ADP Ratio Research Articles

Transcriptional Regulation of Glucose Sensors in Pancreatic β-Cells and Liver: An Update

Pancreatic β-cells and the liver play a key role in glucose homeostasis. After a meal or in a state of hyperglycemia, glucose is transported into the β-cells or hepatocytes where it is metabolized. In the β-cells, glucose is metabolized to increase the ATP:ADP ratio, resulting in the secretion of insulin stored in the vesicle. In the hepatocytes, glucose is metabolized to CO2, fatty acids or stored as glycogen. In these cells, solute carrier family 2 (SLC2A2) and glucokinase play a key role in sensing and uptaking glucose. Dysfunction of these proteins results in the hyperglycemia which is one of the characteristics of type 2 diabetes mellitus (T2DM). Thus, studies on the molecular mechanisms of their transcriptional regulations are important in understanding pathogenesis and combating T2DM. In this paper, we will review a recent update on the progress of gene regulation of glucose sensors in the liver and β-cells.

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Ankyrin regulates KATP channel membrane trafficking and gating in excitable cells

KATP channels play critical roles in many cellular functions by coupling cell metabolic status to electrical activity. First discovered in cardiomyocytes 1, KATP channels (comprised of Kir6.x and SUR subunits) have since been found in many other tissues, including pancreatic beta cells, skeletal muscle, smooth muscle, brain, pituitary, and kidney. By linking cellular metabolic state with membrane potential, KATP channels are able to regulate a number of cellular functions such as hormone secretion, vascular tone, and excitability. Specifically, a reduction in metabolism causes a decrease in the ATP:ADP ratio, opening of KATP channels and allowing K+ efflux, membrane hyperpolarization, and suppression of electrical activity. Conversely, increased cellular metabolism causes a decrease in the ATP:ADP ratio that leads to closure of the KATP channel, membrane depolarization, and stimulation of cell electrical activity.

The increase of adenylate kinase activity in the blood can control aggregation of platelets in coronary or peripheral arterial ischemia

Activation and aggregation of blood platelets is crucial for hemostasis and thrombosis. In the vascular system adenine nucleotides are important signaling molecules playing a key role in hemostasis. ADP was the first low molecular weight agent recognized to cause blood platelets activation and aggregation. NTPDases and adenylate kinase (AK) are the main enzymes involved in metabolism of extracellular adenine nucleotides. The majority of studies concentrated on the role of NTPDase1 (apyrase) in the inhibition of platelets aggregation. Up to now, there are still insufficient data concerning the role of AK in this process. We found that adenylate kinase activity in the serum of patients with myocardial infarction is significantly increased when compared to the healthy volunteers. The elevated activity of AK is connected to appearance of another isoform of that enzyme, expressed in patients with myocardial infarction. The influence of AK on the pig blood platelets aggregation induced by 20 μM ADP or 7.5 μg/ml rat collagen was examined. 1U of adenylate kinase added to platelet-rich plasma (PRP) before ADP or collagen, inhibited the platelets aggregation. One minute after induction of platelets activation by ADP as much as 5U of adenylate kinase was necessary to stop the platelet aggregation. In the case of collagen activated aggregation, only 2U of AK added 1 or 5 minutes after initiation of the aggregation process were sufficient for disaggregation of platelets. The increase of ATP: ADP ratio is probably responsible for the initiation of disaggregation process. We conclude that adenylate kinase is involved in regulation of plate-lets aggregation. Anticoagulative role of AK indicates the possibility of using this enzyme in the treatment of cardiovascular diseases.

Insulin release, peripheral insulin resistance and muscle function in protein malnutrition: a role of tricarboxylic acid cycle anaplerosis

Pancreatic beta-cells and skeletal muscle act in a synergic way in the control of systemic glucose homeostasis. Several pyruvate-dependent and -independent shuttles enhance tricarboxylic acid cycle intermediate (TACI) anaplerosis and increase beta-cell ATP:ADP ratio, triggering insulin exocytotic mechanisms. In addition, mitochondrial TACI cataplerosis gives rise to the so-called metabolic coupling factors, which are also related to insulin release. Peripheral insulin resistance seems to be related to skeletal muscle fatty acid (FA) accumulation and oxidation imbalance. In this sense, exercise has been shown to enhance skeletal muscle TACI anaplerosis, increasing FA oxidation and by this manner restores insulin sensitivity. Protein malnutrition reduces beta-cell insulin synthesis, release and peripheral sensitivity. Despite little available data concerning mitochondrial metabolism under protein malnutrition, evidence points towards reduced beta-cell and skeletal muscle mitochondrial capacity. The observed decrease in insulin synthesis and release may reflect reduced anaplerotic and cataplerotic capacity. Furthermore, insulin release is tightly coupled to ATP:ADP rise which in turn is related to TACI anaplerosis. The effect of protein malnutrition upon peripheral insulin resistance is time-dependent and directly related to FA oxidation capacity. In contrast to beta-cells, TACI anaplerosis and cataplerosis pathways in skeletal muscle seem to control FA oxidation and regulate insulin resistance.

