Abstract
In this study, we demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion. The effect was observed in two different insulin-secreting cell lines and in rat pancreatic islets. Tyrosine phosphorylation was largely calcium independent and occurred within 2 min after stimulation of beta cells with glucose and the muscarinic agonist carbachol. In islets, the effect of glucose was greatly diminished by the addition of mannoheptulose, a seven-carbon sugar that inhibits glucokinase, suggesting that glucose metabolism is required for tyrosine phosphorylation of the protein to occur. Neither insulin nor insulin-like growth factor I significantly increased tyrosine phosphorylation of the 125-kDa protein, suggesting that it was not an autocrine effect. Depolarization of beta cells with glyburide or 50 m potassium dramatically increased insulin secretion but had no significant effect on tyrosine phosphorylation. Addition of phorbol ester caused a less than 2-fold increase in tyrosine phosphorylation, whereas the calcium ionophore A23187 had no effect. Among the various fuel secretagogues tested, only -glucose stimulated tyrosine phosphorylation, both alone and in combination with carbachol. Finally, the tyrosine kinase inhibitor AG879 inhibited both tyrosine phosphorylation and insulin secretion in a dose-dependent manner. Taken together, these data demonstrate the presence of a novel signaling pathway in glucose-induced insulin secretion: tyrosine phosphorylation of beta cell p125, which is a proximal step in insulin secretion. Our current working hypothesis is that glucose stimulation of beta cell p125 tyrosine phosphorylation is an essential step for insulin secretion.
Highlights
We demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion
Exogenous insulin and insulin-like growth factor I (IGF-I)1 and agents that resulted in insulin release such as Kϩ caused the rapid tyrosine phosphorylation of the 97-kDa  subunit of the insulin receptor
We show that stimulation of beta cells with glucose, carbachol, and various fuel secretagogues causes increased tyrosine phosphorylation of a 125-kDa protein
Summary
Increased intracellular Ca2ϩ activates protein kinases such as the Ca2ϩ- and calmodulin-dependent protein kinase [17,18,19,20,21], it remains unclear how increases in intracellular Ca2ϩ lead to insulin release Because of these questions, the search for additional beta cell-specific pathways that result in insulin exocytosis has continued. Rothenberg et al [32] have demonstrated that in late passage -TC3 cells glucoseinduced insulin secretion activates the beta cell insulin receptor tyrosine kinase and its intracellular signal transduction pathway [32] In these experiments, exogenous insulin and insulin-like growth factor I (IGF-I) and agents that resulted in insulin release such as Kϩ caused the rapid tyrosine phosphorylation of the 97-kDa  subunit of the insulin receptor. We show that stimulation of beta cells with glucose, carbachol, and various fuel secretagogues causes increased tyrosine phosphorylation of a 125-kDa protein. The tyrosine kinase inhibitor AG879 dose-dependently inhibited both insulin release and 125-kDa protein tyrosine phosphorylation, suggesting that tyrosine phosphorylation of the protein may be an essential step in insulin secretion
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