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SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation

Abstract

Sirtuins are NAD+-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins3. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

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Figure 1: Fasting induces SIRT3 expression in oxidative tissues.
Figure 2: Abnormal accumulation of acylcarnitines and triglycerides in the livers of mice lacking SIRT3 during fasting.
Figure 3: Defective fatty-acid oxidation in mice lacking Sirt3-/- .
Figure 4: LCAD is hyperacetylated in Sirt3 -/- mice, deacetylated by SIRT3 in vivo and in vitro , and displays increased enzymatic activity when deacetylated.
Figure 5: Mice lacking SIRT3 show reduced ATP production, cold intolerance and hypoglycaemia.

References

  1. Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S. & Verdin, E. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc. Natl Acad. Sci. USA 103, 10224–10229 (2006)

    ADS  CAS  Article  Google Scholar 

  2. Hallows, W. C., Lee, S. & Denu, J. M. Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. Proc. Natl Acad. Sci. USA 103, 10230–10235 (2006)

    ADS  CAS  Article  Google Scholar 

  3. Lombard, D. B. et al. Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Mol. Cell. Biol. 27, 8807–8814 (2007)

    CAS  Article  Google Scholar 

  4. Kim, S. C. et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 23, 607–618 (2006)

    CAS  Article  Google Scholar 

  5. Guarente, L. Sirtuins as potential targets for metabolic syndrome. Nature 444, 868–874 (2006)

    ADS  CAS  Article  Google Scholar 

  6. Schwer, B. & Verdin, E. Conserved metabolic regulatory functions of sirtuins. Cell Metab. 7, 104–112 (2008)

    CAS  Article  Google Scholar 

  7. Schwer, B. et al. Calorie restriction alters mitochondrial protein acetylation. Aging Cell 8, 604–606 (2009)

    CAS  Article  Google Scholar 

  8. Cox, K. B. et al. Gestational, pathologic and biochemical differences between very long-chain acyl-CoA dehydrogenase deficiency and long-chain acyl-CoA dehydrogenase deficiency in the mouse. Hum. Mol. Genet. 10, 2069–2077 (2001)

    CAS  Article  Google Scholar 

  9. Kurtz, D. M. et al. Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation. Proc. Natl Acad. Sci. USA 95, 15592–15597 (1998)

    ADS  CAS  Article  Google Scholar 

  10. Zhang, D. et al. Mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance. Proc. Natl Acad. Sci. USA 104, 17075–17080 (2007)

    ADS  CAS  Article  Google Scholar 

  11. Son, C. G. et al. Database of mRNA gene expression profiles of multiple human organs. Genome Res. 15, 443–450 (2005)

    CAS  Article  Google Scholar 

  12. Ahn, B. H. et al. A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc. Natl Acad. Sci. USA 105, 14447–14452 (2008)

    ADS  CAS  Article  Google Scholar 

  13. Schuler, A. M. & Wood, P. A. Mouse models for disorders of mitochondrial fatty acid beta-oxidation. ILAR J 43, 57–65 (2002)

    CAS  Article  Google Scholar 

  14. Tolwani, R. J. et al. Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice. PLoS Genet. 1, e23 (2005)

    Article  Google Scholar 

  15. Herrema, H. et al. Disturbed hepatic carbohydrate management during high metabolic demand in medium-chain acyl-CoA dehydrogenase (MCAD)-deficient mice. Hepatology 47, 1894–1904 (2008)

    CAS  Article  Google Scholar 

  16. Spiekerkoetter, U. et al. Evidence for impaired gluconeogenesis in very long-chain acyl-CoA dehydrogenase-deficient mice. Horm. Metab. Res. 38, 625–630 (2006)

    CAS  Article  Google Scholar 

  17. Zhang, W., Della-Fera, M. A., Hartzell, D., Hausman, D. & Baile, C. Adipose tissue gene expression profiles in ob/ob mice treated with leptin. Life Sci. 83, 35–42 (2008)

    CAS  Article  Google Scholar 

  18. Yechoor, V. K. et al. Distinct pathways of insulin-regulated versus diabetes-regulated gene expression: an in vivo analysis in MIRKO mice. Proc. Natl Acad. Sci. USA 101, 16525–16530 (2004)

    ADS  CAS  Article  Google Scholar 

  19. Schwer, 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. 158, 647–657 (2002)

    CAS  Article  Google Scholar 

  20. Yang, H. et al. Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell 130, 1095–1107 (2007)

    CAS  Article  Google Scholar 

  21. McGarry, J. D. & Foster, D. W. Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem. 49, 395–420 (1980)

    CAS  Article  Google Scholar 

  22. Eaton, S., Bartlett, K. & Pourfarzam, M. Mammalian mitochondrial β-oxidation. Biochem. J. 320, 345–357 (1996)

    CAS  Article  Google Scholar 

  23. Shibata, M., Kihara, Y., Taguchi, M., Tashiro, M. & Otsuki, M. Nonalcoholic fatty liver disease is a risk factor for type 2 diabetes in middle-aged Japanese men. Diabetes Care 30, 2940–2944 (2007)

    CAS  Article  Google Scholar 

  24. Targher, G. et al. Nonalcoholic fatty liver disease and risk of future cardiovascular events among type 2 diabetic patients. Diabetes 54, 3541–3546 (2005)

