AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.

Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.

Abstract-Ischemic preconditioning confers powerful protection against myocardial infarction through pre-emptive activation of survival signaling pathways, but it remains difficult to apply to patients with ischemic heart disease, and its effects are transient. Promoting a sustained activation of preconditioning mechanisms in vivo would represent a novel approach of cardioprotection. We tested the role of the protein H11 kinase (H11K), which accumulates by 4-to 6-fold in myocardium of patients with chronic ischemic heart disease and in experimental models of ischemia. This increased expression was quantitatively reproduced in cardiac myocytes using a transgenic (TG) mouse model. After 45 minutes of coronary artery occlusion and reperfusion, hearts from TG mice showed an 82Ϯ5% reduction in infarct size compared with wild-type (WT), which was similar to the 84Ϯ4% reduction of infarct size observed in WT after a protocol of ischemic preconditioning. Hearts from TG mice showed significant activation of survival kinases participating in preconditioning, including Akt and the 5ЈAMP-activated protein kinase (AMPK). H11K directly binds to both Akt and AMPK and promotes their nuclear translocation and their association in a multiprotein complex, which results in a stimulation of survival mechanisms in cytosol and nucleus, including inhibition of proapoptotic effectors (glycogen synthase kinase-3␤, Bad, and Foxo), activation of antiapoptotic effectors (protein kinase C⑀, endothelial and inducible NO synthase isoforms, and heat shock protein 70), increased expression of the hypoxia-inducible factor-1␣, and genomic switch to glucose utilization. Therefore, activation of survival pathways by H11K preemptively triggers the antiapoptotic and metabolic response to ischemia and is sufficient to confer cardioprotection in vivo equally potent to preconditioning. (Circ Res. 2006;98:280-288.)

Role of the ␣ 2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia. Am J Physiol Heart Circ Physiol 291: H2875-H2883, 2006. First published July 28, 2006 doi:10.1152/ajpheart.01032.2005.-AMP-activated protein kinase (AMPK) is a major sensor and regulator of the energetic state of the cell. Little is known about the specific role of AMPK␣2, the major AMPK isoform in the heart, in response to global ischemia. We used AMPK␣2-knockout (AMPK␣2 Ϫ/Ϫ ) mice to evaluate the consequences of AMPK␣ 2 deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPK␣ 2 Ϫ/Ϫ mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPK␣ 2 Ϫ/Ϫ mice to no-flow ischemia was characterized by a more rapid onset of ischemia-induced contracture. This ischemic contracture was associated with a decrease in ATP content, lactate production, glycogen content, and AMPK␤ 2 content. Hearts from AMPK␣2 Ϫ/Ϫ mice were also characterized by a decreased phosphorylation state of acetyl-CoA carboxylase during normoxia and ischemia. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPK␣ 2 does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPK␣ 2 is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPK␣ 1 cannot compensate for the absence of AMPK␣ 2.glycogen; glycolysis; acetyl-CoA carboxylase ADENOSINE 5Ј-MONOPHOSPHATE-ACTIVATED protein kinase (AMPK), a ubiquitous serine/threonine protein kinase, senses the energy state of the cell. AMPK is a heterotrimeric enzyme consisting of a catalytic (␣) and two regulatory (␤ and ␥) subunits. Different isoforms for each of these subunits have been identified (43). In the heart, the catalytic ␣ 2 -subunit is predominant and represents 60 -70% of total AMPK activity; the remaining activity is due to the ␣ 1 -subunit (9). Control of AMPK activity is complex and involves allosteric stimulation by AMP as well as AMP-dependent phosphorylation at Thr 172 , a residue located in the activation loop of the ␣-subunit (15). Several protein kinases responsible for this phosphorylation have been identified: LKB1 (33, 39, 50) and the Ca 2ϩ /calmoldulin-dependent protein kinase kinase (16,24,49). The rapid rise in AMP concentration during an ischemic episode explains the activation of cardiac AMPK under this pathological condition (21, 22). Moreover, AMPK can also be activated by antidiabetic drugs (12, 55), osmotic stress (11), leptin (35), and adiponectin (53), probably via AMP-independent pathways. In hearts subjected to ischemia-reperfusion, AMPK can be considered a metabolic master switch (21,42,54). Indeed, once activated, AMPK phosphorylates several downstream targets, switching on ATP-generating pathways and switching off ATP-consuming biosynthetic pathways, thereby moderating the negative effects of ischemia-r...

Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.

Hyperglycemia (HG) stimulates the production of reactive oxygen species in the heart through activation of NADPH oxidase 2 (NOX2). This production is independent of glucose metabolism but requires sodium/glucose cotransporters (SGLT). Seven SGLT isoforms (SGLT1 to 6 and sodium-myoinositol cotransporter-1, SMIT1) are known, although their expression and function in the heart remain elusive. We investigated these 7 isoforms and found that only SGLT1 and SMIT1 were expressed in mouse, rat and human hearts. In cardiomyocytes, galactose (transported through SGLT1) did not activate NOX2. Accordingly, SGLT1 deficiency did not prevent HG-induced NOX2 activation, ruling it out in the cellular response to HG. In contrast, myo-inositol (transported through SMIT1) reproduced the toxic effects of HG. SMIT1 overexpression exacerbated glucotoxicity and sensitized cardiomyocytes to HG, whereas its deletion prevented HG-induced NOX2 activation. In conclusion, our results show that heart SMIT1 senses HG and triggers NOX2 activation. This could participate in the redox signaling in hyperglycemic heart and contribute to the pathophysiology of diabetic cardiomyopathy.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70S6K activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70S6K activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70S6K activity induced by insulin and leucine correlated with changes in phosphorylation of Thr389, the mTOR phosphorylation site on p70S6K, and of Ser2448 on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70S6K, leading to the absence of p70S6K activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70S6K, suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70S6K. We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70S6K pathway requires PDK1 in a way that differs from that of insulin.