Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.     

Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.     

Low-intensity contraction activates the alpha1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle

Skeletal muscle expresses two catalytic subunits, alpha1 and alpha2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the alpha2-containing AMPK complex (AMPKalpha2), but this activation may occur with or without activation of the alpha1-containing AMPK complex (AMPKalpha1), suggesting that AMPKalpha2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPKalpha1, but not AMPKalpha2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPKalpha1 activity increased twofold and AMPKalpha2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr(172) of the alpha-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-d-glucose transport. In contrast, increasing the frequency (>or=5 Hz) or duration (>or=5 min) of contraction activated AMPKalpha1 and AMPKalpha2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPKalpha1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPKalpha2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle.     

Possible involvement of the alpha1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Maternal overnutrition suppresses the phosphorylation of 5'-AMP-activated protein kinase in liver, but not skeletal muscle, in the fetal and neonatal sheep

Epidemiological studies have shown that infants exposed to an increased supply of nutrients before birth are at increased risk of type 2 diabetes in later life. We have investigated the hypothesis that fetal overnutrition results in reduced expression and phosphorylation of the cellular fuel sensor, AMP-activated kinase (AMPK) in liver and skeletal muscle before and after birth. From 115 days gestation, ewes were fed either at or approximately 55% above maintenance energy requirements. Postmortem was performed on lamb fetuses at 139-141 days gestation (n = 14) and lambs at 30 days of postnatal age (n = 21), and liver and quadriceps muscle were collected at each time point. The expression of AMPKalpha1 and AMPKalpha2 mRNA was determined by quantitative RT-PCR (qRT-PCR). The abundance of AMPKalpha and phospho-AMPKalpha (P-AMPKalpha) was determined by Western blot analysis, and the proportion of the total AMPKalpha pool that was phosphorylated in each sample (%P-AMPKalpha) was determined. The ratio of AMPKalpha2 to AMPKalpha1 mRNA expression was lower in fetuses compared with lambs in both liver and muscle, independent of maternal nutrition. Hepatic %P-AMPKalpha was lower in both fetuses and lambs in the Overfed group and %P-AMPKalpha in the lamb liver was inversely related to plasma glucose concentrations in the first 24 h after birth (r = 0.73, P < 0.025). There was no effect of maternal overnutrition on total AMPKalpha or P-AMPKalpha abundance in liver or skeletal muscle. We have, therefore, demonstrated that AMPKalpha responds to signals of increased nutrient availability in the fetal liver. Suppression of hepatic AMPK phosphorylation may contribute to increased glucose production, and basal hyperglycemia, present in lambs of overfed ewes in early postnatal life.     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle

The 5'-AMP-activated protein kinase (AMPK) is proposed to be involved in signaling pathways leading to adaptations in skeletal muscle in response to both a single exercise bout and exercise training. This study investigated the effect of endurance training on protein content of catalytic (alpha1, alpha2) and regulatory (beta1, beta2 and gamma1, gamma2, gamma3) subunit isoforms of AMPK as well as on basal AMPK activity in human skeletal muscle. Eight healthy young men performed supervised one-legged knee extensor endurance training for 3 wk. Muscle biopsies were obtained before and 15 h after training in both legs. In response to training the protein content of alpha1, beta2 and gamma1 increased in the trained leg by 41, 34, and 26%, respectively (alpha1 and beta2 P < 0.005, gamma1 P < 0.05). In contrast, the protein content of the regulatory gamma3-isoform decreased by 62% in the trained leg (P = 0.01), whereas no effect of training was seen for alpha2, beta1, and gamma2. AMPK activity associated with the alpha1- and the alpha2-isoforms increased in the trained leg by 94 and 49%, respectively (both P < 0.005). In agreement with these observations, phosphorylation of alpha-AMPK-(Thr172) and of the AMPK target acetyl-CoA carboxylase-beta(Ser221) increased by 74 and 180%, respectively (both P < 0.001). Essentially similar results were obtained in four additional subjects studied 55 h after training. This study demonstrates that protein content and basal AMPK activity in human skeletal muscle are highly susceptible to endurance exercise training. Except for the increase in gamma1 protein, all observed adaptations to training could be ascribed to local contraction-induced mechanisms, since they did not occur in the contralateral untrained muscle.     

