Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production Enxuan Jinga, Brice Emanuellia, Matthew D. Hirscheyb,c, Jeremie Bouchera, Kevin Y. Leea, David Lombardd,1, Eric M. Verdinb,c, and C. Ronald Kahna,2 aJoslin Diabetes Center, Harvard Medical School, Boston, MA 02215; bGladstone Institute of Virology and Immunology, San Francisco, CA 94158; cUniversity of California, San Francisco, CA 94143; and dDepartment of Genetics, Harvard Medical School, Boston, MA Contributed by C. Ronald Kahn, July 20, 2011 (sent for review April 14, 2011) Sirt3 is a member of the sirtuin family of protein deacetylases that early, or even primary, contributor to development of skeletal is localized in mitochondria and regulates mitochondrial function. muscle insulin resistance and type 2 diabetes. Sirt3 expression in skeletal muscle is decreased in models of type 1 In recent years, the sirtuin family of NAD+-dependent and type 2 diabetes and regulated by feeding, fasting, and caloric deacetylases has emerged as important regulators of metabolism. restriction. Sirt3 knockout mice exhibit decreased oxygen con- Among seven members of the sirtuin family, Sirt3 is of particular sumption and develop oxidative stress in skeletal muscle, leading interest with regard to mitochondrial function because it is lo- to JNK activation and impaired insulin signaling. This effect is mim- calized primarily in mitochondria (16). Sirt3 has been shown to Sirt3 icked by knockdown of in cultured myoblasts, which exhibit deacetylate and thereby regulate several mitochondrial targets, reduced mitochondrial oxidation, increased reactive oxygen spe- including acetyl-CoA synthase 2 and glutamate dehydrogenase cies, activation of JNK, increased serine and decreased tyrosine (17, 18). It has been suggested that Sirt3 is also closely involved in phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 energy homeostasis and regulation of ATP production in various plays an important role in diabetes through regulation of Sirt3 mitochondrial oxidation, reactive oxygen species production, and tissues (19). Recently, we have shown that plays an im- insulin resistance in skeletal muscle. portant role in hepatic lipid metabolism (20). Through these and other effects, Sirt3 has been shown to be involved in mitochon- mitochondrial metabolism | protein acetylation drial function and is associated with aging (21, 22). We previously found that Sirt3 expression is significantly de- creased in muscle of mice with insulin-deficient diabetes (23), nsulin resistance in skeletal muscle is a major and early feature Iin the pathogenesis of type 2 diabetes (1, 2). This pathological suggesting that decreased Sirt3 activity could contribute to the condition has been shown to involve decreased activity of the in- metabolic abnormalities of diabetes. In the present study, we Sirt3 sulin signaling network with reduced tyrosine phosphorylation of demonstrate that skeletal muscle expression is altered in the insulin receptor and its substrates, decreased activation of models of both type 1 and type 2 diabetes and that alteration in phosphatidylinositol 3-kinase (PI 3-kinase), and decreased acti- Sirt3 expression regulates mitochondrial metabolism and pro- vation of Akt/PKB (protein kinase B), leading to reduced glucose duction of ROS, which in turn alters insulin signaling. These uptake and other metabolic abnormalities (3–5). Another early findings suggest a broad-reaching role of Sirt3 in altered muscle feature of type 2 diabetes is altered mitochondrial function in metabolism in both normal and diabetic states. muscle. Reduced expression of multiple nuclear-encoded genes involved in mitochondrial oxidative phosphorylation and alter- Results ations in mitochondrial morphology have been observed in skel- Changes in Sirt3 Expression in Models of Diabetes and Aging. etal muscle of both rodent models of diabetes and humans with Quantitative PCR using muscle from streptozotocin (STZ) di- type 2 diabetes (6–8). Impaired mitochondrial lipid oxidation and abetic mice revealed an w50% decrease of Sirt3 mRNA, similar glycolytic capacity have also been observed in individuals with to previous data from microarrays (23), and Western blot anal- diabetes and obesity, whereas enhanced mitochondrial lipid oxi- ysis confirmed a parallel decrease of Sirt3 protein (Fig. 1A). dation capacity has been associated with improved insulin re- Likewise, analysis of skeletal muscle from mice made obese by sistance (9, 10). This reduced expression and/or activity of a chronic high-fat diet revealed a >50% decrease of Sirt3 mRNA mitochondrial proteins has been closely associated with altered and protein compared