Impairment of Insulin Signaling in Human Skeletal Muscle Cells by Co-Culture With Human Adipocytes

Impairment of Insulin Signaling in Human Skeletal Muscle Cells by Co-Culture With Human Adipocytes Daniela Dietze 1 , Marlis Koenen 1 , Karin Röhrig 2 , Hiroyoshi Horikoshi 3 , Hans Hauner 2 and Jürgen Eckel 1 1 Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes Research Institute, Düsseldorf, Germany 2 Clinical Department, German Diabetes Research Institute, Düsseldorf, Germany 3 Sankyo Research Institute, La Jolla, California Abstract Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-α (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-α were ineffective. After co-culture, TNF-α in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-α and resistin appear not to be involved in this process. Footnotes Address correspondence and reprint requests to Prof. Dr. Jürgen Eckel, German Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail: eckel{at}uni-duesseldorf.de . J.E. has received grant support from Sankyo, Tokyo, Japan. Received for publication 17 December 2001 and accepted in revised form 26 April 2002. ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IRS, insulin receptor substrate; PI, phosphatidylinositol; PPAR-γ, peroxisome proliferator-activated receptor-γ; TBS, Tris-buffered saline; TNF, tumor necrosis factor. DIABETES