Tight Coupling between Glucose and Mitochondrial Metabolism in Clonal β-Cells Is Required for Robust Insulin Secretion

The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic beta-cells are not completely understood. To identify metabolic disturbances in beta-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal beta-cell lines, which are glucose-responsive (832/13 cells) or glucose-unresponsive (832/2 cells). To this end, we analyzed a number of parameters in glycolytic and mitochondrial metabolism, including mRNA expression of genes involved in cellular energy metabolism. We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production, or respiratory chain complex I, III, and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase A, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. This could account for a Warburg-like effect in the 832/2 cell clone, lacking in 832/13 cells as well as primary beta-cells. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with HbA(1c) levels, reflecting perturbed long term glucose homeostasis, whereas that of Slc2a2 (glucose transporter 2) correlated negatively with HbA(1c) and thus better metabolic control. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal beta-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal beta-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human beta-cells. The 832 beta-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 diabetes.

Metabolic acclimation to hypoxia revealed by metabolite gradients in melon fruit

A metabolomics approach using 1H NMR and GC–MS profiling of primary metabolites and quantification of adenine nucleotides with luciferin bioluminescence was employed to investigate the spatial changes of metabolism in melon fruit. Direct 1H NMR profiling of juice collected from different locations in the fruit flesh revealed several gradients of metabolites, e.g. sucrose, alanine, valine, GABA or ethanol, with increase in concentrations from the periphery to the center of the fruit. GC–MS profiling of ground samples revealed gradients for metabolites not detected using 1H NMR, including pyruvic and fumaric acids. The quantification of adenine nucleotides highlighted a strong decrease in both ATP and ADP ratios and the adenylate energy charge from the periphery to the center of the fruit. These concentration patterns are consistent with an increase in ethanol fermentation due to oxygen limitation and were confirmed by observed changes in alanine and GABA concentrations, as well as other markers of hypoxia in plants. Ethanol content in melon fruit can affect organoleptic properties and consumer acceptance. Understanding how and when fermentation occurred can help to manage the culture and limit ethanol production.

Bile Acids Have the Gall to Function as Hormones

In this issue of Cell Metabolism, Thomas et al. (2009) show that specific activation of the bile-acid-activated G protein-coupled receptor TGR5 improves pancreatic and hepatic function and impairs the development of obesity following administration of a high-fat diet.

Moderate-intensity exercise associated with ketogenic diet. A model for the study of ammonia metabolism?

Introduction: Exercise has been used as a model to study the metabolism of ammonia (NH 3 + NH 4 + ), which is highly toxic to the central nervous system. A ketogenic diet leads to several metabolic adaptations to maintain the ATP/ ADP ratio, including a lack of glycogen reservoirs, the use of amino acids as carbon skeleton donors and increased -oxidation of fatty acids. The formation of ammonia during high-intensity exercise is well studied, but its role in moderate-intensity exercise remains unclear. Here we investigate ammonia metabolism during moderate-intensity exercise associated with a ketogenic diet as a model system. Materials and Methods: Athletes (n=7) were physically evaluated and had their maximum oxygen consumption (VO 2max ) and heart rate (HR max ) determined. The subjects remained on a ketogenic diet 72h prior to exercise. They then exercised for 60min at a power output of 60% of that at VO 2max , and at 70%-75% of HR max . Results: Basal ammonemia increased by 35% due to dietetic modifi cations, while exercise caused a 250% increase in ammonemia in parallel with this effect. Uremia was increased by 60% due to the ketogenic diet without response to exercise. We measured a 10% increase in serum urate that did not change during the exercise protocol. No changes were found in glycemia or lactatemia. Discussion: Our data suggest that moderate-intensity exercise associated with a ketogenic diet can be used to study the increase in ammonemia and as a model to understand ammonia metabolism during metabolic stress.

Degenerative diseases, oxidative stress and cytochrome c oxidase function

Aging and degenerative diseases are associated with increased levels of reactive oxygen species (ROS). ROS are mostly produced in mitochondria, and their levels increase with higher mitochondrial membrane potential. Cellular respiratory control is based on inhibition of respiration by high membrane potentials. However, we have described a second mechanism of respiratory control based on allosteric inhibition of cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, at high ATP:ADP ratios. The mechanism is independent of membrane potential. We have proposed that feedback inhibition of CcO by ATP keeps the membrane potential and ROS production at low levels. Various forms of stress switch off allosteric ATP-inhibition via reversible dephosphorylation of CcO, resulting in increased membrane potential and cellular ROS levels. This mechanism is proposed to represent a missing molecular link between stress and degenerative diseases.

