title: Chronic ethanol treatment of human hepatocytes inhibits the activation of the insulin signaling pathway by increasing cytosolic free calcium levels.
abstract: The present study aimed to investigate the effects of ethanol treatment on the induction of intracellular calcium ([Ca(2+)](i)) levels and the inhibition of the activation of the insulin signaling pathway in human hepatocytes. L‑02 cells were treated with various concentrations of ethanol for different periods of time. Cell viability and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) leakage in the culture supernatant were evaluated. Changes in [Ca(2+)](i) levels were detected by flow cytometry and confocal microscopy. Total RNA and protein were extracted to examine the mRNA and protein levels of insulin receptor substrate (IRS)1, IRS2, phosphatidylinositol 3‑kinase (PI3K) and glucose transporter 2 (GLUT2) by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, respectively. Furthermore, insulin was added to the ethanol‑treated L‑02 cells, and the phosphorylation levels of PI3K and protein kinase B (PKB) were determined by western blot analysis before and after Ca(2+) blockage. No significant changes were observed in cell viability, [Ca(2+)](i) levels and in the expression and phosphorylation levels of insulin signal transduction molecules when the L‑02 cells were treated with 0.5 or 1% ethanol. However, treatment with 2 or 4% ethanol resulted in a significant decrease in cell viability and in the mRNA levels of IRS1, IRS2, PI3K (p85α) and GLUT2, as well as in an increase in ALT/AST leakage and in the [Ca(2+)](i) levels (P<0.05). The expression and phosphorylation levels of PI3K (p85α) and PKB were also inhibited by treatment with 2 or 4% ethanol. These cytological effects induced by ethanol treatment were partially reversed by Ca(2+) blockage. These results suggest that ethanol treatment inhibits the activation of the insulin signal transduction pathway in a dose‑, time‑ and Ca(2+)‑dependent manner. The inhibition of IRS1/2, PI3K (p85α), PKB and GLUT2 expression and of PI3K (p85α) and PKB phosphorylation by the high concentrations of ethanol may be the core molecular mechanism of ethanol-induced insulin resistance, and may be related to the induction of [Ca(2+)](i) levels.
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, P.R. China.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, P.R. China.
authors.forename: Jin-Fang
authors.initials: JF
authors.lastname: Zhao
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, P.R. China.
authors.forename: Yong-Lin
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Department of Endocrinology, The First Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Intensive Care Unit, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, P.R. China.
authors.forename: Rong-Lin
authors.initials: RL
authors.lastname: Jiang
mesh_terms: D000410:Alanine Transaminase / Q000378:metabolism; D000428:Alcohol Drinking / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000139:chemically induced / Q000378:metabolism; D000431:Ethanol / Q000009:adverse effects* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D007328:Insulin; D000431:Ethanol; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium