801 Calcium-Dependent Signaling, via Calcineurin and NFAT, Is Key to Steatosis in Alcoholic Liver Disease (ALD) in Mice

Abstract

Background: Alcohol abuse leads to alcoholic liver disease (ALD). In ALD hepatic steatosis is a prerequisite of disease progresses to steatohepatitis (SH) at which stage the liver injury becomes evident. The mechanisms of steatosis in ALD are noit fully understood. Calciumdependent signaling is activated in ALD in mice. The Aim of our study was to evaluate the role of calcium-dependent signaling in development of steatosis in ALD. Methods: We fed alcohol (Lieber-deCarli) or control diet to control C57Bl6 and NFAT-KO mice or cyclosporine-treated C57Bl6 mice. Results: Alcohol, but not control, diet led to significant ALD, revealed by elevated liver Tg content and significant OilRedO liver tissue staining suggestive of steatosis, increased serum ALT suggestive of liver injury, and serum cytokines TNFa, IL1, IL6, suggestive of inflammation, in C57Bl6 mice. There was significant elevation of calcium signaling in livers of alcohol-fed animals compared to control diet, as revealed by higher expression of Calcineurin, PLC, PKC, and MAPKp38 and elevated NFAT activity. Alcohol, but not control, diet lead to significant induction ACS, SCD1, ELOV16, GPAT and DGAT, LDLR HMG-CoA reductase mRNA in the livers of ethanol-fed animals. Further, the amount of mature SREBP-1protein, suggestive of SREBP activation, was increased in liver of alcoholfed animals. Inhibition of calcium signaling by either Cyclosporine treatment (at the level of Calcineurin) or by genetic NFAT deficiency partially prevented alcohol diet-induced upregulation of ACS, SCD1, ELOV16, GPAT and DGAT; more important, inhibition of calcium signaling led to partial protective against alcohol diet-induced liver injury and steatosis. NFAT protein was detected in both KCs and Hpt. In vitro, palmitic acid-exposed Hepa1.6 cells, used as surrogate of Hpt, developed steatosis; this process was significantly impaired when the cells were treated with cyclosporine and in cells made NFAT deficient by specific siRNA treatment. Co-culture of Hepa 1.6 cells+palmitic acid with inflammatory (LPS-pretreated) KCs lead to further upregulation of lipid uptake; sole exposure of KCs to cyclosporine did not prevent steatosis in co-culture. These data suggested that calciumdependent signaling mechanisms are involved in lipid synthesis in hepatocytes at different levels, including lipogenesis and lipolysis, in a KC-dependent manner. In conclusion, we report novel finding that calcium signaling is, in part, responsible for development of steatosis component of ALD in mice. It remains to be determined if inhibition of calcium signaling may be beneficial for delaying of steatosis and/or blunting of progression from HS to SH phases of ALD.