Intracellular signaling analysis
Tissue samples will be obtained at the end of clamp studies to measure in vivo activities of the following insulin/inflammatory signaling proteins: 1) tyrosine/serine phosphorylation of IRS, 2) PI-3 kinase, 3) Akt/PKB, 4) PKC isoforms, 5) STAT3 and 6) IKK. Briefly, PI 3-kinase activity will be assessed by immunoprecipitating tissue lysates with a polyclonal IRS antibody coupled to protein A-sepharose beads and assessing the incorporation of 32P into PI. Akt activity will be determined by immunoprecipitating tissue lysates with a polyclonal Akt antibody, that recognizes both Akt1 and Akt2. PKC isoform expression and activities will be measured using antibodies to PKC-θ, β, λ and ζ. STAT3 protein expression and tyrosine phosphorylation will be measured using phospho-specific STAT3 (Tyr705) polyclonal antibody. Activity of IKK will be measured by immunoprecipitating IKK complex with IKK-γ antibody.