A genetically encoded fluorescent reporter of ATP:ADP ratio

A fluorescent sensor of adenylate nucleotides was constructed by combining a circularly permuted variant of green fluorescent protein with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The affinity for Mg-ATP is below 100 nM, as seen for the other members of the bacterial PII regulator family – a surprisingly high affinity given normal intracellular [ATP] in the millimolar range. ADP binds to the same site, competing with Mg-ATP but producing a smaller change in fluorescence. With normal physiological concentrations of ATP and ADP, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP/ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP/ADP ratio during live cell imaging.

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase.

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.

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Preface

Chronic Suppression of Acetyl-CoA Carboxylase 1 in β-Cells Impairs Insulin Secretion via Inhibition of Glucose Rather Than Lipid Metabolism

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

Tumor cells express the glycolytic regulator pyruvate kinase subtype M2 (M2-PK), which can occur in a tetrameric form with high affinity to its substrate phosphoenolpyruvate (PEP) and a dimeric form with a low PEP affinity. The transition between both conformations contributes to the control of glycolysis and is important for tumor cell proliferation and survival. Here we targeted M2-PK by synthetic peptide aptamers, which specifically bind to M2-PK and shift the isoenzyme into its low affinity dimeric conformation. The aptamer-induced dimerization and inactivation of M2-PK led to a significant decrease in the PK mass-action ratio as well as ATP:ADP ratio in the target cells. Furthermore, the expression of M2-PK-binding peptide aptamers moderately reduced the growth of immortalized NIH3T3 cell populations by decelerating cell proliferation, but without affecting apoptotic cell death. Moreover, the M2-PK-binding peptide aptamers also reduced the proliferation rate of human U-2 OS osteosarcoma cells. In the present study, we developed the first specific inhibitors of the pyruvate kinase isoenzyme type M2 and present evidence that these inhibitors moderately decelerate tumor cell proliferation.

AMPK ‐ the fuel gauge of the eukaryotic cell

The AMP‐activated protein kinase (AMPK) is expressed in essentially all eukaryotic cells and acts as a sensor of cellular energy status. It is a heterotrimer comprised of catalytic alpha subunits and regulatory beta and gamma subunits, and is only active after phosphorylation at Thr‐172 within the kinase domain by upstream kinases. AMPK is activated whenever a metabolic stress causes a decrease in the cellular ATP:ADP ratio: because of the adenylate kinase reaction, this is always accompanied by a much larger increase in the AMP:ATP ratio. Binding of AMP to the gamma subunit of AMPK causes two effects that are antagonized by ATP: (i) it inhibits dephosphorylation of Thr‐172, causing a sensitive switch to the active, phosphorylated form; (ii) it allosterically activates the phosphorylated kinase. The major upstream kinase in most cells is the tumor suppressor LKB1. Since AMPK can cause an arrest of cell growth and the cell cycle, activation of AMPK may explain why LKB1 is a tumor suppressor, although LKB1 also acts upstream of a family of twelve AMPK‐related kinases. In some cell types (e.g. neurones, endothelial cells and T cells), AMPK is also activated by increases in cell calcium, which trigger phosphorylation of Thr‐172 by CaMKK‐beta.The beta subunit of AMPK in all eukaryotes contains a carbohydrate‐binding module (CBM) that in mammalian cells causes AMPK to associate with glycogen. Recent work from our laboratory suggests that the CBM is a regulatory domain that inhibits AMPK when the reserves of energy in the form of glycogen are high.

Respiration in postharvest sugarbeet roots is not limited by respiratory capacity or adenylates

Control of respiration has largely been studied with growing and/or photosynthetic tissues or organs, but has rarely been examined in harvested and stored plant products. As nongrowing, heterotrophic organs that are reliant on respiration to provide all of their metabolic needs, harvested plant products differ dramatically in their metabolism and respiratory needs from growing and photosynthetically active plant organs, and it cannot be assumed that the same mechanism controls respiration in both actively growing and harvested plant organs. To elucidate mechanisms of respiratory control for a harvested and stored plant product, sugarbeet (Beta vulgaris L.) root respiration was characterized with respect to respiratory capacity, adenylate levels and cellular energy status in roots whose respiration was altered by wounding or cold treatment (1 degrees C) and in response to potential effectors of respiration. Respiration rate was induced by wounding in roots stored at 10 degrees C and by cold temperature in roots stored at 1 degrees C for 11-13d. Alterations in respiration rate due to wounding or storage temperature were unrelated to changes in total respiratory capacity, the capacities of the cytochrome c oxidase (COX) or alternative oxidase (AOX) pathways, adenylate concentrations or cellular energy status, measured by the ATP:ADP ratio. In root tissue, respiration was induced by exogenous NADH indicating that respiratory capacity was capable of oxidizing additional electrons fed into the electron transport chain via an external NADH dehydrogenase. Respiration was not induced by addition of ADP or a respiratory uncoupler. These results suggest that respiration rate in stored sugarbeet roots is not limited by respiratory capacity, ADP availability or cellular energy status. Since respiration in plants can be regulated by substrate availability, respiratory capacity or energy status, it is likely that a substrate, other than ADP, limits respiration in stored sugarbeet roots.