Coffee polyphenol caffeic acid but not chlorogenic acid increases 5'AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle

Chlorogenic acid is an ester of caffeic and quinic acids, and is one of the most widely consumed polyphenols because it is abundant in foods, especially coffee. We explored whether chlorogenic acid and its metabolite, caffeic acid, act directly on skeletal muscle to stimulate 5'-adenosine monophosphate-activated protein kinase (AMPK). Incubation of rat epitrochlearis muscles with Krebs buffer containing caffeic acid (≥0.1 mM, ≥30 min) but not chlorogenic acid increased the phosphorylation of AMPKα Thr(172), an essential step for kinase activation, and acetyl CoA carboxylase Ser(79), a downstream target of AMPK, in a dose- and time-dependent manner. Analysis of isoform-specific AMPK activity revealed that AMPKα2 activity increased significantly, whereas AMPKα1 activity did not change. This enzyme activation was associated with a reduction in phosphocreatine content and an increased rate of 3-O-methyl-d-glucose transport activity in the absence of insulin. These results suggest that caffeic acid but not chlorogenic acid acutely stimulates skeletal muscle AMPK activity and insulin-independent glucose transport with a reduction of the intracellular energy status.     

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.     

Rac1 signalling towards GLUT4/glucose uptake in skeletal muscle

Small Rho family GTPases are important regulators of cellular traffic. Emerging evidence now implicates Rac1 and Rac-dependent actin reorganisation in insulin-induced recruitment of glucose transporter-4 (GLUT4) to the cell surface of muscle cells and mature skeletal muscle. This review summarises the current thinking on the regulation of Rac1 by insulin, the role of Rac-dependent cortical actin remodelling in GLUT4 traffic, and the impact of Rac1 towards insulin resistance in skeletal muscle.     

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.     

Cell cycle regulation via p53 phosphorylation by a 5'-AMP activated protein kinase activator, 5-aminoimidazole- 4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is an activator of AMP activated protein kinase (AMPK) and a regulator of de novo purine synthesis. There are several earlier reports indicating that AICAR treatment suppresses cell growth via regulation of AMPK or de novo purine synthesis. We found cell growth to be suppressed by AICAR treatment in HepG2 because of p53 accumulation, which was associated with p53-Ser15 phosphorylation. Moreover, a motif very similar to the consensus motif of AMPK phosphorylation was found around p53-Ser15, and Ser15 phosphorylation was detected in AICAR treated HepG2 as was in vitro phosphorylation by AMPK. Our results suggest that AICAR may regulate cell growth via p53 phosphorylation, and also indicate the possibility of p53 phosphorylation.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Estradiol stimulates Akt, AMP-activated protein kinase (AMPK) and TBC1D1/4, but not glucose uptake in rat soleus

Post-menopausal women exhibit decreases in circulating estrogen levels and whole body insulin sensitivity, suggesting that estrogen regulates skeletal muscle glucose disposal. Thus, we assessed whether estrogen stimulates glucose uptake or enhances insulin sensitivity in skeletal muscle. Ex vivo muscle stimulation with 17β-estradiol (10 nM) resulted in a rapid (?10 min) increase in the phosphorylation of Akt, AMP-activated protein kinase (AMPK), and TBC1D1/4, key signaling proteins that regulate glucose uptake in muscle. Treatment with the estrogen receptor antagonist, ICI 182,780, only partly inhibited signaling, suggesting both an estrogen receptor-dependent and independent mechanism of estradiol action. 17β-Estradiol did not stimulate ex vivo muscle [³H]-2-deoxyglucose uptake or enhance insulin-induced glucose uptake, demonstrating discordance between the estradiol-induced stimulation of signaling proteins and muscle glucose uptake. This study is the first to demonstrate that estradiol stimulates Akt, AMPK, and TBC1D1/4 in intact skeletal muscle, but surprisingly, estradiol does not stimulate muscle glucose uptake.     

Relationship between 5-aminoimidazole-4-carboxamide-ribotide and AMP-activated protein kinase activity in the perfused mouse heart

AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside). Phosphorylated AICA-riboside or AICA-ribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICA-riboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: 1) sufficient ZMP concentration ([ZMP]) forms to increase AMPK activity, and 2) [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 0.15-2 mM AICA-riboside concentration resulted in [ZMP] of 2-8 mM. ZMP accumulation reduced phosphocreatine concentration, which increased cytosolic [AMP]. In hearts with [ZMP] less than approximately 3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 and 5 mM, in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with 0.25 mM AICA-riboside for 48 min and 0.5 mM AICA-riboside for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 +/- 0.6 mM. Hearts with [ZMP] >5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the half-maximal activity of AMP was 5.6 +/- 1.6 microM. Thus, in mouse hearts, AICA-riboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity.