title: Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport.
abstract: The role of atypical protein kinase C (aPKC) in insulin-stimulated glucose transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated glucose transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on glucose transport are particularly relevant for understanding whole body glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-zeta in L6 myotubes and PKC-lambda in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-zeta, but not PKC-lambda, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-lambda. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-lambda, but not PKC-zeta, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-zeta. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated glucose transport in myocytes and adipocytes.
authors.affiliation: James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
authors.forename: Mini P
authors.initials: MP
authors.lastname: Sajan
authors.affiliation: James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
authors.forename: Jose
authors.initials: J
authors.lastname: Rivas
authors.affiliation: James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Standaert
authors.affiliation: James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612. Electronic address: rfarese@hsc.usf.edu.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Farese
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D032342:Muscle Cells / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D005947:Glucose; D007328:Insulin; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D000099221:Protein Kinase C zeta; C115015:PKC-3 protein