GLUCOSE-INDUCED AND INSULIN-INDUCED PHOSPHORYLATION OF THE INSULIN-RECEPTOR AND ITS PRIMARY SUBSTRATES IRS-1 AND IRS-2 IN RAT PANCREATIC-ISLETS

The presence of tyrosine-phosphorylated proteins was studied in cultur ed rat pancreatic islets, Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM gl ucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insuli n led to a substantial increase in the phosphorylation of all three ba nds, with no appearance of novel bands. Immunoprecipitation with speci fic antibodies demonstrated that the signal detected at 95 kDa corresp onds to the beta subunit of the insulin receptor (IR) while the band a t 165-185 kDa corresponds to the early substrates of the insulin recep tor, IRS-1 and IRS-2. Immunoprecipitation with IRS-I or IRS-2 antisera detected their association with the lipid metabolizing enzyme phospha tidylinositol 3-kinase (PI 3-kinase), Thus, this is the first demonstr ation that elements involved in the insulin-signalling pathway of trad itional target tissues are also present in pancreatic islets and are p otentially involved in auto- and paracrine-signalling in this organ.