Phosphotyrosine protein profiles in monocytes after insulin and IGF-1 stimulation.
Mononuclear cells are largely used in clinical studies on insulin action because of their accessibility. Insulin acts in monocytes in different ways than it does in other cells, i.e. adipocytes and muscular cells. 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 have studied the phosphotyrosine protein profiles in intact human monocytes after insulin and IGF-1 stimulation with the aim of identifying substrate/s of these receptors and of comparing them to the substrates already described in major insulin target tissues. Mononuclear cells were prepared from peripheral blood by centrifugation on Ficoll Hypaque and by adhesion to tissue-culture plates. Cell stimulation, lysis, immunoprecipitation 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 rapidly increased the tyrosine phosphorylation of the 95 Kdal beta-subunit of their own receptors. Under our experimental conditions insulin and IGF-1 were not able to stimulate the phosphorylation of IRS-1, a major substrate of the insulin receptor kinase.
Zoppini
G
G
Institute of Metabolic Diseases, University of Verona, Italy.
Galante
P
P
Zardini
M
M
Muggeo
M
M
D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D014443:Tyrosine / Q000187:drug effects / Q000378:metabolism*
C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1