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 (Thr(495), Ser(527), and S-1005). Insulin increased the phosphorylation of Ser(312), Ser(616), Ser(636), Ser(892), Ser(1101), and Ser(1223) (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), Thr(446), Thr(495), and Ser(1005) 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.