Compound C, an inhibitor of AMP-activated protein kinase, inhibits glycolysis in mouse longissimus dorsi postmortem

Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKα1 and α2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the β-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKα1 and α2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKα1 and α2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis.

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Abstract 337: Activation of Myocardial AMP-activated Protein Kinase by Metformin Prevents Progression of Heart Failure in Dogs; Involvement of eNOS Activation

Circulation ◽

10.1161/circ.118.suppl_18.s_286-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Hideyuki Sasaki ◽

Hiroshi Asanuma ◽

Masashi Fujita ◽

Hiroyuki Takahama ◽

Masanori Asakura ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Heart Failure ◽

Cell Death ◽

Protein Kinase ◽

Left Ventricular ◽

Vehicle Group ◽

Mrna Levels ◽

Compound C ◽

Amp Activated Protein Kinase

Background; Several studies have shown that metformin activates AMP-activated protein kinase (AMPK), which mediates potent cardioprotection against ischemia-reperfusion injury. AMPK is also activated in experimental failing myocardium, suggesting that activation of AMPK is beneficial for the pathophysiology of heart failure. We investigated whether metformin prevents oxidative stress-induced cell death in rat cardiomyocytes and attenuates the progression of heart failure in dogs. Methods and Results; The treatment with metformin (10 μmol/L) protected the rat cultured cardiomyocytes against cell death due to H 2 O 2 exposure (50 μmol/L) as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TUNEL staining, and flow cytometry. These effects were blunted by an AMPK inhibitor, compound-C (20 μmol/L), suggesting that the activation of AMPK decreased the extent of apoptosis-induced cell death due to H 2 O 2 exposure. Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused heart failure and the treatment with metformin (100 mg/kg/day PO, n=8) decreased left ventricular (LV) end-diastolic dimension (32.8±0.4 vs. 36.5±1.0 mm, p< 0.01) and pressure (11.8±1.1 vs. 22±0.9 mmHg, p< 0.01), and increased LV fractional shortening (18.6±1.8 vs. 9.6±0.7 %, p< 0.01) along with enhanced phosphorylation of AMPK and the decreased the number of TUNEL-positive cells of the LV myocardium compared with the vehicle group (n=8). Interestingly, metformin increased the protein and mRNA levels of endothelial nitric oxide synthase of the LV myocardium and plasma nitric oxide levels. Metformin improved the plasma insulin resistance without increased myocardial GLUT-4 translocation. Furthermore, the subcutaneous administration of AICAR (50 mg/kg/every other day), another AMPK activator mediated the equivalent effects to metformin, strengthening the pivotal role of AMPK in reduction of apoptosis and prevention of heart failure. Conclusions; Activation of myocardial AMPK attenuated the oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with eNOS activation. Thus, metformin or AICAR may be applicable as a novel therapy for heart failure.

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Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

Journal of Molecular Endocrinology ◽

10.1530/jme-11-0165 ◽

2012 ◽

Vol 49 (1) ◽

pp. 47-55 ◽

Cited By ~ 5

Author(s):

H Tokuda ◽

K Kato ◽

H Natsume ◽

A Kondo ◽

G Kuroyanagi ◽

...

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Interleukin 6 ◽

P38 Map Kinase ◽

Thrombin Time ◽

Rt Pcr ◽

Α Subunit ◽

Compound C ◽

Kinase C ◽

Amp Activated Protein Kinase

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression ofIL6mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. TheIL6mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

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AMP-activated protein kinase agonists increase mRNA content of the muscle-specific ubiquitin ligases MAFbx and MuRF1 in C2C12 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00622.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. E1555-E1567 ◽

Cited By ~ 93

Author(s):