261. Disruption of mitochondrial function in the blastocyst alters expression of the chromatin remodeler ATRX

Exposure of an embryo to suboptimal environments, including poor embryo culture media or inadequate maternal diet, can disrupt fetal and placental development and whilst the exact mechanisms responsible remain unknown, perturbed embryo metabolism has been implicated. We propose that stress applied to an early embryo causes mitochondrial dysfunction, resulting in a permanent epigenetic change. Thus the aim of this study was to determine the affect of directly perturbing mitochondria in the embryo, on development, metabolism and expression of the ATP-dependant chromatin remodelling protein, ATRX. Zygotes collected from gonadotrophin stimulated C57BL/6xCBA mice were cultured to the two-cell stage and then exposed to one of three treatments; control medium (C), medium lacking pyruvate (-P; embryos dependant on the mitochondrial Malate Aspartate Shuttle, MAS) or medium lacking pyruvate plus 5µM amino-oxyacetate (AOA), a specific MAS inhibitor (-P+AOA). Blastocyst development and metabolism were assessed by determining cell number and allocation, glycolysis, and ATP:ADP ratio. Relative gene expression of ATRX, was examined using RT PCR. Embryos dependant on the MAS alone (-P) had significantly decreased blastocyst development (87.1% v. 98.2%, P < 0.05), with a compensatory increase in glycolysis (0.20 v. 0.07 pmol/cell/hr, P < 0.001) despite a decrease in ATP:ADP (0.10 v. 0.13, P < 0.06), relative to the control. Inhibition of the MAS (-P+AOA) further reduced blastocyst development,(77.3%, P < 0.001) and decreased ATP:ADP (0.08, P < 0.004), but there was no change in glycolysis relative to control embryos (0.09 pmol/cell/hr, P = 0.3). Expression of ATRX was significantly increased for –P+AOA embryos relative to the control (1.63 v. 1.0, P < 0.007) but did not differ for –P embryos (1.1). This study demonstrates that direct perturbations of mitochondrial function in the embryo compromises its metabolic regulation and blastocyst development, and the expression of the epigenetic modulator ATRX. Further studies are underway to elucidate the implications of disrupted metabolic control and this epigenetic modulator on pregnancy outcomes.

Low oxygen sensing and balancing in plant seeds: a role for nitric oxide.

Storage product accumulation in seeds of major crop species is limited by their low internal oxygen concentration. Adjustment of energy and storage metabolism to oxygen deficiency (hypoxia) in seeds is highly relevant for agriculture and biotechnology. However, the mechanisms of low-oxygen sensing and balancing remain a mystery.Here, it is shown that normal hypoxia in seeds of soybean (Glycine max) and pea (Pisum sativum) triggers a nitrite-dependent increase in endogenous nitric oxide (NO) concentrations. NO, in turn, reduces the oxygen consumption of seeds, generating a localized decrease in both ATP availability and biosynthetic activity. Increasing oxygen availability reduces endogenous NO concentrations, thereby abolishing mitochondrial and metabolic inhibition.This auto-regulatory and reversible oxygen balancing, via NO, avoids seed anoxia and suggests a key role for NO in regulating storage activity. This hypothesis is reinforced by changes in energy status (ATP:ADP ratio), steady-state metabolite concentrations and biosynthetic fluxes under NO treatment.The proposed mechanism of low-oxygen sensing and balancing in plants offers the prospect of a new field of study in crop biotechnology.New Phytologist (2007) 176: 813–823

An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells

In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. Single channel activity was measured with the patch-clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. The ATP sensitivity of K(ATP) channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K(ATP) channel regulation. The observation that the stimulatory effect of AMP on K(ATP) channels was prevented by the AK inhibitor P (1),P (5)-di(adenosine-5')pentaphosphate (Ap(5)A; 20 micromol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K(ATP) channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K(ATP) channels. Thus, these enzymes determine the open probability of K(ATP) channels and eventually the actual rate of insulin secretion.