Effects Of High Level Free Fatty Acids On The Secretory Function Of Islet Alpha Cells And Its Mechanism

Objective To investigate the secretory function of the islet a cells in rats fed high-fat-diet and the changes of insulin signal transduction molecules in islet a cells in beta-cell-deleting high-fat-diet rat models established by injecting high dose STZ and its underlying mechanism.Methods Thirty SD rats were randomly divided into 2 groups and fed with high-fat-diet (HFD group)or normal diet(ND group).At the end of twenty weeks feeding , we determined serum glucose, FINS, glucagon, FFA, TG. The GIR was measured by using euglycemic hyperinsulinemia clamp to evaluate the perpherial insulin resistance and the islet perfusion assays were performed. At the same time, the beta-cell-deleting rat models were established by injecting large dose STZ (lOOmg/kg) in HFD and ND group. Control the serum glucose by insulin. Five days later, the beta-cell-deleting rat models were acquired and sacrificed, named HFD-B and ND-B group. The levels of insulin and glucagon in the pancreatic homogenate were measured. The HE and immunohistochemistry dyeing was performed to evaluate the areas of islets,beta and alpha cells. Quantitative analysis was executed by image analyzer. And islets were isolated and collected. The expression of glucagon, IRS-1, IRS-2, PI3-K gene in islets was detected by real-time RT-PCR. Results (1) The serum FFA,FINS and glucagon concentration in HFD group were higher than in ND group(FFA 508(394-622) umol/L vs 325(240-410) umol/L, FINS 23.7(14.0-33.4) mIU/L vs 11.5(3.6-19.4)mIU/L;glucagon 345(298.6-391.4) pg/ml vs 256(226.4-285.6) pg/ml;P<0.05).(2)The GIR was decreased significantly in HFD group compared with ND group(5.25mg·min^-1·kg^-1±1.2mg·min^-1·kg^-1 vs 13.56 mg·min^-1·kg^-1±1.7mg·min^-1·kg-1,P<0.01).(3)The basal glucagon secretion in rats fed high-fat-diet was 3 folds of that in ND group, and couldn't be inhibited during the perfusion of 16.7mmol/L glucose. (4)The total pancreas island areas of beta-cell-deleting rats were approximately 1/7 of normal control rats. Moreover, the percentages of beta-cell areas from total pancreas island areas were decreased from 74.3% down to 5.4%. The insulin content in pancreas tissue homogenate of beta-cell-deleting rats did't reach 3% of normal ones, while the glucagon content unexpectedly increased. The aggregation of alpha cells from periphery to centre of pancreas islands was found in beta-cell-deleting groups. Furthermore, the percentages of alpha cells area from total pancreas island areas are promoted from 16.4% to 76.5%. (5)The gene expression of glucagon was significantly increased by 34.2% in HFD-B group than in ND-B group. In contrast, the expression of IRS-2,PI3-K was significantly decreased by 28.5% and 21.3% respectively (P<0.01).(6) There was a significantly negative correlation between the serum FFA concentration and GIR(r=-0.675, P<0.05) as well as IRS-2 gene expression in islet a cells (r=-0.458, P<0.05).Conclusions High-fat-diet induced high level secretion of glucagon in islet alpha cells in rats, which couldn't be inhibited by the elevated insulin level, beta-cell-deleting rat models fed high-fat-died showed an impaired expression of insulin signal transduction molecules in islet a cells which may correlate with the increased serum FFA concentration. Objective To study the the changes of the secretory function and the insulin signaling and inflammatory path molecules in islet alpha cells in obese SD rats induced by a high-fat-diet and the effects of pioglitazone interventionMethods SD rats were randomly divided into 3 groups, i.e., a normal diet group(ND), a high-fat-diet group(HFD), and a pioglitazone treated group (HP, pioglitazone 15mg·kg^-1·d^-1 by orally injection and high-fat-diet). At the end of twenty weeks feeding, serum FINS, glucagon, FFA, hsCRP, TNF-alpha, IL-6; the MDA,GSH, nitrotyrosine(NT) in serum and pancreatic homogenate were measured. The GIR was determined and islet perfusion assays was performed. At the same time, the beta cell-deleting rat models were established by injecting large dose STZ (100 mg/kg) in the three groups, i.e., HFD-B group,HP-B group and ND-B group. Five days later, The mRNA levels of glucagon, IRS-1, IRS-2, PI3-K, NF-κB,IκB-alpha in betacell-deleting rat islets was detected by quantitative real-time RT-PCR.Results Compared with rats fed the normal diet, FINS, glucagon, FFA, hsCRP TNF-alpha and IL-6 was significantly increased in rats fed the high-fat-diet