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AMP-activated Protein Kinase (AMP-activat + protein_kinase)
Selected AbstractsImpaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodellingACTA PHYSIOLOGICA, Issue 1 2010A. Daniels Abstract Aim:, To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. Methods:, Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. Results:, The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or ,1-adrenoceptor (,1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac ,-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. Conclusion:, db/db mice show impaired cardiac functional reserve capacity during maximal ,-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism. [source] Structure and function of AMP-activated protein kinaseACTA PHYSIOLOGICA, Issue 1 2009J. S. Oakhill Abstract AMP-activated protein kinase (AMPK) regulates metabolism in response to energy demand and supply. AMPK is activated in response to rises in intracellular AMP or calcium-mediated signalling and is responsible for phosphorylating a wide variety of substrates. Recent structural studies have revealed the architecture of the ,,, subunit interactions as well as the AMP binding pockets on the , subunit. The , catalytic domain (1,280) is autoinhibited by a C-terminal tail (313,335), which is proposed to interact with the small lobe of the catalytic domain by homology modelling with the MARK2 protein structure. Two direct activating drugs have been reported for AMPK, the thienopyridone compound A769662 and PTI, which may activate by distinct mechanisms. [source] AMP-activated protein kinase: a core signalling pathway in the heartACTA PHYSIOLOGICA, Issue 1 2009A. S. Kim Abstract Over the past decade, AMP-activated protein kinase (AMPK) has emerged as an important intracellular signalling pathway in the heart. Activated AMPK stimulates the production of ATP by regulating key steps in both glucose and fatty acid metabolism. It has an inhibitory effect on cardiac protein synthesis. AMPK also interacts with additional intracellular signalling pathways in a coordinated network that modulates essential cellular processes in the heart. Evidence is accumulating that AMPK may protect the heart from ischaemic injury and limit the development of cardiac myocyte hypertrophy to various stimuli. Heart AMPK is activated by hormones, cytokines and oral hypoglycaemic drugs that are used in the treatment of type 2 diabetes. The tumour suppressor LKB1 is the major regulator of AMPK activity, but additional upstream kinases and protein phosphatases also contribute. Mutations in the regulatory ,2 subunit of AMPK lead to an inherited syndrome of hypertrophic cardiomyopathy and ventricular pre-excitation, which appears to be due to intracellular glycogen accumulation. Future research promises to elucidate the molecular mechanisms responsible for AMPK activation, novel downstream AMPK targets, and the therapeutic potential of targeting AMPK for the prevention and treatment of myocardial ischaemia or cardiac hypertrophy. [source] AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therapeutic perspectivesACTA PHYSIOLOGICA, Issue 1 2009B. Viollet Abstract As the liver is central in the maintenance of glucose homeostasis and energy storage, knowledge of the physiology as well as physiopathology of hepatic energy metabolism is a prerequisite to our understanding of whole-body metabolism. Hepatic fuel metabolism changes considerably depending on physiological circumstances (fed vs. fasted state). In consequence, hepatic carbohydrate, lipid and protein synthesis/utilization are tightly regulated according to needs. Fatty liver and hepatic insulin resistance (both frequently associated with the metabolic syndrome) or increased hepatic glucose production (as observed in type 2 diabetes) resulted from alterations in substrates oxidation/storage balance in the liver. Because AMP-activated protein kinase (AMPK) is considered as a cellular energy sensor, it is important to gain understanding of the mechanism by which hepatic AMPK coordinates hepatic energy metabolism. AMPK has been implicated as a key regulator of physiological energy dynamics by limiting anabolic pathways (to prevent further ATP consumption) and by facilitating catabolic pathways (to increase ATP generation). Activation of hepatic AMPK leads to increased fatty acid oxidation and simultaneously inhibition of hepatic lipogenesis, cholesterol synthesis and glucose production. In addition to a short-term effect on specific enzymes, AMPK also modulates the transcription of genes involved in lipogenesis and mitochondrial biogenesis. The identification of AMPK targets in hepatic metabolism should be useful in developing treatments to reverse metabolic abnormalities of type 2 diabetes and the metabolic syndrome. [source] AMP-activated protein kinase , a sensor of glycogen as well as AMP and ATP?ACTA PHYSIOLOGICA, Issue 1 2009A. McBride Abstract The classical role of the AMP-activated protein kinase (AMPK) is to act as a sensor of the immediate availability of cellular energy, by monitoring the concentrations of AMP and ATP. However, the , subunits of AMPK contain a glycogen-binding domain, and in this review we develop the hypothesis that this is a regulatory domain that allows AMPK to act as a sensor of the status of cellular reserves of energy in the form of glycogen. We argue that the pool of AMPK that is bound to the glycogen particle is in an active state when glycogen particles are fully synthesized, causing phosphorylation of glycogen synthase at site 2 and providing a feedback inhibition of further extension of the outer chains of glycogen. However, when glycogen becomes depleted, the glycogen-bound pool of AMPK becomes inhibited due to binding to ,1,6-linked branch points exposed by the action of phosphorylase and/or debranching enzyme. This allows dephosphorylation of site 2 on glycogen synthase by the glycogen-bound form of protein phosphatase-1, promoting rapid resynthesis of glycogen and replenishment of glycogen stores. This is an extension of the classical role of AMPK as a ,guardian of cellular energy', in which it ensures that cellular energy