Studies on mechanisms of insulin-stimulated glucose transport : analysis of downstream signaling and real-time monitoring of GLUT4 translocation

胰岛素刺激葡萄糖转运机制研究：下游信号分析和GLUT4易位实时监测

• 批准号: 13470226
• 负责人: OKA Yoshitomo
• 金额: $ 10.37万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470226/
• 关键词: Insulin; glucose transport; Diabetes; GLUT4; acyl-coenzyme A dehydrogenase; PKC; PDK1; intracellular signaling; GFP; acy1-CoA dehdrogenase; インスリン反応性アミノペプチダーゼ

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced glucose transport, we expressed several PKC isoforms, conventional PKC-α, novel PKC-δ, and atypical PKC isoforms of PKC-λ and PKC-ζ, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-αand PKC-λ/ζ, but not of PKC-δ, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Atypical PKC-λ/ζ was not significantly activated by insulin, and expression of the wild-type, constitutively active, and domimant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.Insulin-regulated aminopeptidase (IRAP) is known to be localized on the GLUT4-containing vesicles. The region of IRAP fused … More with glutathione-S-transferase [GST-IRAP(55-82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment. Less

为了阐明蛋白激酶C亚型在胰岛素诱导的葡萄糖转运中的作用，我们用腺病毒介导的基因转导系统在3T3-L1脂肪细胞中表达了几种蛋白激酶C亚型，常规的蛋白激酶C-α、新型的蛋白激酶C-δ和非典型的蛋白激酶C亚型λ和蛋白C-ζ及其突变体。在3T3-L1脂肪细胞中检测到内源性PKC-α和PKC-λ/ζ的表达和活性，但没有检测到PKC-δ的内源性表达和活性。每种野生型PKC亚型的过表达均可诱导3T3-L1脂肪细胞中大量的PKC活性。非典型PKC-λ/ζ不能被胰岛素显著激活，并且非典型PKC的野生型、成分活性和控制区阴性突变体的表达既不影响基础葡萄糖转运，也不影响胰岛素刺激的葡萄糖转运。因此，在3T3-L1脂肪细胞中，非典型的PKC酶在胰岛素刺激的葡萄糖转运中并不起主要作用。胰岛素调节氨基肽酶(IRAP)定位于含GLUT4的小泡上。融合…的IRAP区域将3T3-L1脂肪细胞裂解产物与谷胱甘肽-S转移酶共同孵育，鉴定出长链、中链和短链酰基辅酶A脱氢酶(ACDs)是与IRAP相关的蛋白质。对蔗糖梯度超速离心组分和抗GLUT4抗体免疫纯化的囊泡的免疫印迹表明，这些ACDs定位于含GLUT4的囊泡上。此外，长链和中链ACDs的抑制剂3-巯基丙酸和己酰辅酶A分别在体外诱导IRAP中长链酰基辅酶A脱氢酶和/或中链酰基辅酶A脱氢酶的解离以及GLUT4在质膜上的募集，并刺激通透性3T3-L1脂肪细胞的葡萄糖转运活性。这些发现表明，ACDs通过与IRAP结合，以一种依赖于其二亮氨酸基序的方式定位于含GLUT4的囊泡上，并在将含GLUT4的囊泡保留到细胞内的间隔中发挥作用。较少

项目成果

Fujishiro, M., et al.: "MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake, but regulates glucose transporter expression"J. Biol. Chem.. 276. 19800-19806 (2001)

Fujishiro, M. 等人：“MKK6/3 和 p38 MAPK 通路激活对于胰岛素诱导的葡萄糖摄取不是必需的，但可以调节葡萄糖转运蛋白的表达”J.

Yamada T, Katagiri H, Asano T, Tsuru M, Inukai K, Ono H, Kodama T, Kikuchi M, Oka Y: "Role of PDK1 in insulin signaling pathway for glucose metabolism In 3T3-L1 adipocytes"Am J Physiol. 282. E1385-E1394 (2002)

Yamada T、Katagiri H、Asano T、Tsuru M、Inukai K、Ono H、Kodama T、Kikuchi M、Oka Y：“PDK1 在胰岛素信号通路中对 3T3-L1 脂肪细胞葡萄糖代谢的作用”Am J Physiol。

Yujiri T, Nawata R, Takahashi T, Sato Y, Tanizawa Y, Kitamura T, Oka Y.: "MEK kinase 1 interacts with focal adhesion kinase and regulates insulin receptor substrate-1 expression"J Biol Chem.. 278. 3846-3851 (2003)

Yujiri T、Nawata R、Takahashi T、Sato Y、Tanizawa Y、Kitamura T、Oka Y.：“MEK 激酶 1 与粘着斑激酶相互作用并调节胰岛素受体底物 1 表达”J Biol Chem.. 278. 3846-3851

Yamada T, et al.: "Role of PDK1 in insulin signaling pathway for glucose metabolism In 3T3-L1 adipocytes"Am J Physiol.. (in press).

Yamada T 等人：“PDK1 在 3T3-L1 脂肪细胞葡萄糖代谢的胰岛素信号通路中的作用”Am J Physiol..（出版中）。

Tsuru M, Katagiri H, Asano T, Yamada T, Ohno S, Ogihara T, Oka Y: "Role of protein kinase C isoforms in glucose transport in 3T3-L1 adipocytes-insignificance of atypical PKC"Am J Physiol. 283. E338-E345 (2002)

Tsuru M、Katagiri H、Asano T、Yamada T、Ohno S、Ogihara T、Oka Y：“蛋白激酶 C 异构体​​在 3T3-L1 脂肪细胞葡萄糖转运中的作用 - 非典型 PKC 的重要性”Am J Physiol。