Acidosis during ischemia promotes adenosine triphosphate resynthesis in postischemic rat heart. In vivo regulation of 5'-nucleotidase.

Bak, Marianna I; Ingwall, Joanne S.

doi:10.1172/jci116974

Cited by 80 publications

(44 citation statements)

References 42 publications

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“…The supernatant (200 l) was injected into a Waters HPLC system equipped with a Waters 1525 HPLC pump and a 2487 dual wavelength (UV-visible) absorbance detector (Waters Co., Milford MA) to measure nucleotides. ATP, ADP, and AMP were separated through a C-18 reverse phase column (YMC-Pack TM ODS-AQ TM , 150 ϫ 4.6 mm, particle size 3 m, pore size 120 Å) at a flow rate of 0.8 ml/min with 50 mM ammonium dihydrogen phosphate (pH 6.0) as the mobile phase during the initial 50 min (26). Peaks were monitored by UV absorption at 254 nm and identified by comparison with the retention times of known standards.…”

Section: Methodsmentioning

confidence: 99%

Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

Pulinilkunnil

Kong

et al. 2011

Journal of Biological Chemistry

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AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK ␣2 protein and activity were reduced by 45%. In ␤ 1,2,3 -adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr 172 phosphorylation. In contrast, acute ␣-or ␤-adrenergic blockade in WT mice resulted in increased AMPK ␣ Thr 172 phosphorylation and AMPK ␣1 and ␣2 activity in BAT. But short term blockade of ␣-adrenergic signaling in ␤ 1,2,3 -adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser 485/491 , a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser 485/491 phosphorylation resulting from ␣-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of ␣-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr 172 phosphorylation and chronically through changes in the ␣ catalytic subunit protein levels. Furthermore, AMPK ␣ Ser 485/491 phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects. AMP-activated protein kinase (AMPK),4 an evolutionarily conserved serine threonine kinase, is a fuel-sensing enzyme complex activated by cellular stresses that increase AMP or deplete ATP including hypoxia, ischemia, glucose deprivation, uncouplers of oxidative phosphorylation, exercise, and muscle contraction (1, 2). Once activated, AMPK phosphorylates multiple downstream substrates that function to preserve the AMP: ATP ratio through inhibition of ATP-catabolizing pathways and promotion of ATP-generating pathways. Mechanisms involved in AMPK activation include 1) binding of AMP to an allosteric site on the ␥ subunit, which renders the holoenzyme resistant to inactivating serine phosphatases and may also have direct allosteric effects on kinase activity (1, 2) and 2) phosphorylation by upstream AMPK kinases of the ␣ (catalytic) subunits on Thr 172 , which is essential for kinase activity. Recent studies employing INS-1 cells (3), hepatitis C virus harboring replicon cells (4), and isolated heart preparations (5) have demonstrated another potential regulatory mechanism; that is, AMPK may also undergo inhibitory phosphorylation on serine 485 and 491 residues of the ␣1 and ␣2 catalytic subunits, respectively. Some data suggest that upstr...

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Section: Methodsmentioning

confidence: 99%

Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

Pulinilkunnil

Kong

et al. 2011

Journal of Biological Chemistry

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“…Western blotting was done with antibodies against AMPK ␣1, ␣2, pan-␣, HA (Roche Diagnostics), GLUT1, GLUT4 (Chemicon, Intl., Inc., Temecula, CA), SERCA2 (Affinity BioReagents, Golden, CO), and Na ϩ /Ca 2ϩ exchanger (Swant, Bellinzona, Switzerland). HPLC Measurements and Glycogen Assay-Freeze-clamped tissues were used for determination of myocardial content of adenine nucleotides, nucleosides, and purine bases by a HPLC method as reported previously (20). Myocardial glycogen content was determined by measuring the amount of glucose released from glycogen by use of an alkaline extraction to separate glycogen and exogenous glucose (21).…”

Section: Methodsmentioning

confidence: 99%

“…The mean value of [ATP] for WT or TG hearts was used to calibrate the ATP peak area of the base-line 31 P NMR spectrum. Concentrations of other metabolites were calculated using the ratio of their peak areas to the ATP peak area, and intracellular pH (pH i ) was determined by the chemical shift of inorganic phosphate (P i ) relative to PCr (20). The values of [ATP] and [PCr] in the ischemic hearts were obtained from summed spectra of 3-4 hearts.…”

Section: Methodsmentioning

confidence: 99%

Glucose Metabolism and Energy Homeostasis in Mouse Hearts Overexpressing Dominant Negative α2 Subunit of AMP-activated Protein Kinase

