Sato Hiroki, Nagashima Kazuaki, Ogura Masahito, Sato Yuichi, Tahara Yumiko, Ogura Kasane, Yamano Gen, Sugizaki Kazu, Fujita Naotaka, Tatsuoka Hisato, Usui Ryota, Mukai Eri, Fujimoto Shimpei, Inagaki Nobuya
Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
Department of Medical Physiology Graduate School of Medicine, Chiba University Chiba Japan.
J Diabetes Investig. 2016 Mar;7(2):171-8. doi: 10.1111/jdi.12407. Epub 2015 Sep 13.
AIMS/INTRODUCTION: Src, a non-receptor tyrosine kinase, regulates a wide range of cellular functions, and hyperactivity of Src is involved in impaired glucose metabolism in pancreatic β-cells. However, the physiological role of Src in glucose metabolism in normal, unstressed β-cells remains unclear. In the present study, we investigated the role of Src in insulin secretion and glucose metabolism.
Src was downregulated using small interfering ribonucleic acid in INS-1 cells, and glucose-induced insulin secretion, adenosine triphosphate content, intracellular calcium concentration, glucose utilization and glucokinase activity were measured. Expression levels of messenger ribonucleic acid and protein of glucokinase were examined by semiquantitative real-time polymerase chain reaction and immunoblotting, respectively. Cells were fractionated by digitonin treatment, and subcellular localization of glucokinase was examined by immunoblotting. Interaction between glucokinase and neuronal nitric oxide synthase was estimated by immunoprecipitation.
In Src downregulated INS-1 cells, glucose-induced insulin secretion was impaired, whereas insulin secretion induced by high K(+) was not affected. Intracellular adenosine triphosphate content and elevation of intracellular calcium concentration by glucose stimulation were suppressed by Src downregulation. Src downregulation reduced glucose utilization in the presence of high glucose, which was accompanied by a reduction in glucokinase activity without affecting its expression. However, Src downregulation reduced glucokinase in soluble, cytoplasmic fraction, and increased it in pellet containing intaracellular organelles. In addition, interaction between glucokinase and neuronal nitric oxide synthase was facilitated by Src downregulation.
Src plays an important role in glucose-induced insulin secretion in pancreatic β-cells through maintaining subcellular localization and activity of glucokinase.
目的/引言：Src作为一种非受体酪氨酸激酶，可调节多种细胞功能，且Src的过度激活与胰腺β细胞葡萄糖代谢受损有关。然而，Src在正常、未受应激的β细胞葡萄糖代谢中的生理作用仍不清楚。在本研究中，我们探究了Src在胰岛素分泌和葡萄糖代谢中的作用。
使用小干扰核糖核酸下调INS-1细胞中的Src，然后检测葡萄糖诱导的胰岛素分泌、三磷酸腺苷含量、细胞内钙浓度、葡萄糖利用情况以及葡萄糖激酶活性。分别通过半定量实时聚合酶链反应和免疫印迹法检测葡萄糖激酶信使核糖核酸和蛋白质的表达水平。用洋地黄皂苷处理细胞进行分级分离，通过免疫印迹法检测葡萄糖激酶的亚细胞定位。通过免疫沉淀法评估葡萄糖激酶与神经元型一氧化氮合酶之间的相互作用。
在Src下调的INS-1细胞中，葡萄糖诱导的胰岛素分泌受损，而高钾诱导的胰岛素分泌不受影响。Src下调抑制了细胞内三磷酸腺苷含量以及葡萄糖刺激引起的细胞内钙浓度升高。Src下调降低了高糖存在时的葡萄糖利用，这伴随着葡萄糖激酶活性降低但不影响其表达。然而，Src下调减少了可溶性细胞质部分的葡萄糖激酶，增加了含有细胞内细胞器的沉淀部分中的葡萄糖激酶。此外，Src下调促进了葡萄糖激酶与神经元型一氧化氮合酶之间的相互作用。
Src通过维持葡萄糖激酶的亚细胞定位和活性，在胰腺β细胞葡萄糖诱导的胰岛素分泌中起重要作用。
