The Relationship between AMP-activated Protein Kinase Activity and AMP Concentration in the Isolated Perfused Rat Heart

Abstract

AMP-activated protein kinase (AMPK)1 and AMPK kinase comprise a protein kinase cascade that has been highly conserved throughout evolution (1, 2). Increases in AMP concentration ([AMP]) activate this cascade by four mechanisms (3-5). These mechanisms are as follows: 1) an allosteric activation by AMP of AMPK kinase, which then phosphorylates AMPK; 2) the binding of AMP to AMPK, which makes it a poorer substrate for protein phosphatases; 3) the binding of AMP to AMPK, which makes AMPK a better substrate for AMPK kinase; and 4) the allosteric activation by AMP of AMPK. The activating effects of AMP are antagonized by high concentrations of ATP. Since the AMPK is activated when AMP is elevated and ATP is depressed, AMPK is hypothesized to act as cellular "fuel gauge" (1).After AMPK activation, in response to metabolic stress, AMPK phosphorylates enzymes, leading to activation of catabolic pathways to increase ATP synthesis and inhibition of anabolic pathways to limit ATP consumption. For example, AMPK phosphorylation decreases acetyl-CoA-carboxylase (ACC) activity, which decreases malonyl-CoA concentration. Malonyl-CoA inhibits carnitine palmitoyltransferase-1, which transports fatty acids into the mitochondrion (6, 7). Reduction of malonyl-CoA increases fatty acid uptake via carnitine palmitoyltransferase-1 and, thereby, the fatty acid oxidation by the mitochondria, which increases ATP production. In skeletal muscle and hearts, AMPK activity also increases glucose uptake by enhancing GLUT-4 translocation (8 -10). In hearts, AMPK phosphorylation of 6-phosphofructo-2-kinase increases fructose 2,6-bisphosphate, a stimulator of 6-phosphofructo-1-kinase, which accelerates glycolysis (11). AMPK also downregulates ATP-consuming pathways, such as glycogen, cholesterol, and fatty acid synthesis (12, 13). The aim of the present study was to define the relationship between AMPK activation and cytosolic [AMP] in vivo in the isolated perfused rat heart. To accomplish this, we employed an approach that restrains substrate metabolism by use of the inhibitors bromo-octanoate (BrO) to inhibit fatty acid oxidation (16) and amino-oxyacetate (AOA), to inhibit pyruvate oxidation (17). Using this approach together with increased work demand reduced the phosphocreatine (PCr) (18). We used 31 P NMR spectroscopy to measure the PCr and ATP content as well as the intracellular pH (pH i ) of these hearts. The creatine kinase and the adenylate kinase equilibrium expressions were used to calculate [ADP] and [AMP], respectively. The net effect of [PCr] reductions is an increase in [AMP] with a relatively constant [ATP]. Cytosolic [AMP] was therefore manipulated in a relatively controlled manner, and the AMPK activity was measured. EXPERIMENTAL PROCEDURESPreparation of Isolated Perfused Rat Hearts-Hearts of male Sprague-Dawley rats (280 -320 g) were isolated and perfused in the isovolumic Langendorf model (18). The Krebs-Henseleit buffer (KH) perfusate contained 118 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 1.75 mM CaCl 2 , 0.5 mM ...

Experimentally observed phenomena on cardiac energetics in heart failure emerge from simulations of cardiac metabolism

“…The associated model predictions are coincident with several experimentally observed phenomena: (i) the predicted metabolic tipping point coincides with the transition to severe cardiac dysfunction indicated by end-diastolic pressures greater than 15 mmHg in dogs (6,8); (ii) the tipping point is associated with a Ϸ30% reduction of TAN reduction (Fig. 2D), consistent with observations in humans in heart failure (3); (iii) the predicted cytoplasmic AMP at the tipping point is Ϸ1.6 mM and equal to the apparent AMP concentration at half-maximal activation of cardiac AMP-activated protein kinase (17); (iv) oxidative stress in the myocardium is predicted to progressively increase as the metabolic pools are diminished; and (v) at given values of TAN and TEP during hypertrophic remodeling, CR tot attains a value that is associated with optimal ⌬G ATPase . Thus, both increases and decreases to the creatine pool are predicted to result in diminished energetic state unless accompanied by appropriate simultaneous changes in the other pools.…”

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

“…The rapid increase in glycogenolysis in G-replete hearts provides more than 85% of glucose consumed through glycolysis during the initial 10 min of LFI, and the elevation of glucose-6-phosphate availability suppresses hexokinase activity (22) and thereby slows the rate of glucose uptake. Similar observations were reported for skeletal muscles during moderate exercise (20). Because of the finite supply of glycogen, glycogenolysis slows during more prolonged periods of LFI, and, consequently, glucose uptake recovers to maintain substrate availability for glycolysis.…”

“…AMPK is a key kinase involved in the regulation of many aspects of cellular metabolism, including glucose metabolism (20,29,30,51). AMPK activation by AICAR in rat skeletal muscles is accompanied by an activation of glycogen phosphorylase that increases glycogenolysis (51), but no such relationship occurs in rat ventricular papillary muscle (38).…”

AMP-activated protein kinase control of energy metabolism in the ischemic heart

“…Elucidating the role of AMP in the control of AMPK has been hampered by the complexity of accurately measuring cytoplasmic [AMP], as total cellular AMP content does not reflect cytoplasmic [AMP]. However, studies by Frederich et al 25 have carefully examined the relationship between AMPK and AMP, using a 31 P-NMR approach in which cytoplasmic [AMP] concentrations can be calculated. These authors showed that cardiac AMPK is activated by AMP concentrations in the low micromolar range.…”