All abnormal RR intervals were checked for validation and labeling

free fatty acid toxicity, and endoplasmic reticulum stress. G-protein-coupled transmembrane receptor 40 is a membrane-bound FFA receptor mainly expressed in the brain and pancreatic b-cells. Accumulating evidence indicates that GPR40 mediates the majority of both acute and chronic effects of FFAs on insulin secretion, including the amplification of GSIS, and the receptor has been suggested to be involved in the control of cell proliferation via extracellular signal-related kinase, phosphoinositide 3-kinase, and PKB PPAR-c and GPR40 in Pancreatic b-Cells signaling pathways. GPR40 is also expressed in enteroendocrine cells, including cells expressing incretin hormones, glucagon like peptide-1 and glucose-dependent insulinotrophic peptide, and it modulates FFA-stimulated insulin secretion not only from pancreatic b-cells, but also through the regulation of incretin hormones. Recently, it was reported that TZDs may preferentially activate the GPR40 receptor, resulting in Ca2+ mobilization from thapsigargin-sensitive intracellular stores that would induce cell growth, whereas the endogenous PPAR-c ligand, 15-deoxy-D12,14prostaglandin J2, did not induce any Ca2+ signal and inhibited cell growth in nonmalignant human bronchial epithelial cells. Taken together, TZDs increase intracellular Ca2+ from the ER through GPR40 receptor activation in a PPAR-cindependent manner. In this context, we investigated whether PPAR-c activation stimulates insulin secretion through the up-regulation of GPR40 in INS-1 cells. We also explored the GPR40 downstream signaling pathways involved in the role of PPAR-c activation in pancreatic b-cells. Measurement of insulin release The medium was replaced with defined serum-free medium containing RGZ, and other inhibitors at the designated concentrations. After 24 h of treatment, cells were washed with KrebsRinger-bicarbonate-HEPES buffer and incubated for an additional 60 min in 1 ml of KRBH buffer containing 5.6 or 16.7 mM of glucose. Insulin secretion was normalized for cell number by measuring total protein in each AZ-6102 site experiment with the Bradford assay, and was determined using a rat insulin ELISA kit. FFAs including linoleic, oleic and palmitic acid in 0.01 M NaOH was incubated at 60uC for 30 min, and then FFAs were complexed with 5% FFA-free BSA in PBS. The FFA/BSA conjugates were used to treat INS-1 cells. Perifusion for insulin secretion After GPR40 RNAi transfection, secreted insulin was measured in a perifusion system. 16105 INS-1 cells were cultured in small chambers on Millicell culture inserts. INS-1 cells were perifused in 3.3 mM glucose KRBB media for 30 mins at flow rate of 1 ml/min at 37uC chamber. Glucose concentrations were modified at 16.7 mM concentration. Fractions were collected at 1 min intervals during the 1st peak insulin secretion and then collected at 2 min intervals. Collected fractions were stored at 220uC before performing insulin ELISA. Methods Materials Rosiglitazone was obtained from Alexis. The U-73122 and nifedipine were purchased from Calbiochem. All other reagents were purchased from Sigma-Aldrich unless noted. Western blotting Thirty micrograms of proteins per lane were electrophoresed on 10% polyacrylamide gels and electroblotted onto nitrocellulose membranes. Blots were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 40 min and incubated overnight at 4uC with the primary antibodies. Nuclear and cytoplasmic extraction to determine FoxO1 cellular localiza