Cells were induced with the indicated reagents at 24 h after transfection

ncentration. The 19271755 elevation of i in b-cells, which may be achieved by either calcium PTK/ZK chemical information influx through membrane channels or calcium release from intracellular calcium stores, triggers and amplifies the exocytosis of insulin granules. Both intracellular metabolites of FFAs and activation of GPR40 may regulate b-cell function via modulating levels of i in b-cell. FFA metabolites, particularly long-chain acyl-CoA, have stimulatory effects on Ca2+ release from endoplasmic/sarcoplasmic reticulum in muscle cells. In addition, long-chain acyl-CoA induces mitochondrial permeability transition pore formation leading to cell apoptosis of liver cells. The Ca2+mobilizing effects of long-chain acyl-CoA or other metabolites of FFAs in b-cells need to be clarified. On the other hand, GPR40 activation stimulated by FFAs leads to an acute increase in i in b-cells. GPR40 activation by FFAs activates phospholipase C to produce inositol triphosphate and the increase in i through Ca2+ release from IP3-senstive intracellular Ca2+ pools. The relative contribution and significance of this 1 LA Increases i in Beta-Cells two signaling pathways in FFAs-induced increase in i in bcells is unknown. In the present study, we used linoleic acid to observe the effects of FFAs on i levels in primary cultured rat b-cells. The respective effects of the FFA receptor signaling pathway and the intracellular 15120495 FFA metabolite signaling pathway on i were carefully dissected and the multiple pathways for increase in i were demonstrated. 2/AM was purchased from Invitrogen. Fetal calf serum, HEPES and penicillin/streptomycin were from Gibco. Preparation and Culture of Rat Pancreatic b-cells Pancreatic islets were isolated from 1012 week-old male Sprague-Dawley rats as previously described. Briefly, rats were killed by CO2 inhalation and the pancreas of each rat was inflated by injecting 10 ml collagenase solution into it through the bile duct. The collagenase solution was composed of 0.5 mg/ml collagenase, 0.1 mg/ml DNase I and 1 mg/ml BSA in Hank’s Balanced Salt Solution. The pancreases were collected and digested at 37uC for 30 minutes and then were dispersed by shaking. The islets were separated by Histopaque-1077 density gradient centrifugation and collected for cell isolation. The islets were dispersed into single cells by digestion with dispase solution. Dispase solution was composed of 1 mg/ml dispase, 0.1 mg/ml DNase I and 1 mg/ml BSA in Ca2+-free HBSS. The islet cells were plated onto glass cover slips coated with 0.01% poly-L-lysine and then cultured at 37uC in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2. The culture medium was changed every 2 days. The cells were used for i recording during day 36 in culture. Materials and Methods Ethic Statement Sprague-Dawley rats were purchased from the Animal House of the University of Queensland. The animal experiment was reviewed and approved by the Animal Care and Use Committee of UQ. The experiment was performed in compliance with the Animal Welfare Act and the guide to the care and use of laboratory animals in UQ. Every effort was made to alleviate animal discomfort and CO2 inhalation was applied as the appropriate method of sacrifice. Chemicals Measurement of i Islet cells were loaded with 1 mM Fura-2/AM in RPMI-1640 medium for 30 minutes at 37uC. Cells were subsequently rinsed LA Increases i in Beta-Cells wi