Effect of G protein-coupled signaling pathways on insulin receptor tyrosine kinase signaling pathways and insulin's anti-apoptotic function

Abstract

Although the activation of receptor tyrosine kinase (RTK) is crucial to execute a number of physiological response by insulin, the roles of signaling pathways derived by G protein-coupled receptors (GPCRs) are unravelled. We previously showed that insulin suppressed apoptotic cell death through signaling pathways including stimulation of PI3 kinase and ERK. The aim of the present study was to examine whether the activation of cAMP-dependent signaling pathways might affect insulin's anti-apoptotic function and RTK-dependent signaling components including ERK and Akt/PKB upon insulin stimulation in CHO-IR cells. cAMP-dependent signaling pathways were stimulated by treatments with cholera toxin (CTX, an activator of Gs protein), pertussis toxin (PTX, an inhibitor of Gi protein) or 8-bromo-cAMP (a cell membrane-permeable analogue of cAMP) together with - or without insulin in CHO-IR cells. Activities of ERK and Akt/PKB were analyzed by immunoblotting experiments using specific antibodies against activated ERK and Akt/PKB proteins. Apoptotic cell death was evaluated with fluorescence microscopy after H33342 staining and with cell cycle analysis with flow cytometer after propidium iodide staining. Addition of CTX as well as PTX suppressed ERK activity stimulated by insulin treatment. However, insulin-induced Akt/PKB activity was more increased by the addition of PTX or CTX. 8-bromo-cAMP treatment also led to a suppression of ERK activity and an additional increase in Akt/PKB activity stimulated by insulin. Regardless of an additional increase in Akt/PKB activity, insulin's antiapoptotic function was blocked by activations of cAMP-dependent signaling pathways. Taken together, the present study showed that the activation of cAMP-dependent signaling pathways regulated downstream steps of RTK after binding of insulin to its receptor in different manners, down-regulating the ERK activity and up-regulating Akt/PKB activity in CHO-IR cells. From the blockade of insulin's anti-apoptotic function even with the up-regulation of Akt/PKB activity by cAMP pathways, it is suggested that insulin's anti-apoptotic function is not totally dependent on Akt/PKB activity or, at least, is regulated at any downstream signaling steps of Akt/PKB activation.