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（PKC）亚型在胰岛素诱导的葡萄糖转运中的作用，我们利用腺病毒介导的基因转导系统在3 T3-L1脂肪细胞中表达了几种PKC亚型，传统的PKC-α、新型的PKC-δ、非典型的PKC-λ和PKC-β亚型及其突变体。3 T3-L1脂肪细胞内源性表达PKC-α和PKC-λ/β，但不表达PKC-δ。在3 T3-L1脂肪细胞中，每种野生型PKC亚型的过表达诱导了大量的PKC活性。胰岛素不能显著激活非典型PKC-λ/β，非典型PKC的野生型、组成型活性和显性阴性突变体的表达不影响基础或胰岛素刺激的葡萄糖转运。胰岛素调节的氨基肽酶（Insulin-regulated aminopeptidase，IRAP）定位于含GLUT 4的囊泡上。IRAP融合区域 ...更多信息 与谷胱甘肽-S-转移酶[GST-IRAP（55-82）]一起孵育，导致鉴定出长链、中链和短链酰基辅酶A转移酶（ACD）作为与IRAP相关的蛋白。免疫印迹法制备的馏分从蔗糖梯度超离心和囊泡免疫纯化与抗GLUT 4抗体显示这些ACD被定位于GLUT 4-含有囊泡。此外，3-巯基丙酸和己酰辅酶A，长链和中链ACD的抑制剂，分别诱导长链酰基辅酶A脱氢酶和/或中链酰基辅酶A脱氢酶在体外从IRAP中解离，以及GLUT 4向质膜的募集和透化3 T3-L1脂肪细胞中葡萄糖转运活性的刺激。这些研究结果表明，ACD通过与IRAP以依赖于其双亮氨酸基序的方式结合定位于含GLUT 4的囊泡上，并在将含GLUT 4的囊泡保留到细胞内区室中中发挥作用。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