In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, I nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 muM okadaic, acid (OA), which inhibits both PP-1 and PP-2A but not by I nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (I muM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation. (C) 2002 Elsevier Science (USA).