Knockout of the alpha2 but not alpha1 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle

We investigated the importance of the two catalytic alpha-isoforms of the 5'-AMP-activated protein kinase (AMPK) in 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) and contraction-induced glucose uptake in skeletal muscle. Incubated soleus and EDL muscle from whole-body alpha2- or alpha1-AMPK knockout (KO) and wild type (WT) mice were incubated with 2.0 mm AICAR or electrically stimulated to contraction. Both AICAR and contraction increased 2DG uptake in WT muscles. KO of alpha2, but not alpha1, abolished AICAR-induced glucose uptake, whereas neither KO affected contraction-induced glucose uptake. AICAR and contraction increased alpha2- and alpha1-AMPK activity in wild type (WT) muscles. During AICAR stimulation, the remaining AMPK activity in KO muscles increased to the same level as in WT. During contraction, the remaining AMPK activity in alpha2-KO muscles was elevated by 100% probably explained by a 2-3-fold increase in alpha1-protein. In alpha1-KO muscles, alpha2-AMPK activity increased to similar levels as in WT. Both interventions increased total AMPK activity, as expressed by AMPK-P and ACCbeta-P, in WT muscles. During AICAR stimulation, this was dramatically reduced in alpha2-KO but not in alpha1-KO, whereas during contraction, both measurements were essentially similar to WT in both KO-muscles. The results show that alpha2-AMPK is the main donor of basal and AICAR-stimulated AMPK activity and is responsible for AICAR-induced glucose uptake. In contrast, during contraction, the two alpha-isoforms seem to substitute for each other in terms of activity, which may explain the normal glucose uptake despite the lack of either alpha2- or alpha1-AMPK. Alternatively, neither alpha-isoform of AMPK is involved in contraction-induced muscle glucose uptake.     

Role of the alpha2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia

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 AMPKalpha(2), the major AMPK isoform in the heart, in response to global ischemia. We used AMPKalpha(2)-knockout (AMPKalpha(2)(-/-)) mice to evaluate the consequences of AMPKalpha(2) deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPKalpha(2)(-/-) mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPKalpha(2)(-/-) 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 AMPKbeta(2) content. Hearts from AMPKalpha(2)(-/-) 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 AMPKalpha(2) does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPKalpha(2) is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPKalpha(1) cannot compensate for the absence of AMPKalpha(2).     

Alpha2-AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise

A single bout of exercise increases glucose uptake and fatty acid oxidation in skeletal muscle, with a corresponding activation of AMP-activated protein kinase (AMPK). While the exercise-induced increase in glucose uptake is partly due to activation of AMPK, it is unclear whether the increase of fatty acid oxidation is dependent on activation of AMPK. To examine this, transgenic mice were produced expressing a dominant-negative (DN) mutant of alpha(1)-AMPK (alpha(1)-AMPK-DN) in skeletal muscle and subjected to treadmill running. alpha(1)-AMPK-DN mice exhibited a 50% reduction in alpha(1)-AMPK activity and almost complete loss of alpha(2)-AMPK activity in skeletal muscle compared with wild-type littermates (WT). The fasting-induced decrease in respiratory quotient (RQ) ratio and reduced body weight were similar in both groups. In contrast with WT mice, alpha(1)-AMPK-DN mice could not perform high-intensity (30 m/min) treadmill exercise, although their response to low-intensity (10 m/min) treadmill exercise was not compromised. Changes in oxygen consumption and the RQ ratio during sedentary and low-intensity exercise were not different between alpha(1)-AMPK-DN and WT. Importantly, at low-intensity exercise, increased fatty acid oxidation in response to exercise in soleus (type I, slow twitch muscle) or extensor digitorum longus muscle (type II, fast twitch muscle) was not impaired in alpha(1)-AMPK-DN mice, indicating that alpha(1)-AMPK-DN mice utilize fatty acid in the same manner as WT mice during low-intensity exercise. These findings suggest that an increased alpha(2)-AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise.     

