Reduced Activation of Phosphatidylinositol-3 Kinase and Increased Serine 636 Phosphorylation of Insulin Receptor Substrate-1 in Primary Culture of Skeletal Muscle Cells From Patients With Type 2 Diabetes

Reduced Activation of Phosphatidylinositol-3 Kinase and Increased Serine 636 Phosphorylation of Insulin Receptor Substrate-1 in Primary Culture of Skeletal Muscle Cells From Patients With Type 2 Diabetes Karim Bouzakri 1 , Marina Roques 1 , Philippe Gual 2 , Sophie Espinosa 1 , Fitsum Guebre-Egziabher 1 , Jean-Paul Riou 1 3 , Martine Laville 1 3 , Yannick Le Marchand-Brustel 2 , Jean-François Tanti 2 and Hubert Vidal 1 1 INSERM U449 and CRNHL, IFR 62, R. Laennec Medical Faculty, Lyon, France 2 INSERM U568, Medical Faculty, Nice, France 3 Department of Endocrinology, Diabetology and Nutrition, E. Herriot Hospital, Lyon, France Abstract To understand better the defects in the proximal steps of insulin signaling during type 2 diabetes, we used differentiated human skeletal muscle cells in primary culture. When compared with cells from control subjects, myotubes established from patients with type 2 diabetes presented the same defects as those previously evidenced in vivo in muscle biopsies, including defective stimulation of phosphatidylinositol (PI) 3-kinase activity, decreased association of PI 3-kinase with insulin receptor substrate (IRS)-1 and reduced IRS-1 tyrosine phosphorylation during insulin stimulation. In contrast to IRS-1, the signaling through IRS-2 was not altered. Investigating the causes of the reduced tyrosine phosphorylation of IRS-1, we found a more than twofold increase in the basal phosphorylation of IRS-1 on serine 636 in myotubes from patients with diabetes. Concomitantly, there was a higher basal mitogen-activated protein kinase (MAPK) activity in these cells, and inhibition of the MAPKs with PD98059 strongly reduced the level of serine 636 phosphorylation. These results suggest that IRS-1 phosphorylation on serine 636 might be involved in the reduced phosphorylation of IRS-1 on tyrosine and in the subsequent alteration of insulin-induced PI 3-kinase activation. Moreover, increased MAPK activity seems to play a role in the phosphorylation of IRS-1 on serine residue in human muscle cells. Footnotes Address correspondence and reprint requests to Dr. Marina Roques, INSERM U449, Faculté de Médecine RTH Laennec, F-69370 Lyon Cedex 08, France. E-mail: roques{at}laennec.univ-lyon1.fr . Received for publication 1 August 2002 and accepted in revised form 19 February 2003. K.B. and M.R. contributed equally to this work. ERK, extracellular signal-related kinase; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; PI, phospha