Brian J. Krawiec ◽

Gerald J. Nystrom ◽

Robert A. Frost ◽

Leonard S. Jefferson ◽

Charles H. Lang

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Mrna Expression ◽

Muscle Atrophy ◽

Ubiquitin Ligases ◽

C2c12 Cells ◽

Skeletal Muscle Atrophy ◽

Compound C ◽

Mrna Content ◽

Amp Activated Protein Kinase

The hypothesis of the present study was that exposure of differentiated muscle cells to agonists of the AMP-activated protein kinase (AMPK) would increase the mRNA content of the muscle-specific ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). C2C12 cells were incubated with incremental doses of 5-aminoimidazol-4-carboximide ribonucleoside (AICAR) or metformin for 24 h. Both MAFbx and MuRF1 mRNA increased dose dependently in response to these AMPK activators. AICAR, metformin, and 2-deoxy-d-glucose produced time-dependent alterations in ubiquitin ligase expression, typified by a biphasic pattern of expression marked by an acute repression followed by a sustained induction. AMPK-activating treatments in conjunction with dexamethasone produced a pronounced synergistic effect on ligase mRNA expression at later time points. This cooperative response occurred in the absence of a dexamethasone-dependent increase in AMPK expression or activity, as determined by immunoblotting for phosphorylation and expression of AMPKα and its downstream target acetyl-CoA carboxylase (ACC). These responses elicited by AMPK activation singly or in combination with dexamethasone did not extend to the mRNA expression of the UBR box family E3s UBR1/E3αI and UBR2/E3αII. Treatment with the AMPK inhibitor compound C prevented increases in MAFbx and MuRF1 mRNA in response to serum deprivation, as well as AICAR and dexamethasone treatment individually or jointly. Stimulation of AMPK activity in vivo via AICAR injection increased both MAFbx and MuRF1 mRNA in murine skeletal muscle. These data suggest that activation of AMPK in skeletal muscle results in a specific upregulation of MAFbx and MuRF1, responses that are reminiscent of the proposed atrophic transcriptional program executed under various conditions of skeletal muscle wasting. Therefore, AMPK may be a critical component of the intercalated network of signaling pathways governing skeletal muscle atrophy, where its input acts to modify anti- and proatrophic signals to influence gene expression in reaction to catabolic perturbations.

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Abstract 565: The Activation of AMP-Activated Protein Kinase Ameliorates the Severity of Heart Failure in Dogs

Circulation ◽

10.1161/circ.116.suppl_16.ii_102-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hideyuki Sasaki ◽

Hiroshi Asanuma ◽

Masashi Fujita ◽

Hiroyuki Takahama ◽

Masakatsu Wakeno ◽

...

Keyword(s):

Heart Failure ◽

Protein Kinase ◽

Cardiac Myocytes ◽

Left Ventricular ◽

Neonatal Rat ◽

Cellular Damage ◽

Control Group ◽

Compound C ◽

Rapid Pacing ◽

Amp Activated Protein Kinase

Backgrounds; Since AMP-activated protein kinase (AMPK) is activated in the pressure-overloaded hypertrophic hearts, we investigated whether the activation of AMPK caused by metformin attenuates the progression of heart failure induced by rapid pacing in dogs and decreases cellular damage caused by oxidative stress in neonatal rat cardiac myocytes. Methods and Results; Heart failure was induced by right ventricular (RV) pacing at 230 bpm for 4 weeks in dogs. Treatment of dogs with metformin (100mg/kg/day, orally, n=8, Met group) for 4 weeks prevented significantly the progression of pacing-induced heart failure evaluated by echocardiographical and hemodynamic measurement compared with the control group (n=8). Left ventricular (LV) diastolic and systolic dimension (LVDd and LVDs) were smaller (32.8±0.4 and 26.7±0.9 mm, respectively) and fractional shortening (FS) and ejection fraction (EF) were preserved in Met group (18.6±1.8 and 45.5±3.5 %, respectively) compared with the control group (LVDd and LVDs; 36.5±1.0 and 33.0±1.0 mm, FS and EF; 9.6±0.7 and 27.0±1.9 %, p<0.05 vs. Met group each). Furthermore, both pulmonary capillary wedge pressure (PCWP) and mean pulmonary arterial pressure (mPA) were significantly lower in Met group (11.1±0.9 and 18.1±1.4 mmHg, respectively) compared with the control group (21.0±2.2 and 26.8±2.8 mmHg, respectively). Treatment of cultured cardiac myocytes with a maximal physiological concentration of metformin (10μmol/L) attenuated the cellular damage against H 2 O 2 exposure (50μmol/L). These effects were blunted by an AMPK inhibitor, compound-C (20μmol/L), suggesting that the activation of AMPK increased the cellular viability during H 2 O 2 exposure. Conclusions; Metformin that activates AMPK prevented the progression of heart failure induced by rapid pacing in dogs and attenuated the cellular damage against H 2 O 2 exposure in cardiac myocytes. AMPK may be one of new targets for preventing heart failure in clinical settings.

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PRKAA1 Promotes Proliferation and Inhibits Apoptosis of Gastric Cancer Cells Through Activating JNK1 and Akt Pathways

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504019x15668125347026 ◽

2020 ◽

Vol 28 (3) ◽

pp. 213-223 ◽

Cited By ~ 2

Author(s):

Yangmei Zhang ◽

Xichang Zhou ◽

Long Cheng ◽

Xiang Wang ◽

Qinglin Zhang ◽

...

Keyword(s):

Gastric Cancer ◽

Protein Kinase ◽

Cancer Cells ◽

Akt Signaling ◽

Catalytic Subunit ◽

Gastric Cancer Cells ◽

In Vivo Experiments ◽

Compound C ◽

Amp Activated Protein Kinase

PRKAA1 (protein kinase AMP-activated catalytic subunit α 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC-823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.

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AMP-activated protein kinase influences metabolic remodeling in H9c2 cells hypertrophied by arginine vasopressin

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00396.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. H1822-H1832 ◽

Cited By ~ 16

Author(s):

Ramesh Saeedi ◽

Varun V. Saran ◽

Sherry S. Y. Wu ◽

Erika S. Kume ◽

Kim Paulson ◽

...

Keyword(s):

Protein Kinase ◽

Arginine Vasopressin ◽

Heart Muscle ◽

Muscle Cells ◽

H9c2 Cells ◽

Hypertrophied Heart ◽

Compound C ◽

Metabolic Remodeling ◽

Heart Muscle Cells ◽

Amp Activated Protein Kinase

Substrate use switches from fatty acids toward glucose in pressure overload-induced cardiac hypertrophy with an acceleration of glycolysis being characteristic. The activation of AMP-activated protein kinase (AMPK) observed in hypertrophied hearts provides one potential mechanism for the acceleration of glycolysis. Here, we directly tested the hypothesis that AMPK causes the acceleration of glycolysis in hypertrophied heart muscle cells. The H9c2 cell line, derived from the embryonic rat heart, was treated with arginine vasopressin (AVP; 1 μM) to induce a cellular model of hypertrophy. Rates of glycolysis and oxidation of glucose and palmitate were measured in nonhypertrophied and hypertrophied H9c2 cells, and the effects of inhibition of AMPK were determined. AMPK activity was inhibited by 6-[4-(2-piperidin-1- yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo-[1,5-a]pyrimidine (compound C) or by adenovirus-mediated transfer of dominant negative AMPK. Compared with nonhypertrophied cells, glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation in hypertrophied cells, a metabolic profile similar to that of intact hypertrophied hearts. Inhibition of AMPK resulted in the partial reduction of glycolysis in AVP-treated hypertrophied H9c2 cells. Acute exposure of H9c2 cells to AVP also activated AMPK and accelerated glycolysis. These elevated rates of glycolysis were not altered by AMPK inhibition but were blocked by agents that interfere with Ca2+ signaling, including extracellular EGTA, dantrolene, and 2-aminoethoxydiphenyl borate. We conclude that the acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK, whereas the acute glycolytic effects of AVP are AMPK independent and at least partially Ca2+ dependent.

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Pyruvate prevents cardiac dysfunction and AMP-activated protein kinase activation by hydrogen peroxide in isolated rat hearts

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-050 ◽

2004 ◽

Vol 82 (6) ◽

pp. 409-416 ◽

Cited By ~ 19

Author(s):

Hernando Leon ◽

Laura L Atkinson ◽

Jolanta Sawicka ◽

Ken Strynadka ◽

Gary D Lopaschuk ◽

...

Keyword(s):

Protein Kinase ◽

Cardiac Dysfunction ◽

Mechanical Function ◽

Activity Increase ◽

Enhanced Production ◽

Compound C ◽

Rat Hearts ◽

Ampk Activity ◽

Amp Activated Protein Kinase ◽

Isolated Rat

Ischemia-reperfusion injury in the heart results in enhanced production of H2O2 and activation of AMP-activated protein kinase (AMPK). Since mutations in AMPK result in cardiovascular dysfunction, we investigated whether the activation of AMPK mediates the H2O2-induced reduction in cardiac mechanical function. Isolated working rat hearts were perfused at 37 °C with Krebs-Henseleit solution. Following a 20-minute equilibration period, a single bolus of H2O2 (300 µmol/L) was added and the hearts were perfused for an additional 5 min. H2O2 induced a dramatic and progressive reduction in cardiac function. This was accompanied by rapid and significant activation of AMPK, an increase in Thr-172 phosphorylation of AMPK, and an increase in the creatine to phosphocreatine (Cr/PCr) ratio. Addition of pyruvate (5 mmol/L) to the perfusate prevented the H2O2-mediated reduction in cardiac mechanical dysfunction, activation of myocardial AMPK activity, increase in AMPK phosphorylation and the increase in the Cr/PCr ratio. Hearts challenged with H2O2 (300 µmol/L) in presence of either AMPK inhibitor Compound C (10 µmol/L) or its vehicle (dimethyl sulfoxide (DMSO), 0.1%) showed reduced impairment in cardiac mechanical function. Compound C but not its vehicle significantly inhibited myocardial AMPK activity. Thus, H2O2 induces cardiac dysfunction via both AMPK-dependent and independent mechanisms.Key words: oxidative stress, AMPK, antioxidant, isolated rat heart, pyruvate.

