Regulation of XXXX , @entity8277 substrate-1 and @entity4357 3-kinase in 3T3-F442A adipocytes. Effects of differentiation, insulin, and @entity619 .

@entity10175 @entity8277 @entity619 @entity669 @entity4357
Insulin rapidly stimulates @entity669 kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate @entity8277 substrate 1 ( @entity10175 ), which in turn associates with and activates the enzyme @entity4357 3-kinase (PI 3-kinase). In the present study we have examined these three initial steps in insulin action during the differentiation of 3T3-F442A adipocytes and after treatment with @entity619 or insulin. The differentiation of 3T3-F442A cells was characterized by a 13-fold increase in @entity8277 protein, a 9-fold increase in @entity10175 , and a 10- and 4.5-fold increase in their insulin-stimulated phosphorylation, respectively. The mRNA expression of these two proteins showed a similar 8-fold increase during differentiation. In addition there was a 3.5-fold increase in PI 3-kinase protein [85 kilodalton (kDa) subunit] and a 16-fold increase in @entity10175 -associated PI 3-kinase activity between day 0 and day 8 of differentiation. @entity619 (1 microM) treatment of differentiated cells induced a further 48% (P < 0.05) increase in @entity8277 level, but the autophosphorylation of the receptor was decreased by 31 +/- 1% (P < 0.02). At the same time there was a decrease by 56 +/- 4% (P < 0.005) in @entity10175 protein and by 31 +/- 1% (P < 0.001) in @entity10175 phosphorylation. The expression of @entity8277 mRNA was unchanged, but the expression of @entity10175 mRNA was decreased by approximately 75% after @entity619 . By contrast, @entity619 induced a 69% increase in the level of PI 3-kinase as determined by immunoblotting. The combined effect of decreased @entity10175 phosphorylation and increased PI 3-kinase protein was a minimal change (15% decrease) in the association/activation between @entity10175 and PI 3-kinase. Chronic treatment with 100 nM insulin induced a time- and dose-dependent decrease in @entity8277 and @entity10175 protein levels reaching a nadir of 34 +/- 5% (P < 0.005) and 39 +/- 5% (P < 0.01) of control levels after 24 h, respectively. There was an even more marked decrease in the phosphorylation level of these proteins. Chronic insulin treatment also produced a 30% decrease in PI 3-kinase protein levels and a approximately 50% decrease in the association/activation between @entity10175 /PI 3-kinase. The expression of @entity8277 and @entity10175 mRNA was unchanged during chronic insulin treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