with samples from mice fed a normal skeletal muscle physiology and metabolism. Some, but not all, chow diet (Fig. 1B). Conversely, caloric restriction of mice for 12 studies have found similar alterations in skeletal muscle of indi- wk resulted in a 2.5-fold increase of Sirt3 mRNA and protein in viduals with a family history positive for type 2 diabetes (8, 11). skeletal muscle (Fig. 1C). By contrast, there was no change in Reduced oxidative capacity and reduced ATP synthesis rates have muscle Sirt3 expression level with aging as demonstrated by a also been shown in individuals with type 2 diabetes and in some nondiabetic individuals with a family history for diabetes (12). comparison of 3- and 24-mo-old mice (Fig. S1). In addition to its role in substrate metabolism, the mito- chondrion is the major production site of reactive oxygen species (ROS). When ROS level is excessive or there is impaired ROS Author contributions: E.J., E.M.V., and C.R.K. designed research; E.J., B.E., M.D.H., J.B., and K.Y.L. performed research; D.L. contributed new reagents/analytic tools; E.J., B.E., and clearance, the oxidative stress response can activate serine/ M.D.H. analyzed data; and E.J. and C.R.K. wrote the paper. threonine kinases such as protein kinase C, S6 kinase, and Jun The authors declare no conflict of interest. N-terminal kinase (JNK), which can phosphorylate the insulin 1Present address: Department of Pathology and Institute of Gerontology, University of receptor (IR) and/or insulin receptor substrate (IRS) proteins Michigan, Ann Arbor, MI 48109. – (13 15), leading to a decrease in their tyrosine phosphorylation, 2To whom correspondence should be addressed. E-mail: [email protected]. decreased activation of PI 3-kinase and Akt, and resistance to edu. the metabolic actions of insulin. Collectively, these data suggest This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that altered mitochondrial oxidative phosphorylation may be an 1073/pnas.1111308108/-/DCSupplemental. 14608e14613 | PNAS | August 30, 2011 | vol. 108 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1111308108 Downloaded by guest on October 1, 2021 A B A B 1.2 1.6 25 3.0 1.4 WT 1.0 20 2.5 * 1.2 ) g 0.8 ( S3KO 2.0 1.0 s 15 * s 0.6 0.8 a 1.5 0.6 M 10 0.4 y 1.0 b 0.4 o 5 0.2 B 0.5 Sirt3 mRNA 0.2 0.0 0 0.0 0.0 Total Lean Total Fat Control STZ Diab Chow HFD Food intake/mouse/day(g) WT S3KO Sirt3 C D Tubulin 9500 1.0 9000 * 8500 * 0.9 C D 80002 4.0 2.5 7500 0.8 * * 7000 RER 2.0 * 6500 3.0 VO Mean 6000 0.7 1.5 5500 2.0 5000 0.6 1.0 WT S3KO WT S3KO WT S3KO WT S3KO 1.0 Light Cycle Dark Cycle Light Cycle Dark Cycle Sirt3 mRNA 0.5 0.0 0.0 Control CR Fed Fasted Refed E F 2.5 * WT S3KO Sirt3 2.0 Tubulin TBARS 1.5 p-JNK 1.0 Fig. 1. Sirt3 expression in different states of diabetes and aging. (A) Skel- JNK Muscle etal muscles from the hind limbs of STZ diabetic mice and controls (8-wk-old 0.5 C57BL/6) were collected. RNA and protein was extracted and analyzed by 0.0 either quantitative PCR or Western blotting. (B) Skeletal muscles from the WT S3KO hind limb of obese high fat diet (HFD) and control mice were collected and processed for quantitative PCR and Western blot analysis for Sirt3 expres- G _ WT_ S3KO H 350 Ins ++ ++ * sion. (C) Hind-limb skeletal muscle of CR mice along with controls was col- WT pY-IR lected and processed for quantitative PCR and Western blot analysis. (D) S3KO IR 250 Mice were either randomly fed or fasted for 24 h; half of the fasted animals p-IRS1 Y612 were then refed for 16 h. After each treatment, quadriceps muscles from 150 fed, fasted, and refed groups were collected and processed for detection of p-Akt Glucose (mg/dl) Sirt3 expression as above. Akt p-Erk 50 0306090120150 MEDICAL SCIENCES Erk Time (min) Changes in Sirt3 expression can also occur rapidly as illus- trated by the effects of fasting and refeeding. After 24 h of Fig. 2. Sirt3 knockout mice have increased oxidative stress and insulin re- fasting, Sirt3 mRNA and protein expression in quadriceps muscle sistance in skeletal muscle accompanied by decreased respiration. (A) A DXA were decreased by w50% and were reversed after refeeding for scan was performed using 16-wk-old male C57BL/6 WT and KO mice (n =4), 16 h (Fig. 1D). The other mitochondrial sirtuin, Sirt4, was also body composition was measured, and Student’s t test was performed for fi down-regulated in ob/ob mice. During fasting, however, STZ- signi cance. (B) CLAMS analysis was performed using 16-wk-old male C57BL/ 6 WT and KO mice. Daily food intake per mouse over 48 h of CLAMS study induced diabetes and caloric restriction had no effect on ex- was calculated and Student’s t test was performed for significance.