Palmitic Acid-Induced Insulin Resistance

The treatment with palmitic acid (PA) was conducted on serum-starved cells as previously described [18 (link)]. Before the addition of palmitate to the medium, it was conjugated with fatty acid-free bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO). Briefly, palmitate stock solution was prepared by dissolving PA in a mixture of absolute ethanol and 1 M NaOH, heating to 70°C and conjugating with 10% BSA. Next, stock solution was diluted in serum free-DMEM, containing 10mM Hepes. Next the cells were incubated in the presence or absence of the palmitic acid at the concentration of 0.75 mM for 16h and 40h. At the end of each experimental set cells morphology as well as viability was assessed (i.e. Trypan blue staining). In selected sets (as indicated in Figures) the hepatocytes were chased by 100nM insulin (NovoRapid, Novo Nordisk, Ontario, Canada) for 15 min at 37°C then washed three times with PBS, harvested and homogenized in ice cold RIPA lysis buffer containing protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Other sets of palmitate induced insulin resistant hepatocytes were transferred for further analysis of glycogen synthesis or sphingolipid content.