipitates was normalized to total PDH E1a (n = 4 separate experiments; *P < 0.05, Student t test). B: Total PDH activity was assessed in conﬂuent control and shSirt3 myoblasts using PDH activity microplate assay kit and nor- malized to total protein from detergent extraction (n = 5 separate experiments; †P < 0.05, paired t test). C: Phosphorylation of PDH E1a and total Sirt3 levels were determined by Western blot analysis of whole cell lysates from conﬂuent shGFP and shSirt3 C2C12 myoblasts. D: Densitometry of Western blots from C (n = 3 separate experiments, *P < 0.05, Student t test). E: ECAR was measured in shSirt3 and control myoblasts using aSeahorse ﬂux analyzer after incubation in glucose-free Seahorse running media for 1 h at 37°C. A representative tracing of basal and 3412 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES In the current study, we ﬁnd that Sirt3 is an important Another interesting aspect of Sirt3 physiology is the mitochondrial factor in the regulation of skeletal muscle differential regulation and physiological effects of this metabolic ﬂexibility by targeting the enzymatic deacetyla- enzyme in different tissues. For example, during fasting tion of PDH E1a. Thus, when nutrients are abundant, Sirt3 Sirt3 expression is decreased in muscle but increased in deacetylation promotes PDH activity and postprandial liver (25,30). Mitochondrial protein acetylation patterns glucose metabolism (Fig. 7A). During fasting, there is conﬁrm that Sirt3 is more active in the fed state in skeletal a decrease of Sirt3 in skeletal muscle, which leads to muscle, whereas in liver Sirt3 is more active during fasting. hyperacetylation of PDH E1a and promotes a metabolic Sirt3 also has different tissue-speciﬁc metabolic effects. switch from glucose to fatty acids as a predominant sub- Sirt3 deletion in liver suppresses lipid oxidation via sup- strate (Fig. 7B). This can be mimicked by Sirt3 deletion, pression of LCAD activity (25), whereas deletion of Sirt3 in which results in decreased catalytic activity of PDH, de- muscle decreases glucose oxidation and increases lipid creased glucose oxidation, and an accumulation of pyru- oxidation, at least in part to compensate for PDH inhibition. vate and lactate levels even in the fed state. Impaired This dichotomous role of Sirt3 in muscle versus liver may glucose utilization in Sirt3 KO muscle induces a reliance help to explain the report showing minimal changes in on fatty acid b-oxidation for energy production. This global metabolism when Sirt3 is deleted in a tissue-speciﬁc results in increased breakdown of acylcarnitines and ROS manor (49). Muscle-speciﬁcdeletionof Sirt3 maylead to generation and over time leads to insulin resistance (30). changes in mitochondrial substrate choice, which are Previous reports have suggested that 24 h of fasting compensated for by opposing Sirt3 action in liver, resulting increases Sirt3 levels in muscle (27), but in our hands, 24 h in a balance at the whole-body level. Nonetheless, this of fasting leads to a consistent decrease in Sirt3 in each differential regulation of Sirt3 expression and function in a muscle group tested. The difference between these two tissue-dependent manner would allow Sirt3 to coordi- observations may be explained by the timing of the study nately regulate divergent pathways of substrate utilization (mice were killed at 6 P.M. in the former study and at 9 A.M. in different organs based on nutrient availability. in our study) and the duration of fasting in the different fed Reversible phosphorylation of PDH E1a by PDH kinases and fasted groups. Peak feeding in mice occurs at the inhibits enzyme activity and, in starvation and diabetes, beginning of the nocturnal phase. Thus, fed mice killed at levels of PDH kinases increase in skeletal muscle (14,16,50). 9:00 A.M. are only a few hours after peak feeding time. On PDH kinases can be activated by a decrease in the NAD - the other hand, when mice are killed at 6:00 P.M., even the to-NADH ratio, providing feedback inactivation of the PDH “fed” mice are likely to have fasted or partially fasted for as complex (51). Our studies demonstrate that increased many as 10–12 h prior to study, and the “24-h fasted” mice acetylation of PDH E1a is associated with decreased PDH may have gone without food for as long as 36 h, resulting complex activity, leading us to speculate that PDH acety- in dramatic wasting and changes in muscle protein compo- lation also regulates complex activity. Indeed, PDH activity sition (46). Alternatively, there may be circadian changes in was decreased in both Sirt3 KO muscle in vivo and Sirt3 expression of Sirt3 or some of its regulators, which is altered knockdown myoblasts in vitro; the former was associated 232 300 during prolonged periods of fasting. with increased phosphorylation of Ser and Ser , Metabolic ﬂexibility is the ability of an organism to adapt whereas the latter was associated with decreased serine fuel oxidation in response to fuel availability (47). In both phosphorylation. However, the degree of PDH activity insulin resistance and type 2 diabetes, this metabolic decrease is different in the two models—55–70% in Sirt3 ﬂexibility is compromised in that skeletal muscle fails to KO in vivo compared with 27% in shSirt3 in vitro—which switch substrate utilization from lipid metabolism to could be related to phosphorylation status. Lastly, the insulin-stimulated glucose oxidation (5,48). The current observation that incubation of Sirt3 KO muscle homoge- study demonstrates that mitochondrial Sirt3 can help op- nate in the absence of deacetylase inhibitors partially re- timize the switch of substrate oxidation toward glucose stored PDH activity supports the notion that acetylation of utilization by deacetylating PDH, a vital enzyme complex PDH inhibits its activity. Regulation of enzyme activity by in glucose oxidation. Acute regulation of metabolic ﬂexi- acetylation may also help explain the observed accumula- bility by Sirt3 upon refeeding is not due changes in Sirt3 tion of a-KG, as a-KG dehydrogenase is similar in structure levels, since 16 h of refeeding is required for full recovery and regulation to PDH, in that both use an E1 component of mRNA and protein levels. These acute changes in sub- that may be a target for acetylation and Sirt3-catalyzed strate ﬂexibility going from the fed to the fasted state are deacetylation. Other mechanisms may also contribute to more likely coordinated by a complex network of signaling changes in PDH activity. For example, PDH and a-KG de- cascades including insulin and nutrient-dependent phos- hydrogenase contain many thiol groups, which are sensitive phorylation events. This acute regulation may still be to ROS, and we have previously shown that deletion of Sirt3 affected by Sirt3 intracellular partitioning or possible post- causes increased ROS production in muscle from 24-week- translational modiﬁcation affecting its deacetylase activity. old animals (30). However, our data support the conclusion that Sirt3 plays Mass spectrometry–based analysis revealed that PDH E1a an important role in regulating muscle metabolic ﬂexibility is acetylated on multiple lysine residues. We focused our upon refeeding by deacetylating PDH and, with dephos- attention on lysine 336, since it exhibited one of the greatest phorylation, allows for maximal enzyme activity for glucose levels of differential acetylation in Sirt3-depleted skeletal oxidation. muscle. Our attempts to determine whether acetylation at glucose-stimulated ECARs recorded before and after addition of 25 mmol/L glucose (ﬁnal concentration) is shown. At the end of the glucose metabolism period, 2-deoxyglucose was injected to give a ﬁnal concentration of 25 mmol/L. F: AUC calculation of glucose-stimulated ECAR from E. G: A representative tracing of palmitate OCR measured in control and Sirt3 knockdown cells after incubation with substrate-free buffer for 1 h at 37°C. Basal and palmitate-BSA–stimulated OCRs were recorded and plotted as a percentage over basal OCR. Finally, 50 mmol/L etomoxir was injected. H: AUC of palmitate-stimulated OCR from G (n =3; *P < 0.05, Student t test). mOD, milli optical density. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3413 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 6. Expression of a K336Q and K336R mutant of PDH E1a in C2C12 myoblasts decreases phosphorylation but does not affect PDH activity. A: Phosphorylation of PDH E1a levels determined by Western blot analysis of whole cell lysates from conﬂuent C2C12 myoblasts expressing WT PDH E1a, K336Q, or K336R mutant of PDH E1a. B: Densitometry of Western blots from A (n = 3 separate experiments, #P < 0.05, ANOVA). C: Total PDH activity was assessed in conﬂuent WT, K336Q, or K336R myoblasts using detergent extracts either with PIs 2 and 3 (Sigma) or 20 mmol/L DCA added to the PDH activity assay as described in RESEARCH DESIGN AND METHODS (n =2–4 separate experiments). D: Western blot analysis of de- tergent extracts from C either with PI (Phos Inhib) or with DCA after 3 h incubation at room temperature in buffer 1 of the PDH activity assay kit. E: A representative example of Seahorse analysis of glucose-induced ECAR with PDH E1a WT, K336Q, and K336R mutants. F: AUC quantiﬁcation after addition of glucose showed no statistical difference between mutants (n = 4 separate experiments). 