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.


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.
Yi
Zhengping
Z
Center for Metabolic Biology, Arizona State University, Tempe, AZ 85287-3704, USA.
Langlais
Paul
P
De Filippis
Elena A
EA
Luo
Moulun
M
Flynn
Charles R
CR
Schroeder
Stefanie
S
Weintraub
Susan T
ST
Mapes
Rebekka
R
Mandarino
Lawrence J
LJ
D000328:Adult; D001706:Biopsy; D001786:Blood Glucose / Q000032:analysis; D005260:Female; D015309:Glucose Clamp Technique; D005951:Glucose Tolerance Test; D006706:Homeostasis; D006801:Humans; D007328:Insulin / Q000097:blood / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D008297:Male; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D012016:Reference Values
D001786:Blood Glucose; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine