Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK.
Diabetes. 2010 May;59(5):1192-201. doi: 10.2337/db09-0791. Epub 2010 Feb 11.
The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks.
C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration (Ca(2+)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked Ca(2+). The functional data were complemented by analyses of histology and gene transcription.
After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or Ca(2+) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed.
HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.
本研究旨在阐明高脂饮食喂养 15 周的小鼠中葡萄糖诱导的胰岛素分泌受到抑制的细胞机制。
将 C57BL6J 小鼠用高脂饮食或正常饮食喂养 3 或 15 周。通过腹腔内葡萄糖耐量试验评估体内胰岛素和血糖水平。使用静态孵育和胰腺灌注制备研究体外胰岛素分泌。通过膜片钳技术测量测量膜电流、电活动和胞吐作用。通过微荧光法测量细胞内钙离子浓度(Ca(2+))。总内反射荧光显微镜（TIRFM）用于胞吐作用和亚膜去极化诱导的Ca(2+)的光学成像。功能数据通过组织学和基因转录分析进行补充。
高脂饮食喂养 15 周而非 3 周后，小鼠出现高血糖和胰岛素血症。胰岛含量和β细胞面积分别增加了 2 倍和 1.5 倍。这些变化与葡萄糖诱导的胰岛素分泌减少 20-50%（与胰岛素含量归一化）相关。后一种作用与电活动或Ca(2+)信号的受损无关。单细胞电容和 TIRFM 测量的胞吐作用显示，短（50ms）去极化诱发的胞吐作用选择性抑制（>70%），而较长（500ms）去极化的反应则受影响较小。快速胞吐作用的丧失与 HFD β细胞中 Ca(2+)进入的弥散相关。未观察到关键胞吐蛋白的基因转录发生变化。
HFD 通过使电压门控 Ca(2+)内流与胞吐作用功能分离导致胰岛素分泌减少。这些观察结果为肥胖与糖尿病之间的既定联系提供了新的解释。
