Progression of diet-induced diabetes in C57BL6J mice involves functional dissociation of Ca2(+) channels from secretory vesicles.
Progression of diet-induced diabetes in C57BL6J mice involves functional dissociation of Ca2(+) channels from secretory vesicles.
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作者：
Collins SC;Hoppa MB;Walker JN;Amisten S;Abdulkader F;Bengtsson M;Fearnside J;Ramracheya R;Toye AA;Zhang Q;Clark A;Gauguier D;Rorsman P
该研究的目的是阐明在喂食高脂饮食（HFD）15周的小鼠中葡萄糖诱导的胰岛素分泌抑制的细胞机制。给C57 BL 6 J小鼠喂食HFD或正常饮食（ND）3或15周。通过腹腔葡萄糖耐量试验测定体内血浆胰岛素和葡萄糖水平。采用静态孵育和灌注胰腺制备研究体外胰岛素分泌。膜电流，电活动，和胞吐进行了检查，膜片钳技术测量。细胞内钙离子浓度（[Ca 2 +]i）用显微荧光法测定。采用全内反射荧光显微镜（TIRFM）对胞吐和膜下去极化诱发的[Ca 2 +]i进行光学成像。组织学和基因转录分析补充了功能数据。15周后，但不是3周后，HFD小鼠表现出高血糖症和低胰岛素血症。胰岛含量和β细胞面积分别增加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%相关（标准化为胰岛素含量）。后一种效应与受损的电活动或[Ca 2 +]i信号传导无关。胞吐的单细胞电容和TIRFM测量揭示了由短（50 ms）去极化引起的胞吐的选择性抑制（>70%），而对较长去极化（500 ms）的响应受影响较小。HFD β细胞快速胞吐的丧失与Ca 2+内流的分散相关。未观察到关键胞吐蛋白基因转录的变化。HFD通过引起电压门控Ca 2+进入与胞吐作用的功能性解离而导致胰岛素分泌减少。这些观察结果为肥胖和糖尿病之间的既定联系提供了新的解释。
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 ([Ca2+]i) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca2+]i. 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 β-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 [Ca2+]i 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 Ca2+ entry in HFD β-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 Ca2+ entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.