PMID- 8665940 OWN - NLM STAT- MEDLINE DCOM- 19960802 LR - 20211203 IS - 0014-2956 (Print) IS - 0014-2956 (Linking) VI - 238 IP - 1 DP - 1996 May 15 TI - Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells. PG - 214-20 AB - 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. FAU - Begum, N AU - Begum N AD - Diabetes Research Laboratory, Winthrop University Hospital, Mineola, NY 11501, USA. FAU - Ragolia, L AU - Ragolia L FAU - Srinivasan, M AU - Srinivasan M LA - eng PT - Journal Article PL - England TA - Eur J Biochem JT - European journal of biochemistry JID - 0107600 RN - 0 (Ceramides) RN - 0 (Enzyme Inhibitors) RN - 0 (Insulin) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Tumor Necrosis Factor-alpha) RN - EC 2.7.- (Protein Kinases) RN - EC 2.7.1.- (Mitogen-Activated Protein Kinase 10) RN - EC 2.7.10.1 (Protein-Tyrosine Kinases) RN - EC 2.7.11.1 (Protein Serine-Threonine Kinases) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-raf) RN - EC 2.7.11.24 (Mitogen-Activated Protein Kinases) RN - EC 2.7.12.2 (Mitogen-Activated Protein Kinase Kinases) RN - EC 3.1.3.16 (Phosphoprotein Phosphatases) RN - EC 3.1.3.16 (Protein Phosphatase 1) RN - EC 3.1.3.16 (Protein Phosphatase 2) RN - EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras)) SB - IM MH - Animals MH - Cell Membrane Permeability MH - Cells, Cultured MH - Ceramides/pharmacology MH - Enzyme Inhibitors/pharmacology MH - Insulin/metabolism/*pharmacology MH - Mitogen-Activated Protein Kinase 10 MH - Mitogen-Activated Protein Kinase Kinases MH - *Mitogen-Activated Protein Kinases MH - Muscle, Skeletal/cytology/metabolism MH - Phosphoprotein Phosphatases/antagonists & inhibitors/drug effects/metabolism MH - Phosphorylation/drug effects MH - Protein Kinases/drug effects/metabolism MH - Protein Phosphatase 1 MH - Protein Phosphatase 2 MH - Protein Serine-Threonine Kinases/*drug effects/*metabolism MH - Protein-Tyrosine Kinases/*drug effects/*metabolism MH - Proto-Oncogene Proteins/drug effects/metabolism MH - Proto-Oncogene Proteins c-raf MH - Proto-Oncogene Proteins p21(ras)/drug effects/metabolism MH - Rats MH - Signal Transduction MH - Tumor Necrosis Factor-alpha/*pharmacology EDAT- 1996/05/15 00:00 MHDA- 1996/05/15 00:01 CRDT- 1996/05/15 00:00 PHST- 1996/05/15 00:00 [pubmed] PHST- 1996/05/15 00:01 [medline] PHST- 1996/05/15 00:00 [entrez] AID - 10.1111/j.1432-1033.1996.0214q.x [doi] PST - ppublish SO - Eur J Biochem. 1996 May 15;238(1):214-20. doi: 10.1111/j.1432-1033.1996.0214q.x.