Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

Denise R. Cooper, Ph.D.

Co-Major Professor

George Blanck, Ph.D.

Committee Member

Duane Eichler, Ph.D.

Committee Member

Niketa A. Patel, Ph.D.

Committee Member

Eric Bennett, Ph.D.

Keywords

PGC1α, PPARγ, GLUT4, Akt, mTORC2

Abstract

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.

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