Caffeine-induced Ca(2+) release increases AMPK-dependent glucose uptake in rodent soleus muscle

Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.     

Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that plays a pivotal role in regulating cellular metabolism for sustaining energy homeostasis under stress conditions. Activation of AMPK has been observed in the heart during acute and chronic stresses, but its functional role has not been completely understood because of the lack of effective activators and inhibitors of this kinase in the heart. We generated transgenic mice (TG) with cardiac-specific overexpression of a dominant negative mutant of the AMPK alpha2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia. In isolated perfused hearts subjected to a 10-min ischemia, AMPK alpha2 activity in wild type (WT) increased substantially (by 4.5-fold), whereas AMPK alpha2 activity in TG was similar to the level of WT at base line. Basal AMPK alpha1 activity was unchanged in TG and increased normally during ischemia. Ischemia stimulated a 2.5-fold increase in 2-deoxyglucose uptake over base line in WT, whereas the inactivation of AMPK alpha2 in TG significantly blunted this response. Using 31P NMR spectroscopy, we found that ATP depletion was accelerated in TG hearts during no-flow ischemia, and these hearts developed left ventricular dysfunction manifested by an early and more rapid increase in left ventricular end-diastolic pressure. The exacerbated ATP depletion could not be attributed to impaired glycolytic ATP synthesis because TG hearts consumed slightly more glycogen during this period of no-flow ischemia. Thus, AMPK alpha2 is necessary for maintaining myocardial energy homeostasis during ischemia. It is likely that the functional role of AMPK in myocardial energy metabolism resides both in energy supply and utilization.     

Interleukin-6 release from human skeletal muscle during exercise: relation to AMPK activity.

We tested the hypothesis that IL-6 release from muscle during exercise may be related to muscle activity of 5'-AMP-activated protein kinase (AMPK). Eight healthy, well-trained young men completed two 60-min trials on a bicycle ergometer at 70% of their peak oxygen uptake in either a glycogen-depleted or a glycogen-loaded state. IL-6 was released from the leg already after 10 min of exercise in the glycogen-depleted state, whereas no significant release was observed at any time in the loaded state. Nevertheless, plasma IL-6 increased similarly in the two trials from approximately 0.8 pg/ml at rest to approximately 4.5 pg/ml after 60 min of exercise. Activity of alpha1-AMPK (160%) and alpha2-AMPK (145%) was increased at rest in the glycogen-depleted compared with the loaded situation. During exercise, alpha1-AMPK activity did not change from resting levels in both trials, whereas alpha2-AMPK activity increased only in the glycogen-depleted state. After 60 min of exercise in the glycogen-depleted state, individual values of alpha2-AMPK activity correlated significantly (r = 0.87, P < 0.006) with individual values of IL-6 release as well as with average IL-6 release over the entire 60 min (r = 0.86, P < 0.006). The present data are compatible with a role for AMPK in IL-6 release during exercise or a role for IL-6 in activating AMPK. Alternatively, both AMPK and IL-6 are independent sensors of a low muscle glycogen concentration during exercise. In addition, leg release of IL-6 cannot alone explain the increase in plasma IL-6 during exercise.     

Restricted feeding improves postischemic recovery of Langendorff-perfused rat hearts

The goal of the present study was to assess the effects of a restricted feeding schedule (RFS) on postischemic contractile recovery in relation to triacylglycerol (TAG), glycogen, and ATP content. Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced/oxidized glutathione ratio (GSH/GSSG), and thiobarbituric acid reactive substances (TBARS) levels were also determined. Isolated rat hearts entrained to daily RFS (2 h food access starting at 1200) or fed ad libitum (FED) for 3 weeks were Langendorff-perfused (25 min ischemia, 30 min reperfusion) with Krebs-Ringer bicarbonate solution (10?mmol/L glucose). RFS improved the recovery of contractility and reduced creatine kinase (CK) release upon reperfusion. Further, at the end of reperfusion, RFS hearts exhibited increased G6PDH activity and repletion of tissue glycogen, TAG, and ATP that was not observed in the FED hearts. GSH/GSSG at the end of reperfusion fell to the same value in both nutritional states, and TBARS levels were higher in the RFS hearts. In conclusion, RFS improved postischemic functional recovery, which was accompanied by a reduction in CK release and a striking energy recovery. Although enhanced G6PDH activity was displayed, RFS was unable to reduce lipid peroxidation, supporting a clear dissociation between protection against mechanical dysfunction and CK release on the one hand and oxidative damage on the other.     

