As an Endocrinologist, I see daily the devastating impacts obesity, hyperlipidemia, and diabetes mellitus have on patients. These three diseases are reaching pandemic status in the United States, and novel therapeutic candidates are needed to comabt [sic] these grave risks to the public health. In order to identify pharmacologically amenable targets in these three related diseases, I propose to use zebrafish in forward genetic and unbiased proteomic studies of lipid absorption, transport, and metabolism. In the first part of this project, I shall develop a simple and reliable method for staining and tracking yolk-derived and dietary lipids in the transparent zebrafish embryo. With this method, I shall confirm the suitability of the model organism for studying lipid metabolism by examining the effect of targeted knock-down of the evolutionary central and physiologically critical lipoprotein particle-packaging enzyme microsomal triglcyeride [sic] transfer protein (Mtp). Biochemical, microscopic, and physiologic assays shall be developed in order to compare the effects of decreasing zebrafish Mtp on development and dietary lipid absorption with those of abetalipoproteinemia, the human hereditary disease in which Mtp is mutated. In the second part of the project, an unbiased, forward genetic screen shall be performed to identify mutants that have alterations in lipid uptake, transport, and storage. The genetic screen shall rely on the methods developed in the first part of the project to identify and fully characterize the defects in lipid metabolism. Mutants that model human diseas [sic] states characterized by malabsorption of dietary lipid, altered kinetics of lipid consumption, and inappropriate deposition of lipids in organs (i.e., hepatic steatosis and intramyocellular liposis) shall be identified, and the genes giving rise to these mutant phenotypes shall be identified using standard positional cloning methods. The final arm of the study shall examine global changes in expression of proteins following high fat feeding. I shall use proteomic methods to find proteins whose expression is altered by high fat feeding, and employ mass spectrometric methods to identify the differentially regulated proteins. As with the genes identified in the forward genetic screen, the proteins identified in these proteomic studies shall be studied in order to develop novel strategies to treat obesity, hyperlipidemia and diabetes mellitus. These studies are well suited to the development of my career as an academic physician-scientist and they shall help combat the looming public health crisis.
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