2-DG, 2-deoxyglucose. lysine 336 alone could alter PDH activity using the K336Q of diabetes has been proposed as a way to increase glu- (which is regarded as an acetylation mimic) and K336R cose disposal, thereby decreasing circulating glucose lev- (which is regarded as a deacetylation mimic) mutations els (15). However, many of these treatments induce fasting demonstrated that changing the ability of this lysine to hypoglycemia as a result of PDH activation during fasting. become acetylated can cause alterations in phosphoryla- Since Sirt3 levels decrease in muscle with fasting, phar- tion of PDH E1a but did not change catalytic function or macologic activation of Sirt3 in muscle should be beneﬁ- substrate metabolism in vitro. It is likely that acetylation of cial, as it would have maximal effect in the fed state, when other lysine residues in the PDH complex contribute to the glucose levels are highest in diabetes. observed changes in PDH activity. In summary, our study demonstrates that Sirt3 plays an Identifying a role of Sirt3 in regulating metabolic ﬂexi- important role in control of substrate metabolism and adds bility and PDH activity in muscle may provide a new target another layer of regulation to the multiple pathways that for preventing and treating metabolic syndrome. Evidence control metabolic ﬂexibility in skeletal muscle. In the fed for Sirt3 impacting human metabolic disease is accumu- state, Sirt3 expression/activity in muscle is high, resulting lating. We have recently identiﬁed a single nucleotide in deacetylation of many mitochondrial proteins, including polymorphism in the human Sirt3 gene that decreases its PDH E1a. This enhances PDH complex activity and post- activity and is associated with metabolic syndrome (29). prandial glucose metabolism. Thus, Sirt3 helps orchestrate Indeed, targeting PDH activity in muscle for the treatment the efﬁcient use of available nutrients in skeletal muscle by 3414 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 7. Model for the role of Sirt3 in control of skeletal muscle substrate metabolism. Sirt3 regulates PDH E1a subunit deacetylation and activates PDH activity. A: In the fed state, Sirt3 skeletal muscle expression is abundant and leads to deacetylation of PDH E1a. This is associated with dephosphorylation of PDH allowing for maximal enzyme activation, enhanced glucose utilization, and increased ﬂux of pyruvate to acetyl-CoA used by the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) to generate ATP. B: In contrast, decreased Sirt3 expression in muscle by fasting or genetic deletion leads to PDH E1a hyperacetylation and decreased PDH complex activity, which is correlated with increased PDH E1a phosphorylation in vivo. The activity of PDH controls the substrate inﬂux to the TCA cycle from glycolysis. In the case of Sirt3 deletion, inactivation of the PDH caused by hyperacetylation leads to metabolic inﬂexibility as evidenced by an inability to fully oxidize glucose, a shunt of excess pyruvate toward lactate production, and increased lipid oxidation even in the fed state. CPT, carnitine palmitoyl transferase; FFA, free fatty acid; Mito, mitochondrial; OxPhos, oxidative phosphorylation. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3415 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY 14. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM. Evidence for promoting metabolic ﬂexibility through enzymatic deacet- existence of tissue-speciﬁc regulation of the mammalian pyruvate de- ylation of PDH. Modulation of mitochondrial acetylation hydrogenase complex. Biochem J 1998;329:191–196 may offer a new therapeutic target for correcting some of 15. Mayers RM, Leighton B, Kilgour E. PDH kinase inhibitors: a novel therapy the metabolic defects of obesity and type 2 diabetes. for Type II diabetes? Biochem Soc Trans 2005;33:367–370 16. Wu P, Inskeep K, Bowker-Kinley MM, Popov KM, Harris RA. Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes. Diabetes 1999;48:1593–1599 ACKNOWLEDGMENTS 17. Zhao S, Xu W, Jiang W, et al. Regulation of cellular metabolism by protein The primary funding for this project was National Insti- lysine acetylation. Science 2010;327:1000–1004 tutes of Health Grant R24 DK085610. B.T.O. was funded by 18. Choudhary C, Kumar C, Gnad F, et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009;325:834–840 Joslin Training Grant T32DK007260. A.K. was funded by 19. Schwer B, Eckersdorff M, Li Y, et al. Calorie restriction alters mitochon- German Research Foundation project KL2399/1-1. S.U. drial protein acetylation. Aging Cell 2009;8:604–606 was supported by a Human Frontier Science Program 20. Michan S, Sinclair D. Sirtuins in mammals: insights into their biological Long-Term fellowship. The authors acknowledge the function. Biochem J 2007;404:1–13 support of National Center for Research Resources shared 21. Milne JC, Denu JM. The Sirtuin family: therapeutic targets to treat diseases instrumentation grant S10 RR024615 (to B.W.G.) for the of aging. Curr Opin Chem Biol 2008;12:11–17 22. Cooper HM, Spelbrink JN. The human SIRT3 protein deacetylase is ex- QSTAR Elite mass spectrometer used in these studies. clusively mitochondrial. 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Alpha-keto acid dehydrogenase complexes. X. production. Oncogene 2011;30:2986–2996 Regulation of the activity of the pyruvate dehydrogenase complex from 45. Yang H, Yang T, Baur JA, et al. Nutrient-sensitive mitochondrial NAD+ beef kidney mitochondria by phosphorylation and dephosphorylation. levels dictate cell survival. Cell 2007;130:1095–1107 Proc Natl Acad Sci USA 1969;62:234–241 46. Jagoe RT, Lecker SH, Gomes M, Goldberg AL. Patterns of gene expression 51. Harris RA, Bowker-Kinley MM, Huang B, Wu P. Regulation of the activity in atrophying skeletal muscles: response to food deprivation. FASEB J of the pyruvate dehydrogenase complex. Adv Enzyme Regul 2002;42: 2002;16:1697–1712 249–259 diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3417 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central http://www.deepdyve.com/lp/pubmed-central/sirt3-regulates-metabolic-flexibility-of-skeletal-muscle-through-1TD1DRQ5JB

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Publisher: Pubmed Central
Copyright: © 2013 by the American Diabetes Association.
ISSN: 0012-1797
eISSN: 1939-327X
DOI: 10.2337/db12-1650
Publisher site: See Article on Publisher Site

Abstract

ORIGINAL ARTICLE Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation 1 1 2 1 3 Enxuan Jing, Brian T. O’Neill, Matthew J. Rardin, André Kleinridders, Olga R. Ilkeyeva, 1 3 1 4,5 3 Siegfried Ussar, James R. Bain, Kevin Y. Lee, Eric M. Verdin, Christopher B. Newgard, 2 1 Bradford W. Gibson, and C. Ronald Kahn Recent reports have shown that mitochondrial dys- Sirt3 is an NAD -dependent deacetylase that regulates mitochon- function is a major contributor to the development of in- drial function by targeting metabolic enzymes and proteins. In sulin resistance and diabetes (6,7). Transcription factors fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion regulating mitochondrial function and biogenesis, such of Sirt3 led to impaired glucose oxidation in muscle, which was as peroxisome proliferator–activated receptor (PPAR)g associated with decreased pyruvate dehydrogenase (PDH) activ- coactivator-1a, nuclear respiratory factor-1, and PPARa ity, accumulation of pyruvate and lactate metabolites, and an in- play critical roles in insulin sensitivity, glucose metabo- ability of insulin to suppress fatty acid oxidation. Antibody-based lism, and lipid metabolism in muscle (8–11). Mutations of acetyl-peptide enrichment and mass spectrometry of mitochon- key metabolic enzymes and subunits of the electron trans- drial lysates from WT and Sirt3 KO skeletal muscle revealed that porter chain can also lead to mitochondrial dysfunction and a major target of Sirt3 deacetylation is the E1a subunit of PDH various degrees of myopathy and neuropathology. Among (PDH E1a). Sirt3 knockout in vivo and Sirt3 knockdown in myo- these, pyruvate dehydrogenase (PDH) complex deﬁciency blasts in vitro induced hyperacetylation of the PDH E1a subunit, altering its phosphorylation leading to suppressed PDH enzy- due to mutations of the E1a subunit gene (PDHA1) that matic activity. The inhibition of PDH activity resulting from reduced encodes the catalytic subunit of PDH is a genetic cause of levels of Sirt3 induces a switch of skeletal muscle substrate uti- mitochondrial dysfunction and inherited neurodegenerative lization from carbohydrate oxidation toward lactate production disease in humans, implicating this subunit’scritical rolein and fatty acid utilization even in the fed state, contributing to metabolism (12,13). a loss of metabolic ﬂexibility. Thus, Sirt3 plays an important role The PDH complex catalyzes the rate-limiting step in in skeletal muscle mitochondrial substrate choice and metabolic aerobic carbohydrate metabolism and mediates the efﬁcient ﬂexibility in part by regulating PDH function through deacetyla- conversion of pyruvate from glycolysis to energy in cells. tion. Diabetes 62:3404–3417, 2013 The activity of this multienzyme complex is regulated, at least in