Abstract 3783: An Insulin-independent AMPK-dependent Survival Pathway Is Activated In Glucose-starved Cardiac Myocytes through IRS-1/PI3-kinase and Rictor-mediated Activation of PDK1 and PDK2
INTRODUCTION: Akt (PKB) is the downstream effector of insulin and IGF-1 and regulates multiple targets that control cell survival and growth. Akt is phosphorylated at Thr-308 (activating) and Ser-473 (regulatory) sites by PI3-kinase dependent kinase-1 (PDK1) and TOR2-rictor/PDK2 respectively. When energy is abundant Akt supports cell growth by stimulating TORC1 and inhibiting GSK3β. When energy and insulin are low such as during ischemia, AMPK is activated and depresses TORC1 by activating the repressive TSC1/2 complex. It is not known how AMPK affects TORC2 in this setting.
HYPOTHESIS: Under low energy states AMPK promotes survival by differentially regulating TORC1 and 2 thereby maintaining Akt phosphorylation and survival in the absence of insulin.
METHODS: Cardiac myocytes (CM) were subjected to glucose and insulin-free incubation for 4h and the activities of insulin signaling components were measured by western blot and co-IP. Survival was measured ± siRNAs by Hoechst/PI staining.
RESULTS: After 4h of glucose/insulin deprivation there was increased phosphorylation of Akt-Thr-308 (11.2±2.4; n=6, p<0.001) and Akt-Ser-473 (9.7± 2; n=6, p<0.001). Phosphorylation of GSK-3β (Ser-9) also increased but there was decreased phosphorylation of p70S6-kinase (Thr389) and 4EBP (Ser65) indicating global down-regulation of TORC1. We identified 2 separate pathways for the insulin-independent phosphorylation of Akt at both sites in glucose-depleted CM. Activated AMPK promoted phosphorylation of insulin receptor substrate-1 (IRS-1) on Ser-789. AraA or a Ser-789 decoy peptide blocked this, and IRS-1-P-Ser-789 co-IP’d with PI3-kinase suggesting positive regulation of PDK1. Glucose depletion did not change the levels of rictor or raptor or their relative binding to TOR. However TSC1 and rictor were quantitatively associated with PI3-kinase selectively under glucose/insulin depletion and this correlated with Akt phosphorylation. CM survival under glucose/insulin depletion was impaired by siRNA-mediated knockdown of IRS-1 or rictor (n=3; P<0.05).
CONCLUSIONS: AMPK maintains phosphorylation of Akt under low energy states in the absence of insulin. The mechanism involves IRS-1 phosphorylation by AMPK and enhanced binding of rictor and TSC1 to PI3K.
This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).