Co-Transcriptional Splicing and Functional Role of PKCβ in Insulin-Sensitive L6 Skeletal Muscle Cells and 3T3-L1 Adipocytes

PKC βII is alternatively spliced during acute insulin stimulation in L6 skeletal muscle cells. This PKC βII isoform is critical in propagating GLUT4 translocation. PKC β protein and promoter dysfunction correlate with human insulin resistance. TZD treatment ameliorates whole-body insulin-resistance. Its primary target is adipocyte PPAR γ, 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 PKC βII by a process that involves co-transcriptional splicing. PGC1 α overexpression most closely resembled TZD treatment by increasing PKCβII protein levels and keeping PKC βI levels relatively constant. Use of a heterologous PKCβ promoter driven PKC β minigene demonstrated that PPARγ could regulate the PKCβ promoter, but whether this is direct or indirect is unclear. SRp40 splicing factor has been shown to dock onto the PGC1 α CTD and influence splicing. SRp40, through overexpression and silencing, appears to play a part in PKC β promoter regulation. PKC β promoter regulation was also studied in 3T3-L1 cells. TZDs were experimentally shown to have no role in PKC β promoter regulation despite PPARγ activation. Chromatin immunoprecipitation assays revealed PU.1 as a putative PKC β transcription factor that can cross-talk with the spliceosome, possibly through SRp40 which was also associated with the PKC β promoter. 3T3-L1 adipocyte differentiation revealed a novel developmentally-regulated switch from PKC βI to PKCβ II, using western blot and Real-Time PCR analysis. Pharmacological inhibition of PKC β II using CGP53353 and LY379196 blocked [ 3 H]2-deoxyglucose uptake and revealed a functional role for PKC β II in adipocyte ISGT. CGP53353 specifically inhibited phosphorylation of PKC β II Serine 660 and not other critical upstream components of the insulin signaling pathway. Subcellular fractionation and PM sheet assay pointed to PKC β II-mediated regulation of GLUT4 translocation to the PM. Co-immunoprecipitation between PKC β II and GLUT4 allude to possible direct interaction. Western blot and immunofluorescence assays show PKC β II 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 PKC βII. Data support a model whereby PKCβ II is downstream of mTORC2 yet upstream of Akt, thereby regulating GLUT4 translocation