part, by reversible phosphorylation of serine resi- dues of the E1a subunit through PDH kinases (PDHKs) and PDH phosphatases whose enzymatic functions are keletal muscle is the major oxidative tissue in mammals. Metabolic ﬂexibility, i.e., the ability to regulated by cellular nutrient cues (14). Phosphorylation switch between glucose and lipid oxidation, in by PDHKs inhibits the E1a subunit, decreasing PDH ac- Smuscle is essential to maintain normal energy tivity; accordingly, inhibition of PDHKs is a potential metabolism and physiology. In the fed state, the main fuel therapeutic target for diabetes (15). Nutrient deprivation, source in muscle is insulin-induced glucose metabolism such as starvation or diabetes, leads to increased NAD -to- (1,2); during fasting, muscle switches its fuel utilization NADH ratio and increases PDHK expression and activity, from glucose to lipid oxidation (3). Insulin resistance, type thereby inhibiting PDH in muscle; this is reversible with 2 diabetes, and obesity are strongly associated with im- refeeding or insulin treatment (16). Besides phosphor- paired skeletal muscle substrate metabolism including ylation, recent studies suggest that reversible acetylation/ decreased fasting lipid oxidation, impaired postprandial deacetylation may also regulate PDH catalytic subunit E1a glucose oxidation, and reduced capacity for lipid oxidation (PDH E1a) function (17–19), although the pathways con- during exercise (4,5). Thus, the ﬂexibility and capacity of trolling this process have not been fully elucidated. In recent years, NAD -dependent deacetylases called substrate metabolism are compromised in muscle in these states. sirtuins (Sirt) have been shown to play important roles in metabolism (20,21). Among seven members of this protein family, Sirt3 is identiﬁed as the major mitochondrial From the Section on Integrative Physiology and Metabolism, Joslin Diabetes deacetylase (22,23). Several recent studies have shown Center, Harvard Medical School, Boston, Massachusetts; the Buck Institute 3 that Sirt3 regulates lipid metabolism, energy production, and for Research on Aging, Novato, California; the Department of Medicine, stress response in different tissues through its deacetylase Duke University Medical Center, Durham, North Carolina; the Gladstone Institute of Virology and Immunology, San Francisco, California; and the activity (24–26). In muscle, Sirt3 expression is regulated Department of Medicine, University of California, San Francisco, San Fran- by nutrient signals and contractile activity and impacts cisco, California. downstream signaling events through AMP-activated pro- Corresponding author: C. Ronald Kahn, [email protected]. Received 27 November 2012 and accepted 26 June 2013. tein kinase activation and PPARg coactivator-1a expres- DOI: 10.2337/db12-1650 sion (27,28). Sirt3 was implicated in the development of This article contains Supplementary Data online at http://diabetes metabolic disease in humans when a commonly identiﬁed .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1650/-/DC1. E.J. and B.T.O. contributed equally to this study. polymorphism that decreases Sirt3 activity was found to 2013 by the American Diabetes Association. Readers may use this article as be associated with the development of metabolic syndrome long as the work is properly cited, the use is educational and not for proﬁt, (29). We previously demonstrated that skeletal muscle Sirt3 and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. expression is downregulated in rodent models of diabetes 3404 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES reversed-phase nano-high-performance liquid chromatography electrospray and upregulated by caloric restriction and that decreased tandem mass spectrometry (HPLC-ESI-MS/MS) on a QSTAR Elite mass spec- Sirt3 expression induces oxidative stress and impairs trometer. The resulting MS/MS datasets were analyzed using Mascot 2.3.2 insulin signaling in muscle (30). Sirt3 also regulates levels (Matrix Sciences) and Protein Pilot 4.1 (AB Sciex, Foster City, CA) searched of reactive oxygen species (ROS) through deacetylation of against the mouse SwissProt database (SwissProt 2011_04). Peptide scores SOD2 (26,31). In the current study, using a combination of outside of the 5% local false discovery rate were excluded. Skyline MS1 Fil- proteomic, metabolomic, and functional approaches, we tering (34) was used to quantify changes in the MS1 ion abundances among acetylated peptides between WT and KO mice. demonstrate that skeletal muscle Sirt3 regulates substrate PDH activity assay. PDH activity was measured using two methods. The ﬁrst metabolism by targeting mitochondrial PDH E1a subunit used the Mitosciences Pyruvate Dehydrogenase Enzyme Activity Microplate and PDH enzyme activity and thus optimizes the complex Assay Kit (Abcam) in which PDH complexes from isolated mitochondria or and intricate switch of substrate utilization between glu- cellular lysates are immunocaptured on a microplate. A reaction medium cose and lipid oxidation and substrate ﬂexibility. containing pyruvate and NAD is then added. The readout is the rate of pro- duction of NADH, which is coupled to a dye whose formation is monitored on a spectrophotometric plate reader, and activity is calculated from the rate of RESEARCH DESIGN AND METHODS change in optical density at 410 nm. For in vivo experiments, muscle mito- Animal studies were performed according to protocols approved by the In- chondria were isolated as previously described (35), and equal amounts of 2/2 stitutional Animal Care and Use Committee. Male C57Bl/6 mice or Sirt3 and mitochondrial protein were loaded into the immunocapture plate. For in vitro wild-type (WT) littermate controls backcrossed onto a C57Bl/6 background experiments, conﬂuent myoblasts were treated as speciﬁed in the manu- and maintained on a standard chow diet were used. Fed mice were allowed ad facturer’s protocol. For speciﬁed experiments, PI cocktails 2 and 3 (Sigma) or libitum access to food and killed at 9:00 A.M. For fasting studies, mice were 20 mmol/L dichloroacetate (DCA) was added to detergent-soluble fraction and transferred to a new cage without food for 24 h and then killed or refed for buffer 1 prior to microplate incubation. Activity was normalized to protein 4 or 16 h prior to sacriﬁce. content of the detergent-soluble fraction. For Western blots of extracts from Insulin signaling in muscle strips. Extensor digitorum longus (EDL) or PDH activity experiments, an aliquot of detergent soluble extracts was in- hemidiaphragms were dissected and incubated for 30 min in Krebs-Henseleit cubated at room temperature for 3 h to mimic microplate conditions and buffer (KHB) at 37°C and then transferred to KHB with or without 1 or 10 mU/mL blotted using phospho-speciﬁc PDH E1a antibodies. insulin for 10 min. Muscle strips were blotted and snap frozen in liquid PDH activity was also measured in whole muscle homogenates based on 14 14 the evolution of CO from C-labeled pyruvate (36). Brieﬂy, gastrocnemius nitrogen. muscle from randomly fed mice was snap frozen until use. Muscle homoge- Glycolysis, glycogen synthesis, glucose oxidation, and palmitate nates (50 mg wet weight/mL) were incubated for 15 min at 37°C with either 1) oxidation in muscle strips. Glycolysis, glycogen synthesis, glucose oxida- “inactivation” buffer containing 50 mmol/L NaF and 8 mmol/L ATP to prevent tion, and palmitate oxidation were measured in isolated muscle strips as dephosphorylation of the PDH complex with or without the addition of previously described with the following modiﬁcations (32). Brieﬂy, hemi- deacetylase inhibitors (1 mmol/L nicotinamide and 1 mmol/L trichostatin A) diaphragms and EDL muscles were dissected and incubated for 30 min in KHB or 2) “activation” buffer containing 1 mg recombinant PDH phosphatase 1 gassed with 95% O and 5% CO . Muscle strips were transferred to gassed KHB 2 2 (Abcam), 5 mmol/L DCA, 9 mmol/L MgCl , and 0.1 mmol/L CaCl with or containing 5 mmol/L glucose and 0.2 mmol/L palmitate bound to 3% fatty acid– 2 2 free BSA, with or without 1 mU/mL insulin, and with radiolabeled tracers. For without deacetylase inhibitors. Activity was then measured in 20 mL homo- glycolysis, glycogen synthesis, and glucose oxidation, incubation buffer con- genates diluted in reaction buffer for 10 min and terminated with 20% tri- 14 3 tained 20 mCi/mmol [U- C]glucose and 80 mCi/mmol [5- H]glucose, and chloroacetate and 30 mmol/L pyruvate as described in the protocol. Evolved muscles were incubated for 1 h with shaking at 37°C. Reactions were termi- CO was collected in suspended center wells containing 1 mol/L hyamine nated by removal of the tissue from the incubation medium, followed by in- hydroxide in methanol and counted in 4 mL Cytoscint scintillation ﬂuid. Ac- jection of 0.2 mL hyamine hydroxide into the center wells and 0.1 mL 70% tivity of homogenates treated with inactivating solution was termed “PDHa,” (w/v) HClO into 1 mL of the contents of the ﬂask. The rate of glucose oxi- as this likely represents the native activation of PDH in muscle. Activity of dation was determined from the production of CO . Glycogen was puriﬁed homogenates treated with activating solution was termed “PDHt,” represent- by digestion of tissue in 1 mol/L NaOH and then precipitation in 66% ethanol ing total activity of PDH present in muscle. with 100 mg unlabeled glycogen to determine the amount of glycogen syn- ATP and glycogen measurement. ATP levels were measured by an enzymatic 14 3 3 thesized from [U- C]glucose and [5- H]glucose incorporation. H Oformation coupled assay as previously described (37). Brieﬂy, metabolites were isolated was measured by separating H O from 0.5 mL incubation buffer containing from pulverized muscle in 6% perchloric acid and neutralized with KOH [5- H]glucose on Poly-Prep Columns AG 1-X8-731-6212 (Bio-Rad) pretreated with and imidazole, and then ATP was measured as the change in optical 1 mol/L NaOH and then 0.3 mol/L Boric acid. The rate of glycolysis was de- density at 340 nm after addition of glucose, NADP , glucose-6-phosphate termined from thedifferencebetween therateof H O formed and the rate of dehydrogenase, and hexokinase. Glycogen content was measured as substrate recycling determined from the difference between the rates of previously described (38). 