Impact of the regulation of PI3-kinase product on insulin action and its role in the disease state.

PI3-激酶产物的调节对胰岛素作用的影响及其在疾病状态中的作用。

• 批准号: 11671110
• 负责人: SASAOKA Toshiyasu
• 金额: $ 2.3万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671110/
• 关键词: Insulin; Insulin action; PI3-kinase; Lipid phosphatase; SHIP2; Glucose uptake; Glycogen synthesis

PI3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI3-kinase product PI (3, 4, 5) P3 on these biological responses is unknown. We have cloned rat SHIP2 cDNA which possesses the 5'-phosphatase activity to hydrolyze PI (3, 4, 5) P3 to PI (3, 4) P2 and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type (WT)- and 5'-phosphatase defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes and L6 myocytes by means of adenovirus mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor p-subunit and IRS-1, IRS-1 association with p85 subunit, and PI3-kinase activity were not affected by expression of either WT- … More or ΔIP-SHIP2. As expected from possessing the 5'-phosphatase catalytic region, insulin-induced PI (3, 4, 5) P3 production was markedly decreased by overexpression of WT-SHIP2. In contrast, the amount of PI (3, 4, 5) P3 was oppositely increased by expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant negative manner in 3T3-L1 adipocytes. Both PI (3, 4, 5) P3 and PtdIns (3, 4) P2 were known to possibly activate downstream targets Akt and PKCλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and PKCλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2, and these insulin actions were increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity. Less

PI3-激酶在胰岛素的各种代谢活动中起着重要作用，包括葡萄糖摄取和糖原合成。虽然PI3-激酶的主要功能是作为一种脂酶，优先将磷脂的D-3位磷酸化，但关键的PI3-激酶产物PI(3，4，5)P3的水解对这些生物反应的影响尚不清楚。我们克隆了具有5‘-磷酸酶活性的大鼠SHIP2基因，该基因主要在胰岛素靶组织中表达，能将PI(3，4，5)P3水解为PI(3，4)P2。为研究SHIP2在胰岛素信号转导中的作用，用腺病毒介导的基因转移方法在3T3-L1脂肪细胞和L6心肌细胞中过表达野生型(WT)和5‘-磷酸酶缺陷型SHIP2(ΔIP-SHIP2)。WT-…的表达不影响胰岛素信号转导的早期事件，包括胰岛素诱导的胰岛素受体p-亚基和胰岛素受体-1的酪氨酸磷酸化、胰岛素受体-1与p85亚基的结合以及PI3-激酶的活性更多或ΔIP-SHIP2.正如预期的那样，由于具有5‘-磷酸酶催化区，WT-SHIP2的过表达显著减少了胰岛素诱导的PI(3，4，5)P3的产生。相反，ΔIP-SHIP2的表达增加了PI(3，4，5)P3的数量，表明ΔIP-SHIP2在3T3-L1脂肪细胞中以显性负向方式发挥作用。已知PI(3，4，5)P3和PtdIns(3，4)P2在体外可能激活下游靶标Akt和PKCλ。重要的是，WT-SHIP2的表达抑制了胰岛素诱导的AKT和PKCλ的激活，而体内表达的ΔIP-SHIP2则增强了这些激活。与PI3K下游分子的调节一致，WT-SHIP2的表达降低了胰岛素诱导的2-脱氧葡萄糖摄取和GLUT4的转位，而Δ的IP-SHIP2的表达增加了胰岛素诱导的2-脱氧葡萄糖摄取和GLUT4转位。此外，WT-SHIP2的表达降低了胰岛素诱导的GSK-3GSK-3β的磷酸化和糖原合成酶的激活以及糖原合成的激活，而Δ的表达增强了这一作用。这些结果表明SHIP2通过5‘-磷酸酶活性负性调节胰岛素的代谢信号。较少

项目成果

Hajime Ishihara Toshiyaso Sasaoka et al.: "Molecular Cloning of Rat SH2-Containing Inositol Phasphatase 2 (SHIP2) and Its Rols in the Regulation of Insulin Signaling."Biochemical and Biophyoical Research Communications. 260. 265-272 (1999)

Hajime Ishihara Toshiyaso Sasaoka 等人：“含有肌醇磷酸酶 2 (SHIP2) 的大鼠 SH2 的分子克隆及其在胰岛素信号传导调节中的作用。”生物化学和生物生理研究通讯。

I.Usui,T.Sasaoka et al: "Retinoblastoma protein phosphorylation via PI3-kinase and mTOR pathway regulates adipocyte differentiation."Biochem.Biophys.Res.Commun.. 275. 115-120 (2000)

I.Usui、T.Sasaoka 等人：“视网膜母细胞瘤蛋白磷酸化通过 PI3 激酶和 mTOR 途径调节脂肪细胞分化。”Biochem.Biophys.Res.Commun.. 275. 115-120 (2000)

Tsutomu Wada,Toshiyasu Sasaoka et al.: "Overexpression of SH2-containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metaboli Actims in 3T3-L1 Adiposytes via Its 5'-phosphatase catalytic Activity."Molecular and Cellular Biolog

Tsutomu Wada、Toshiyasu Sasaoka 等人：“含有 SH2 的肌醇磷酸酶 2 的过度表达通过其 5-磷酸酶催化活性导致 3T3-L1 脂肪细胞中胰岛素诱导的代谢活动的负调节。”分子和细胞生物学