VDAC itself binds ANT in the inner mitochondrial membrane to form the mPTP, a pore that allows the exchange of ADP and ATP across the mitochondrial membrane (28,40). respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function. Introduction Lysine acetylation has recently emerged as an important post-translational modification. Consisting of the reversible transfer of the acetyl group from acetyl-CoA to a lysine residue, this highly regulated event modulates diverse cellular processes, including protein-protein interactions, protein subcellular localization, protein stability, and enzymatic activity (1C3). Lysine acetylation is also increasingly linked to metabolism regulation and energy balance (4), and it has been shown that high-fat (HF) feeding induces a shift in the acetylation balance, causing protein hyperacetylation in liver (5). This dysregulation of the 4E2RCat acetylation balance could be due to diet-induced increases in acetyl-CoA levels, as observed in liver and muscle (6,7). 4E2RCat In a setting of overnutrition, deacetylases such as sirtuins (SIRTs) should play a key protective role by counterbalancing increased protein acetylation. SIRTs are NAD+-dependent deacetylases and as such are metabolic sensors (8). The SIRT family is comprised of seven members that differ by their subcellular distribution, substrate specificity, and cellular 4E2RCat functions (SIRT1C7) (9). Recent studies have highlighted the importance of mitochondrial SIRTs in 4E2RCat the regulation of metabolism (10). Indeed, numerous mitochondrial proteins involved in metabolism are regulated by acetylation (11). It now appears that lysine acetylation could be the most important regulatory protein modification event within the mitochondria. Recent evidence suggests that mitochondrial protein acetylation is solely regulated by deacetylases, with acetylation being the consequence of unregulated nonenzymatic reactions, driven by acetyl-CoA levels (12). SIRT3 in particular has been identified as the primary mitochondrial deacetylase (13) and has been linked to the regulation of lipid metabolism, reactive oxygen species levels, and energy production (14,15). SIRT3 has specifically been shown to direct mitochondrial respiration, and its targets include subunits of the respiratory chain complex (16,17). Importantly, SIRT3 has been implicated in the development of metabolic disease in both humans and rodents (5). SIRT3 activity is increased by nutrient distress such as fasting, caloric restriction, and forced exercise, while SIRT3 activity is decreased in skeletal muscle of models of type 1 and type 2 diabetes (18). 4E2RCat Given that SIRT3 is implicated in mitochondrial function, understanding how SIRT3 is involved in the regulation of metabolism is critical in understanding the link between mitochondrial function and metabolic disease. Mice lacking SIRT3 exhibit a decrease in oxygen consumption in isolated mitochondria from liver (16) and reduced glucose tolerance when placed on a high-fat diet (HFD) (5). However, the in vivo contribution of distinct peripheral tissues to this phenotype remains to be elucidated, as the results of efforts Ntrk1 to decipher the distinct roles of SIRT3 in tissue-specific models have remained inconclusive. Indeed, while various global SIRT3 knockout (KO) models consistently exhibit metabolic impairments (5,16,18), liver and muscle-specific KO models have no overt metabolic phenotype (19) as assessed by primary screening tools (20). Here, we report, for the first time, the assessment of insulin sensitivity in chow and HF-fed SIRT3 KO mice using the hyperinsulinemic-euglycemic clamp. In this study, we show that HF-fed SIRT3 KO mice have exacerbated insulin resistance compared with their wild-type (WT) littermates and that this phenotype is due to defects in skeletal muscle metabolism. Furthermore, we show that SIRT3-deficient muscle exhibits profound.