14 3 glycogen synthesis from [U- C]glucose and [5- H]glucose. Immunoprecipitation and Western analysis. Powdered muscle tissue, Cell culture maintenance. C2C12 cells (American Type Culture Collection, isolated muscle mitochondria, or conﬂuent myoblasts were homogenized in Manassas, VA) were maintained in high-glucose Dulbecco’s modiﬁed Eagle’s radioimmunoprecipitation assay buffer (Millipore) with protease and PIs medium (DMEM) (Invitrogen) containing 10% FBS (Gemini Bioproducts) un- (Sigma) and deacetylase inhibitors (10 mmol/L nicotinamide/1 mmol/L tri- less otherwise indicated. Sirt3 short hairpin RNA (shRNA) and short hairpin chostatin A). Lysates were subjected to SDS-PAGE and blotted using PDH green ﬂuorescent protein (shGFP) control lentiviral constructs were pur- E1a, PDHK4, LCAD, glyceraldehyde-3-phosphate dehydrogenase, insulin re- chased from Open Biosystems (Huntsville, AL). Stable Sirt3 knockdown and ceptor b (Santa Cruz), phospho-IR/IGFR, phospho-Akt, phospho-ERK, Akt, shGFP control cell lines were generated by viral transduction of C2C12 extracellular signal–related kinase (ERK), VDAC, Sirt3, (Cell Signaling), or myoblasts and selection with puromycin. Wild-type PDH E1a cDNA lentiviral acetyl-lysine (Immunechem or Cell Signaling) antibodies. For immunoprecip- construct (Genecopeia) was used for site-directed mutagenesis (Stratagene) itation assays, mitochondria were isolated in the presence of protease and to generate K336Q and K336R mutations and further sequenced. WT, K336Q, deacetylase inhibitors as previously described (35), resuspended in IP lysis and K336R overexpressing stable C2C12 cell lines were generated. buffer (Pierce), and immunoprecipitated with anti–acetyl-lysine (AcK) agarose Proteomic analysis of acetylated skeletal muscle mitochondrial beads (Immunechem) with streptavidin beads (Pierce) as a control. peptides. Mitochondria were isolated from hindlimb muscle of WT and Oxygen consumption rate and extracellular acidiﬁcation rate assays. SIRT3 KO mice as previously described (33) with the following adaptations: Cellular oxygen consumption rate (OCR) was measured using a Seahorse Muscle was homogenized with teﬂon-pestle in Medium 1 with deacetylase Bioscience XF24 ﬂux analyzer. Cells were seeded at 30,000 cells/well 24 h prior inhibitors (250 mmol/L sucrose, 1 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH to the analysis in low-glucose (100 mg/dL) DMEM containing 10% FBS. Each 7.4; protease and phosphatase inhibitors [PIs] [Sigma]; 10 mmol/L nicotinamide; experimental condition was analyzed using four to six biological replicates. For and 1 mmol/L trichostatin A). Mitochondria were enriched by differential cen- OCR experiments using palmitate, KHB buffer (pH 7.4) was added to each well trifugation and puriﬁed by centrifugation at 59,800g on 5–25% (w/v) linear Ficoll and measurements were performed every 3 min with 2 min intermeasurement (Sigma) gradients. Puriﬁed mitochondria were lysed in radioimmunoprecipitation mixing. BSA-conjugated palmitate (ﬁnal concentration 200 mmol/L) and assay buffer containing protease, phosphatase, and deacetylase inhibitors. etomoxir (ﬁnal concentration 50 mmol/L) were injected sequentially. For ex- Approximately 1 mg total mitochondrial protein was digested with trypsin, and tracellular acidiﬁcation rate (ECAR) experiments with glucose as a substrate, an acetylated peptide fraction was prepared using a combination of anti-AcK sodium carbonate and glucose/pyruvate-free DMEM was used. Glucose and antibodies from ImmuneChem (cat. no. ICP0380-100) and Cell Signaling 2-deoxyglucose were injected sequentially to give ﬁnal concentrations of (cat. no. 9441) as previously described (34). Peptides were analyzed by 25 mmol/L. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3405 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY Metabolomic assays for skeletal muscle amino acids, organic acids, and KO mice to fully characterize muscle substrate metabolism acylcarnitines. Amino acids, acylcarnitines, and organic acids were analyzed in the absence of Sirt3. Glucose oxidation in EDL muscles using stable isotope dilution techniques. Amino acids and acylcarnitine (composed primarily of glycolytic ﬁbers) in the presence measurements were made by ﬂow injection MS/MS using sample preparation of 5 mmol/L glucose, 0.2 mmol/L palmitate, and 1 mU/mL methods described previously (39,40). The data were acquired using a Micro- insulin was signiﬁcantly decreased in Sirt3 KO mice com- mass Quattro MicroTM system equipped with a model 2777 autosampler, a model 1525 HPLC solvent delivery system, and a data system controlled by pared with controls (Fig. 2A). However, glycogen synthe- MassLynx 4.1 operating system (Waters, Millford, MA). Organic acids were sis rates responded normally to insulin, and glycolytic quantiﬁed using methods described previously with Trace Ultra GC coupled to rates were unchanged in Sirt3 KO, suggesting that glucose a Trace DSQ MS operating under Xcalibur 1.4 (Thermo Fisher Scientiﬁc, uptake was normal but that the end products of glycolysis Austin, TX) (41). were not fully oxidized in Sirt3 KO muscle generating Statistical analyses. All data are means 6 SEM. Student t test was performed lactate. Palmitate oxidation rate was quite low in EDL for comparison of two groups or ANOVA was performed for comparison of three or more groups to determine signiﬁcance. muscle and was unchanged between KO and WT. To de- termine whether these changes were due to insulin re- sistance, as we have observed in older 24-week animals RESULTS (30), we measured insulin signaling in muscle strips from Sirt3 expression and mitochondrial acetylation is 8- to 16-week-old Sirt3 KO mice and controls. Interestingly, regulated by fasting. Quantitative real-time PCR using in this ex vivo experiment in EDL strips from younger mice in the fed or fasted state, we found no difference in insulin quadriceps (Quad), EDL, and soleus muscles from 8-week- old male WT C57Bl/6 mice revealed that 24 h of fasting signaling between Sirt3 KO mice and controls (Fig. 2B). In hemidiaphragms (composed of more oxidative ﬁbers) suppressed Sirt3 mRNA expression in hindlimb muscles (Fig. 1A). All of these reductions returned to close to fed palmitate oxidation was increased in the presence of in- levels by 16 h of refeeding and the EDL muscle showed sulin, and glucose oxidation was not signiﬁcantly changed some recovery as early as 4 h after refeeding (Supplemen- in Sirt3 KO but tended to decrease (Fig. 2C). Again, there tary Fig. 1A). Western blotting analysis conﬁrmed a parallel was no change in insulin signaling, glycolysis, or glycogen ;50% decrease of Sirt3 protein level in soleus and gas- synthesis rates in hemidiaphragms from Sirt3 KO com- trocnemius muscle from fasted mice, which increased after pared with WT controls (Supplementary Fig. 2A and B). refeeding to near-normal levels, as we have previously de- These changes in substrate metabolism did not affect total scribed in quadriceps (30) (Fig. 1B). There was some vari- glycogen content or ATP levels in tissues isolated in the fed ability in the recovery of Sirt3 levels from experiment to or fasted states, and AMP-activated protein kinase phos- experiment, but a decrease in Sirt3 levels in the fasted phorylation was decreased in Sirt3 KO muscle upon fasting state was observed in all muscle groups tested (Supple- as previously described (Supplementary Fig. 2D and E)(27). mentary Fig. 1A and C). In the fasted state, levels of ex- The decreased glucose oxidation rate in the insulin- stimulated state in muscle samples from Sirt3 KO mice pression also varied, with muscle ﬁber type being highest in red soleus and lower in white EDL muscle. indicated a potential regulatory role for Sirt3 on mito- Western analysis of isolated mitochondria from skeletal chondrial glucose oxidation. Since PDH E1a function can muscle using an antibody against AcK revealed a general be regulated by phosphorylation on three serine residues increase of mitochondrial protein acetylation after 24 h of (42), we assessed phosphorylation of PDH E1a in WT and fasting, coinciding with decreased Sirt3 levels. We ob- Sirt3 KO skeletal muscle to determine whether deletion