Intracellular sorting of glucose transporter and insulin action

葡萄糖转运蛋白的细胞内分选和胰岛素作用

• 批准号: 10044296
• 负责人: OKA Yoshitomo
• 金额: $ 2.62万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044296/
• 关键词: Insulin; Glucose transport; GLUT4; PI3 kinase; Akt; PH domain; Grp1; PDK1; P13キナーゼ; ARNO

Insulin rapidly stimulates glucose transport activity in muscle and adipose tissue. This increase in glucose transport activity is primarily due to translocation of glucose transporter GLUT4 from intracellular compartment to the plasma membrane. We have shown that activation of PI3 kinase is crucial for insulin-stimulation of glucose transport, based on our findings that overexpression of p110a catalytic subuniti of PI3 kinase and dominant negative p85 subunit mimic and inhibit, respectively, this insulin action. We have also studied signals downstream of PI3 kinase. Insulin stimulation phosphorylates AKt1 at both Thr308 and Ser473, leading to activation of various signals including those towards glucose metabolism. To investigate the role of PDK1 on the phosphorylation state of AKt1, the wild-type PDK1 (wt-PDK1) and its kinase dead mutant (kd-PDK1) were expressed in CHO-IR cells using adenovirus gene transduction system. Immunoblotting using antiphosphorylated AKt1 antibody revealed that Thr308 was maximally phosphorylated already at 1 min by insulin stimulation but almost completely dephosphorylated at 5 min. Insulin-stimulated phosphorylation state of Thr308 was markedly increased in CHO-IR cells overexpressing wt-PDK1 at 1 min, but it returned to the basal level at 5 min. On the contrary, insulin-stimulated phosphorylation state of Thr308 was maintained even at 15 min in cells overexpressing kd-PDK1, suggesting that kd-PDK1 overexpression inhibited insulin-stimulated phosphatase activity. Insulin-stimulated Ser473 phosphorylation was not affected by overexpression of we-PDK1 or kd-PDK1.

胰岛素快速刺激肌肉和脂肪组织中的葡萄糖转运活性。这种葡萄糖转运活性的增加主要是由于葡萄糖转运蛋白GLUT 4从细胞内区室易位到质膜。我们已经表明，PI 3激酶的激活是至关重要的胰岛素刺激葡萄糖转运，基于我们的研究结果，过度表达PI 3激酶的p110 a催化亚单位和显性负性p85亚单位模拟和抑制，分别，这种胰岛素作用。我们还研究了PI 3激酶下游的信号。胰岛素刺激使AKt 1在Thr 308和Ser 473处磷酸化，导致各种信号的激活，包括那些朝向葡萄糖代谢的信号。为研究PDK 1对AKt 1磷酸化状态的影响，利用腺病毒基因转导系统在CHO IR细胞中表达野生型PDK 1（wt-PDK 1）及其激酶死亡突变体（kd-PDK 1）。使用抗磷酸化AKt 1抗体的免疫印迹显示，Thr 308在胰岛素刺激1分钟时已经最大程度地磷酸化，但在5分钟时几乎完全去磷酸化。在过表达wt-PDK 1的CHO-IR细胞中，胰岛素刺激的Thr 308磷酸化状态在1分钟时显著增加，但在5分钟时恢复到基础水平。胰岛素刺激的Thr 308的磷酸化状态在过表达kd-PDK 1的细胞中甚至在15分钟时仍保持，表明kd-PDK 1过表达抑制胰岛素刺激的磷酸酶活性。胰岛素刺激的Ser 473磷酸化不受we-PDK 1或kd-PDK 1过表达的影响。

项目成果

Ishihara H,et al.: "Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C β1 or δ1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells."Biochem Biophys Res Commun. 254. 77-82 (1999)

Ishihara H 等人：“通过磷脂酶 C β1 或 δ1 的过度表达增强磷酸肌醇水解抑制胰岛素瘤 MIN6 细胞中刺激诱导的胰岛素释放。”Biochem Biophys Res Commun. 254. 77-82 (1999)。

HOSAKA, T, et al.: "Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes."Endocrine Journal. 46(3). 349-357 (1999)

HOSAKA, T 等人：“通过 3T3-L1 脂肪细胞中的慢性葡萄糖暴露调节胰岛素刺激的葡萄糖转运。”内分泌杂志。

Ishihara H,et al.: "Type I Phosphatidylinositol-4-phosphate 5-kinases.Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family"J Biol Chem. 273. 8741-8748 (1998)

Ishihara H,et al.：“I 型磷脂酰肌醇-4-磷酸 5-激酶。第三亚型的克隆以及该新型脂质激酶家族成员的删除/取代分析”J Biol Chem。

Funaki M,et al.: "p85/p110-type phophatidylinositol kinase phosphorylates not only the D-3,but also the D-4 position of the inositol ring"J Biol Chem. 274. 22019-22024 (1999)

Funaki M,et al.：“p85/p110 型磷脂酰肌醇激酶不仅磷酸化肌醇环的 D-3 位点，而且磷酸化肌醇环的 D-4 位点”J Biol Chem.

Ueda K,et al.: "Overexpression of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase does not correct glucose-stimulated insulin secretion from diabetic GK rat pancreatic islets." Diabetologia. 41. 649-653 (1998)

Ueda K 等人：“线粒体 FAD 连接的 3-磷酸甘油脱氢酶的过度表达并不能纠正糖尿病 GK 大鼠胰岛中葡萄糖刺激的胰岛素分泌。”