Store-operated calcium channels and the development of liver steatosis

Author: Eunus Ali

Ali, Eunus, 2015 Store-operated calcium channels and the development of liver steatosis, Flinders University, School of Medicine

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Abstract

Obesity, the development of a fatty liver growth, and insulin resistance in hepatocytes are some of the critical hallmarks of type 2 diabetes. Lipid accumulation (steatosis) in hepatocytes may lead to non-alcoholic fatty liver disease (NAFLD), which can progress to non-alcoholic steatohepatitis and type 2 diabetes. The mechanisms by which steatosis leads to NAFLD and insulin resistance are not well understood. Moreover, intracellular Ca2+ signalling and endoplasmic reticulum function are known to be altered in steatotic hepatocytes. Therefore the aims of this thesis were to determine if store-operated Ca2+ entry (SOCE) is altered in steatotic liver cells, to evaluate the mechanisms involved, and to evaluate the effects of impaired SOCE on glucose and lipid metabolism in steatotic liver cells. Lipid accumulation in vitro was induced in cultured liver cells by amiodarone or palmitate, and in vivo in hepatocytes isolated from obese Zucker rats. Intracellular lipid accumulation was determined by Nile red fluorescent stain and Ca2+ entry through store-operated Ca2+ channels (SOCs) was measured using fura-2 fluorescence imaging. It was found that the rates of store-operated Ca2+ entry and Ca2+ release from intracellular stores were substantially reduced in lipid loaded cells. Inhibition of protein kinase C reversed the impairment of Ca2+ entry in lipid-loaded cells. The lipid-induced inhibition of SOCE was substantially reversed by exendin-4, a member of the glucagon like peptide-1 (GLP-1) analogue family of anti-diabetic drugs in frequent use for the treatment of insulin resistance. Inhibition of Ca2+ entry was associated with reduced hormone-initiated intracellular Ca2+ signalling, enhanced lipid accumulation and altered glucose metabolism. It is concluded that these results provide evidence that steatosis leads to substantial inhibition of store-operated Ca2+ entry through a mechanism involving the activation of one or more isoforms of protein kinase C. The inhibition of SOCE enhances the accumulation of intracellular lipids and may contribute to the development of non-alcoholic steatohepatitis and insulin resistance. In addition, the finding that the lipid-induced inhibition of SOCE is reversed by antidiabetic drug exendin-4 may indicate to a novel site of action for this drug.

Keywords: Steatosis, non-alcoholic steatohepatitis, intracellular calcium, hepatocytes, store-operated calcium entry, STIM1, Orai1, protein kinase C, exendin-4
Subject: Medical Biochemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Prof Greg Barritt