Investigation On Relationship Between GPR40 And Lipotoxicity Of The Pancreatic β-cells As Well As The Effect Of Pioglitazone

Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreaticβ-cells, whereas impairβ-cell function following long term exposure. The FFAs receptor, G-protein-coupled receptor 40 (GPR40) has been proposed to mediate insulin secretion, hence to play an important pathophysiological role in type 2 diabetes mellitus. Despite intensive research efforts, the physiological role of GPR40 still remains unclear. In order to gain a better understanding for GPR40, this study was conducted to investigate the role of GPR40 in pancreatic islet cells, including the effect of GPR40 on both short and long-term FFAs on GSIS, the relationship between GPR40 and FFAs mediated expression of PDX-1 and GLUT2 as well as the GPR40 contribution to the thiazolidinedione reverse role on lipotoxicity ofβ-cells.The first part of this study was to establishβTC6 cell line stably expressing GPR40shRNA. Therefore two complementary 55-mer siRNA template oligonucleotides encoding GPR40 short hairpin RNAs (shRNAs) were designed and ligated with linearized pSilencer 4.1-CMV neo siRNA expression vector. Then the sequence was further identified by sequencing from both sides. The plasmid was transfected intoβTC6 cells using lipofectamine method. The clonedβTC6 cells were selected by G418. RT-PCR and western blot were used to detecte the expression of GPR40. The results showed that the recombinant plasmid knocked down GPR40 mRNA and protein inβTC6 cells.βTC6 cell line stably expressing GPR40shRNA were established.The second part of this study was to determine the contribution of GPR40 to short- or long-term effects of FFAs and pioglitazone on glucose stimulated insulin secretion (GSIS) inβTC6 cells. Cells were then incubated in FFAs (0.25,0.5,or 1 mmol/L, a 2:1 mixture of oleate: palmitate) or/and pioglitazone(0.1, 1, or 10μmol/l) for 1 h or 48 h. Results showed that 1-h exposure to FFAs significantly enhanced GSIS in pSilencer-control transfected cells, but not in the cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly impaired GSIS in pSilencer-control transfected cells as well as the cells transfected with GPR40shRNA. Furthermore, pioglitazone enhanced insulin secretion in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in the cells transfected with GPR40shRNA.The third part of this study was to determine the contribution of GPR40 to short- or long-term effects of FFAs and pioglitazone on the expression of PDX-1 and GLUT2 inβTC6 cells. Cells were incubated in 0.5 mmol/L FFAs for 1 h or 48 h, or 10μmol/l pioglitazone or combination of 0.5 mmol/L FFAs and 10μmol/l pioglitazone for 48 h. Results showed that 1-h exposure to FFAs significantly increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells, but not in the cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly decreased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells as well as the cells transfected with GPR40shRNA. Furthermore, pioglitazone increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in the cells transfected with GPR40shRNA.These results indicate that GPR40 mediates the short-term effects of FFAs on GSIS and the expression of PDX-1 and GLUT2, but does not mediate the chronic lipotoxicity onβ-cells. The reverse role of pioglitazone on lipotoxicity ofβ-cells may be related to GPR40.