PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of liver abnormalities from the relatively benign storage of excess fat in nonalcoholic fatty liver (NAFL) to the more ominous nonalcoholic steatohepatitis (NASH), characterized by hepatocellular injury, necroinflammation, and fibrosis. Liver damage in NASH is the fastest growing cause of hepatocellular carcinoma and is now the most common reason for liver transplantation in women. The prevalence of NAFLD parallels that of obesity, insulin resistance and type 2 diabetes (T2D), and patients with these comorbidities are at greater risk of life-threatening liver outcomes. However, there are no approved treatments for NAFLD and there is an urgent need for new strategies to suppress this emerging epi- demic. Our goal is to employ innovative and highly integrative approaches in human subjects to gain mechanistic insight into the pathogenesis of NAFLD that will foster the development of new therapeutics that address the underlying pathological processes. Early in the course of NAFL, hepatic lipid delivery is increased as a conse- quence of high fat diets, adipose tissue insulin resistance and increased uptake and de novo synthesis of lipids. This metabolic milieu increases mitochondrial density, oxidative capacity and TCA cycle flux in NAFL livers, which eventually may overwhelm antioxidant defense mechanisms to promote the development of NASH. In preclinical studies we observed that the insulin sensitizer and PPARγ agonist pioglitazone directly suppressed elevated hepatic mitochondrial oxidative capacity and TCA cycle activity in these NAFL livers. Therefore, in the following three specific aims we will utilize stable isotope tracer methodology to test the central hypothesis that hepatic mitochondrial function is a targetable feature of NAFLD in humans. In Specific Aim 1 we will first quanti- tate hepatic mitochondrial fluxes across the NAFLD spectrum using a triple stable isotope tracer approach ([U- 13C]propionate, [3,4-13C2]glucose and 2H2O) coupled to 2H and 13C NMR analysis to simultaneously interrogate hepatic glucose metabolism and mitochondrial fluxes in well characterized patients with NAFL and NASH. Next, in Specific Aim 2 we will evaluate hepatic mitochondrial function as a target of insulin sensitizers in humans and will interrogate the contribution of peripheral PPARy agonism on hepatic mitochondrial fluxes and glucose me- tabolism in NAFLD. Finally, in Specific Aim 3 we will determine the mitochondrial mechanisms governing the effect of pioglitazone on NAFLD: In mice with NAFLD using a combination of single-nuclei genomics and shotgun lipidomics approaches to examine parenchymal and non-parenchymal cell transcriptomics and to quantitate mi- tochondrial lipid remodeling, respectively, in liver biopsies obtained following chronic pioglitazone treatment. Collectively, these studies will identify mechanisms that can be exploited in the future to foster the design of novel therapies that safely target T2D and NAFLD.

NARRATIVE Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity of type 2 diabetes (T2D) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. The increased prevalence of these metabolic disorders presents a significant threat to public health worldwide, and treatment strategies that address the underlying pathological processes in liver are urgently needed. In the current proposal we will reveal novel physiological and molecular mechanisms of insulin sensitization with the goal of identifying novel strategies to target these metabolic diseases in the future.