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.     

C1q/Tumor Necrosis Factor-Related Protein 9 Protects against Acute Myocardial Injury through an Adiponectin Receptor I-AMPK-Dependent Mechanism

Obesity is a risk factor for cardiovascular disease. C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine that is downregulated by obesity. We investigated the role of CTRP9 in cardiac injury with loss-of-function genetic manipulations and defined the receptor-mediated signaling pathway downstream of this adipokine. CTRP9-knockout (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from wild-type (WT) mice under basal conditions. CTRP9-KO mice had exacerbated contractile left ventricle dysfunction following intraperitoneal injection of lipopolysaccharide (LPS) compared to WT mice. Administration of LPS to CTRP9-KO mice also resulted in increased expression of proinflammatory cytokines and oxidative stress markers in the heart compared to WT mice. Likewise, CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory mediators in ischemic heart following ischemia and reperfusion compared to WT mice. Treatment of cardiac myocytes with CTRP9 protein led to suppression of LPS-induced expression of proinflammatory genes, which was reversed by blockade of AMPK or ablation of adiponectin receptor I (AdipoR1). Systemic delivery of CTRP9 attenuated LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK or in AdipoR1-knockout mice. CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AdipoR1/AMPK-dependent mechanisms.     

Ischemia-induced activation of AMPK does not increase glucose uptake in glycogen-replete isolated working rat hearts

Alterations in myocardial glucose metabolism are a key determinant of ischemia-induced depression of left ventricular mechanical function. Since myocardial glycogen is an important source of endogenous glucose, we compared the effects of ischemia on glucose uptake and utilization in isolated working rat hearts in which glycogen content was either replete (G replete, 114 micromol/g dry wt) or partially depleted (G depleted, 71 mumol/g dry wt). The effects of low-flow ischemia (LFI, 0.5 ml/min) on glucose uptake, glycogen turnover (glycogenolysis and glycogen synthesis), glycolysis, adenosine 5'-monophosphate-activated protein kinase (AMPK) activity, and GLUT4 translocation were measured. Relative to preischemic values, LFI caused a time-dependent reduction in glycogen content in both G-replete and G-depleted groups due to an acceleration of glycogenolysis (by 12-fold and 6-fold, respectively). In G-replete hearts, LFI (15 min) decreased glucose uptake (by 59%) and did not affect GLUT4 translocation. In G-depleted hearts, LFI also decreased initially glucose uptake (by 90%) and glycogen synthesis, but after 15 min, when glycogenolysis slowed due to exhaustion of glycogen content, glucose uptake increased (by 31%) in association with an increase in GLUT4 translocation. After 60 min of LFI, glucose uptake, glycogenolysis, and glycolysis recovered to near-preischemic values in both groups. LFI increased AMPK activity in a time-dependent manner in both groups (by 6-fold and 4-fold, respectively). Thus, when glycogen stores are replete before ischemia, ischemia-induced AMPK activation is not sufficient to increase glucose uptake. Under these conditions, an acceleration of glycogen degradation provides sufficient endogenous substrate for glycolysis during ischemia.     

Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle.

Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 muM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased alpha(2)-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and alpha(2)-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced alpha(2)-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced alpha(2)-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca(2+)-independent activation of ERK1/2 as well as Ca(2+)-dependent activation of CaMKII and AMPK.     

