title: Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells.
abstract: Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
authors.affiliation: Diabetes Research Laboratory, Winthrop University Hospital, Mineola, NY 11501, USA.
authors.forename: N
authors.initials: N
authors.lastname: Begum
authors.forename: L
authors.initials: L
authors.lastname: Ragolia
authors.forename: M
authors.initials: M
authors.lastname: Srinivasan
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D048057:Mitogen-Activated Protein Kinase 10; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000187:drug effects* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002518:Ceramides; D004791:Enzyme Inhibitors; D007328:Insulin; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; D011494:Protein Kinases; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D016283:Proto-Oncogene Proteins p21(ras)