探讨应力纤维在糖毒性抑制胰岛β细胞分泌功能中的作用及GLP-1的干预机制

It has been revealed that glucotoxicity inhibits insulin secretion via blunting pancreatic β-cell functions, which exacerbates type 2 diabetes. However, the mechanisms underlying remain largely unknown. As a cytoskeleton, F-actin is organized as a web beneath the plasma membrane. It is widely accepted that F-actin remodeling is essential for insulin release from pancreatic β-cells. Our data show that β-cells at glucotoxicity have abundant stress fibers, accompanied by loss of glucose-induced F-actin depolymerization and insulin secretion. Treatment with GLP-1 resulted in F-actin depolymerization and restored glucose-stimulated insulin secretion. By employing molecular biological, electrophyisological and immunocytochemistry approaches, we would therefore explore whether ① Rho A-ROCK/Formins would be involved in glucotoxicity-increased stress fibers in β-cells;② stress fibers would impede priming and exocytosis of insulin containing granules;③ GLP-1 would induced F-actin depolymerization via activation of ERK1/2 in β-cells at glucotoxicity. The results will shed light on revealing the cellular mechanisms underlying glucotoxicity-suppressed β-cell functions, as well as the protective effects of GLP-1 against glucotoxicity.

糖毒性损坏胰岛β细胞功能及其所致的胰岛素分泌降低是2型糖尿病发生发展的重要环节。然而糖毒性抑制β细胞功能的机理迄今仍不清楚。作为细胞骨架成分，微丝在β细胞胞膜下形成网状结构，微丝的解聚是胰岛素囊泡出胞的前提。我们的前期工作发现糖毒性导致β细胞内微丝聚集，形成长且稳固的应力纤维；在该条件下葡萄糖失去解聚微丝的能力，也失去刺激胰岛素分泌的能力。GLP-1处理糖毒性β细胞导致应力纤维解聚，恢复葡萄糖刺激的胰岛素分泌。以此为基础，我们将采用分子生物学、电生理和免疫细胞化学等方法探讨①糖毒性是否通过Rho A-ROCK/Formins促进应力纤维的形成；②应力纤维是否通过干扰囊泡的出胞机制抑制胰岛素分泌；③GLP-1是否通过激活ERK1/2导致应力纤维解聚并恢复葡萄糖刺激的胰岛素分泌。研究结果将为揭示糖毒性抑制β细胞功能的机理提供新理论，也将为阐明GLP-1改善糖毒性β细胞功能提供新的科学依据。

糖尿病慢性高糖（糖毒性）导致胰岛细胞功能损伤，特别是葡萄糖诱导的胰岛素分泌减弱或丧失，后者是糖尿病餐后高血糖的直接原因。为揭示糖毒性损伤胰岛细胞分泌能力的细胞机制，我们建立了糖尿病糖毒性细胞模型和胰岛模型。我们发现慢性高糖导致胰岛细胞内微丝聚集，形成贯穿全细胞的应力纤维，相伴随的是细胞丧失对葡萄糖刺激的反应能力。用微丝解聚因子如Cytochalasin B 或Latrunculin B解聚应力纤维，则能恢复糖毒性细胞对葡萄糖刺激的反应，恢复葡萄糖诱导的胰岛素分泌。进一步探讨微丝应力纤维形成的细胞机制，我们发现抑制小分子G蛋白Rho及其Rho相关螺旋卷曲蛋白激酶ROCK是糖毒性应力纤维形成的关键分子，抑制Rho/ROCK导致应力纤维解聚，进而增进葡萄糖刺激的胰岛素分泌。我们还发现GLP-1通过激活cAMP-PKA信号通路，阻断糖毒性诱导的Rho/ROCK的激活，从而阻断应力纤维的形成，恢复细胞的分泌能力。我们分别用PKA激动剂和阻断剂进一步验证了上述结果。此外，我们还利用糖尿病动物模型检验细胞实验的结果；用生物信息学与临床糖尿病患者的胰岛损伤资料比较，证实了我们的实验结果揭示糖尿病胰岛细胞损伤的新机制。为进一步开展以Rho/ROCK为靶点的抗糖尿病治疗手段提供了科学依据和新思路。