EFFECT OF TUMOR-NECROSIS-FACTOR-ALPHA ON INSULIN-STIMULATED MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE IN CULTURED RAT SKELETAL-MUSCLE CELLS

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insu lin resistance in obese/diabetic animals through its effects on tyrosi ne 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 e xamined in cultured rat skeletal muscle cell line, L6. Insulin treatme nt of Lb cells resulted in a rapid increase in MAPK activity (> twofol d 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- a nd time-dependent manner. Metabolic labelling studies with inorganic [ P-32]phosphate followed by immunoprecipitation of MAPK and its upstrea m activator, mitogen-activated protein kinase kinase, indicated decrea sed phosphorylation of MAPK and its kinase in response to insulin in c ells exposed to TNF-alpha. This effect of TNF-alpha was nor due to inh ibition of insulin-stimulated p21(ras)-GTP loading or Raf-1 phosphoryl ation, Low concentrations 12 nM) of okadaic acid, a serine/threonine p hosphatase inhibitor. prevented TNF-alpha-induced inhibition of MAPK a nd 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 s uggested 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 p rotein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activit ies. As reported by us earlier, insulin rapidly stimulated PP-1 and co ncomitantly inhibited PP-2A activities in control cells, TNF-alpha tre atment 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-permea ble 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 kin ase via an activated phosphatase.