of served a speciﬁc increase in acetylation of bands at 97, 72, Sirt3 mimics the effect of fasting on posttranslational and 47 kDa (Fig. 1C). The lower hyperacetylated band was modiﬁcation of PDH E1a. Knockout of Sirt3 resulted in an in the migration position of PDH catalytic subunit E1a.We increase in the phosphorylation of PDH E1a on S300 and immunoprecipitated muscle mitochondrial lysates isolated tended to increase S232 (but not S293) in muscle of fed from either fed or fasted mice using anti-AcK antibody and mice (Fig. 2D and Supplementary Fig. 2F), mimicking the subjected the immunoprecipitates to Western blotting increased phospho–PDH E1a observed in fasted WT mice. analysis with anti–PDH E1a antibody. The abundance of the As observed in fasting, increased PDH E1a acetylation and PDH E1a protein from the immunoprecipitates represents phosphorylation in Sirt3 KO occurred without changes in its acetylation status. This analysis conﬁrmed hyperacet- the total level of PDH E1a or PDHK4 in either the fed or ylation of PDH E1a in fasted skeletal muscle (Fig. 1D). the fasted state (Fig. 2D and F). Posttranslational modiﬁcations known to suppress PDH Posttranslational modiﬁcations like phosphorylation activity include PDH kinase–mediated phosphorylation of PDH E1a are known to have effects on PDH enzymatic targeting three serine residues of PDH E1a (S232, S293, activity (14,43); thus, we investigated whether the in- and S300) (42). We tested serine phosphorylation of PDH creased acetylation and phosphorylation of PDH E1a re- E1a in muscle mitochondria using phospho-speciﬁc anti- lated to Sirt3 deletion would change catalytic activity of bodies and found that S232 and S300 phosphorylation was the PDH enzyme complex. Indeed, PDH activity measured increased during fasting coincident with the PDH E1a by the immunocapture assay (see RESEARCH DESIGN AND hyperacetylation (Fig. 1D). The changes in PDH E1a acet- METHODS) was signiﬁcantly reduced in Sirt3 KO mitochon- drial lysates compared with WT. Quantiﬁcation of the ylation/phosphorylation after fasting were not explained by changes in PDH E1a total protein levels (Fig. 1D). slopes of the linear regressions demonstrated a .70% in- Sirt3 deletion induces a shift in substrate utilization hibition of PDH activity in KO mice (Fig. 2E). To further away from glucose oxidation and increases PDH E1a elucidate the possible regulatory role of acetylation on phosphorylation suppressing PDH activity. We hy- PDH activity, we determined active and total PDH activity pothesized that Sirt3 has its most prominent role in met- in muscle homogenates from fed Sirt3 KO and WT mice as abolic regulation in the fed state, when it is present at well as fasted C57Bl/6 mice by measurement of CO highest levels. To test this hypothesis, we assessed glucose production from C-labeled pyruvate in the presence or and palmitate oxidation, as well as glycolysis and glycogen absence of deacetylase inhibitors (see RESEARCH DESIGN AND synthesis, in isolated muscle strips from fed WT and Sirt3 METHODS). As in the mitochondrial isolates, PDHa activity 3406 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 1. Skeletal muscle Sirt3 expression and mitochondrial protein acetylation are regulated by fasting. Wild-type 8-week-old male C57Bl/6 mice were fed ad libitum, fasted for 24 h, or fasted and then refed for 16 h. After each treatment, RNA and protein were extracted and analyzed by real- time quantitative PCR (A) or Western blotting (B) for assessment of Sirt3 expression in quadriceps (Quad), EDL, soleus (Sol), and gastrocnemius (Gast) muscle groups (n =3–5, #P < 0.05 vs. fed, ANOVA). C: Mixed hindlimb muscles (gastrocnemius and soleus) were collected from mice in the fed or fasted state. Muscle mitochondria were isolated in the presence of protease and deacetylase inhibitors as described in RESEARCH DESIGN AND METHODS. Mitochondrial protein lysates from each animal were subjected to SDS-PAGE and Western blotting using an antibody against AcK. The intensity of speciﬁed bands was quantiﬁed with ImageJ software (n =3,*P < 0.05, Student t test). D: PDH E1a acetylation in muscle of fed or fasted mice was measured by immunoprecipitation (IP) of mitochondrial lysates using anti-AcK antibody. Immunoprecipitates were subjected to Western blotting analysis (IB) using anti–PDH E1a antibody. The same muscle mitochondrial lysates were directly subjected to SDS-PAGE electrophoresis and Western blotting using antibodies against phosphorylated serine sites p-232, p-293, and p-300 of the PDH E1a subunit and total protein of PDH E1a. Autoradiography of Western blots was quantiﬁed with ImageJ software (n =3,*P < 0.05, Student t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. was signiﬁcantly decreased in Sirt3 KO muscle to the level act on PDH, partially restored PDH activity from Sirt3 KO of fasted C57Bl/6 mice (Fig. 2G) without any changes in mice. Since inhibition of PDH activity in fasting muscle has total PDH (Supplementary Fig. 2C). Remarkably, incubation profound effects on substrate metabolism, the observed of the muscle homogenates in the absence of deacetylase reduction of PDH activity induced by Sirt3 deletion inhibitors, which likely allows other deacetylases outside demonstrates that Sirt3 is an important regulator of the the mitochondria but present in the tissue homogenate to metabolic changes in fed skeletal muscle. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3407 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 2. Sirt3 deletion induces synergistic switch from glucose oxidation toward fatty acid oxidation and suppresses PDH activity by inducing PDH E1a hyperacetylation and hyperphosphorylation in fed Sirt3 KO skeletal muscle. A: Basal and insulin-stimulated (1 mU/mL) glucose oxidation, glycogen synthesis, glycolysis, and palmitate oxidation were measured in EDL muscles isolated from randomly fed 8- to 16-week-old WT and Sirt3 KO mice (n =9;§P < 0.05 vs. basal, #P < 0.05 vs. WT, ANOVA). B: Basal and insulin-stimulated (10 mU/mL) phosphorylation of insulin/IGF-1 receptor, Akt, and ERK in tissue lysates of EDL muscles from fed and fasted 8- to 16-week-old WT and Sirt3 KO mice. C: Insulin-stimulated (1 mU/mL) glucose and palmitate oxidation were measured as described in RESEARCH DESIGN AND METHODS in diaphragms isolated from randomly fed 8- to 16- week-old WT and Sirt3 KO mice (n =9; *P < 0.05, Student t test). D: Mitochondrial lysates were subjected to Western blot analysis using anti- bodies against p-232, p-293, and p-300 serine phosphorylation sites on PDH E1a, total PDH E1a, and Sirt3 (densitometry in Supplementary Fig. 2F). E: PDH activity was measured in 20 mg mitochondrial lysate from hindlimb of fed WT and Sirt3 KO mice using a PDH activity microplate kit as described in RESEARCH DESIGN AND METHODS. The PDH activities were calculated by linear regression of the steady-state kinetics and quantiﬁed (n =4–5, 3408 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES PDH E1a subunit is a Sirt3 substrate. Using anti-AcK Knockdown of Sirt3 in C2C12 myoblasts decreases antibody to detect protein acetylation, we observed that PDH activity. To further elucidate the role of Sirt3 in PDH Sirt3 KO muscle mitochondria displayed an elevation in function, we determined PDH E1a acetylation and enzyme acetylation similar to that seen in fasted WT mice (Fig. activity in C2C12 myoblasts in which Sirt3 was knocked 3A), supporting the idea that Sirt3 is the primary mito- down using shRNA (shSirt3). This resulted in a .90% de- chondrial deacetylase and consistent with our previous crease in Sirt3 protein by Western analysis (Fig. 5A, C, and report in Sirt3 KO fed mice (23,30). To identify the po- D). Immunoprecipitation of mitochondrial lysates from tential Sirt3 targets in skeletal muscle during fasting (when shGFP control and shSirt3 myoblasts using anti-AcK anti- Sirt3 levels are low) and in Sirt3 KO muscle, we performed body revealed signiﬁcantly increased acetylated PDH E1a a global acetyl-proteomic analysis (34). To this end, acet- in knockdown cells (Fig. 5A). The increased acetylation of ylated peptides were enriched from trypsinized mito- PDH E1a in vitro was associated with a 27% decrease in chondrial protein lysates of WT and Sirt3 KO muscle using PDH activity in shSirt3 myoblasts measured by microplate AcK antibody immunoprecipitation and subjected to mass immunocapture of PDH (Fig. 5B). However, in contrast to spectrometry analysis. A total of 549 acetylated peptides results in skeletal muscle in vivo from Sirt3 KO mice, shSirt3 were identiﬁed in the combined WT and KO muscle mi- myoblasts showed decreased phosphorylation at both S232 tochondria. Among these, 299 acetylated peptides were and S300 sites (Fig. 5C and D), suggesting that PDH E1a present in both WT and KO samples, only 49 of which were acetylation may inhibit PDH activity independent of detected exclusively in WT, whereas 201 acetylated pep- phosphorylation. tides were exclusive to the Sirt3 KO, indicating increased Previous bioenergetic proﬁling using Seahorse XF ﬂux acetylation induced by Sirt3 deletion (Fig. 3B). These 549 analyzer with glucose and pyruvate as substrates showed acetylated peptides corresponded to a total of 147 acety- that shSirt3 myoblasts had signiﬁcantly