The purpose of this application is to train the applicant to become a productive, independent investigator in obesity-related diabetes research. The applicant's long-term goal is to elucidate the molecular mechanisms by which fatty acids are partitioned in cells and the effects of fatty acid on regulating obesity associated insulin resistance. The proposed studies will provide the applicant with the opportunity to build on existing knowledge in lipid metabolism, insulin action, rodent and human physiology and biochemistry. Specifically the applicant will obtain expertise in cell culture and microscopy techniques, protein kinase signaling, lipid metabolism, molecular biological techniques, and glucose clamp analyses. The training plan will also provide training and experience in writing manuscripts, grant proposal development, group presentations, and increased scientific independence. In concordance with a goal of NIDDK funded obesity research, the long term objective of the proposal is to understand at the cellular level mechanisms linking obesity and overnutrition to insulin resistance. Perilipin is a protein that localizes at the surface of intracellular lipid droplets (LD) where triacylglycerol (TAG) is stored and regulates fatty acid release from stored TAG. Although perilipin is abundantly expressed in human hepatocytes within steatotic livers it is not expressed within steatotic livers of obese mice. Preliminary data from our laboratory confirm this finding, and also that ectopic expression of perilipin in isolated mouse hepatocytes induces and enhances formation of hepatic lipid droplets. The proposed project will test the hypothesis that perilipin expression in human hepatocytes acts to sequester fatty acids as TAG into LD, thereby reducing TAG secretion and preventing the effects of fatty acids to promote insulin resistance. The applicant will test this hypothesis, in Aim 1, using primary human hepatocytes and HepaRG human hepatoma cells, which express perilipin, and examining the effects of perilipin knockdown;as well as using adenoviruses used to express perilipin with isolated mouse hepatocytes. These hepatocytes will be incubated with oleic and/or palmitic acid to mimic in vivo trafficking of fatty acids to the liver and facilitate TAG accumulation.
In Aim 2 we will extend our studies to examine the effects of perilipin expression within hepatocytes in vivo, by creating a transgenic mouse with hepatocyte-specific expression of perilipin and inducing obesity with a high fat diet. This hypothesis driven proposal will produc novel insights and understanding about the role of perilipin in regulating hepatic lipid metabolism and the pathogenesis of insulin resistance.
Obesity is associated with increased storage of fat within the liver that can alter the regulation of glucose in the blood. The proposed experiments will expand our understanding of the mechanisms that regulate fat storage within the liver and blood glucose levels. These studies will guide the design of new therapeutic targets to prevent insulin resistance and treat type 2 diabetes.