Mechanisms of NAT2 Regulation of Insulin Resistance and Mitochondrial Dysfunction

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PROJECT SUMMARY/ABSTRACT Decreased insulin sensitivity (insulin resistance, IR) is a fundamental abnormality in patients with type 2 diabetes (T2D), and a major risk factor for cardiovascular disease (CVD). We led a genome wide association study (GWAS) for direct measures of IR and identified a novel IR gene, N-acetyl transferase 2 (NAT2). Non- synonymous coding variants in NAT2 were associated with increased IR independently of body mass index as well as IR-related traits. Knockdown and overexpression of the mouse ortholog Nat1 led to changes in glucose homeostasis in adipocytes and myoblasts. Nat1 deficient mice (Nat1 KO) had decreased insulin sensitivity and elevations in fasting blood glucose, insulin and triglycerides. Nat1 is highly co-regulated with key mitochondrial genes and RNA-interference mediated silencing of Nat1 leads to mitochondrial dysfunction characterized by increased intracellular reactive oxygen species and mitochondrial fragmentation as well as decreased mitochondrial membrane potential, biogenesis, mass, cellular respiration and ATP generation. Nat1 KO mice have a decrease in basal metabolic rate and exercise capacity without altered thermogenesis versus Nat1 wild type (Nat1 WT) mice. Nat1 KO mice also have changes in plasma metabolites and lipids, such as decreased levels of acylcarnitines, and indirect calorimetry data shows decreased utilization of fats for energy, suggesting that Nat1 deficiency is associated with an impaired fatty acid oxidation (FAO). New data indicate that supernatant from Nat1 deficient liver cells results in IR in adipocytes. Our overall hypothesis is that Nat1 binds to and regulates key mediators of mitochondrial function and energy balance in the liver ultimately leading to IR. Using our unique resources including a liver specific knockout mouse (Nat1 LKO), we will test this hypothesis and elucidate the mechanisms of insulin resistance caused by Nat1 deficiency. Nat1 is known to acetylate certain drugs and carcinogens but the endogenous substrate/s are unknown. Studies in Aim 1 will identify Nat1 protein-protein interactions and Nat1 acetylation substrates that regulate energy balance and metabolism. Our hypothesis is that Nat1 binds key regulators of mitochondrial function. In Aim 2 we will define the specific mitochondrial defects in Nat1 deficiency. Our hypothesis is that Nat1 deficiency causes impaired FAO and that this can be rescued by augmenting β-oxidation. In Aim 3 we will define mediators of local and systemic effects of Nat1 deficiency. Nat1 is highly expressed in the liver with more modest expression in insulin-sensitive tissues. We believe that hepatic Nat1 mediates whole body insulin sensitivity specifically through signaling intermediates that act through effects on adipose and skeletal muscle. We will confirm this through detailed phenotyping, including euglycemic clamp, of liver specific Nat1 KO. We will also identify secreted factors that impair insulin sensitivity in Nat1 deficiency building on our co-culture data from Nat1 deficient liver cells and adipocytes. These aims will define the pathophysiological role of the novel IR gene Nat1, thereby increasing our understanding of IR, which is a necessary step towards new therapies.