Global IRS-1 phosphorylation analysis in insulin resistance

IRS-1 serine phosphorylation is often elevated in insulin resistance models, but confirmation in vivo in humans is lacking. We therefore analysed IRS-1 phosphorylation in human muscle in vivo. We used HPLC-electrospray ionisation (ESI)-MS/MS to quantify IRS-1 phosphorylation basally and after insulin infusion in vastus lateralis muscle from lean healthy, obese non-diabetic and type 2 diabetic volunteers. Basal Ser323 phosphorylation was increased in type 2 diabetic patients (2.1 +/- 0.43, p a parts per thousand currency signaEuro parts per thousand 0.05, fold change vs lean controls). Thr495 phosphorylation was decreased in type 2 diabetic patients (p a parts per thousand currency signaEuro parts per thousand 0.05). Insulin increased IRS-1 phosphorylation at Ser527 (1.4 +/- 0.17, p a parts per thousand currency signaEuro parts per thousand 0.01, fold change, 60 min after insulin infusion vs basal) and Ser531 (1.3 +/- 0.16, p a parts per thousand currency signaEuro parts per thousand 0.01, fold change, 60 min after insulin infusion vs basal) in the lean controls and suppressed phosphorylation at Ser348 (0.56 +/- 0.11, p a parts per thousand currency signaEuro parts per thousand 0.01, fold change, 240 min after insulin infusion vs basal), Thr446 (0.64 +/- 0.16, p a parts per thousand currency signaEuro parts per thousand 0.05, fold change, 60 min after insulin infusion vs basal), Ser1100 (0.77 +/- 0.22, p a parts per thousand currency signaEuro parts per thousand 0.05, fold change, 240 min after insulin infusion vs basal) and Ser1142 (1.3 +/- 0.2, p a parts per thousand currency signaEuro parts per thousand 0.05, fold change, 60 min after insulin infusion vs basal). We conclude that, unlike some aspects of insulin signalling, the ability of insulin to increase or suppress certain IRS-1 phosphorylation sites is intact in insulin resistance. However, some IRS-1 phosphorylation sites do not respond to insulin, whereas other Ser/Thr phosphorylation sites are either increased or decreased in insulin resistance.