The relationship between AMP-activated protein kinase activity and AMP concentration in the isolated perfused rat heart.
The objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of beta-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by (31)P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 microm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol.min(-1).microg of protein(-1). The half-maximal AMPK activation (A(0.5)) was 1.8 +/- 0.3 microm AMP. Measurements in vitro have reported similar AMPK A(0.5) at 0.2 mm ATP, but found that A(0.5) increased 10-20-fold at 4 mm ATP. The low A(0.5) of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart.
Frederich
Markus
M
NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Balschi
James A
JA
D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002210:Caprylates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002210:Caprylates; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D000249:Adenosine Monophosphate; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase; C031492:octanoic acid