    CAS  Article  Google Scholar 

  25. Hsiao, P. J. et al. Significant correlations between severe fatty liver and risk factors for metabolic syndrome. J. Gastroenterol. Hepatol. 22, 2118–2123 (2007)

    CAS  Article  Google Scholar 

  26. Kim, J. Y., Hickner, R. C., Cortright, R. L., Dohm, G. L. & Houmard, J. A. Lipid oxidation is reduced in obese human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 279, E1039–E1044 (2000)

    CAS  Article  Google Scholar 

  27. Frerman, F. E. & Goodman, S. I. Fluorometric assay of acyl-CoA dehydrogenases in normal and mutant human fibroblasts. Biochem. Med. 33, 38–44 (1985)

    CAS  Article  Google Scholar 

  28. Bennett, M. J. Assays of fatty acid β-oxidation activity. Methods Cell Biol. 80, 179–197 (2007)

    CAS  Article  Google Scholar 

  29. Srere, P. A. Citrate synthase. Methods Enzymol. 13, 3–11 (1969)

    CAS  Article  Google Scholar 

  30. Wu, J. Y. et al. ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease. J. Clin. Invest. 113, 434–440 (2004)

    CAS  Article  Google Scholar 

  31. Jensen, M. V. et al. Compensatory responses to pyruvate carboxylase suppression in islet β-cells. Preservation of glucose-stimulated insulin secretion. J. Biol. Chem. 281, 22342–22351 (2006)

    CAS  Article  Google Scholar 

  32. Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957)

    CAS  PubMed  Google Scholar 

  33. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959)

    CAS  Article  Google Scholar 

  34. Morrison, W. R. & Smith, L. M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5, 600–608 (1964)

    CAS  PubMed  Google Scholar 

  35. Oliver, H. & Lowry, J. V. P. A Flexible System of Enzymatic Analysis (Academic Press, 1972)

    Google Scholar 

  36. Saha, A. K. et al. Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo. Biochem. Biophys. Res. Commun. 314, 580–585 (2004)

    CAS  Article  Google Scholar 

  37. Stremmel, W. & Berk, P. D. Hepatocellular influx of [14C]oleate reflects membrane transport rather than intracellular metabolism or binding. Proc. Natl Acad. Sci. USA 83, 3086–3090 (1986)

    ADS  CAS  Article  Google Scholar 

  38. Rikova, K. et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131, 1190–1203 (2007)

    CAS  Article  Google Scholar 

  39. Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nature Biotechnol. 23, 94–101 (2005)

    CAS  Article  Google Scholar 

  40. Graham, J. M. Isolation of mitochondria from tissues and cells by differential centrifugation. Curr. Protocols Cell Biol. (suppl. 4), unit 3.3 (1999)

  41. Graham, J. M. Purification of a crude mitochondrial fraction by density-gradient centrifugation. Curr. Protocols Cell Biol. (suppl. 4), unit 3.4 (1999)

  42. Hirschey, M. D., Shimazu, T., Huang, J. & Verdin, E. Acetylation of mitochondrial proteins. Methods Enzymol. 457, 137–147 (2009)

    CAS  Article  Google Scholar 

  43. Taniguchi, C. M., Ueki, K. & Kahn, R. Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J. Clin. Invest. 115, 718–727 (2005)

    CAS  Article  Google Scholar 

  44. Ueki, K., Kondo, T., Tseng, Y. H. & Kahn, C. R. Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse. Proc. Natl Acad. Sci. USA 101, 10422–10427 (2004)

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

We thank T. Canavan and J. J. Maher for primary hepatocyte preparation (P30 DK026743); C. Harris, L. Swift and the Mouse Metabolic Phenotyping Center (DK59637) for analysis of plasma and tissue lipid analysis; J. Wong for electron microscopy studies, S. Mihalik and D. Cuebas for synthesis of the 2,6-dimethylheptanoyl-CoA; A.-W. Mohsen for purification of recombinant pig ETF; Y.-C. Si for assistance with adenoviral studies; A. Wilson and J. Carroll for preparation of figures; and G. Howard and S. Ordway for editorial review. This work was supported in part by a Senior Scholarship in Aging from the Elison Medical Foundation to E.V. and by institutional support from the J. David Gladstone Institutes. F.W.A. is an Investigator of the Howard Hughes Medical Institute and recipient of an Ellison Medical Foundation Senior Scholar Award. D.B.L. is supported by a K08 award from the National Institute on Aging and the National Institutes of Health. B.S. is supported by an Ellison Medical Foundation/AFAR Senior Postdoctoral Research Grant. N.B.R. and A.K.S. are supported by National Institutes of Health (NIH) grants PO1 HL068758-06A1, DK019514-29 and R01 DK067509-04.

Author Contributions M.D.H., T.S., E.G., E.J., B.S., C.A.G., C.H., S.B. and A.K.S. performed in vitro, in vivo and biochemical studies; O.R.I., R.D.S. and J.R.B. performed metabolomic studies; D.B.L. and Y.L. performed mass spectrometry studies; M.D.H. and E.V. designed the studies, analysed the data and wrote the manuscript; all other authors reviewed and commented on the manuscript.

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Hirschey, M., Shimazu, T., Goetzman, E. et al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature 464, 121–125 (2010). https://doi.org/10.1038/nature08778

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