title: Src regulates insulin secretion and glucose metabolism by influencing subcellular localization of glucokinase in pancreatic β-cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Src plays an important role in glucose-induced insulin secretion in pancreatic β-cells through maintaining subcellular localization and activity of glucokinase.
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sato
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nagashima
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Ogura
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Tahara
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Kasane
authors.initials: K
authors.lastname: Ogura
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Yamano
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Kazu
authors.initials: K
authors.lastname: Sugizaki
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Naotaka
authors.initials: N
authors.lastname: Fujita
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Hisato
authors.initials: H
authors.lastname: Tatsuoka
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Usui
authors.affiliation: Department of Medical Physiology Graduate School of Medicine, Chiba University Chiba Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Mukai
authors.affiliation: Department of Endocrinology, Metabolism, and Nephrology Kochi Medical School Kochi University Nankoku Japan.
authors.forename: Shimpei
authors.initials: S
authors.lastname: Fujimoto
authors.affiliation: Department of Diabetes, Endocrinology and Nutrition Graduate School of Medicine Kyoto University Kyoto Japan.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Inagaki
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D005941:Glucokinase / Q000032:analysis / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D051273:Glucose Transporter Type 2 / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000502:physiology*
chemical_list: D051273:Glucose Transporter Type 2; D007328:Insulin; D017382:Reactive Oxygen Species; C495683:Slc2a2 protein, rat; D000255:Adenosine Triphosphate; D052248:Nitric Oxide Synthase Type I; D005941:Glucokinase; D019061:src-Family Kinases; D005947:Glucose
keywords: Glucokinase; Insulin secretion; Src