Role of AMPKalpha2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle

We investigated the role of AMPKalpha2in basal, exercise training-, and AICAR-induced protein expression of GLUT4, hexokinase II (HKII), mitochondrial markers, and AMPK subunits. This was conducted in red (RG) and white gastrocnemius (WG) muscle from wild-type (WT) and alpha2-knockout (KO) mice after 28 days of activity wheel running or daily AICAR injection. Additional experiments were conducted to measure acute activation of AMPK by exercise and AICAR. At basal, mitochondrial markers were reduced by approximately 20% in alpha2-KO muscles compared with WT. In both muscle types, AMPKalpha2 activity was increased in response to both stimuli, whereas AMPKalpha1 activity was increased only in response to exercise. Furthermore, AMPK signaling was estimated to be 60-70% lower in alpha2-KO compared with WT muscles. In WG, AICAR treatment increased HKII, GLUT4, cytochrome c, COX-1, and CS, and the alpha2-KO abolished the AICAR-induced increases, whereas no AICAR responses were observed in RG. Exercise training increased GLUT4, HKII, COX-1, CS, and HAD protein in WG, but the alpha2-KO did not affect training-induced increases. Furthermore, AMPKalpha1, -alpha2, -beta1, -beta2, and -gamma3 subunits were reduced in RG, but not in WG, by 30-60% in response to exercise training. In conclusion, the alpha2-KO was associated with an approximately 20% reduction in mitochondrial markers in both muscle types and abolished AICAR-induced increases in protein expression in WG. However, the alpha2-KO did not reduce training-induced increases in HKII, GLUT4, COX-1, HAD, or CS protein in WG, suggesting that AMPKalpha2 may not be essential for metabolic adaptations of skeletal muscles to exercise training.     

mu-Opiate receptor activation and increase in heart resistance to ischemic and reperfusion injury

Stimulation of mu-opioid receptors was found to contribute to prevention of myocardial contractile dysfunction and ventricular arrhythmias in ischemia and reperfusion of the rat isolated heart. Endogenous agonists of the mu-opioid receptors were not involved in tonic regulation of the heart resistance against reperfusion disturbances of the rhythm and contractility. On the other hand, mu-opioid receptors are important for development of postischemic contracture.     

Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca²⁺ handling), ultrastructural disarray, apoptosis, O₂⁻, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser⁷⁵⁷ and Ser⁷⁷⁷), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.     

p38 mitogen-activated protein kinase mediates adenosine-induced alterations in myocardial glucose utilization via 5'-AMP-activated protein kinase

Adenosine-induced acceleration of glycolysis in hearts stressed by transient ischemia is accompanied by suppression of glycogen synthesis and by increases in activity of adenosine 5'-monophosphate-activated protein kinase (AMPK). Because p38 mitogen-activated protein kinase (MAPK) may regulate glucose metabolism and may be activated downstream of AMPK, this study determined the effects of the p38 MAPK inhibitors SB202190 and SB203580 on adenosine-induced alterations in glucose utilization and AMPK activity. Studies were performed in working rat hearts perfused aerobically following stressing by transient ischemia (2 x 10-min ischemia followed by 5-min reperfusion). Phosphorylation of AMPK and p38 MAPK each were increased fourfold by adenosine, and these effects were inhibited by either SB202190 or SB203580. Neither of these inhibitors directly affected AMPK activity. Attenuation of the adenosine-induced increase in AMPK and p38 MAPK phosphorylation by SB202190 and SB203580 occurred independently of any change in tissue ATP-to-AMP ratio and did not alter glucose uptake, but it was accompanied by an increase in glycogen synthesis and glycogen content and by inhibition of glycolysis and proton production. There was a significant inverse correlation between the rate of glycogen synthesis and AMPK activity and between AMPK activity and glycogen content. These data demonstrate that AMPK is likely downstream of p38 MAPK in mediating the effects of adenosine on glucose utilization in hearts stressed by transient ischemia. The ability of p38 MAPK inhibitors to relieve the inhibition of glycogen synthesis and to inhibit glycolysis and proton production suggests that these agents may restore adenosine-induced cardioprotection in stressed hearts.     

Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I

Aims

The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes.

Main methods

Mechanical properties and intracellular Ca^2 + properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis.

Key findings

AMPK activator, A-769662 induced maximal velocity of shortening (+ dL/dt) and relengthening (− dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca^2 + levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser¹⁴⁹ site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662.

Significance

This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.     

Metabolic activation of AMP kinase in vascular smooth muscle.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.     

Effects of adenosine on myocardial glucose and palmitate metabolism after transient ischemia: role of 5'-AMP-activated protein kinase

Loss of cardioprotection by adenosine in hearts stressed by transient ischemia may be due to its effects on glucose metabolism. In the absence of transient ischemia, adenosine inhibits glycolysis, whereas it accelerates glycolysis after transient ischemia. Inasmuch as 5'-AMP-activated protein kinase (AMPK) is implicated as a regulator of glucose and fatty acid utilization, this study determined whether a differential alteration of AMPK activity contributes to acceleration of glycolysis by adenosine in hearts stressed by transient ischemia. Studies were performed in working rat hearts perfused aerobically under normal conditions or after transient ischemia (two 10-min periods of ischemia followed by 5 min of reperfusion). LV work was not affected by adenosine. AMPK phosphorylation was not affected by transient ischemia; however, phosphorylation and activity were increased nine- and threefold, respectively, by adenosine in stressed hearts. Phosphorylation of acetyl-CoA carboxylase and rates of palmitate oxidation were unaltered. Glycolysis and calculated proton production were increased 1.8- and 1.7-fold, respectively, in hearts with elevated AMPK activity. Elevated AMPK activity was associated with inhibition of glycogen synthesis and unchanged rates of glucose uptake and glycogenolysis. Phentolamine, an alpha-adrenoceptor antagonist, which prevents adenosine-induced activation of glycolysis in stressed hearts, prevented AMPK phosphorylation. These data demonstrate that adenosine-induced activation of AMPK after transient ischemia is not sufficient to alter palmitate oxidation or glucose uptake. Rather, activation of AMPK alters partitioning of glucose away from glycogen synthesis; the increase in glycolysis may in part contribute to loss of adenosine-induced cardioprotection in hearts subjected to transient ischemia.     

Double knockout of Akt2 and AMPK predisposes cardiac aging without affecting lifespan: Role of autophagy and mitophagy

Increased age often leads to a gradual deterioration in cardiac geometry and contractile function although the precise mechanism remains elusive. Both Akt and AMPK play an essential role in the maintenance of cardiac homeostasis. This study examined the impact of ablation of Akt2 (the main cardiac isoform of Akt) and AMPKα2 on development of cardiac aging and the potential mechanisms involved with a focus on autophagy. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated in young (4-month-old) and old (12-month-old) wild-type (WT) and Akt2-AMPK double knockout mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy were evaluated using western blot. Our results revealed that increased age (12 months) did not elicit any notable effects on cardiac geometry, contractile function, morphology, ultrastructure, autophagy and mitophagy, although Akt2-AMPK double knockout predisposed aging-related unfavorable changes in geometry (heart weight, LVESD, LVEDD, cross-sectional area and interstitial fibrosis), TEM ultrastructure, and function (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, intracellular Ca²⁺ release and clearance rate). Double knockout of Akt2 and AMPK unmasked age-induced cardiac autophagy loss including decreased Atg5, Atg7, Beclin1, LC3BII-to-LC3BI ratio and increased p62. Double knockout of Akt2 and AMPK also unmasked age-related loss in mitophagy markers PTEN-induced putative kinase 1 (Pink1), Parkin, Bnip3, and FundC1, the mitochondrial biogenesis cofactor PGC-1α, and lysosomal biogenesis factor TFEB. In conclusion, our data indicate that Akt2-AMPK double ablation predisposes cardiac aging possibly related to compromised autophagy and mitophagy. This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.