Role And Mechanism Of Uncoupling Protein 2 On Secretory Dysfunction Of α Cells Induced By The Free Fatty Acid

Partâ… Effects of uncoupling protein 2 on secretory dysfunction of a cell induced by free fatty acidsObjective To investigate the effects of inhibition of uncoupling protein 2 on the secretory function of pancreatic a cells cultured with free fatty acids (FFA).Methods The pancreatic a cell line TC1-6 cells were cultured in normal medium or with palmitate and/or UCP2 inhibitor genipin for 72h. The mRNA expression, the content and the secretion of glucagon in cells was detected. The UCP2 expression was suppressed by RNA interference and after that the same index was measured as above.Results 1.After 72h culture, the glucagon secretion of TC1-6 cells with palmitate supplement was higher than that without it. In context of palmitate, genipin or si-UCP2 decreased the elevation of glucagon.2. Cultured with palmitate and genipin or si-UCP2, glucagon content and mRNA of TC1-6 cells did not change significantly. 3. When UCP2 in normal TC1-6 cells was knocked down by si-RNA, glucagon secretion was decreased markedly. It suggested that UCP2 involved in normal a cell secretory function maintaining. Part II The oxidative stress on a cell secretory dysfunction induced by free fatty acids:the role of uncoupling protein 2Objective To examine the state of oxidative stress and intracellular Ca2+level in a cells cultured with FFA and the role of UCP2 on the oxidative stress.Methods The TC1-6 cells were cultured in palmitate with or without genipin and the siRNA against UCP2 for 72h. The expression of UCP2 and peroxisome proliferator activated receptor co-activator la (PGC-la) were examined by RT-PCR and western blot. The nitrotyrosine (NT) concentration of medium was detected by ELISA. The free intracellular Ca2+was labeled with fluorescent marker and measured by flow cytometer. Mean fluorescence intensity (MFI) was used to represent the intracellular Ca2+level.Results 1. Compared with normal cultured TC1-6 cells, the mRNA and protein expressions of UCP2 were higher in cells cultured with palmitate. Genipin suppressed UCP2 and siRNA suppressed it much efficiently.2. Palmitate stimulated PGC-la. Genipin inhibited it in some degree and siRNA against UCP2 had no significant effect. 3. The NT concentration in palmitate cultured group was much higher than that in normal medium. There was no discernible effect of genipin on the NT concentration. Inhibition of UCP2 by siRNA against UCP2 increased NT concentration slightly in normal medium but markedly in palmitate medium.4. The MFI in cells cultured with palmitate was significantly higher that in cells cultured with normal medium. Cells cultured with palmitate based on knockdown of UCP2 had lower MFI than that cultured with palmitate only.Conclusions The activation of oxidative stress by FFA may be regulated by UCP2. The effect of suppression on oxidative stress of UCP2 may be not the major aspact of a cell secretory function. The influence of FFA and UCP2 on intracellular Ca2+ concentration may contribute to the change of a cell secretory function. Part III Effects of UCP2 on the insulin signal pathway of pancreatic a cellsObjective To investigating the effects of high level FFA on insulin signal pathway in cultured a cell and the role of UCP2 in that pathway.Methods The TC1-6 cells were cultured with normal medium, palmitate or palmitate with siRNA against UCP2 for 72h, and the insulin induced IRS-1 phosphorylation was examined by western blot. To explore the phosphoinositide 3-kinases (PI3-K)/Akt pathway in the secretion of glucagon, the TC1-6 cells were cultured with or without palmitate for 72h. Then cells were treated with genipin, insulin or PI3-K inhibitor wortmannin at various combinations. Insulin and/or wortmannin were added into the media 1h before the termination of culture. The glucagon secretion was measured by RIA, and the phosphorylated Akt and Akt levels were determined by western blotting.Results 1. Insulin induced IRS-1 phosphorylation in a cells. Compared with normal culture, the IRS-1 phosphorylation of cells was suppressed significantly by palmitate supplement, which was attenuated by the siRNA against UCP2.2. The glucagon secretion of subgroups with palmitate was generally higher than that without palmitate. Insulin suppressed glucagon secretion, which attenuated by wortmannin. Glucagon secretion in cells with genipin supplement was slightly lower than those without it, which was not influenced by insulin and/or wortmannin supplement. In cells cultured with palmitate, insulin slightly inhibited the glucagon secretory. Wortmannin blocked the effect of insulin and attenuated the suppression of genipin on glucagon secretion. 3. The baseline phosphorylated Akt was at low level. Akt phosphorylation was induced by insulin and much higher without palmitate than with it. With wortmannin, Akt phosphorylation induced by insulin decreased, but suppression with palmitate much lower. The Akt phosphorylation in subgroups with genipin was higher than without it.Conclusions High level FFA suppressed the key point of insulin signal pathway INS-1 and Akt phosphorylation in a cells. The inhibition of UCP2 partly reversed the suppression. UCP2 action pathway may crosstalk with the insulin signal pathway.