lower uncoupled lated proteins, indicating that there were multiple acety- respiration (30). Since changes in the metabolomic proﬁle lation sites for each protein (Fig. 3B). Our analysis revealed suggested a switch in substrate utilization in Sirt3 KO mus- several lysine acetylation sites on PDH E1a (Fig. 3C). cles, we measured glucose and fatty acid oxidation occurred Among the peptides showing signiﬁcantly increased acet- in a cell autonomous manner using shSirt3 knockdown ylation in Sirt3 KO muscle mitochondria was one peptide myoblasts. Basal rates of lactate production, as indicated representing acetylated lysine 336 (K336) of PDH E1a by ECAR, were similar in control and shSirt3 myoblasts. (Supplementary Fig. 3A). Quantitation of the MS1 pre- When given glucose as substrate, shSirt3 cells displayed cursor signals of this peptide between the four WT and a greater increase in ECAR compared with control (Fig. 5E). four KO mice showed a threefold increase in acetylation at The area under the curve (AUC) quantiﬁcation of ECAR K336 in the Sirt3 KO animals (Fig. 3D), indicating that was signiﬁcantly higher in shSirt3 after glucose stimulation K336 is a substrate of Sirt3. (Fig. 5F). As previously reported, OCRs were lower in To conﬁrm that Sirt3 directly acetylates PDH, we immu- shSirt3 myoblasts (Supplementary Fig. 4A and B) (30). noprecipitated WT and Sirt3 KO muscle mitochondrial OCR in the presence of 200 mmol/L palmitate was signiﬁ- lysates from both fed and fasted mice using anti-AcK anti- cantly higher in shSirt3 cells, and this was inhibited by body and subjected the immunoprecipitates to Western addition of etomoxir, indicating higher rates of fatty acid blotting analysis with anti-PDH E1a antibody. This revealed b-oxidation in shSirt3 compared with control cells. AUC that PDH E1a acetylation was increased signiﬁcantly in calculation after palmitate showed a ;35% increase in OCR Sirt3 KO muscle mitochondria (Fig. 3E), and this was in shSirt3 (Fig. 5G and H). Together, these data demon- speciﬁc, as no enrichment was observed using streptavidin strate that decreased Sirt3 expression induces a switch in beads as a control (Supplementary Fig. 3). muscle substrate utilization from glucose to fatty acid oxi- Sirt3 deletion in skeletal muscle induces an altered dation to compensate for decreased carbohydrate oxidation metabolic proﬁle. To fully assess the metabolic effects caused by inhibition of PDH activity. of PDH E1a inhibition in Sirt3 KO skeletal muscle, we Overexpression of K336Q or K336R mutants of PDH performed targeted gas chromatography/mass spectrome- E1a decreases phosphorylation at S232 and S300 but try and MS/MS metabolomic analysis on samples of skel- does not affect PDH activity or substrate metabolism. etal muscle from fed WT and Sirt3 KO mice. We found To determine whether acetylation at lysine 336 of PDH E1a signiﬁcantly elevated levels of lactate, pyruvate, and a- was sufﬁcient to affect PDH activity, we created C2C12 ketoglutarate (a-KG) in Sirt3 KO skeletal muscle (Fig. 4A). myoblast cell lines with stable overexpression of WT PDH The increase in lactate and pyruvate in Sirt3 KO muscles is E1a or with either a K336Q or a K336R mutation. In some consistent with suppression of PDH activity and decreased molecules, the lysine (K) to glutamine (Q) mutation can carbohydrate oxidation. We also observed a signiﬁcant mimic the acetylated state, while the K to arginine (R) decrease in levels of acylcarnitines measured in Sirt3 KO prevents acetylation and mimics the deacetylated state. muscle, suggestive of increased fatty acid use (Fig. 4B). Both K336Q and the K336R mutations caused a reduction in Additionally, amino acids levels were decreased in Sirt3 phosphorylation of S232 and S300 compared with over- KO muscle (Fig. 4C). Taken together, these metabolomic expression of WT PDH E1a (Fig. 6A and B), indicating an data indicate that Sirt3 deletion induces a metabolic de- important role of lysine 336 in control of serine phosphor- rangement in skeletal muscle in which impaired PDH ac- ylation. To dissect the effect of phosphorylation status tivity suppresses glucose oxidation and enhances lipid and versus K336 mutation on PDH activity, we determined PDH amino acid catabolism. activity in the presence of PIs to promote phosphorylation *P < 0.05, Student t test). F: Western blot analysis was performed on tissue lysates from gastrocnemius muscle from fed and fasted WT and Sirt3 KO for PDHK4, Sirt3, and glyceraldehyde-3-phosphate dehydrogenase. G: Native PDH activity (PDHa) was measured in gastrocnemius muscle 14 14 homogenate from WT fed, Sirt3 KO fed, and fasted C57Bl/6 mice by collection of CO release from C-pyruvate. Prior to PDH assay, aliquots of homogenates were incubated in the presence of 50 mmol/L NaF with or without deacetylase inhibitors (1 mmol/L nicotinamide and 1 mol/L tri- chostatin A) as described in RESEARCH DESIGN AND METHODS (n =6–7; #P < 0.05 vs. WT plus deacetylase inhibitors, ANOVA). OD, optical density. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3409 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 3. Discovery of PDH E1a subunit as a target of Sirt3 in skeletal muscle. A: Muscle mitochondrial protein lysates from WT and Sirt3 KO mice in either the fed or fasted state were subjected to Western blotting analysis using anti-AcK antibody. B: Proteomic analysis using anti-AcK antibody– based acetylated peptide enrichment and mass spectrometry discovered a total of 549 acetylated peptides present in WT and Sirt3 KO skeletal muscle mitochondria. These peptides represented a total of 147 proteins. Venn diagrams show overlapping and distinctive patterns of distribution of acetylated peptides and proteins between WT and Sirt3 KO skeletal muscle. C: Schematic diagram of lysine acetylation sites and previously 3+ reported serine phosphorylation sites on PDH E1a. D: A representative MS1 chromatogram of the triply charged precursor peak at m/z 772.7303 (324-MVNSNLASVEELacKEIDVEVR-343) of PDH E1a was highly increased in Sirt3 KO skeletal muscle mitochondria compared with WT (n =4; P < 0.05, Student t test). E: Skeletal muscle mitochondrial lysates from WT and Sirt3 KO mice in both fed and fasted states were immunoprecipitated (IP) with AcK antibody–bound beads. Immunoprecipitates of the mitochondrial lysate were subjected to Western blotting (IB) using an anti–PDH E1a antibody. Densitometry of either fed or fasted animals was calculated (n =4;*P < 0.05, Student t test). 3410 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 4. Sirt3 deletion induces a substrate switch and derangement of metabolites in skeletal muscle. Quadriceps muscles from fed male WT and Sirt3 KO mice were collected and subjected to metabolomic analysis as described in RESEARCH DESIGN AND METHODS. Relative levels of organic acids and Kreb cycle intermediates are shown in A, levels of acylcarnitines in B, and amino acid levels in C (n =4–5; *P < 0.05, Student t test). Asx, asparagine and aspartic acid; Cit, citrulline; Glx, glutamine and glutamic acid; Orn, ornithine. or DCA, a PDH kinase speciﬁc inhibitor, to decrease phos- recapitulate the effects of decreased Sirt3 expression on phorylation of PDH E1a. PDH activity was unchanged in PDH complex activity and substrate metabolism. myoblasts harboring either of the K336Q or K336R mutations compared with WT controls (Fig. 6C) with or without ad- DISCUSSION dition of PI to preserve S293 and S300 phosphorylation or addition of DCA to maximally dephosphorylate PDH E1a Mitochondrial sirtuins are uniquely positioned to regulate (Fig. 6D). energy metabolism via protein deacetylation. Although To assess whether K336Q or K336R mutations could Sirt3, Sirt4, and Sirt5 have all been localized to mito- recapitulate the substrate switch observed in shSirt3 chondria, previous reports have shown that only Sirt3 cells, we analyzed glucose-induced ECAR in WT, K336Q, deletion induces mitochondrial protein hyperacetylation, or K336R myoblasts. No signiﬁcant differences in glucose- suggesting that Sirt3 is the major mitochondrial protein stimulated ECAR were observed in the K336Q or K336R deacetylase (23). Indeed, recent studies have shown that mutant cells compared with control (Fig. 6E and F). Thus, altered Sirt3 expression can have effects on lipid metab- altering the ability of K336 to undergo acetylation did olism, ROS production, oxidative stress response, and cell modify phosphorylation of PDH E1a but could not fully survival (25,26,30,44,45). diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3411 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 5. Sirt3 knockdown in C2C12 myoblasts impairs PDH activity despite decreased phosphorylation of PDH E1a and leads to a substrate switch toward fatty acid utilization. A: Total mitochondrial protein lysates from shGFP control and shSirt3 myoblasts were immunoprecipitated (IP) with AcK (AcLys) antibody and subjected to Western blot analysis (IB) using an anti–PDH E1a antibody. The same mitochondrial lysates were directly subjected to Western blot analysis using antibodies against PDH E1a, Sirt3, and voltage-dependent anion channel (VDAC) as a mitochondrial loading control. Densitometry of PDH E1a from AcK immunoprecipitates was normalized to total PDH E1a (n = 4 