Palmitate and GPR40 agonist effects

A stock solution (100 mM) of palmitic acid (#P0500, Sigma-Aldrich, Munich, Germany) was prepared in DMSO and was added at a final concentration of 6 mM to FCS (containing 5% serum albumin) or modified Krebs-Ringer buffer (KRB) supplemented with 5% BSA. The GPR40 agonist TUG-469 (a kind gift from Prof. T. Ulven, Southern University of Denmark, Denmark) was applied at concentrations of 3–10 μM in culture medium supplemented with 1% FCS (containing 0.05% BSA) [2 (link), 18 (link)]. The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO. Inhibitors were applied 1 h prior to palmitate and kept throughout the treatment.