Insulin‐mediated redox regulation of cyclic AMP‐dependent protein kinase (PKA) in rat adipocytes

Abstract

Lipolysis and lipogenesis are two highly regulated pathways. Catecholamines promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. H2O2 mimics insulin action inhibiting lipolysis, and insulin stimulates NADPH oxidase in adipocytes and transiently generates H2O2. Therefore, the possibility that H2O2 might regulate cAMP‐dependent protein kinase (PKA) was explored.Results show that H2O2 generated by insulin in rat adipocytes impaired cAMP‐mediated amplification cascade of lipolysis. Micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of both type IIß or type IIα cyclic AMP‐dependent protein kinase (PKA) holoenzyme. However, H2O2 was ineffective on reconstituted PKA holoenzyme preincubated with cAMP, or if added to the catalytic αsubunit alone. The same results were obtained if the catalytic αsubunit was substituted by its C199A mutant. H2O2 inhibition of PKA activation remained after H2O2 elimination but was reverted with thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing PKA activation. Phylogenetic analysis showed that Cys‐97 is conserved only in type II (but not type I) regulatory subunits. Supported by DGAPA‐UNAM grant IN208308