Co-transcriptional splicing and functional role of PKCbeta in insulin-sensitive L6 skeletal muscle cells and 3T3-L1 adipocytes

PKCbetaII is alternatively spliced during acute insulin stimulation in L6 skeletal muscle cells. This PKCbetaII isoform is critical in propagating GLUT4 translocation. PKCbeta protein and promoter dysfunction correlate with human insulin resistance. TZD treatment ameliorates whole-body insulin-resistance. Its primary target is adipocyte PPARgamma, which it activates upon binding. This causes both altered circulating serum FFA concentrations and adipokine secretion profile. How TZDs affect the intracellular signaling of skeletal muscle cells is unknown. RT-PCR and Western blot analysis showed that TZDs elevated PKCbetaII by a process that involves co-transcriptional splicing. PGC1alpha overexpression most closely resembled TZD treatment by increasing PKCbetaII protein levels and keeping PKCbetaI levels relatively constant. Use of a heterologous PKCbeta promoter driven PKCbeta minigene demonstrated that PPARgamma could regulate the PKCbeta promoter, but whether this is direct or indirect is unclear. SRp40 splicing factor has been shown to dock onto the PGC1alpha CTD and influence splicing. SRp40, through overexpression and silencing, appears to play a part in PKCbeta promoter regulation.;PKCbeta promoter regulation was also studied in 3T3-L1 cells. TZDs were experimentally shown to have no role in PKCbeta promoter regulation despite PPARgamma activation. Chromatin immunoprecipitation assays revealed PU.1 as a putative PKCbeta transcription factor that can cross-talk with the spliceosome, possibly through SRp40 which was also associated with the PKCbeta promoter. 3T3-L1 adipocyte differentiation revealed a novel developmentally-regulated switch from PKCbetaI to PKCbetaII, using western blot and Real-Time PCR analysis. Pharmacological inhibition of PKCbetaII using CGP53353 and LY379196 blocked [3H]2-deoxyglucose uptake and revealed a functional role for PKCbetaII in adipocyte ISGT. CGP53353 specifically inhibited phosphorylation of PKCbetaII Serine 660 and not other critical upstream components of the insulin signaling pathway. Subcellular fractionation and PM sheet assay pointed to PKCbetaII-mediated regulation of GLUT4 translocation to the PM. Co-immunoprecipitation between PKCbetaII and GLUT4 allude to possible direct interaction. Western blot and immunofluorescence assays show PKCbetaII activity is linked with Akt Serine 473 phosphorylation, thus full Akt activity. Western blot and co-immunoprecipitation suggested that insulin caused active mTORC2 to directly activate PKCbetaII. Data support a model whereby PKCbetaII is downstream of mTORC2 yet upstream of Akt, thereby regulating GLUT4 translocation.