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

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

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