Calorie Restriction Reduces Oxidative Stress by SIRT3-Mediated SOD2 Activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases. A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases. CR reduces oxidative stress by inducing SIRT3 SIRT3 activates SOD2 via deacetylation SIRT3 reduces cellular ROS and promotes stress resistance by deacetylating SOD2 SOD2 is activated by SIRT3 via deacetylation during CR The free radical theory of aging postulates that the production of reactive oxygen species (ROS) is the major determinant of life span (Balaban et al., 2005Balaban R.S. Nemoto S. Finkel T. Mitochondria, oxidants, and aging.Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3039) Google Scholar, Merry, 2004Merry B.J. 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Mitochondria, oxidants, and aging.Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3039) Google Scholar, Merry, 2004Merry B.J. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction.Aging Cell. 2004; 3: 7-12Crossref PubMed Scopus (152) Google Scholar). Consistent with this, animals with reduced levels of oxidative stress, such as animals fed a calorie restriction (CR) diet or overexpressing mitochondrially targeted catalase, have extended life spans, while animals with high levels of oxidative stress, such as mice lacking SODs, have shortened life spans (Merry, 2004Merry B.J. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction.Aging Cell. 2004; 3: 7-12Crossref PubMed Scopus (152) Google Scholar, Schriner et al., 2005Schriner S.E. Linford N.J. Martin G.M. Treuting P. Ogburn C.E. Emond M. Coskun P.E. Ladiges W. Wolf N. 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Falcone S. Valerio A. Cantoni O. Clementi E. et al.Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.Science. 2005; 310: 314-317Crossref PubMed Scopus (870) Google Scholar). Thus, the molecular mechanism underlying CR-induced reduction of oxidative stress remains elusive. In the budding yeast Saccharomyces cerevisiae and the fruit fly Drosophila melanogaster, CR extends life span by increasing the activity of the Sir2 protein (Lin et al., 2000Lin S.J. Defossez P.A. Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1429) Google Scholar, Rogina and Helfand, 2004Rogina B. Helfand S.L. Sir2 mediates longevity in the fly through a pathway related to calorie restriction.Proc. Natl. Acad. Sci. USA. 2004; 101: 15998-16003Crossref PubMed Scopus (1011) Google Scholar). 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Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase.Science. 2004; 305: 390-392Crossref PubMed Scopus (1589) Google Scholar, Cohen et al., 2009Cohen D.E. Supinski A.M. Bonkowski M.S. Donmez G. Guarente L.P. Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction.Genes Dev. 2009; 23: 2812-2817Crossref PubMed Scopus (146) Google Scholar, Li et al., 2008Li Y. Xu W. McBurney M.W. Longo V.D. SirT1 inhibition reduces IGF-I/IRS-2/Ras/ERK1/2 signaling and protects neurons.Cell Metab. 2008; 8: 38-48Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Given the role of SIR2 in mediating the CR response and the observation that animals fed a CR diet have reduced oxidative stress, we speculate that mammalian SIR2 homologs might play a role in regulating oxidative stress. Since 90% of cellular ROS are produced in the mitochondria (Balaban et al., 2005Balaban R.S. Nemoto S. Finkel T. Mitochondria, oxidants, and aging.Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3039) Google Scholar), we set out to investigate whether SIRT3, a mammalian SIR2 homolog localized in the mitochondria (Onyango et al., 2002Onyango P. Celic I. McCaffery J.M. Boeke J.D. Feinberg A.P. SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria.Proc. Natl. Acad. Sci. USA. 2002; 99: 13653-13658Crossref PubMed Scopus (397) Google Scholar, Schwer et al., 2002Schwer B. North B.J. Frye R.A. Ott M. Verdin E. The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.J. Cell Biol. 2002; 158: 647-657Crossref PubMed Scopus (425) Google Scholar), mediates the CR response and reduces oxidative stress. CR induces an increase in the expression of SIRT3 (Palacios et al., 2009Palacios O.M. Carmona J.J. Michan S. Chen K.Y. Manabe Y. Ward 3rd, J.L. Goodyear L.J. Tong Q. Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1alpha in skeletal muscle.Aging (Albany NY). 2009; 1: 771-783Crossref PubMed Scopus (367) Google Scholar, Shi et al., 2005Shi T. Wang F. Stieren E. Tong Q. SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes.J. Biol. Chem. 2005; 280: 13560-13567Crossref PubMed Scopus (540) Google Scholar) and mitochondrial NAD+ levels (Nakagawa et al., 2009Nakagawa T. Lomb D.J. Haigis M.C. Guarente L. SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle.Cell. 2009; 137: 560-570Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar), suggesting that SIRT3 activity is likely to be upregulated during CR. To investigate whether SIRT3 is required for CR to reduce oxidative stress, we fed SIRT3 knockout (KO) mice (Lombard et al., 2007Lombard D.B. Alt F.W. Cheng H.L. Bunkenborg J. Streeper R.S. Mostoslavsky R. Kim J. Yancopoulos G. Valenzuela D. Murphy A. et al.Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation.Mol. Cell. Biol. 2007; 27: 8807-8814Crossref PubMed Scopus (888) Google Scholar) and wild-type (WT) littermates a CR diet for 6 months. We compared oxidative stress and damage between WT and SIRT3 KO mice fed ad libitum (AL) or CR diets. Consistent with earlier reports, CR significantly reduced oxidative stress and damage in WT mice, as shown by levels of 4-hydroxy-2-nonenal (HNE), a marker for lipid peroxidation (Figure 1A ), protein carbonyl content, a protein oxidative modification (Figure 1B), and the GSH:GSSG ratio, a common measure of oxidative stress (Figure 1C) (Merry, 2004Merry B.J. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction.Aging Cell. 2004; 3: 7-12Crossref PubMed Scopus (152) Google Scholar, Rebrin et al., 2003Rebrin I. Kamzalov S. Sohal R.S. Effects of age and caloric restriction on glutathione redox state in mice.Free Radic. Biol. Med. 2003; 35: 626-635Crossref PubMed Scopus (209) Google Scholar). However, the reduction in oxidative stress and damage under CR was not observed in SIRT3 KO mice (Figures 1A–1C), suggesting that SIRT3 is required for reducing oxidative stress during CR. In mammals, there are three forms of SODs localized in various cellular compartments (Balaban et al., 2005Balaban R.S. Nemoto S. Finkel T. Mitochondria, oxidants, and aging.Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3039) Google Scholar). SOD2 is located in the mitochondria. To determine whether SOD2 plays a role in the reduction of cellular ROS mediated by SIRT3, we overexpressed SIRT3 in WT and SOD2 KO mouse embryonic fibroblasts (MEFs) via lentiviral transduction and assessed cellular ROS levels. Overexpression of SIRT3 in WT MEFs reduced cellular ROS by 40%. However, reduction of cellular ROS mediated by SIRT3 was blunted in SOD2 KO MEFs (Figure 2A ), indicating that SOD2 is the major downstream mediator of SIRT3 in reducing cellular ROS. SOD2 was identified in mass spectrometry-based screens for acetylated peptides (Choudhary et al., 2009Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (2914) Google Scholar, Kim et al., 2006Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. et al.Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar, Kim et al., 2010Kim H.S. Patel K. Muldoon-Jacobs K. Bisht K.S. Aykin-Burns N. Pennington J.D. van der Meer R. Nguyen P. Savage J. Owens K.M. et al.SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress.Cancer Cell. 2010; 17: 41-52Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, Schwer et al., 2009Schwer B. Eckersdorff M. Li Y. Silva J.C. Fermin D. Kurtev M.V. Giallourakis C. Comb M.J. Alt F.W. Lombard D.B. Calorie restriction alters mitochondrial protein acetylation.Aging Cell. 2009; 8: 604-606Crossref PubMed Scopus (196) Google Scholar). We tested the possibility that SIRT3 regulates the acetylation state of SOD2. To test whether SIRT3 interacts with SOD2, we overexpressed Flag-tagged SIRT3 in 293T cells and SIRT3-associated proteins were immunopurified (anti-Flag). The