This is a competitive renewal of an R24 grant focused on SIRT3, the major mitochondrial protein deacetylase. Our initial funding was limited to two years due to NIH budget constraints, but we believe that during the first 18 months of this grant we have made great progress in forming and an interdisciplinary team that has defined novel biological functions of SIRT3 and its role in regulation of systemic metabolism. We have found that SIRT3 expression is differentially regulated in liver and muscle in fasting and diabetes. In liver, increased SIRT3 during fasting leads to deacetylation of key mitochondrial enzymes in the fatty acid oxidation pathway and to an increase in their enzymatic activities. In contrast, SIRT3 expression decreases in muscle during fasting, leading to hyperacetylation of pyruvate dehydrogenase and several components of the electron transport chain. Metabolomic analysis of SIRT3KO reveals possible tissue-specific differences in regulation of fatty acid, glucose, and amino acid oxidation that will be explored further in the current application. These studies clearl show that SIRT3KO mice have defective fatty acid oxidation in liver and reduced metabolic flexibility and reduced ATP levels in muscle, as well as insulin resistance due to increased ROS and activation of stress kinases. Furthermore, SIRT3KO mice placed on a high fat diet show accelerated development of a syndrome that mimics human metabolic syndrome with obesity, type 2 diabetes, lipid abnormalities, and steatohepatitis. Together these findings point to a role of reversible mitochondrial protein acetylation as a key regulator of mitochondrial metabolism and SIRT3 as a potentially important factor in the pathogenesis of type 2 diabetes and the metabolic syndrome. The overall goal of this proposal is to extend these studies to completely define the role of protein acetylation and SIRT3 in mitochondrial function and control of metabolism. We will take advantage of our highly collaborative and multidisciplinary team harnessing the power of mass spectrometry-based proteomics, metabolomics, molecular biology, extensive physiological testing and unique animal models to further expand our understanding of this important process in regulation of mitochondrial function and metabolism.

Public Health Relevance

Mitochondria are key players in the pathogenesis of several metabolic disorders. The recent identification of reversible lysine acetylation on a large number of mitochondrial proteins and its regulation by SIRT3 indicates that reversible mitochondrial acetylation plays a role in the pathogenesis of metabolic disorders. Understanding the role of SIRT3 in the pathogenesis of the metabolic syndrome and other metabolic disorders could yield novel therapeutic opportunities for the treatment of these disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Resource-Related Research Projects (R24)
Project #
5R24DK085610-04
Application #
8496766
Study Section
Special Emphasis Panel (ZDK1-GRB-7 (M2))
Program Officer
Sechi, Salvatore
Project Start
2010-06-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$1,553,752
Indirect Cost
$320,516
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
AbouElfetouh, Alaa; Kuhn, Misty L; Hu, Linda I et al. (2015) The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites. Microbiologyopen 4:66-83
Kuhn, Misty L; Zemaitaitis, Bozena; Hu, Linda I et al. (2014) Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation. PLoS One 9:e94816
Newman, John C; Verdin, Eric (2014) ?-hydroxybutyrate: much more than a metabolite. Diabetes Res Clin Pract 106:173-81
Rardin, Matthew J; Held, Jason M; Gibson, Bradford W (2013) Targeted quantitation of acetylated lysine peptides by selected reaction monitoring mass spectrometry. Methods Mol Biol 1077:121-31
Jing, Enxuan; O'Neill, Brian T; Rardin, Matthew J et al. (2013) Sirt3 regulates metabolic flexibility of skeletal muscle through reversible enzymatic deacetylation. Diabetes 62:3404-17
Wirth, Martina; Karaca, Samir; Wenzel, Dirk et al. (2013) Mitochondrial SIRT4-type proteins in Caenorhabditis elegans and mammals interact with pyruvate carboxylase and other acetylated biotin-dependent carboxylases. Mitochondrion 13:705-20
Rardin, Matthew J; Newman, John C; Held, Jason M et al. (2013) Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways. Proc Natl Acad Sci U S A 110:6601-6
Schilling, Birgit; Rardin, Matthew J; MacLean, Brendan X et al. (2012) Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteomics 11:202-14
Hirschey, Matthew D; Shimazu, Tadahiro; Jing, Enxuan et al. (2011) SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome. Mol Cell 44:177-90
Jing, Enxuan; Emanuelli, Brice; Hirschey, Matthew D et al. (2011) Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production. Proc Natl Acad Sci U S A 108:14608-13