separate experiments; *P < 0.05, Student t test). B: Total PDH activity was assessed in conﬂuent control and shSirt3 myoblasts using PDH activity microplate assay kit and nor- malized to total protein from detergent extraction (n = 5 separate experiments; †P < 0.05, paired t test). C: Phosphorylation of PDH E1a and total Sirt3 levels were determined by Western blot analysis of whole cell lysates from conﬂuent shGFP and shSirt3 C2C12 myoblasts. D: Densitometry of Western blots from C (n = 3 separate experiments, *P < 0.05, Student t test). E: ECAR was measured in shSirt3 and control myoblasts using aSeahorse ﬂux analyzer after incubation in glucose-free Seahorse running media for 1 h at 37°C. A representative tracing of basal and 3412 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES In the current study, we ﬁnd that Sirt3 is an important Another interesting aspect of Sirt3 physiology is the mitochondrial factor in the regulation of skeletal muscle differential regulation and physiological effects of this metabolic ﬂexibility by targeting the enzymatic deacetyla- enzyme in different tissues. For example, during fasting tion of PDH E1a. Thus, when nutrients are abundant, Sirt3 Sirt3 expression is decreased in muscle but increased in deacetylation promotes PDH activity and postprandial liver (25,30). Mitochondrial protein acetylation patterns glucose metabolism (Fig. 7A). During fasting, there is conﬁrm that Sirt3 is more active in the fed state in skeletal a decrease of Sirt3 in skeletal muscle, which leads to muscle, whereas in liver Sirt3 is more active during fasting. hyperacetylation of PDH E1a and promotes a metabolic Sirt3 also has different tissue-speciﬁc metabolic effects. switch from glucose to fatty acids as a predominant sub- Sirt3 deletion in liver suppresses lipid oxidation via sup- strate (Fig. 7B). This can be mimicked by Sirt3 deletion, pression of LCAD activity (25), whereas deletion of Sirt3 in which results in decreased catalytic activity of PDH, de- muscle decreases glucose oxidation and increases lipid creased glucose oxidation, and an accumulation of pyru- oxidation, at least in part to compensate for PDH inhibition. vate and lactate levels even in the fed state. Impaired This dichotomous role of Sirt3 in muscle versus liver may glucose utilization in Sirt3 KO muscle induces a reliance help to explain the report showing minimal changes in on fatty acid b-oxidation for energy production. This global metabolism when Sirt3 is deleted in a tissue-speciﬁc results in increased breakdown of acylcarnitines and ROS manor (49). Muscle-speciﬁcdeletionof Sirt3 maylead to generation and over time leads to insulin resistance (30). changes in mitochondrial substrate choice, which are Previous reports have suggested that 24 h of fasting compensated for by opposing Sirt3 action in liver, resulting increases Sirt3 levels in muscle (27), but in our hands, 24 h in a balance at the whole-body level. Nonetheless, this of fasting leads to a consistent decrease in Sirt3 in each differential regulation of Sirt3 expression and function in a muscle group tested. The difference between these two tissue-dependent manner would allow Sirt3 to coordi- observations may be explained by the timing of the study nately regulate divergent pathways of substrate utilization (mice were killed at 6 P.M. in the former study and at 9 A.M. in different organs based on nutrient availability. in our study) and the duration of fasting in the different fed Reversible phosphorylation of PDH E1a by PDH kinases and fasted groups. Peak feeding in mice occurs at the inhibits enzyme activity and, in starvation and diabetes, beginning of the nocturnal phase. Thus, fed mice killed at levels of PDH kinases increase in skeletal muscle (14,16,50). 9:00 A.M. are only a few hours after peak feeding time. On PDH kinases can be activated by a decrease in the NAD - the other hand, when mice are killed at 6:00 P.M., even the to-NADH ratio, providing feedback inactivation of the PDH “fed” mice are likely to have fasted or partially fasted for as complex (51). Our studies demonstrate that increased many as 10–12 h prior to study, and the “24-h fasted” mice acetylation of PDH E1a is associated with decreased PDH may have gone without food for as long as 36 h, resulting complex activity, leading us to speculate that PDH acety- in dramatic wasting and changes in muscle protein compo- lation also regulates complex activity. Indeed, PDH activity sition (46). Alternatively, there may be circadian changes in was decreased in both Sirt3 KO muscle in vivo and Sirt3 expression of Sirt3 or some of its regulators, which is altered knockdown myoblasts in vitro; the former was associated 232 300 during prolonged periods of fasting. with increased phosphorylation of Ser and Ser , Metabolic ﬂexibility is the ability of an organism to adapt whereas the latter was associated with decreased serine fuel oxidation in response to fuel availability (47). In both phosphorylation. However, the degree of PDH activity insulin resistance and type 2 diabetes, this metabolic decrease is different in the two models—55–70% in Sirt3 ﬂexibility is compromised in that skeletal muscle fails to KO in vivo compared with 27% in shSirt3 in vitro—which switch substrate utilization from lipid metabolism to could be related to phosphorylation status. Lastly, the insulin-stimulated glucose oxidation (5,48). The current observation that incubation of Sirt3 KO muscle homoge- study demonstrates that mitochondrial Sirt3 can help op- nate in the absence of deacetylase inhibitors partially re- timize the switch of substrate oxidation toward glucose stored PDH activity supports the notion that acetylation of utilization by deacetylating PDH, a vital enzyme complex PDH inhibits its activity. Regulation of enzyme activity by in glucose oxidation. Acute regulation of metabolic ﬂexi- acetylation may also help explain the observed accumula- bility by Sirt3 upon refeeding is not due changes in Sirt3 tion of a-KG, as a-KG dehydrogenase is similar in structure levels, since 16 h of refeeding is required for full recovery and regulation to PDH, in that both use an E1 component of mRNA and protein levels. These acute changes in sub- that may be a target for acetylation and Sirt3-catalyzed strate ﬂexibility going from the fed to the fasted state are deacetylation. Other mechanisms may also contribute to more likely coordinated by a complex network of signaling changes in PDH activity. For example, PDH and a-KG de- cascades including insulin and nutrient-dependent phos- hydrogenase contain many thiol groups, which are sensitive phorylation events. This acute regulation may still be to ROS, and we have previously shown that deletion of Sirt3 affected by Sirt3 intracellular partitioning or possible post- causes increased ROS production in muscle from 24-week- translational modiﬁcation affecting its deacetylase activity. old animals (30). However, our data support the conclusion that Sirt3 plays Mass spectrometry–based analysis revealed that PDH E1a an important role in regulating muscle metabolic ﬂexibility is acetylated on multiple lysine residues. We focused our upon refeeding by deacetylating PDH and, with dephos- attention on lysine 336, since it exhibited one of the greatest phorylation, allows for maximal enzyme activity for glucose levels of differential acetylation in Sirt3-depleted skeletal oxidation. muscle. Our attempts to determine whether acetylation at glucose-stimulated ECARs recorded before and after addition of 25 mmol/L glucose (ﬁnal concentration) is shown. At the end of the glucose metabolism period, 2-deoxyglucose was injected to give a ﬁnal concentration of 25 mmol/L. F: AUC calculation of glucose-stimulated ECAR from E. G: A representative tracing of palmitate OCR measured in control and Sirt3 knockdown cells after incubation with substrate-free buffer for 1 h at 37°C. Basal and palmitate-BSA–stimulated OCRs were recorded and plotted as a percentage over basal OCR. Finally, 50 mmol/L etomoxir was injected. H: AUC of palmitate-stimulated OCR from G (n =3; *P < 0.05, Student t test). mOD, milli optical density. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3413 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY FIG. 6. Expression of a K336Q and K336R mutant of PDH E1a in C2C12 myoblasts decreases phosphorylation but does not affect PDH activity. A: Phosphorylation of PDH E1a levels determined by Western blot analysis of whole cell lysates from conﬂuent C2C12 myoblasts expressing WT PDH E1a, K336Q, or K336R mutant of PDH E1a. B: Densitometry of Western blots from A (n = 3 separate experiments, #P < 0.05, ANOVA). C: Total PDH activity was assessed in conﬂuent WT, K336Q, or K336R myoblasts using detergent extracts either with PIs 2 and 3 (Sigma) or 20 mmol/L DCA added to the PDH activity assay as described in RESEARCH DESIGN AND METHODS (n =2–4 separate experiments). D: Western blot analysis of de- tergent extracts from C either with PI (Phos Inhib) or with DCA after 3 h incubation at room temperature in buffer 1 of the PDH activity assay kit. E: A representative example of Seahorse analysis of glucose-induced ECAR with PDH E1a WT, K336Q, and K336R mutants. F: AUC quantiﬁcation after addition of glucose showed no statistical difference between mutants (n = 4 separate experiments). 