谷氨酰胺抑制MIN6细胞AMPK活性并促进其胰岛素分泌

Glutamine Inhibits AMPK and Increases Insulin Secretion From MIN6 Cell Line Culture

目的：观察谷氨酰胺对MIN6细胞胰岛素分泌和AMPK磷酸化水平的影响,以探讨谷氨酰胺影响胰岛素分泌的可能机理.方法：将MIN6细胞接种于24孔或6孔细胞培养板,实验前用含0.2%BSA和2.8mmol/L葡萄糖KRB预培养30min,分别换入含不同浓度葡萄糖和谷氨酰胺的KRB缓冲液,培养1h,收集上清,待测胰岛素;抽提蛋白,检测蛋白磷酸化水平.结果：葡萄糖在（2.8～22.5）mmol/L浓度下能剂量依赖性地降低AMPK蛋白磷酸化,抑制其活性,胰岛素分泌和AMPK活性呈负相关.10mmol/L谷氨酰胺孵育MIN6细胞1h可降低AMPK蛋白磷酸化,胰岛素分泌增加35%.结论：抑制AMPK活性可以促进胰岛素分泌,谷氨酰胺可能通过抑制AMPK活性促进胰岛素分泌.

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 100μm β - mercaptoethanol at 37℃ 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 dected 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 ceils, 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 10mmol/L glutamine could furthur inhibit AMPK phosphorylation and increase GSIS. Conclusion AMPK activity was involved in the regulation of GSIS. Clutamine might regulate GSIS through the inhibition of AMPK activity.