Aims/hypothesis Muscle may experience hypoglycaemia during ischaemia or insulin infusion. During severe hypoglycaemia energy production is blocked, and an increase of AMP:ATP activates the energy sensor and putative insulinsensitiser AMP-activated protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defence pathway involving AMPK-dependent, insulin-independent activation of the insulin signalling pathway. Methods Cardiac or skeletal myocytes were subjected to glucose and insulin-free incubation for increasing intervals up to 20 h. AMPK, and components of the insulin signalling pathway and their targets were quantified by western blot using phosphor-specific antibodies. Phosphomimetics were used to determine the function of IRS-1 Ser789 phosphorylation and in vitro [ 32 P]ATP kinase assays were used to measure the phosphorylation of the purified insulin receptor by AMPK. Results Glucose deprivation increased Akt-Thr308 and Akt-Ser473 phosphorylation by almost tenfold. Phosphorylation of glycogen synthase kinase 3 beta increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase and mammalian target of rapamycin decreased. AMPK inhibitors blocked and aminoimidazole carboxamide ribonucleotide (AICAR) mimicked the effects of glucose starvation. Glucose deprivation increased the phosphorylation of IRS-1 on serine-789, but phosphomimetics revealed that this conferred negative regulation. Glucose deprivation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Conclusions/interpretation AMPK phosphorylates and activates the insulin receptor, providing a direct link between AMPK and the insulin signalling pathway; this pathway promotes energy conservation and survival of muscle exposed to severe glucose deprivation.

Brief periods of ischemia do not damage the heart and can actually protect against reperfusion injury caused by extended ischemia. It is not known what causes the transition from protection to irreversible damage as ischemia progresses. c-Jun N-terminal kinase-1 (JNK-1) is a stress-regulated kinase that is activated by reactive oxygen and thought to promote injury during severe acute myocardial infarction. Because some reports suggest that JNK-1 can also be protective, we hypothesized that the function of JNK-1 depends on the metabolic state of the heart at the time of reperfusion, a condition that changes progressively with duration of ischemia. Mice treated with JNK-1 inhibitors or transgenic mice wherein the JNK-1 gene was ablated were subjected to 5 or 20 min of ischemia followed by reperfusion. When JNK-1 was inactive, ischemia of only 5 min duration caused massive apoptosis, infarction, and negative remodeling that was equivalent to or greater than extended ischemia. Conversely, when ischemia was extended JNK-1 inactivation was protective. Mechanisms of the JNK-1 switch in function were investigated in vivo and in cultured cardiac myocytes. In vitro there was a comparable switch in the function of JNK-1 from protective when ATP levels were maintained during hypoxia to injurious when reoxygenation followed glucose and ATP depletion. Both apoptotic and necrotic death pathways were affected and responded reciprocally to JNK-1 inhibitors. JNK-1 differentially regulated Akt phosphorylation of the regulatory sites Ser-473 and Thr-450 and the catalytic Thr-308 site in vivo. The studies define a novel role for JNK-1 as a conditional survival kinase that protects the heart against brief but not protracted ischemia.There are more than 1,000,000 cases of acute myocardial infarction (AMI) 3 in the United States each year and many progress to heart failure (1, 2). Timely reperfusion by angioplasty, coronary artery bypass grafting, or thrombolytic therapy is the most effective treatment for limiting the extent of infarction and improving clinical outcome. However, reperfusion itself after protracted ischemia is known to cause additional irreversible injury (2). Myocardial infarction involves overlapping contributions of programmed death (apoptosis) and necrosis (3-5). During reperfusion, mitochondria are rapidly re-energized, and intracellular levels of reactive oxygen species (ROS) and calcium increase favoring an open state of mitochondrial death channels (3-8). Reperfusion also activates the so-called death and survival kinases that contribute importantly to cell fate and degree of infarction (reviewed in Refs. 1, 9 -11). When the heart is reperfused after a short period of ischemia, irreversible injury is avoided, and the consequences of ischemia reperfusion are limited to reversible defects of contractility known as myocardial stunning. Stunning is probably caused by elevations of reactive oxygen species (ROS) and calcium that are generated during reperfusion. Stunning varies in intensity, and although it usually r...

RationaleThe adult myocardium has been reported to harbor several classes of multipotent progenitor cells (CPCs) with tri-lineage differentiation potential. It is not clear whether c-kit+CPCs represent a uniform precursor population or a more complex mixture of cell types.ObjectiveTo characterize and understand vasculogenic heterogeneity within c-kit+presumptive cardiac progenitor cell populations.Methods and Resultsc-kit+, sca-1+ CPCs obtained from adult mouse left ventricle expressed stem cell-associated genes, including Oct-4 and Myc, and were self-renewing, pluripotent and clonogenic. Detailed single cell clonal analysis of 17 clones revealed that most (14/17) exhibited trilineage differentiation potential. However, striking morphological differences were observed among clones that were heritable and stable in long-term culture. 3 major groups were identified: round (7/17), flat or spindle-shaped (5/17) and stellate (5/17). Stellate morphology was predictive of vasculogenic differentiation in Matrigel. Genome-wide expression studies and bioinformatic analysis revealed clonally stable, heritable differences in stromal cell-derived factor-1 (SDF-1) expression that correlated strongly with stellate morphology and vasculogenic capacity. Endogenous SDF-1 production contributed directly to vasculogenic differentiation: both shRNA-mediated knockdown of SDF-1 and AMD3100, an antagonist of the SDF-1 receptor CXC chemokine Receptor-4 (CXCR4), reduced tube-forming capacity, while exogenous SDF-1 induced tube formation by 2 non-vasculogenic clones. CPCs producing SDF-1 were able to vascularize Matrigel dermal implants in vivo, while CPCs with low SDF-1 production were not.ConclusionsClonogenic c-kit+, sca-1+ CPCs are heterogeneous in morphology, gene expression patterns and differentiation potential. Clone-specific levels of SDF-1 expression both predict and promote development of a vasculogenic phenotype via a previously unreported autocrine mechanism.

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).