Xing

Musi

Fujii

et al. 2003

Journal of Biological Chemistry

206

215

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AMP-activated protein kinase (AMPK) is an energysensing 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 ␣2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia. In isolated perfused hearts subjected to a 10-min ischemia, AMPK ␣2 activity in wild type (WT) increased substantially (by 4.5-fold), whereas AMPK ␣2 activity in TG was similar to the level of WT at base line. Basal AMPK ␣1 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 ␣2 in TG significantly blunted this response. Using 31 P 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 ␣2 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.The AMP-activated protein kinase (AMPK) 1 functions as a fuel gauge, such that when the cell is exposed to stresses associated with energy depletion, it switches off ATP-utilizing pathways and switches on ATP-generating pathways to restore energy homeostasis (1, 2). This kinase is activated by decreases in ATP/AMP and in phosphocreatine (PCr)/creatine through Thr 172 phosphorylation by one or more upstream kinases (AMPK kinase) and through allosteric modification by AMP (1, 2). AMPK is a heterotrimeric protein consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (1, 3). Each subunit has two or more different isoforms; the ␣1 subunit is widely expressed, whereas the ␣2 subunit is expressed primarily in liver, heart, and skeletal muscle (2, 4, 5).It has been suggested that AMPK regulates glucose and fatty acid metabolism in striated muscles (6, 7). Studies from our groups and others showed increased AMPK activity during acute and chronic stresses, such as hypoxia and exercise in skeletal muscle and ischemia and pressure overload in the heart (8 -12). Activation of AMPK in the heart is associated with enhanced glucose uptake and glycolysis (10, 11, 13). As glycolysis is a major source of ATP during ischemia, stimulation of glucose uptake and glycolysis by AMPK in the ischemic heart is consistent ...

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“…23 31 P nuclear magnetic resonance (NMR) spectra were obtained as previously described 24 at 161.94 MHz with a GE-400 wide-bore spectrometer. Hearts were placed in a 10-mm glass NMR tube and inserted into a custom-made, 1 H/ 31 P doubletuned probe situated in an 89-mm bore, 9.4-T superconducting magnet.…”

Section: Energetics Assaysmentioning

confidence: 99%

Increased α2 Subunit–Associated AMPK Activity and PRKAG2 Cardiomyopathy

et al. 2005

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Background-AMP-activated protein kinase (AMPK) regulatory ␥2 subunit (PRKAG2) mutations cause a human cardiomyopathy with cardiac hypertrophy, preexcitation, and glycogen deposition. PRKAG2 cardiomyopathy is recapitulated in transgenic mice overexpressing mutant PRKAG2 N488I in the heart (TG␥2 N488I ). AMPK is a heterotrimeric kinase consisting of 1 catalytic (␣) and 2 regulatory (␤ and ␥) subunits. Two ␣-subunit isoforms, ␣1 and ␣2, are expressed in the heart; however, the contribution of AMPK utilization of these subunits to PRKAG2 cardiomyopathy is unknown. Mice overexpressing a dominant-negative ␣2 subunit of AMPK (TG␣2 DN ) provide a tool for selectively inhibiting ␣2, but not ␣1, subunit-associated AMPK activity. Methods and Results-In compound-heterozygous TG␥2N488I /TG␣2 DN mice, AMPK activity associated with ␣2 but not ␣1 was decreased compared with TG␥2 N488I . The TG␣2 DN transgene reduced the disease phenotype of TG␥2 N488I , partially or completely normalizing the ECG, cardiac function, cardiac morphology, and exercise capacity in compoundheterozygous mice. TG␥2 N488I hearts had normal resting levels of high-energy phosphates and could improve cardiac performance during exercise. Cardiac glycogen content decreased in TG␥2 N488I mice after exercise stress, indicating availability of the stored glycogen for metabolic utilization. No differences in glycogen-metabolizing enzymes were observed. Conclusions-The PRKAG2 N488I mutation causes inappropriate AMPK activation, which leads to glycogen accumulation and conduction system disease. The accumulated glycogen can serve as an energy source, and the animals have contractile reserve during exercise. Because the dominant-negative ␣2 subunit attenuates the mutant PRKAG2 phenotype, AMPK complexes containing the ␣2 rather than the ␣1 subunit are the primary mediators of the effects of PRKAG2 mutations. (Circulation. 2005;112:3140-3148.)

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Acidosis during ischemia promotes adenosine triphosphate resynthesis in postischemic rat heart. In vivo regulation of 5'-nucleotidase.

Cited by 80 publications

References 42 publications

Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

Glucose Metabolism and Energy Homeostasis in Mouse Hearts Overexpressing Dominant Negative α2 Subunit of AMP-activated Protein Kinase

Increased α2 Subunit–Associated AMPK Activity and PRKAG2 Cardiomyopathy

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