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Pioglitazone Attenuates Injury-Induced Neointima Formation in Mouse Femoral Artery Partially through the Activation of AMP-Activated Protein Kinase

Pharmacology ◽

10.1159/000471769 ◽

2017 ◽

Vol 100 (1-2) ◽

pp. 64-73 ◽

Cited By ~ 2

Author(s):

Islam Osman ◽

Arwa Fairaq ◽

Lakshman Segar

Keyword(s):

Protein Kinase ◽

Femoral Artery ◽

Immunoblot Analysis ◽

Inhibitory Effect ◽

Aortic Tissue ◽

Neointima Formation ◽

Vsmc Proliferation ◽

Compound C ◽

Amp Activated Protein Kinase

Background/Aims: Pioglitazone (PIO), an antidiabetic drug, has been shown to attenuate vascular smooth muscle cell (VSMC) proliferation, which is a major event in atherosclerosis and restenosis after angioplasty. Till date, the likely contributory role of AMP-activated protein kinase (AMPK) toward PIO inhibition of VSMC proliferation has not been examined in vivo. This study is aimed at determining whether pharmacological inhibition of AMPK would prevent the inhibitory effect of PIO on neointima formation in a mouse model of arterial injury. Methods: Male CJ57BL/6J mice were subjected to femoral artery injury using guidewire. PIO (20 mg/kg/day) was administered orally 1 day before surgery and for 3 weeks until sacrifice in the absence or presence of compound C (an AMPK inhibitor). Injured femoral arteries were used for morphometric analysis of neointima formation. Aortic tissue lysates were used for immunoblot analysis of phosphorylated AMPK. Results: PIO treatment resulted in a significant decrease in intima-to-media ratio by ∼50.3% (p < 0.05, compared with vehicle control; n = 6), which was accompanied by enhanced phosphorylation of AMPK by ∼85% in the vessel wall. Compound C treatment led to a marked reduction in PIO-mediated inhibition of neointima formation. Conclusion: PIO attenuates injury-induced neointima formation, in part, through the activation of AMPK.

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AMP-activated protein kinase facilitates avian reovirus to induce mitogen-activated protein kinase (MAPK) p38 and MAPK kinase 3/6 signalling that is beneficial for virus replication

Journal of General Virology ◽

10.1099/vir.0.013953-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 3002-3009 ◽

Cited By ~ 22

Author(s):

Wen T. Ji ◽

Long H. Lee ◽

Feng L. Lin ◽

Lai Wang ◽

Hung J. Liu

Keyword(s):

Protein Kinase ◽

Vero Cells ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Avian Reovirus ◽

Mtor Inhibition ◽

Mapk Kinase ◽

Compound C ◽

Mitogen Activated Protein ◽

Amp Activated Protein Kinase

Stimulated by energetic stress, AMP-activated protein kinase (AMPK) controls several cellular functions. It was discovered here that infection of Vero cells with avian reovirus (ARV) upregulated AMPK and mitogen-activated protein kinase (MAPK) p38 phosphorylation in a time- and dose-dependent manner. Being an energy status sensor, AMPK is potentially an upstream regulator of MAPK p38. Treatment with 5-amino-4-imidazolecarboxamide ribose (AICAR), a well-known activator of AMPK, induced phosphorylation of MAPK p38. Unlike AICAR, wortmannin or rapamycin did not induce phosphorylation of MAPK p38, suggesting that mTOR inhibition is not a determining factor in MAPK p38 phosphorylation. Inhibition of AMPK by compound C antagonized the effect of AICAR on MAPK p38 in Vero cells. Specific inhibition of AMPK by small interfering RNA or compound C also suppressed ARV-induced phosphorylation of MAPK kinase (MKK) 3/6 and MAPK p38 in Vero and DF-1 cells, thereby providing a link between AMPK signalling and the MAPK p38 pathway. The mechanism of ARV-enhanced phosphorylation of MKK 3/6 and MAPK p38 in cells was not merely due to glucose deprivation, a probable activator of AMPK. In the current study, direct inhibition of MAPK p38 by SB202190 decreased the level of ARV-induced syncytium formation in Vero and DF-1 cells, and decreased the protein levels of ARV σA and σC and the progeny titre of ARV, suggesting that activation of MAPK p38 is beneficial for ARV replication. Taken together, these results suggested that AMPK could facilitate MKK 3/6 and MAPK p38 signalling that is beneficial for ARV replication. Although well studied in energy metabolism, this study provides evidence for the first time that AMPK plays a role in modulating ARV and host-cell interaction.

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