association of SOD2 with SIRT3 was detected by western blotting with SOD2 antibody (Figure 2B). Additionally, we also transfected Flag-tagged SOD2 into 293T cells and the presence of SIRT3 in the immunopurified SOD2 complex was confirmed by western blotting with SIRT3 antibody (Figure 2C). Finally, we examined whether the interaction between SIRT3 and SOD2 is physiologically relevant by carrying out immunoprecipitation with liver extracts. SIRT3 was coimmunoprecipitated with SOD2 antibody (Figure 2D). To test whether SIRT3 deacetylates SOD2, we cotransfected Flag-tagged SOD2 with SIRT3 or enzymatically inactive SIRT3-H248Y into 293T cells. Acetylation levels for SOD2 were measured after immunoprecipitation by western blotting with anti-acetyl-lysine antibody. SIRT3, but not SIRT3-H248Y, reduced acetylation levels of SOD2 (Figure 2E). To determine whether the acetylation state of SOD2 modifies its enzymatic activity, we overexpressed Flag-tagged SOD2 in 293T cells with SIRT3 or SIRT3-H248Y, immunopurified SOD2, and determined its enzymatic activity by measuring superoxide conversion colorimetrically (Schisler and Singh, 1985Schisler N.J. Singh S.M. Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice.Biochem. Genet. 1985; 23: 291-308Crossref PubMed Scopus (17) Google Scholar). Overexpression of WT SIRT3 decreased the acetylation of SOD2 (Figure 2E) and significantly increased its enzymatic activity (Figure 2F). In contrast, overexpression of SIRT3-H248Y had the opposite effect, suggesting that SIRT3 activates SOD2 via deacetylation. To identify which lysine residue(s) on SOD2 are targeted by SIRT3 for deacetylation, we mutated lysines (K68 and K130) that have been shown to be acetylated in mass spectrometry-based acetylation proteomic surveys (Choudhary et al., 2009Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (2914) Google Scholar, Kim et al., 2006Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. et al.Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar, Schwer et al., 2009Schwer B. Eckersdorff M. Li Y. Silva J.C. Fermin D. Kurtev M.V. Giallourakis C. Comb M.J. Alt F.W. Lombard D.B. Calorie restriction alters mitochondrial protein acetylation.Aging Cell. 2009; 8: 604-606Crossref PubMed Scopus (196) Google Scholar). Surprisingly, mutating these lysine residues did not significantly reduce the overall acetylation levels of SOD2 (Figure 3A ). Additionally, SIRT3 reduced acetylation levels of these SOD2 mutants, indicating that these lysines are unlikely to be the major acetylation sites on SOD2. Protein sequence alignment studies showed that two lysines (K53 and K89) adjacent to the active site of SOD2 are highly conserved across species (Figure 3B). Acetylation levels of SOD2 were decreased when these two lysines were mutated individually (K53R and K89R) or simultaneously (K53/89R) (Figures 3A and 3C). SIRT3 further decreased acetylation levels of K53R and K89R, but not K53/89R. Collectively, these studies identified K53 and K89 as the acetylation sites on SOD2 targeted by SIRT3. To determine how deacetylation of these two lysines on SOD2 affects its antioxidative activity, we determined the antioxidative activity of K53/89R, which mimics the constitutively deacetylated state. The enzymatic activity of SOD2 K53/89R was 100% higher than the WT control and SIRT3 did not further increase its enzymatic activity (Figure 3D). Thus, SIRT3 promotes the enzymatic activity of SOD2 by deacetylating two critical lysine residues adjacent to the active site. Conceivably, these two lysine residues, when exposed, increase the positive charge around the active site and improve the efficiency of trapping the negatively charged superoxide. To determine whether SIRT3 reduces cellular ROS levels by deacetylating SOD2, we overexpressed SOD2 or SOD2 K53/89R with or without SIRT3 into SIRT3 KO MEFs and assessed cellular ROS levels. We used SIRT3 KO MEFs in this assay to avoid the influence of endogenous SIRT3. Surprisingly, overexpression of SOD2 6-fold above the endogenous levels only marginally decreased cellular ROS (10%) (Figure 3E). However, coexpression