Objective

Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells.

Methods

A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation.

Results

The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice.

Conclusion

Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting.

中文翻译：

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ARFRP1依赖性高尔基体支架蛋白GOPC是胰腺β细胞分泌胰岛素所必需的

客观的

代谢活跃组织（如胰岛）的激素分泌受特定且高度调节的信号通路控制。胰岛素分泌缺陷是糖尿病的主要原因之一。然而，调节胰岛素分泌的分子机制尚未完全了解。在这项工作中，我们研究了 GTPase ARFRP1 对胰腺 β 细胞分泌胰岛素的作用。

方法

对 β 细胞特异性Arfrp1基因敲除小鼠进行表型表征。下拉实验和质谱分析用于筛选新的 ARFRP1 相互作用蛋白。共免疫沉淀测定以及超分辨率显微镜用于验证。

结果

GTPase ARFRP1 与高尔基体相关的 PDZ 和包含卷曲螺旋基序的蛋白质 (GOPC) 相互作用。这两种蛋白质共同定位于反式高尔基体网络，并通过控制 SNARE 蛋白 SNAP25 的质膜定位来调节胰岛素分泌的第一和第二阶段。Min6 细胞中 GOPC 和 ARFRP1 的下调干扰 SNAP25 的质膜定位并增强其降解，从而削弱葡萄糖刺激的胰岛素从 β 细胞释放。反过来，SNAP25 和 GOPC 的过表达恢复了 β 细胞特异性Arfrp1敲除小鼠胰岛中的胰岛素分泌。

结论

我们的结果确定了迄今为止尚未识别的在反式高尔基体分选水平上胰岛素分泌所需的途径。