Protein Kinase C-Independent Effects of Protein Kinase D3 in Glucose Transport in L6 Myotubes

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Abstract
Protein kinase C (PKC) and protein kinase D (PKD) coordinate and regulate many fundamental cellular processes. In this study, we evaluate the role of classic and novel PKC (c/nPKC) and PKD in glucose transport in L6 myotubes. c/nPKC is either activated by short-term phorbol 12-myristate 13-acetate (PMA) treatment or down-regulated by prolonged PMA treatment at a high dose in L6 myotubes. Our results indicate that PMA treatments have little impact on basal and insulin-stimulated glucose uptake and insulin-induced Akt activation. In contrast, the PKC inhibitors Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c] carbazole], Go6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], GF 109203X [bisindolylmaleimide I; 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide], and Ro 31-8220 [bisindolylmaleimide IX; 2-{1-[3-(amidinothio)propyl]-1H-indol3-yl}-3-(1-methylindol-3-yl)maleimide] block basal and insulin-stimulated glucose uptake, and their inhibitory effects persist upon down-regulation of c/nPKC by PMA, implying the presence of PKC-independent effectors in mediating their inhibition of glucose uptake. Go6976, the potent cPKC inhibitor that also effectively inhibits PKD, dose-dependently blocks basal glucose uptake in L6 myotubes, whereas Go6983, the nonselective PKC inhibitor that is ineffective for PKD, has little effect on basal glucose uptake, implying the involvement of PKD in this process. Most prominently, adenoviral gene expression of a dominant-negative PKD isoform, PKD3, primarily inhibits basal glucose uptake and, to a lesser extent, insulin-stimulated glucose uptake, whereas overexpression of wild-type PKD3 significantly enhances basal glucose uptake. Moreover, expression of a PKD3-targeted siRNA significantly inhibits basal glucose uptake. Taken together, our results indicate that PKD, specifically PKD3, directly contributes to insulin-independent basal glucose uptake in L6 skeletal muscle cells.