Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport.
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.
Sajan
Mini P
MP
James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
Rivas
Jose
J
James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
Li
Pengfei
P
James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
Standaert
Mary L
ML
James A. Haley Veterans Medical Center and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
Farese
Robert V
RV
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.
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
D005947:Glucose; D007328:Insulin; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D000099221:Protein Kinase C zeta; C115015:PKC-3 protein