| BackgroundIn islet, a cells and β cells communicate and interact with each other closely. Diabetes is not only characterized by β cell dysfunction, but also closely related with a cell function. Glucagon-like peptide (GLP-1) secreted mainly by intestinal L cells can promote the proliferation and regeneration of β cells, facilitate insulin secretion.. Studies have confirmed that a cells can secrete GLP-1 to improve islet function. This is considered as a self-protective physical reaction in the occurrence and development of diabetes mellitus.So far, little is know about the influence factors of a cell secreting GLP-1. According to the previous study in intestinal L cells, insulin has a promoting effect to the GLP-1 secretion, however, how insulin influences GLP-1 secretion in a cell remainns unclear.ObjectiveInvestigate whether insulin promotes a cell secreting GLP-1 and the related mechanisms.Methods(1) In-Rl-G9 cells, a pancreatic alpha cell line, was divided into low glucose group (control group+insulin group) and high glucose group (control group+Insulin group), respectively cultivated in 1640 medias containing 5.2 mmol/L and 25 mmol/L glucose. Added 10-7 mol/L insulin to insulin group and the same amount of PBS to the control group. After the 6-h treatment, peptides in supernatants were collected to detect the level of GLP-1 and glucagon.(2) Under high glucose conditions (25 mmol/L glucose), insulin of 10"7mol/L concentrations respectively stimulated INR1G9 cells for both 2 hours,6 hours and 24 hours, supernatants was collected to detect the level of GLP-1 and glucagon as previously described.(3) INR1G9 cells were incubated in the high glucose (25 mmol/L) media and given various concentrations (10-11 mol/L,10-10mol/L,10-9 mol/L,10"8 mol/L,10-7 mol/L,10-6 mol/L) of insulin (insulin group) or PBS (control group) stimulation for 2 hours, GLP-1 and glucagon was detected.(4) Under the condition of 25 mmol/L glucose along with 10’7 mol/L insulin, PI3K/AKT pathway inhibitor LY294002, RAS/MAPK pathway inhibitor PD980595 were added. Supernatants was collected to detect the level of GLP-1 and glucagon and cellular proteins were extracted to detect the level of p-AKT, AKT, p-ERK, ERK, PC1/3 and PC2.Results(1) There is no significantly difference of GLP-1 secretion between the low glucose+insulin group and low glucose+PBS group. GLP-1 secretion of the high glucose+PBS group increased markedly than the low glucose+PBS group by 217.41%[(178.67±11.89) pmol/L vs (56.29±4.21) pmol/L,p<0.05]. The secretion of GLP-1 of the high glucose+insulin group increased significantly than the low glucose+insulin group by 96.54%[(351.15±26.77)pmol/L vs (178.67±11.89)pmol/L, p<0.05]. Glucagon levels of both insulin groups decreased markly than control groups.(2) After 2-hour treatment by insulin, the levels of GLP-1 secreted by INR1G9 cells increased significantly by 97.89%[(316.84±13.2) pmol/L vs (160.11±8.76) pmol/L,p<0.05], After 6-hour treatment by insulin, the levels of GLP-1 increased significantly by 87.2%[(346.02±15.8) pmol/L vs (184.84±11.44) pmol/L,p<0.05], After 24-hour treatment by insulin, the levels of GLP-1 increased significantly by 76.02%[(387.98±31.61) pmol/L vs (220.42±18.28) pmol/L,p<0.05]. The obvious variation between the experimental group and control group existed after 2-hour treatment by insulin. Whereas glucagon secretion decreased gradually along the time course.(3) As the concentrations of insulin increased, the secretion of GLP-1 in each group increased at first and then followed by a downward tendency. The secretion of GLP-1 reached a peak when insulin concentration in the media was 10-7 mol/L, which was significantly higher than control group by 94.39%[(349.16±23.63) pmol/L vs (179.62±9.81) pmol/L,p<0.05]. The secretion of glucagon showed a downward trend overall, GLP-1/glucagon ratio was on the increase.(4) The expression of p-AKT, p-ERK increased by 743.8% and 160.14% respectively. When given PI3K/AKT pathway inhibitor LY294002, the phosphorylation of AKT was inhibited and the GLP-1 secretion reduced by 55.95% [(158.31±12.39) pmol/L vs(359.42±31.92) pmol/L,p<0.05]. When given RAS/MAPK pathway inhibitor PD980595 to INR1G9 cells, the phosphorylation of ERK was suppressed and the secretion of GLP-1 reduced by 36.37%[(228.7±12.52) pmol/L vs (359.42±31.92) pmol/L,p<0.05].The expression of glucagon both increased compared to the only insulin group.(5) Under the condition of high glucose, the expression level of PC 1/3 increase by 41.97%, whereas PC2 level decreased by 42.08% after 2-hour stimulated by 10-7mol/L insulin. When added into LY294002, PD98059 separately, the expression of PCI/3 declined markedly and the expression of PC2 increased significantly.ConclusionsUnder high glucose condition, insulin facilitated the secretion of GLP-1 by INR1G9 cells, which might be associated with the activation of PI3K/AKT pathway. |
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