Alcoholic liver disease (ALD) continues to be a major public health problem and remains the leading cause of mortality in patients with chronic liver disease. Ethanol disrupts the pathways of energy metabolism by inhibiting AMP kinase (AMPK), and interferes with the activity of sterol response element binding protein (SREBP) and peroxisome proliferator activated receptor (PPAR), leading to alcoholic steatosis. Ethanol can induce endotoxemia. Lipopolysaccharide (LPS), a component of endotoxin, can stimulate Kupffer cells (KC), to produce cytokines. We hypothesize that LPS stimulates KC to release TNFa, which is central to the pathogenesis of alcoholic fatty liver disease through its effect on PPARa, SREBP, and AMPK. We will also study the role of other cytokines released by the macrophages (i.e., MCP-1) on these regulatory proteins. We plan to test our hypothesis by pursuing the following specific aims. 1) Determine the effect of exogenous TNFa or LPS in the presence or absence of ethanol on the function of PPARa, SREBP-1, and AMPK in McA-RH7777 hepatoma cells and isolated hepatocytes, 2) Determine the effect of LPS, in the presence or absence of ethanol, on the ability of macrophages, RAW 264.7 cells or KC, to modify the function of PPARa, SREBP-1, and AMPK in hepatoma cells and hepatocytes, 3) Determine the effects of co-culture of RAW 264.7 cells (or KC) with McA-RH7777 cells (or isolated hepatocytes) on lipid metabolism of the hepatoma cells (or isolated hepatocytes), and 4) Determine the ability of hepatoma cells or hepatocytes to modify the response of RAW cells or KC to release TNFa when stimulated by LPS, in the presence or absence of ethanol. The candidate is as Assistant Professor of Clinical Medicine. To date the candidate has trained in the laboratory of Dr. David Crabb, acquiring basic molecular biology knowledge and laboratory skills. This application is a logical extension of the work in this laboratory designed to allow the candidate to develop a basic understanding of the roles of cytokines in the pathogenesis of ALD. The candidate will develop new research skills by working with a number of experts in Kupffer cell biology, lipid metabolism, and TNF signaling, as well as through course work in molecular biology and immunology. By acquiring the knowledge and skills outlined in this proposal, the candidate hopes to fulfill his career goal of becoming a well-trained and funded physician scientist.
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