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.
Highlights
Transport is the initial rate-limiting step for cellular uptake and utilization of glucose in skeletal muscle and adipocytes
In these gene knockout studies, insulin-stimulated glucose transport was markedly diminished in the absence of these protein kinases, and restored by their expression. These methods, while highly suggestive, have inherent caveats, e.g., expression of inhibitory kinases may have untoward effects, and genetically manipulated and subsequently selected cells may have little relevance to native adipocytes and myocytes. As another experimental approach for evaluating the importance of atypical protein kinase C (aPKC) and PKB during insulin-stimulated glucose transport, whole body knockout of PKC-λ, the major aPKC in many mouse tissues, has not been feasible, as this aPKC is required for embryonic survival; in the case of PKBα or PKBβ, effects of maximally effective concentrations of insulin on glucose transport are not compromised in muscles of these knockout mice [16,17], perhaps reflecting the continued function of remaining untargeted PKB isoforms that may be sufficient or compensatorially increased
Initial studies with RNAi have suggested that PKBβ/Akt2 is required for insulin-stimulated glucose transport in 3T3/L1 adipocytes [18], whereas, in L6 myotubes, the impaired activation of PKBβ resulting from an RNAi-induced knockdown of insulin receptor substrate (IRS)-2 does not inhibit insulin-stimulated glucose transport [19]
Summary
Transport is the initial rate-limiting step for cellular uptake and utilization of glucose in skeletal muscle and adipocytes. In these gene knockout studies, insulin-stimulated glucose transport was markedly diminished in the absence of these protein kinases, and restored by their expression These methods, while highly suggestive, have inherent caveats, e.g., expression of inhibitory kinases may have untoward effects, and genetically manipulated and subsequently selected cells may have little relevance to native adipocytes and myocytes. As another experimental approach for evaluating the importance of aPKC and PKB during insulin-stimulated glucose transport, whole body knockout of PKC-λ, the major aPKC in many mouse tissues, has not been feasible, as this aPKC is required for embryonic survival; in the case of PKBα or PKBβ, effects of maximally effective concentrations of insulin on glucose transport are not compromised in muscles of these knockout mice [16,17], perhaps reflecting the continued function of remaining untargeted PKB isoforms that may be sufficient or compensatorially increased. Since these aPKCs have have substantially different mRNA and amino acid compositions (30% non-homology), but function interchangeably during insulin-stimulated glucose transport [8], this difference in aPKC composition in these two cell types allowed us to (a) examine the specificity of the presently used RNAi forms for the targeted endogenous aPKC, and (b), most importantly, rescue insulin-stimulated glucose transport in RNAi-treated cells by expression of an exogenous untargeted aPKC to substitute for the depleted endogenous aPKC
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