JNK activation in muscle from insulin resistant, non-obese subjects accompanies reduced insulin signaling and enhanced phosphorylation of IRS-1 on Serine³¹².

<p>Solubilized muscle biopsies obtained prior to insulin infusion were subject to SDS-PAGE, followed by Western blotting with phospho-specific antibodies against JNK Thr¹⁸³/Tyr¹⁸⁵ (<b>A</b>), p38 MAPK Thr¹⁸⁰/Tyr¹⁸² (<b>B</b>), and IKKβ Ser¹⁷⁷ (<b>C</b>). The predominant band (55 kD) recognized by the anti-JNK antibody is shown and was used for quantification. Optical density values were normalized to the content values for each protein as determined by Western blots for JNK, p38 MAPK and IKKβ, and are presented as mean ± SEM of the ratios of phospho- to total protein. *<i>P</i><0.05 RES <i>vs.</i> SEN groups. (<b>D</b>) Muscle biopsies obtained after 2 hours of insulin infusion were solubilized and assayed for phosphorylation state of key second messengers of insulin signal transduction. Phosphorylation of Akt on Thr³⁰⁸ was determined by specific ELISA and normalized to total Akt content, also determined by ELISA. Values shown are mean ± SEM. *<i>P</i><0.05, RES <i>vs.</i> SEN groups. Lysate volumes from insulin-stimulated muscle biopsies (4 SEN, 3 RES subjects) containing equivalent amounts of IRS-1 protein (as determined by ELISA) were immunoprecipitated with an antibody against IRS-1. Following SDS-PAGE, Western blotting was performed with a phospho-specific antibody against IRS-1 Ser³¹² (<b>E</b>), or with an anti-phosphotyrosine antibody (<b>F</b>). Blots were quantified by scanning densitometry and results expressed as mean ± SEM. *<i>P</i><0.0005, RES <i>vs.</i> SEN groups.