oa Effects of lipotoxicity and diabetes on hepatocyte function

Background and Objectives: Obesity is a major risk factor for the development of liver disease and diabetes and there is currently a pandemic of diabetes that is associated with a very high incidence of non-alcoholic fatty liver disease and consequent deregulation of liver function. The overall objective of this project is to determine whether non-alcoholic fatty liver disease and hyperglycemia affect calcium homeostasis via altering the expression of the plasma membrane calcium release-activated calcium channel protein, Orai1, the endoplasmic reticulum calcium sensor protein, STIM1, and store-operated Ca2+ entry, SOCE, in rat hepatocytes. Methods: Rat H4IIE liver cells exposed to amiodarone were used as a cell culture model of steatosis and Western Blot, RT-PCR and fura-2 techniques were used to assess protein, mRNA and changes in Ca2+ homeostasis, respectively. Rat H4IIE liver cells were grown either in normal glucose (5.5 mM) or high glucose (25 mM) for 24 hours, then each group was treated with either vehicle (methanol), amiodarone for 24 hours, or amiodarone for 24 hours followed by normal glucose for another 24 hours. Intracellular calcium [Ca2+]i was measured using fura-2am. Results: Expression of both STIM1 and Oai1 increases after treating with amiodarone for 24 hours in both normal and high glucose, but decreases after treating with amiodarone for 24 hours when followed by normal glucose treatment. In addition high glucose enhances SOCE whereas amiodarone treatment suppresses SOCE. Conclusion: Changes in the concentration of intracellular Ca2+ in hepatocytes play a central role in mediating the actions of insulin, glucagon, catecholamines and other hormones and growth factors on carbohydrate, lipid and protein metabolism in the liver. Both hyperglycemia and steatosis result in alterations in Ca2+ homeostasis suggesting that such changes may be contributing factors by which hyperglycemia, obesity and fatty liver result in insulin resistance in the liver. The results from this UREP project may lead to a better understanding of the mechanisms whereby diabetes, obesity and fatty liver disease result in liver malfunction.