Department of Pharmaceutical Sciences, Wayne State University and Veterans Affairs Medical Center, Detroit, MI 48202, USA.
Am J Physiol Endocrinol Metab. 2010 Aug;299(2):E276-86. doi: 10.1152/ajpendo.00091.2010. Epub 2010 May 25.
We report localization of a cytosolic protein histidine phosphatase (PHP; approximately 16 kDa) in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knockdown of PHP markedly reduced glucose- or mitochondrial fuel-induced but not KCl-induced insulin secretion. siRNA-mediated knockdown of PHP also attenuated mastoparan-induced insulin secretion, suggesting its participation in G protein-sensitive signaling steps, leading to insulin secretion. Functional assays revealed that the beta-cell PHP catalyzes the dephosphorylation of ATP-citrate lyase (ACL). Silencing of PHP expression markedly reduced ACL activity, suggesting functional regulation of ACL by PHP in beta-cells. Coimmunoprecipitation studies revealed modest effects of glucose on the interaction between PHP and ACL. Confocal microscopic evidence indicated that glucose promotes association between ACL and nm23-H1, a known kinase histidine kinase, but not between PHP and ACL. Furthermore, metabolic viability of INS 832/13 cells was resistant to siRNA-PHP, suggesting no regulatory roles of PHP in cell viability. Finally, long-term exposure (24 h) of INS 832/13 cells or rat islets to high glucose (30 mM) increased the expression of PHP. Such increases in PHP expression were also seen in islets derived from the Zucker diabetic fatty rat compared with islets from the lean control animals. Together, these data implicate regulatory roles for PHP in a G protein-sensitive step involved in nutrient-induced insulin secretion. In light of the current debate on putative regulatory roles of ACL in insulin secretion, additional studies are needed to precisely identify the phosphoprotein substrate(s) for PHP in the cascade of events leading to nutrient-induced insulin secretion.
我们报告了细胞质蛋白组氨酸磷酸酶（PHP；约 16 kDa）在 INS 832/13 细胞、正常大鼠胰岛和人胰岛中的定位。siRNA 介导的 PHP 敲低显著降低了葡萄糖或线粒体燃料诱导的但不是 KCl 诱导的胰岛素分泌。siRNA 介导的 PHP 敲低也减弱了 mastoparan 诱导的胰岛素分泌，表明其参与 G 蛋白敏感信号步骤，导致胰岛素分泌。功能测定显示，β细胞 PHP 催化 ATP-柠檬酸裂解酶（ACL）的去磷酸化。沉默 PHP 表达显著降低 ACL 活性，表明 PHP 在β细胞中对 ACL 的功能调节。共免疫沉淀研究显示葡萄糖对 PHP 和 ACL 之间相互作用的适度影响。共聚焦显微镜证据表明，葡萄糖促进 ACL 与 nm23-H1（一种已知的激酶组氨酸激酶）之间的结合，但不促进 PHP 和 ACL 之间的结合。此外，INS 832/13 细胞的代谢活力对 siRNA-PHP 具有抗性，表明 PHP 在细胞活力中没有调节作用。最后，长期暴露（24 小时）于高葡萄糖（30 mM）的 INS 832/13 细胞或大鼠胰岛增加了 PHP 的表达。与来自瘦对照动物的胰岛相比，来自 Zucker 糖尿病肥胖大鼠的胰岛中也观察到 PHP 表达的这种增加。总之，这些数据表明 PHP 在参与营养诱导的胰岛素分泌的 G 蛋白敏感步骤中具有调节作用。鉴于目前关于 ACL 在胰岛素分泌中潜在调节作用的争论，需要进行额外的研究以精确确定 PHP 在导致营养诱导的胰岛素分泌的级联反应中的磷酸蛋白底物。