2-DG, 2-deoxyglucose. lysine 336 alone could alter PDH activity using the K336Q of diabetes has been proposed as a way to increase glu- (which is regarded as an acetylation mimic) and K336R cose disposal, thereby decreasing circulating glucose lev- (which is regarded as a deacetylation mimic) mutations els (15). However, many of these treatments induce fasting demonstrated that changing the ability of this lysine to hypoglycemia as a result of PDH activation during fasting. become acetylated can cause alterations in phosphoryla- Since Sirt3 levels decrease in muscle with fasting, phar- tion of PDH E1a but did not change catalytic function or macologic activation of Sirt3 in muscle should be beneﬁ- substrate metabolism in vitro. It is likely that acetylation of cial, as it would have maximal effect in the fed state, when other lysine residues in the PDH complex contribute to the glucose levels are highest in diabetes. observed changes in PDH activity. In summary, our study demonstrates that Sirt3 plays an Identifying a role of Sirt3 in regulating metabolic ﬂexi- important role in control of substrate metabolism and adds bility and PDH activity in muscle may provide a new target another layer of regulation to the multiple pathways that for preventing and treating metabolic syndrome. Evidence control metabolic ﬂexibility in skeletal muscle. In the fed for Sirt3 impacting human metabolic disease is accumu- state, Sirt3 expression/activity in muscle is high, resulting lating. We have recently identiﬁed a single nucleotide in deacetylation of many mitochondrial proteins, including polymorphism in the human Sirt3 gene that decreases its PDH E1a. This enhances PDH complex activity and post- activity and is associated with metabolic syndrome (29). prandial glucose metabolism. Thus, Sirt3 helps orchestrate Indeed, targeting PDH activity in muscle for the treatment the efﬁcient use of available nutrients in skeletal muscle by 3414 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES FIG. 7. Model for the role of Sirt3 in control of skeletal muscle substrate metabolism. Sirt3 regulates PDH E1a subunit deacetylation and activates PDH activity. A: In the fed state, Sirt3 skeletal muscle expression is abundant and leads to deacetylation of PDH E1a. This is associated with dephosphorylation of PDH allowing for maximal enzyme activation, enhanced glucose utilization, and increased ﬂux of pyruvate to acetyl-CoA used by the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) to generate ATP. B: In contrast, decreased Sirt3 expression in muscle by fasting or genetic deletion leads to PDH E1a hyperacetylation and decreased PDH complex activity, which is correlated with increased PDH E1a phosphorylation in vivo. The activity of PDH controls the substrate inﬂux to the TCA cycle from glycolysis. In the case of Sirt3 deletion, inactivation of the PDH caused by hyperacetylation leads to metabolic inﬂexibility as evidenced by an inability to fully oxidize glucose, a shunt of excess pyruvate toward lactate production, and increased lipid oxidation even in the fed state. CPT, carnitine palmitoyl transferase; FFA, free fatty acid; Mito, mitochondrial; OxPhos, oxidative phosphorylation. diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3415 Sirt3 REGULATES MUSCLE METABOLIC FLEXIBILITY 14. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM. Evidence for promoting metabolic ﬂexibility through enzymatic deacet- existence of tissue-speciﬁc regulation of the mammalian pyruvate de- ylation of PDH. Modulation of mitochondrial acetylation hydrogenase complex. Biochem J 1998;329:191–196 may offer a new therapeutic target for correcting some of 15. Mayers RM, Leighton B, Kilgour E. PDH kinase inhibitors: a novel therapy the metabolic defects of obesity and type 2 diabetes. for Type II diabetes? Biochem Soc Trans 2005;33:367–370 16. Wu P, Inskeep K, Bowker-Kinley MM, Popov KM, Harris RA. Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes. Diabetes 1999;48:1593–1599 ACKNOWLEDGMENTS 17. Zhao S, Xu W, Jiang W, et al. Regulation of cellular metabolism by protein The primary funding for this project was National Insti- lysine acetylation. Science 2010;327:1000–1004 tutes of Health Grant R24 DK085610. B.T.O. was funded by 18. Choudhary C, Kumar C, Gnad F, et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009;325:834–840 Joslin Training Grant T32DK007260. A.K. was funded by 19. Schwer B, Eckersdorff M, Li Y, et al. Calorie restriction alters mitochon- German Research Foundation project KL2399/1-1. S.U. drial protein acetylation. Aging Cell 2009;8:604–606 was supported by a Human Frontier Science Program 20. Michan S, Sinclair D. Sirtuins in mammals: insights into their biological Long-Term fellowship. The authors acknowledge the function. Biochem J 2007;404:1–13 support of National Center for Research Resources shared 21. Milne JC, Denu JM. The Sirtuin family: therapeutic targets to treat diseases instrumentation grant S10 RR024615 (to B.W.G.) for the of aging. Curr Opin Chem Biol 2008;12:11–17 22. Cooper HM, Spelbrink JN. The human SIRT3 protein deacetylase is ex- QSTAR Elite mass spectrometer used in these studies. clusively mitochondrial. 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Metabolism grams in skyline: application to protein acetylation and phosphorylation. 2010;59:70–85 Mol Cell Proteomics 2012;11:202–214 8. Espinoza DO, Boros LG, Crunkhorn S, Gami H, Patti ME. Dual modulation 35. Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mito- of both lipid oxidation and synthesis by peroxisome proliferator-activated chondria from mouse liver, muscle and cultured ﬁbroblasts. Nat Protoc receptor-gamma coactivator-1alpha and -1beta in cultured myotubes. 2007;2:287–295 FASEB J 2010;24:1003–1014 36. Kerr D, Grahame G, Nakouzi G. Assays of pyruvate dehydrogenase com- 9. Finck BN, Bernal-Mizrachi C, Han DH, et al. A potential link between plex and pyruvate carboxylase activity. Methods Mol Biol 2012;837:93–119 muscle peroxisome proliferator- activated receptor-alpha signaling and 37. Goodyear LJ, Giorgino F, Balon TW, Condorelli G, Smith RJ. Effects of obesity-related diabetes. 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FASEB J Nat Med 2004;10:268–274 2003;17:1666–1673 40. Wu JY, Kao HJ, Li SC, et al. ENU mutagenesis identiﬁes mice with mito- 12. Rahman S, Blok RB, Dahl HH, et al. Leigh syndrome: clinical features and chondrial branched-chain aminotransferase deﬁciency resembling human biochemical and DNA abnormalities. Ann Neurol 1996;39:343–351 maple syrup urine disease. J Clin Invest 2004;113:434–440 13. Cameron JM, Levandovskiy V, Mackay N, Tein I, Robinson BH. Deﬁciency 41. Jensen MV, Joseph JW, Ilkayeva O, et al. Compensatory responses to py- of pyruvate dehydrogenase caused by novel and known mutations in the ruvate carboxylase suppression in islet beta-cells. Preservation of glucose- E1alpha subunit. Am J Med Genet A 2004;131:59–66 stimulated insulin secretion. J Biol Chem 2006;281:22342–22351 3416 DIABETES, VOL. 62, OCTOBER 2013 diabetes.diabetesjournals.org E. JING AND ASSOCIATES 42. Rardin MJ, Wiley SE, Naviaux RK, Murphy AN, Dixon JE. Monitoring 47. Galgani JE, Moro C, Ravussin E. Metabolic ﬂexibility and insulin re- phosphorylation of the pyruvate dehydrogenase complex. Anal Biochem sistance. Am J Physiol Endocrinol Metab 2008;295:E1009–E1017 2009;389:157–164 48. DeFronzo RA, Tripathy D. Skeletal muscle insulin resistance is the 43. Akhmedov D, De Marchi U, Wollheim CB, Wiederkehr A. Pyruvate de- primary defect in type 2 diabetes. Diabetes Care 2009;32(Suppl. 2): hydrogenase E1a phosphorylation is induced by glucose but does not S157–S163 control metabolism-secretion coupling in INS-1E clonal b-cells. Biochim 49. Fernandez-Marcos PJ, Jeninga EH, Canto C, et al. Muscle or liver-speciﬁc Biophys Acta 2012;1823:1815–1824 Sirt3 deﬁciency induces hyperacetylation of mitochondrial proteins with- 44. Bell EL, Emerling BM, Ricoult SJ, Guarente L. SirT3 suppresses hypoxia out affecting global metabolic homeostasis. Sci Rep 2012;2:425 inducible factor 1a and tumor growth by inhibiting mitochondrial ROS 50. Linn TC, Pettit FH, Reed LJ. Alpha-keto acid dehydrogenase complexes. X. production. Oncogene 2011;30:2986–2996 Regulation of the activity of the pyruvate dehydrogenase complex from 45. Yang H, Yang T, Baur JA, et al. Nutrient-sensitive mitochondrial NAD+ beef kidney mitochondria by phosphorylation and dephosphorylation. levels dictate cell survival. Cell 2007;130:1095–1107 Proc Natl Acad Sci USA 1969;62:234–241 46. Jagoe RT, Lecker SH, Gomes M, Goldberg AL. Patterns of gene expression 51. Harris RA, Bowker-Kinley MM, Huang B, Wu P. Regulation of the activity in atrophying skeletal muscles: response to food deprivation. FASEB J of the pyruvate dehydrogenase complex. Adv Enzyme Regul 2002;42: 2002;16:1697–1712 249–259 diabetes.diabetesjournals.org DIABETES, VOL. 62, OCTOBER 2013 3417

Journal

Diabetes – Pubmed Central

Published: Sep 17, 2013

References

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Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

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Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation

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