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

  1. Zhengping Yi12,
  2. Paul Langlais13,
  3. Elena A. De Filippis12,
  4. Moulun Luo12,
  5. Charles R. Flynn1,
  6. Stefanie Schroeder1,
  7. Susan T. Weintraub4,
  8. Rebekka Mapes15 and
  9. Lawrence J. Mandarino123
  1. 1Center for Metabolic Biology, Arizona State University, Tempe, Arizona
  2. 2School of Life Sciences, Arizona State University, Tempe, Arizona
  3. 3Department of Kinesiology, Arizona State University, Tempe, Arizona
  4. 4Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas
  5. 5Department of Physiology, The University of Texas Health Science Center, San Antonio, Texas
  1. 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

Abstract

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.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 15 March 2007. DOI: 10.2337/db06-1355.

  • Z.Y., P.L., E.A.D., and M.L. contributed equally to this work.

  • Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1355.

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted March 8, 2007.
    • Received September 22, 2006.
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  1. Published online before print March 14, 2007, doi: 10.2337/db06-1355 Diabetes June 2007 vol. 56 no. 6 1508-1516
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