[en] Objective: To study the insulin secretion responsiveness induced by glucose stimulation in isolated rat islets. Methods: Rat islets were isolated after in situ collagenase digestion through pancreatic duct perfusion. The islets were usually cultured overnight (16-20h) in Rmi 1640 media with 0.5% BSA and 11.1 mmol/L glucose. The test began with a pre-culture of the islets for 30 min in KRB buffer with 0.2% BSA, 3.3mmol/L glucose, then the amounts of insulin produced after exposure to different strengths of glucose (3.3, 5.6, 8.7, 11.1, 16.7 and 22.5mmol/L) in KRB buffer for 1h were assayed. Islets were also cultured over-night in DMEM media with different strengths of glucose to examine the possible toxic injury, with high concentrations of glucose ( 16.7 and 22.5 mmol/L). For viability test of the islets (1wk or longer), they were cultured in DMEM media with 5.5mmol/L glucose. Results: The amounts of insulin produced after exposure to KRB buffer for 1h with basal (3.3mmol/L) and high (16.7mmol/L) strength of glucose were (12.4 ± 3.2) and (45.2 ± 4.2) μU/ml/islets/h respectively. Islets cultured overnight in media with low concentration (5.5 or 11.1 mmol/L) glucose secreted significantly more insulin than those cultured in media with higher concentration (16.7 and 22.5 mmol/L) glucose (P<0.05). After cultured for 5 days, the islets retained excellent response to glucose stimulation with increased insulin secretion (4.28 ± 0.67 fold of basal output of insulin in culture with high concentration of glucose). Conclusion: Isolated rat islets could retain high responsiveness to glucose stimulation for 1-5 days under optimal culture condition. (authors)

[en] Objective: To observe the effects of glutamine on AMPK phosphorylation and insulin secretion from MIN6 cell line culture. Methods: MIN6 cells (passage 22-30) were cultured in Dulbecco's modified Eagle's medium with 25 mmol/L glucose and 15% FBS, and 100 μm β-mercaptoethanol at 37 degree C and 5% CO₂ for 24h and then were cultured for 20h in medium containing 0. 2% BSA. The cell were then incubated in 0.2% BSA KRB medium with different concentrations of glucose(5.6mmol/L, 8.7mmoL/ L, 11.1 mmoL/L and 25mmoL/L), with or without 10mmol/L glutamine respectively for 1 h and the insulin secreted was measured with RIA. Cell extraction was detected for AMPK phosphorylation. Results: Our findings showed that increase of glucose concentration from 2.8mmol/L to 22.5mmol/L could markedly reduce the phosphorylation of AMPK at Thr-172 in MIN6 cells, which was associated with a progressive decline of phosphorylation of acetyl-CoA carboxylase (ACC). Thr-172 phosphorylation is very important for the activity of AMPK. AMPK activity is negatively related to insulin secretion. Adding 10 mmol/L glutamine could further inhibit AMPK phosphorylation and increase GSIS. Conclusion: AMPK activity was involved in the regulation of GSIS. Glutamine might regulate GSIS through the inhibition of AMPK activity. (authors)