PHOSPHOTYROSINE PROTEIN PROFILES IN MONOCYTES AFTER INSULIN AND IGF-1STIMULATION

Mononuclear cells are largely used in clinical studies on insulin acti on because of their accessibility. Insulin acts in monocytes in differ ent ways than it does in other cells, i.e. adipocytes and muscular cel ls. Therefore, it still remains unclear whether monocytes reflect the same changes that occur in insulin receptors at the level of the major insulin target tissues during different pathophysiologic states. We h ave studied the phosphotyrosine protein profiles in intact human monoc ytes after insulin and IGF-1 stimulation with the aim of identifying s ubstrate/s of these receptors and of comparing them to the substrates already described in major insulin target tissues. Mononuclear cells w ere prepared from peripheral blood by centrifugation on Ficoll Hypaque and by adhesion to tissue-culture plates. Cell stimulation, lysis, im muno-precipitation and western blotting were carried out following the protocol described by P. L. Rothenberg in 1991 and the immunoreactive proteins visualized on film by chemiluminescence. Insulin and IGF-1 r apidly increased the tyrosine phosphorylation of the 95 Kdal beta-subu nit of their own receptors. Under our experimental conditions insulin and IGF-1 were not able to stimulate the phosphorylation of IRS-1, a m ajor substrate of the insulin receptor kinase.