Nonalcoholic fatty liver disease (NAFLD), affecting ~30% of the U.S. population, is projected to replace hepatitis C as the leading cause of liver transplantation by 2020. Developing effective NAFLD treatments is hampered by a poor understanding of its underlying mechanisms, including complex interactions between genetic and environmental factors. Glycerophosphodiester phosphodiesterase domain-containing protein 3 (GDPD3) is a newly discovered enzyme containing lysophospholipase D activity that converts lysophospholipid to lysophosphatidic acid (lysoPA) in non-hepatic cells. Mammalian GDPD3 has not previously been implicated in hepatic lipid metabolism. Our preliminary data indicates a positive correlation between human GDPD3 expression and triglyceride (TG) accumulation in hepatocytes and mouse livers, suggesting a novel gene in the regulation of hepatic TG homeostasis. Nonetheless, the intracellular locations, substrate specificity, physiological function, and molecular mechanisms of human GDPD3 in hepatocytes/livers are unknown. Therefore, in this study, with the guidance of a highly experienced multi-disciplinary mentoring group, we propose to investigate enzymatic properties of human GDPD3 and explore whether human GDPD3 is a causal gene for hepatic steatosis. More specifically, we are asking three questions: 1) Is human GDPD3 an endoplasmic reticulum membrane-associated enzyme containing lysophospholipase D activity? 2) Does human GDPD3 increase lysoPA production resulting in increased hepatic TG synthesis and accumulation via the glycerol phosphate pathway? 3) Does human GDPD3-produced lysoPA activate peroxisome proliferator- activated receptor gamma (PPAR?) which enhances hepatic steatosis via increased fatty acid (FA) uptake and TG synthesis? To answer these questions, we will overexpress human GDPD3 in hepatoma cell lines and in mouse liver to determine: a) the effect of human GDPD3 overexpression on oleic acid-induced TG accumulation in hepatoma cells and diet-induced hepatic steatosis in mice; b) the subcellular localization of human GDPD3 in hepatoma cells and mouse primary hepatocytes; and c) the amount and molecular species of GDPD3 lipid substrates and products in mouse livers. Liver-specific human GDPD3 overexpressing mice with loss-of-function or gain-of-function in PPAR? will be fed chow or a Western-type diet to induce hepatic steatosis. We will perform comprehensive hepatic and systemic metabolic phenotyping on these mice. Primary hepatocytes will be used to investigate de novo lipogenesis, FA uptake and incorporation into TG, FA oxidation, and very low density lipoprotein-TG secretion using radioactive isotopes. When the proposed aims are achieved, we will have a better mechanistic understanding of the relationship between human GDPD3 and hepatic steatosis, to address the gap in knowledge regarding NAFLD pathogenesis and inform strategies for its treatment. Finally, this proposal provides the necessary training and mentored guidance for the applicant to transition to a successful career as an independent investigator in lipid and glucose metabolism.
There has been no standard treatment or drug approved by the U.S. Food and Drug Administration to treat hepatosteatosis due to a poor understanding of disease mechanisms. In this proposal, we developed a multidisciplinary approach to functionally annotate a newly discovered enzyme GDPD3 (glycerophosphodiester phosphodiesterase domain-containing protein 3), including mapping its subcellular localization, determining its substrate specificity, and linking its expression to altered hepatic triglyceride metabolism, with the long-term goal of identifying therapeutic targets to improve human hepatic steatosis.