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.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-4890 |
| Date | 29 September 2009 |
| Creators | Kleiman, Eden |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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