Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle

OBJECTIVE - Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans. RESEARCH DESIGN AND METHODS - In this study, we describe our use of capillary high-performance liquid chromotography electrospray tandem mass spectrometry to identify/quantify site-specific phosphorylation of IRS-1 in human vastus lateralis muscle obtained by needle biopsy basally and after insulin infusion in four healthy volunteers. RESULTS - Twenty-two serine/threonine phosphorylation sites were identified; 15 were quantified. Three sites had not been previously identified (Thr⁴⁹⁵, Ser ⁵²⁷, and S¹⁰⁰⁵). Insulin increased the phosphorylation of Ser³¹², Ser⁶¹⁶, Ser⁶³⁶, Ser⁸⁹², Ser¹¹⁰¹, and Ser¹²²³ (2.6 ± 0.4, 2.9 ± 0.8, 2.1 ± 0.3, 1.6 ± 0.1, 1.3 ± 0.1, and 1.3 ± 0.1-fold, respectively, compared with basal; P < 0.05); phosphorylation of Ser ³⁴⁸, Thr⁴⁴⁶, Thr⁴⁹⁵, and Ser¹⁰⁰⁵ decreased (0.4 ± 0.1, 0.2 ± 0.1, 0.1 ± 0.1, and 0.3 ± 0.2-fold, respectively; P < 0.05). CONCLUSIONS - These results provide an assessment of IRS-1 phosphorylation in vivo and show that insulin has profound effects on IRS-1 serine/threonine phosphorylation in healthy humans.