Abstract 18367: G-protein-coupled Receptor Kinase-2 induces Insulin Resistance In Cardiac Myocytes
We have previously shown that GRK2 overexpression in cardiomyocytes produces an attenuation of insulin's biological effects. The aim of the present study was to evaluate the mechanisms by which GRK2 may impart insulin resistance in myocytes. Cultured adult rat ventricular myocytes (ARVMs) were infected with an adenovirus encoding for GRK2 (ADGRK2) or with a GRK2 kinase-dead dominant-negative mutant (ADGRK2-DN) and stimulated with insulin (Ins). Ins induced phosphorylation of Akt is reduced by ADGRK2 with respect to ADGRK2-DN treated cells (1.5±0.05 and 3±0.10 fold over basal, p< 0.05). Therefore, we pursued the mechanism of how the kinase activity of GRK2 may inhibit Ins signaling. Since Ins receptor activation promotes a feedback loop that occurs through Ser phosphorylation of its substrate, IRS1, we evaluated whether GRK2 activity participates in this negative loop. In ARVMs, ADGRK2 significantly increases Ser307 phosphorylation of IRS1 under basal condition as well after Ins stimulation compared to ADGFP treated myocytes (0.7±0.01 vs 0.2±0.01, p<0.05). To further study this novel role of GRK2 in Ins signaling, we have studied cardiac-specific GRK2 knockout (KO) and βARKct overexpressing (Tg-βARKct) mice. βARKct is a peptide inhibitor of GRK2 activity. Reduced levels of GRK2 leads to a significant attenuation of insulin-induced phosphorylation of IRS1 at Ser307 (0.05±0.005 vs 0.1±0.01, GRK2KO vs NLC, p<0.05), as well as GK2 inhibition by βARKct (0.34±0.01 vs 0.67±0.05, Tg-βARKct vs NLC, p<0.01). Reciprocally, GRK2 overexpression in the heart leads to increased IRS1 phosphorylation in vivo (0.18±0.02 vs 0.05±0.005, Tg-GRK2 vs NLC, p<0.05). Further, an in vitro kinase assay shows that purified GRK2 significantly increases basal phosphorylation of IRS1 in Ser307. Increased Ser307 phosphorylation of IRS1 is also accompanied by reduced phospho-Tyr612. In fact, we have found novel data on this site using GRK2 and the GRK2-DN mutant in ARVMs (data not shown). In conclusion, GRK2 activity regulates Ins signaling in cardiomyocytes through increased phosphorylation at the inhibitory site of IRS1 (ser307) and offers novel insight into how increased GRK2 can impart cardiac dysfunction through a negative impact on cardiac metabolic signaling.
- © 2010 by American Heart Association, Inc.