胰淀素对格列苯脲刺激INS-1细胞胰岛素分泌作用的影响及其机制

The effects and mechanism of islet amyloid polypeptide on insulin secretion in INS-1 cells stimulated by glibenclamide

目的 研究胰淀素短时间条件下对格列苯脲刺激INS-1细胞胰岛素分泌的影响及机制.方法 分别采用全细胞膜片钳技术、激光共聚焦显微镜技术及ELISA分析不同浓度胰淀素短时间孵育前后格列苯脲刺激下INS-1细胞ATP敏感性钾通道（KATP通道）电生理学特性、细胞内钙离子浓度（[Ca2＋]i）及胰岛素含量变化.结果 （1） ELISA结果显示：与1μmol/L格列苯脲刺激下胰岛素释放量（11.43 ±1.22） μg/L比较,0.1μmol/L胰淀素预处理组[（11.45 ±1.20） μg/L]无明显差异,而1 μmol/L及10 μmol/L胰淀素预处理组均显著下降,分别为（9.40±0.87）μg/L和（7.11±0.85） μg/L,且呈剂量相关性（P＜0.05）;（2）激光共聚焦显微镜测钙结果显示：1μmol/L格列苯脲刺激后[Ca2＋]i荧光强度变化的AUC为427.78±2.32,0.1μmol/L胰淀素预处理组（424.09 ±2.21）与之差异无统计学意义;而1 μmol/L及10 μmol/L胰淀素预处理组均可使其AUC显著降低（分别为380.59 ±1.49和246.53±8.41）,并呈剂量相关性（P＜0.05）;（3）全细胞膜片钳结果显示：在1μmol/L、10 μmol/L胰淀素预处理组中,1μmol/L格列苯脲刺激下KATP通道半数激活电压（V0.5）分别为（28.75 ±0.77）mV和（46.95 ±1.81）mV,均显著高于单纯格列苯脲组（14.59±0.69）mV,并呈剂量相关性（P＜0.05）.结论 高浓度胰淀素短时间作用可抑制格列苯脲对KATP通道的关闭,进而抑制[Ca2＋]i增加,推测这种作用是INS-1细胞胰岛素分泌减少的机制之一.

Objective To observe the effects,and study the mechanism of islet amyloid polypeptide （IAPP） on insulin secretion in INS-1 cells stimulated by glibenclamide.Methods Whole cell patch clamp technique was employed to study the influences of short exposure to IAPP on electrophysiological characteristics of ATP-sensitive K＋ channel（KATP channel） upon sulfonylurea stimulation.Intracellular free calcium changes in this process was observed by laser scanning confocal microscope.Insulin was measured by enzyme-linked immunoassay.Results （1）Insulin secretion stimulated by 1 μmol/L glibenclamide was significantly decreased from （11.43 ± 1.22） μg/L to （9.40 ± 0.87） μg/L and to （7.11 ± 1.85） μg/L after 1 μmol/L and 10 μmol/L IAPP incubation,respectively.（2）Glibenclamide-stimulated calcium influx was dose dependently inhibited by IAPP from 1 μmol/L to 10 μmol/L,with the AUC of fluorescence intensity-time reduced from 427.78 ± 2.32 to 380.59 ± 1.49,and to 246.53 ± 8.41,respectively.（3） Compared with that in control cells （14.59 ± 0.69） mV,the half activation voltage of KATP channel in response to glibenclamide was significantly increased to （28.75 ±0.77） mV and to （46.95 ± 1.81） mV in cells pretreated with 1 μmol/L and 10 μmol/L IAPP,implicating an inhibitory effect of IAPP on activation of KATP channel.Conclusion Short-term exposure to high concentration of IAPP inhibited glibenclamideinduced closure of KATP,channels and decreased calcium influx,which may ultimately lead to the reduction of insulin secretion in INS-1 cells.