for 20 weeks( P<0.01 or 0.05), the MDA,NT in plasma and pancreatic homogenate in HFD group were increased but the GSH level was decreased significantly. The GIR was significantly decreased by the high-fat-diet. The glucagon secretion couldn't be inhibited in rats fed the high-fat-diet during the perfusion of 16.7mmol/L glucose(see partⅠ), pioglitazone intervention can reverse all these effects. In beta cell-deleting rats fed the high-fat-diet ,the mRNA levels of glucagon was significantly increased, while IRS-2, PI3-K was significantly decreased(see partⅠ); The mRNA levels of NF-κB was significantly increased by 20.5%,IκB-alpha was significantly decreased by 24.3% (P<0.01). In pioglitazone intervention group, when compared with rats fed the high-fat-diet alone, the levels of mRNA were improved 40.6%, 57.2%, 60.6%, 78.3%, 58.8%, respectively. There was a significantly negative correlation between the serum FFA concentration and GIR as well as the mRNA level of IRS-2 (see partⅠ), but a significantly positive correlation between FFA level and the mRNA level of NF-κB in islet alpha cells (r=0.775 P<0.05). Conclusions There was a high level secretion of glucagon in rats fed the high-fat-diet, an impaired expression of insulin signal transduction molecules and an activation of inflammatory path in islet alpha cells at the same time, which may correlate with the elevated plasma FFA concentration. The elevated FFA may activate inflammatory path by inducing oxidative stress, results in insulin resistance in islet alpha cells. Pioglitazone intervention could lower FFA level and lessen oxidative stress, inhibit the activation of inflammatory path, improve insulin resistance in alpha cells. Objective This experiment aims at investigating the effects of palmitate in various time and concentrations on glucagon secretory function of hamster glucagonoma alpha cell line INR1-G9 and its underlying mechanism.Methods Clonal INR1-G9 alpha cells were cultured with palmitate in various gradient concentrations(0.125mmol/L, 0.25mmol/L, 0.5mmol/L)for 24,48 and 72 hours, then the glucagon release potentia of alpha cell was evaluated and the glucagon content was measured. The TG content of various concentrations group cultured 72h were detected, and the mRNA levels of glucagon in INR1-G9 cells was detected by quantitative real-time RT-PCR. The serine phosphorylation of PKB/Akt in alpha cells cultured 72h were determined by western blotting and the effects of insulin and PI3-K inhibitor wortmannin on it were investigated.Results (1)Palmitate enhanced glucagon secretion of alpha cells in a time and dose-dependent manner, There was significant difference in the 24h/0.5mM palmitate group, 48/72h/every concentrations palmitate group compared with normal control without palmitate(P<0.05). (2)The glucagon content in INR1-G9 was decreased gradually when the action time prolonged and the concentration of palmitate elevated and eventually reached a statisticly difference in the 48h/0.5mM palmitate group, 72h/0.25mM,72h/0.5mM palmitate group(P<0.05).(3) There was no difference of mRNA levels of glucagon in each 72h cultured group, only showed a trend of increasing by elevated palmitate. (4)The palmitate increased the accumulation of TG in INR1-G9 cells in a dose-dependent manner, all 72h cultured group reached a statisticly difference (P<0.05) and only 0.5mM palmitate increased TG content significantly(P<0.01). (5)PI3-K inhibitor and palmitate reduced the serine phosphorylation of Akt but didn't reach a statisticly difference; Insulin increased the phosphorylation level of Akt in every group, but the rising degree in palmitate group was lower than that in cells without palmitate. Furtherly, when wortmannin were added in these cells, the phosphorylation level of Akt were all decreased in every group, but the inhibition ratio in palmitate group was lower than in normal controlcells without palmitate.Conclusions Our results demonstrated that elevated palmitate enhanced glucagon secretion of INR1-G9 glucagonoma alpha cells in a time and dose-dependent manner, The glucagon content in INR1-G9 was decreased gradually when the action time prolonged and the concentration of palmitate elevated, but didin't impact the mRNA level of glucagon, which indicates fatty acids mainly enhanced the release of glucagon. The effects of palmitate on the secretory function of alpha cell may relate with the increased accumulation of TG and the suppression of the insulin signaling P13K/Akt pathway mediated by insulin.