| Co-Investigator(Kenkyū-buntansha) |
HINOKIO Yoshinori Tohoku University Hospital, Department of Diabetes and Metabolism, Research Associate, 医学部附属病院, 助手 (10282071)
HIRAI Masashi Tohoku University Hospital, Department of Diabetes and Metabolism, Research Associate, 医学部附属病院, 助手 (80312578)
KATAGIRI Hideki Tohoku University Graduate School of Medicine, Division of Advanced Therapeutics for Metabolic Diseases, Professor, 大学院・医学系研究科, 教授 (00344664)
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| Research Abstract |
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
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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
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