of SIRT3 and SOD2 depleted 90% of cellular ROS. Additionally, constitutively deacetylated SOD2 K53/89R alone also diminished cellular ROS by 90%. These results indicate that increasing SOD2 expression alone can only modestly reduce cellular ROS. SIRT3 deacetylation significantly enhances the ability of SOD2 to reduce cellular ROS. To determine whether SIRT3-mediated SOD2 activation results in increased oxidative stress resistance, we overexpressed SIRT3 in WT or SOD2 KO MEFs, treated the cells with paraquat, a superoxide-generating compound, and assayed cell survival. SIRT3 overexpression in WT MEFs doubled the number of surviving cells (Figures 3F and S1). However, SIRT3 overexpression in SOD2 KO MEFs had no effect on cell survival, indicating that SIRT3 promotes oxidative stress resistance and SOD2 is the mediator of this process. To determine whether SIRT3 increases oxidative stress resistance by deacetylating SOD2, we overexpressed WT SOD2 or SOD2 K53/89R with or without SIRT3 into SIRT3 KO MEFs, and assessed oxidative stress resistance of these cells. Although overexpression of SOD2 alone only modestly reduced cellular ROS (10%) (Figure 3E), it increased the cell survival rate by 50% upon paraquat treatment (Figure 3G). It is worth noting that the condition for measuring cellular ROS level (Figure 3E) is different from the condition for assessing oxidative stress resistance (Figures 3F and 3G). The former is for untreated cells, while the latter is for cells treated with paraquat when ROS levels are significantly higher than physiological levels. Importantly, coexpression of SOD2 and SIRT3 resulted in a nearly 3-fold increase in cell survival (Figure 3G). Moreover, constitutively deacetylated SOD2 K53/89R increased cell survival to the same level as coexpression of SOD2 and SIRT3. Coexpression of SIRT3 did not further increase cell survival mediated by SOD2 K53/89R. These results indicate that SIRT3 promotes oxidative stress resistance by deacetylating SOD2. Given that SIRT3 is induced by CR and that SIRT3 activates SOD2 via deacetylation, we speculated that SIRT3 might deacetylate SOD2 and increase its antioxidative activity in CR animals. We compared acetylation levels of SOD2 and its antioxidative activity in WT and SIRT3 KO mice fed AL or CR diets. To assess acetylation levels of SOD2 in mouse tissues, endogenous proteins were immunoprecipitated with anti-SOD2 antibody and analyzed by western blotting using acetyl-lysine antibody. Endogenous SOD2 was acetylated and became deacetylated during CR in WT mice (Figure 4A ). However, CR-induced SOD2 deacetylation was not observed in SIRT3 KO mice, demonstrating that SIRT3 is necessary for SOD2 deacetylation during CR. We next determined endogenous SOD2 activity using tissue lysates as described (Schisler and Singh, 1985Schisler N.J. Singh S.M. Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice.Biochem. Genet. 1985; 23: 291-308Crossref PubMed Scopus (17) Google Scholar). CR induced a 50% increase in SOD2 activity in the white adipose tissues of WT mice (Figure 4B). Importantly, this increase in SOD2 activity under CR was lost in SIRT3 KO mice. These results suggest that during CR, SIRT3 reduces oxidative stress by activating SOD2 and promoting the detoxification of ROS. It is worth noting that SIRT3-mediated SOD2 deacetylation and activation was not observed in mice fed AL (Figures 4A and 4B). Consistent with this, oxidative stress and damage were comparable in WT and SIRT3 KO mice fed AL (Figure 1), suggesting that SIRT3 is not active under AL conditions. Our studies identify an active defense program CR provokes to reduce oxidative stress. CR induces an increase in SIRT3 expression (Palacios et al., 2009Palacios O.M. Carmona J.J. Michan S. Chen K.Y. Manabe Y. Ward 3rd, J.L. Goodyear L.J. Tong Q. Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1alpha in skeletal muscle.Aging (Albany NY). 