This proposal will evaluate critical-interactions of cytokines and nutritional/ metabolic abnormalities of the hepatic transmethylation/transsulfuration pathways in the development of ALD using novel human, animal, and in vitro models, with the ultimate goal of developing specific therapy for ALD. Abnormal metabolism of tumor necrosis factor-alpha (TNF) is well documented in both experimental models of ALD and human ALD, and TNF plays a critical role in the development of experimental ALD. Abnormal methionine metabolism also is well documented in ALD, with patients frequently having elevated plasma methionine concentrations which correlate with prognosis. MAT1 A, the enzyme responsible for the initial conversion of methionine to S- adenosylmethionine (SAMe) is decreased in ALD. Depressed SAMe levels are observed in most forms of experimental liver injury including ALD, and SAMe therapy is an effective hepatoprotective agent in experimental liver injury. Data from our group show that one potential beneficial effect of SAMe is its ability to inhibit endotoxin stimulated TNF production in monocytes/Kupffer cells. Moreover, MAT1A """"""""knockout"""""""" mice have recently been generated, and these SAMe deficient mice develop spontaneous steatohepatitis and are much more sensitive to a second insult. While SAMe levels are depleted in ALD, both S-adenosylhomocysteine (SAH) and homocysteine levels are elevated. The SAMe:SAH ratio and the cellular concentration of SAH critically control most methyltransferase reactions in the body, and we recently reported that increased SAH sensitized to TNF induced hepatotoxicity. It is our working hypothesis that increased TNF in conjunction with altered SAMe/SAH metabolism play an etiologic role in the development/progression of liver injury in ALD. The specific objectives of this proposal are to: 1. Document that SAH sensitizes to TNF hepatotoxicity in vitro and in vivo. 2. Evaluate the effects of alcohol/SAH on induction of mitochondrial dysfunction and hepatocyte death. 3. Determine mechanisms whereby SAMe and SAH modulate LPS-stimulated cytokine production. 4. Evaluate beneficial effects of SAMe therapy on critical metabolic functions in patients with alcoholic cirrhosis. These studies utilize state-of-the-art models/techniques in a translational approach to develop an enhanced understanding of mechanisms involved in ALD and potential new therapy for ALD.
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