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

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 insulininduced 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.

Isoform-specific defects of insulin stimulation of Akt/protein kinase B (PKB) in skeletal muscle cells from type 2 diabetic patients

“…Four days after initiation of the differentiation, cells showed polynucleated status and produced specific markers of human skeletal muscle. In agreement with previous studies [20,29,30], the rates of myoblasts' growth and fusion into myotubes were similar, and there was no morphological difference between cultured skeletal muscle cells from controls and type 2 diabetic patients.…”

“…Differentiated myotubes were prepared as previously described [20,28]. Four days after initiation of the differentiation, cells showed polynucleated status and produced specific markers of human skeletal muscle.…”

“…PI3K-associated activity was measured in IRS-1 immunocomplexes using phosphatidylinositol (10 µg per reaction; Sigma-Aldrich, St Louis, MO, USA) and 10 μmol/l ATP (supplemented with 32 P-labelled γ-ATP, 185 kBq per reaction; Perkin Elmer, Waltham, MA, USA) as described [20,31]. After the reaction, phosphoinositides were separated by thin-layer chromatography on silica plates (Merck, Darmstadt, Germany) and labelled products were visualised and quantified using Phosphor-Imager SI and Image Quant Software (Molecular Dynamics, Sunnyvale, CA, USA).…”

“…Reduced insulin-stimulated PI3K was observed in muscle biopsies [16][17][18][19] and in primary culture of human skeletal muscle established from type 2 diabetic patients [20,21]. However, deregulation of Akt is more controversial, with studies reporting significant reductions of insulin-stimulated Ser 473 or Thr 308 phosphorylations [22,23], and others showing no difference in phosphorylation or enzymatic activity of Akt between control participants and type 2 diabetic patients [18,24].…”

Increased Protein Tyrosine Phosphatase 1B (PTP1B) Activity and Cardiac Insulin Resistance Precede Mitochondrial and Contractile Dysfunction in Pressure‐Overloaded Hearts

“…However, the regulation of IRS1 activity is highly complex and may occur through phosphorylation at more than 10 serine/threonine residues 20. We chose to assess the phosphorylation at Ser636/639 because increased phosphorylation at this site has repeatedly been shown to inhibit insulin signaling in diabetes mellitus 22, 23. However, IRS1 phosphorylation at Ser636/639 was normal as shown by unchanged ratio of P‐IRS1/total IRS1 at all 3 time points (Figure 4B).…”