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-in L6 myotubes and PKC-in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-, but not PKC-, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-, but not PKC-, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-. 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.Transport is the initial rate-limiting step for cellular uptake and utilization of glucose in skeletal muscle and adipocytes. Insulin is a major controller of glucose transport in adipocytes and skeletal muscle, and defects in insulin-stimulated glucose transport, particularly in skeletal muscle, contribute importantly to the development of insulin resistance in obesity and type 2 diabetes mellitus.Insulin regulates glucose transport in adipocytes and skeletal muscle through the activation of insulin receptor substrate (IRS) 2 -1/2-dependent phosphatidylinositol 3-kinase. Distal effectors of phosphatidylinositol 3-kinase that are postulated to mediate insulininduced increases in glucose transport include protein kinase B (PKB/Akt) and atypical protein kinase C (aPKC) isoforms, and . The initial evidence suggesting requirements for PKB (1-4) and aPKC (5-13) in insulin-stimulated glucose transport was based largely upon inhibitory effects ensuing from expression of kinaseinactive (dominant-negative) forms of these protein kinases in adipocytes and muscle cells. More recently, genetically manipulated adipocytes deficient in either PKB (14) or aPKC (15) have been used to further implicate these protein kinases in insulin-stimulated glucose transport. In these gene knock-out studies, insulin-stimulate...

Cellular depletion of atypical PKCλ is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells

“…able to offer a satisfactory explanation for this discrepancy in results but can only support suggestions put forward previously to explain similar discrepancies (31), namely that variations in the introduction of small-interfering RNA/shRNA in the cells may have an impact on the results observed. Our reported levels of RNAi-mediated knockdown at both mRNA and protein levels are similar to that reported in other studies, so it is unlikely that differing levels of knockdown are at the root of the different responses observed.…”

“…Studies using RNAi techniques in 3T3-L1 adipocytes have suggested that depletion of aPKC has no discernible effects on insulin-stimulated glucose uptake (43), whereas other groups have demonstrated that a similar depletion results in loss of insulin-stimulated glucose uptake (31). Currently, we are un- Fig.…”

Focal adhesion kinase regulates insulin resistance in skeletal muscle

“…Therefore, it is possible that FAK differentially regulates insulin signalling under normal and insulinresistant conditions. Sajan et al [39] recently reported a differential role of the atypical PKC isoforms ζ and λ in insulin-stimulated glucose transport. Regulation of GLUT4 translocation in skeletal muscle of high-fat-diet-fed rats by PKC (ζ/λ) has also been reported [40].…”

From signal transduction to signal interpretation: An alternative model for the molecular function of insulin receptor substrates

“…As in the case of JNK a significant number of IRS kinases are also required for insulin-dependent metabolic control. For example, ERK1/2 are believed to link insulin with cell proliferation, differentiation and the regulation of lipid metabolism, glycogen synthase kinase (GSK)-3 is required to regulate glycogen stores in muscle and liver, S6K to control protein synthesis, and isoforms of PKC may be required for insulin-induced glucose transport (Avruch, 1998;Bandyopadhyay et al, 2000;Bandyopadhyay et al, 2002;Copps et al, 2010;Kotzka et al, 2004;Martin and Parton, 2006;Roth et al, 2000;Ryden et al, 2004;Sajan et al, 2006).…”