The Effect Of Conditioned Medium From Hypoxic Adipocytes On Glucose And Lipid Metabolism In Hepatocytes

Objective:Adipose tissue hypoxia is early phenotype in obesity. Hypoxia is associated with elevated gene expression of pro-inflammatory cytokines such as TNF-a, IL-6among others, infiltration by macrophages. Previously, we have shown that conditioned medium collected from hypoxia-treated adipocytes (CM-H) renders muscle cells insulin resistant. Here, we test the effect of CM-H on lipid accumulation, insulin signaling and gluconeogenic genes expression in liver cells.Methods:1. Adipocytes were incubated at37℃in21%O2and5%CO2(normoxic condition) or1%O2,94%N2and5%CO2(hypoxic condition). Conditioned media (CM) were collected after24h.2. The secretion levels of TNFα、IL-1β and MCP-1in the conditioned medium were determined by ELISA.3. mRNA levels of gluconeogenic genes expression PEPCK and G6Pase were determined by real-time PCR.4. Lipid accumulation level was detected by oil red O staining.5. Phosphorylation of insulin signaling molecules in liver cells were detected by Western Blot.6. The effects of CM on gluconeogenic genes expression PEPCK and phosphorylation of insulin signaling molecules in liver cells were detected after neutralization of TNFa with specific antibody.7. The effects of CM on phosphorylation of insulin signaling molecules in liver cells were detected after neutralization of IL-1β with specific antibody.Results:1. Hypoxia increases the secretion of TNFα IL-1β and MCP-1in adipocytes.2. CM-N does not effect the expression of the gluconeogenic gene, CM-H increases the expression of the gluconeogenic genes G6Pase and PEPCK in hepatocytes.3. Both CM-N and CM-H induce lipid accumulation in hepatocytes. But CM-H significantly induces lipid accumulation in hepatocytes4. CM-H treatment markedly reduces IRS2protein expression and insulin-stimulated IRS phosphotyrosine levels, reduces phospho-Akt at the pT308and pS473sites, as well as phospho-S9-GSK3and phospho-S256-FoxO1in hepatocytes.5. Neutralization of TNFa in CM partly reverses the effect of CM on IRS phosphotyrosine, Akt phosphorylation and PEPCK gene expression.6. Neutralization of IL-1β does not reverses the effect of CM on Akt phosphorylationConclusion:1. Hypoxia increases the secretion of TNFa, IL-1β and MCP-1in adipocytes.2. CM-N does not effect the expression of the gluconeogenic genes, CM-H increases the expression of the gluconeogenic genes G6Pase and PEPCK in hepatocytes.3. CM-N causes slight lipid accumulation, whereas CM-H significantly induces lipid accumulation in hepatocytes.4. CM-N reduces insulin-stimulated IRS, Akt, GSK3β and FoxO1phosphorylation and induces slight insulin resistance in hepatocytes. CM-H markedly reduces insulin-stimulated IRS, Akt, GSK3β and FoxO1phosphorylation and induces insulin resistance in hepatocytes.5. Neutralization of TNFa, but not IL-1β in CM-H partly reverses the effect of CM on IRS phosphotyrosine, Akt phosphorylation and PEPCK gene expression. TNFa but not IL-1β plays a role in CM-induced insulin resistance in hepatocytes.