2009; 1: 771-783Crossref PubMed Scopus (367) Google Scholar, Shi et al., 2005Shi T. Wang F. Stieren E. Tong Q. SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes.J. Biol. Chem. 2005; 280: 13560-13567Crossref PubMed Scopus (540) Google Scholar). Activation of SIRT3 during CR reduces oxidative stress by activating the mitochondrial antioxidant enzyme SOD2. SOD2 activation and increased oxidative stress resistance have been linked to numerous long-lived mouse models (Baba et al., 2005Baba T. Shimizu T. Suzuki Y. Ogawara M. Isono K. Koseki H. Kurosawa H. Shirasawa T. 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Chem. 2005; 280: 38029-38034Crossref PubMed Scopus (478) Google Scholar). We speculate that the loss of protection from oxidative stress in the SIRT3 KO mice could abrogate CR-induced life-span extension. Despite the prevalence of the free radical theory of aging, it is intriguing that SOD2 transgenic mice do not have increased life spans (Pérez et al., 2009Pérez V.I. Bokov A. Van Remmen H. Mele J. Ran Q. Ikeno Y. Richardson A. Is the oxidative stress theory of aging dead?.Biochim. Biophys. Acta. 2009; 1790: 1005-1014Crossref PubMed Scopus (454) Google Scholar). Our studies suggest that increasing SOD2 expression alone can only modestly reduce cellular ROS. The ability of SOD2 to reduce cellular ROS is greatly enhanced by SIRT3 deacetylation. Transgenic mice overexpressing both SIRT3 and SOD2 might have extended life spans. Although mitochondrial activity is increased by CR (Nisoli et al., 2005Nisoli E. Tonello C. Cardile A. Cozzi V. Bracale R. Tedesco L. Falcone S. Valerio A. Cantoni O. Clementi E. et al.Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.Science. 2005; 310: 314-317Crossref PubMed Scopus (870) Google Scholar), oxidative stress and damage are reduced in CR animals (Merry, 2004Merry B.J. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction.Aging Cell. 2004; 3: 7-12Crossref PubMed Scopus (152) Google Scholar). Evidence is emerging to support the mitochondrial hormesis or “mitohormesis” concept, which hypothesizes that induction of mitochondrial metabolism and the increased formation of ROS trigger an active defense program, resulting in increased stress resistance and possibly extended life span (Schulz et al., 2007Schulz T.J. Zarse K. Voigt A. Urban N. Birringer M. Ristow M. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress.Cell Metab. 2007; 6: 280-293Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, Sinclair, 2005Sinclair D.A. Toward a unified theory of caloric restriction and longevity regulation.Mech. Ageing Dev. 2005; 126: 987-1002Crossref PubMed Scopus (451) Google Scholar). Perhaps activation of SIRT3 in CR animals is a key step of this defense program against oxidative stress. In light of the importance of mitochondrial ROS production in the onset and progression of diverse diseases associated with aging (Wallace and Fan, 2009Wallace D.C. Fan W. The pathophysiology of mitochondrial disease as modeled in the mouse.Genes Dev. 2009; 23: 1714-1736Crossref PubMed Scopus (158) Google Scholar), our findings have important implications for developing CR mimetics for treating the diseases of aging. Notably, SIRT3 has been shown to protect mice from developing cancer and cardiac hypertrophy (Kim et al., 2010Kim H.S. Patel K. Muldoon-Jacobs K. Bisht K.S. Aykin-Burns N. Pennington J.D. van der Meer R. Nguyen P. Savage J. Owens K.M. et al.SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress.Cancer Cell. 2010; 17: 41-52Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, Sundaresan et al., 2009Sundaresan N.R. Gupta M. Kim G. Rajamohan S.B. Isbatan A. Gupta M.P. Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice.J. Clin. Invest. 2009; 119: 2758-2771Crossref PubMed Scopus (704) Google Scholar). Future studies will determine the impact of SIRT3 in other pathological conditions and explore therapeutic strategies to treat human diseases with SIRT3 activators or SOD2 variants. SIRT3−/− mice have been described (Lombard et al., 2007Lombard D.B. Alt F.W. Cheng H.L. Bunkenborg J. Streeper R.S. Mostoslavsky R. Kim J. Yancopoulos G. Valenzuela D. Murphy A. et al.Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation.Mol. Cell. Biol. 2007; 27: 8807-8814Crossref PubMed Scopus (888) Google Scholar). All mice were housed on a 12:12 hr light:dark cycle at 25°C. Six-month-old animals (n = 8) were either fed AL or a 30% CR diet, which was provided daily for six months. All animal procedures were in accordance with the animal care committee at the University of California, Berkeley. Proteins from mouse tissues were extracted in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM MgCl2, 1 mM DTT and 1% NP40) supplemented with a complete protease inhibitor cocktail (Roche), trichostatin A, and nicotinamide. Protein extracts were subjected to centrifugation at 14,000 rpm for 10 min. Protein lysates were precleared with protein A beads for 30 min before immunoprecipitation with specified antibodies for 2 hr or overnight. Immunoprecipitates were extensively washed with lysis buffer and eluted with 100 mM glycine, pH 3.0, or Flag peptides. Acetyl-lysine antibody was provided by Cell Signaling, BioLegend; SOD2 antibody was provided by Santa Cruz Biotechnology; Flag antibody was provided by Sigma. SIRT3 and SOD2 were cloned into pFUGW lentiviral vector. Lentiviruses were produced by transient transfection of pFUGW and packaging vectors into 293T cells with lipofectamine. Lentiviruses were harvested 48 hr posttransfection and filtered through 0.45-μm-pore cellulose acetate filters. Virus containing media was mixed with fresh media (1:1) and added to MEF cells in the presence of 8 μg/ml polybrene. Cells were incubated with 3 μM of Mito-SOX at 37°C for 15 min prior to flow cytometry analysis. Superoxide dismutase activity was measured as the inhibition of nitroblue tetrazolium (NBT) reduction in a xanthine-xanthine oxidase system. The assay was performed as described (Schisler and Singh, 1985Schisler N.J. Singh S.M. Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice.Biochem. Genet. 1985; 23: 291-308Crossref PubMed Scopus (17) Google Scholar), and SOD2 specific activity was determined in the presence of 5 mM sodium cyanide. Protein carbonyls were spectrophotometrically quantified with a carbonyl specific reagent, 2,4-dinitrophenylhydrazine (DNPH) (Levine et al., 1994Levine R.L. Williams J.A. Stadtman E.R. Shacter E. Carbonyl assays for determination of oxidatively modified proteins.Methods Enzymol. 1994; 233: 346-357Crossref PubMed Scopus (2152) Google Scholar). Briefly, 1 ml of 0.5 mg protein was treated with 200 μl of 10 mM of DNPH (dissolved in 2M HCl) for 1 hr, and then precipitated by 10% trichloroacetic acid. The pellets were washed with 1:1 (v/v) ethanol:ethyl acetate for three times, and solublized in 0.5 ml 0.2% SDS, 20 mM Tris-Cl, pH 6.8. Protein concentration in the final solution was then determined with a BCA kit (Piercenet), and the absorbance at 360 nm was measured to calculate the carbonyl content. Protein samples treated with HCl but not with DNPH were used as blanks. Glutathione was measured in mitochondrial fractions isolated from liver (Merry, 2004Merry B.J. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction.Aging Cell. 2004; 3: 7-12Crossref PubMed Scopus (152) Google Scholar, Rebrin et al., 2003Rebrin I. Kamzalov S. Sohal R.S. Effects of age and caloric restriction on glutathione redox state in mice.Free Radic. Biol. Med. 2003; 35: 626-635Crossref PubMed Scopus (209) Google Scholar). The GSH: GSSG ratio was determined by Glutathione Assay Kit (BioVision), following the manufacturer's instructions. HNE levels were measured in indicated liver samples with an OxiSelect HNE-His Adduct ELISA Kit (Cell Biolabs, Inc. San Diego, CA) following the instructions. Ten thousand cells were incubated with increasing doses of paraquat (Sigma) (0, 50, 100, 200, 400 μM) for 48 hr. Remaining adherent cells were trypsinized and counted using a hemocytometer. A dose-curve response was determined (Figure S1A). Student's t test was used for statistic analysis and null hypotheses were rejected at 0.05. We thank F. W. Alt, H. Cheng, and T. Huang for reagents. We thank P. Zhang and Y. Liu for comments. Financial support by Searle Scholars Program (D.C.). Download .pdf (.15 MB) Help with pdf files Document S1. One Figure