Phorbol esters inhibit insulin-induced receptor down-regulation in cultured human lymphocytes: association with diminished insulin receptor autophosphorylation

Abstract

Exposure of cells to phorbol 12-myristate 13-acetate (PMA) has been reported to result in resistance to the acute biological effects of insulin and an associated reduction in insulin-receptor tyrosine kinase activity. To investigate the relationship of insulin receptor autophosphorylation with a longer-term action of insulin the effect of PMA on insulin-stimulated receptor down-regulation was examined in cultured human lymphocytes (IM-9). Lymphocytes bound [3H]phorbol dibutyrate specifically with characteristics typical of binding to protein kinase C (PKC). Acute exposure (30 min) to PMA resulted in a transient decrease of insulin binding which is consistent with a decrease in receptor number. Chronic (18 h) exposure to PMA (5 nM) resulted in inhibition of insulin-induced down-regulation of its cognate receptor. Sphingosine, an inhibitor of PKC, or chronic pre-exposure to a high concentration of PMA (1 microM), which is known to inactivate PKC, blocked the effect of PMA. PMA inhibited insulin-stimulated receptor internalization by 26% and receptor degradation by 82%. Exposure of intact cells to PMA followed by insulin treatment inhibited insulin-receptor autophosphorylation subsequently assayed in vitro, as well as beta-subunit tyrosine phosphorylation in situ. In summary, PMA inhibited insulin-stimulated receptor down-regulation via activation of PKC. This was associated with an inhibition of both receptor internalization and receptor degradation. There was a concomitant inhibition of receptor tyrosine autophosphorylation consistent with a requirement of receptor kinase activation for both short-term and long-term biological effects of insulin.