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Global Assessment of Regulation of Phosphorylation of Insulin Receptor Substrate-1 by Insulin In Vivo in Human Muscle

¹ Center for Metabolic Biology, Arizona State University, Tempe, Arizona
² School of Life Sciences, Arizona State University, Tempe, Arizona
³ Department of Kinesiology, Arizona State University, Tempe, Arizona
⁴ Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas
⁵ Department of Physiology, The University of Texas Health Science Center, San Antonio, Texas

Address correspondence and reprint requests to Lawrence J. Mandarino, PhD, Director, Center for Metabolic Biology, College of Liberal Arts and Sciences, P.O. Box 873704, Tempe, AZ 85287-3704. E-mail: lawrence.mandarino{at}asu.edu

Abbreviations: Ang II, angiotensin II; CID, collision-induced dissociation; FTICR, Fourier transform ion cyclotron resonance; hIRS, human insulin receptor substrate; HPLC, high-performance liquid chromatography; IRS, insulin receptor substrate; PI, phosphatidylinositol; PKC, protein kinase C; PTB, phosphotyrosine binding

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, Ser⁵²⁷, 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 Ser³⁴⁸, 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.

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