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Reduced expression of the NAD+-dependent deacetylase, SIRT3, has been associated with insulin resistance and metabolic dysfunction in humans and rodents. In this study, we investigated whether specific overexpression of SIRT3 in vivo in skeletal muscle could prevent high-fat diet (HFD)-induced muscle insulin resistance. To address this, we used a muscle-specific adeno-associated virus (AAV) to overexpress SIRT3 in rat tibialis and extensor digitorum longus (EDL) muscles. Mitochondrial substrate oxidation, substrate switching and oxidative enzyme activity were assessed in skeletal muscles with and without SIRT3 overexpression. Muscle-specific insulin action was also assessed by hyperinsulinaemic-euglycaemic clamps in rats that underwent a 4-week HFD-feeding protocol. Ex vivo functional assays revealed elevated activity of selected SIRT3-target enzymes including hexokinase, isocitrate dehydrogenase and pyruvate dehydrogenase that was associated with an increase in the ability to switch between fatty acid- and glucose-derived substrates in muscles with SIRT3 overexpression. However, during the clamp, muscles from rats fed an HFD with increased SIRT3 expression displayed equally impaired glucose uptake and insulin-stimulated glycogen synthesis as the contralateral control muscle. Intramuscular triglyceride content was similarly increased in the muscle of high-fat-fed rats, regardless of SIRT3 status. Thus, despite SIRT3 knockout (KO) mouse models indicating many beneficial metabolic roles for SIRT3, our findings show that muscle-specific overexpression of SIRT3 has only minor effects on the acute development of skeletal muscle insulin resistance in high-fat-fed rats.

The mitochondrial enzyme SIRT3 is an NAD + -dependent deacetylase important in cell metabolism, and a decline in its protein expression or activity has been linked with insulin resistance in obesity, ageing and type 2 diabetes. While studies in SIRT3 knockout mice have dramatically improved our understanding of the function of SIRT3, the impact of increasing SIRT3 levels remains under-examined. In this study we investigated the effects of liver-specific SIRT3 overexpression in mice on mitochondrial function and metabolic profile in both isolated hepatocytes and in vivo. Primary hepatocytes overexpressing SIRT3 displayed increased oxygen consumption and a reduction in triglyceride accumulation. In mice with hepatic SIRT3 overexpression, increased fasting β-hydroxybutyrate levels were observed, coupled with an increase in oxygen consumption in isolated mitochondria and increased substrate utilization in liver homogenates. However, metabolic profiling of mice exposed to either chow or high-fat diet revealed no effect of hepatic SIRT3 overexpression on glucose tolerance, body composition or tissue triglyceride accumulation. These findings suggest limited whole-body benefit of increasing hepatic SIRT3 during the development of diet-induced insulin resistance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: D.A.S. is a founder, equity owner, advisor to, director of, board member of, consultant to, investor in and/or inventor on patents licensed to RevereBiosensors, UpRNA, GlaxoSmithKline, Wellom-ics, DaVinci Logic, InsideTracker (Segterra), Caudalie, Animal Biosciences, Longwood Fund, Catalio Capital Management, Frontier Acquisition Corporation, AFAR (American Federation for Aging Research), Life Extension Advocacy Foundation (LEAF), Cohbar, Galilei, EMD Millipore, Zymo Research, Immetas, Bayer Crop Science, EdenRoc Sciences (and affiliates Arc-Bio, Dovetail Genomics, Claret Bioscience, MetroBiotech, and Liberty Biosecurity), LifeBiosciences (and affili-ates Iduna, Senolytic Therapeutics, Selphagy, Jumpstart Fertility, Continuum Biosciences, and Al-terity), ATAI Life Sciences, Levels Health, and Bold Capital. D.A.S. is an inventor on a patent ap-plication filed by Mayo Clinic and Harvard Medical School that has been licensed to Elysium Health. More information at https://sinclair.hms.harvard.edu/david-sinclairs-affiliations. The remaining authors note no conflicts of interest., (Copyright © 2022. Published by Elsevier Inc.)