reserves are adequate for medium-term requirements. The literature concerning AMPK, glycogen structure and glycogen-binding proteins that led us to this concept is reviewed. [source] Insulin resistance and fuel homeostasis: the role of AMP-activated protein kinaseACTA PHYSIOLOGICA, Issue 1 2009B. D. Hegarty Abstract The worldwide prevalence of type 2 diabetes (T2D) and related disorders of the metabolic syndrome (MS) has reached epidemic proportions. Insulin resistance (IR) is a major perturbation that characterizes these disorders. Extra-adipose accumulation of lipid, particularly within the liver and skeletal muscle, is closely linked with the development of IR. The AMP-activated protein kinase (AMPK) pathway plays an important role in the regulation of both lipid and glucose metabolism. Through its effects to increase fatty acid oxidation and inhibit lipogenesis, AMPK activity in the liver and skeletal muscle could be expected to ameliorate lipid accumulation and associated IR in these tissues. In addition, AMPK promotes glucose uptake into skeletal muscle and suppresses glucose output from the liver via insulin-independent mechanisms. These characteristics make AMPK a highly attractive target for the development of strategies to curb the prevalence and costs of T2D. Recent insights into the regulation of AMPK and mechanisms by which it modulates fuel metabolism in liver and skeletal muscle are discussed here. In addition, we consider the arguments for and against the hypothesis that dysfunctional AMPK contributes to IR. Finally we review studies which assess AMPK as an appropriate target for the prevention and treatment of T2D and MS. [source] AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?ACTA PHYSIOLOGICA, Issue 1 2009T. E. Jensen Abstract In skeletal muscle, the contraction-activated heterotrimeric 5,-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved in mitochondrial biogenesis and other aspects of promoting an oxidative muscle phenotype. Here, the current knowledge on the expression of AMPK subunits in human quadriceps muscle and evidence from rodent studies suggesting distinct AMPK subunit expression pattern in different muscle types is reviewed. Then, the intensity and time dependence of AMPK activation in human quadriceps and rodent muscle are evaluated. Subsequently, a major part of this review critically examines the evidence supporting a necessary and/or sufficient role of AMPK in a broad spectrum of skeletal muscle contraction-relevant processes. These include glucose uptake, glycogen synthesis, post-exercise insulin sensitivity, fatty acid (FA) uptake, intramuscular triacylglyceride hydrolysis, FA oxidation, suppression of protein synthesis, proteolysis, autophagy and transcriptional regulation of genes relevant to promoting an oxidative phenotype. [source] Cell hydration and mTOR-dependent signallingACTA PHYSIOLOGICA, Issue 1-2 2006F. Schliess Abstract Insulin- and amino acid-induced signalling by the mammalian target of rapamycin (mTOR) involves hyperphosphorylation of the p70 ribosomal S6 protein kinase (p70S6-kinase) and the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 and contributes to regulation of protein metabolism. This review considers the impact of cell hydration on mTOR-dependent signalling. Although hypoosmotic hepatocyte swelling in some instances activates p70S6-kinase, the hypoosmolarity-induced proteolysis inhibition in perfused rat liver is insensitive to mTOR inhibition by rapamycin. Likewise, swelling-dependent proteolysis inhibition by insulin and swelling-independent proteolysis inhibition by leucine, a potent activator of p70S6-kinase and 4E-BP1 hyperphosphorylation, in perfused rat liver is insensitive to rapamycin, indicating that at least rapamycin-sensitive mTOR signalling is not involved. Hyperosmotic dehydration in different cell types produces inactivation of signalling components around mTOR, thereby attenuating insulin-induced glucose uptake, glycogen synthesis, and lipogenesis in adipocytes, and MAP-kinase phosphatase MKP-1 expression in hepatoma cells. Direct inactivation of mTOR, stimulation of the AMP-activated protein kinase, and the destabilization of individual proteins may impair mTOR signalling under dehydrating conditions. Further investigation of the crosstalk between the mTOR pathway(s) and hyperosmotic signalling will improve our understanding about the contribution of cell hydration changes in health and disease and will provide further rationale for fluid therapy of insulin-resistant states. [source] The role of intramuscular lipid in insulin resistanceACTA PHYSIOLOGICA, Issue 4 2003B. D. Hegarty Abstract There is interest in how altered lipid metabolism could contribute to muscle insulin resistance. Many animal and human states of insulin resistance have increased muscle triglyceride content, and there are now plausible mechanistic links between muscle lipid accumulation and insulin resistance, which go beyond the classic glucose,fatty acid cycle. We postulate that muscle cytosolic accumulation of the metabolically active long-chain fatty acyl CoAs (LCACoA) is involved, leading to insulin resistance and impaired insulin signalling or impaired enzyme activity (e.g. glycogen synthase or hexokinase) either directly or via chronic translocation/activation of mediators such as a protein kinase C (particularly PKC , and ,). Ceramides and diacylglycerols (DAGs) have also been implicated in forms of lipid-induced muscle insulin resistance. Dietary lipid-induced muscle insulin resistance in rodents is relatively easily reversed by manipulations that lessen cytosolic lipid accumulation (e.g. diet change, exercise or fasting). PPAR agonists (both , and ,) also lower muscle LCACoA and enhance insulin sensitivity. Activation of AMP-activated protein kinase (AMPK) by AICAR leads to muscle enhancement (especially glycolytic muscle) of insulin sensitivity, but involvement of altered lipid metabolism is less clear cut. In rodents there are similarities in the pattern of muscle lipid accumulation/PKC translocation/altered insulin signalling/insulin resistance inducible by 3,5-h acute free fatty acid elevation, 1,4 days intravenous glucose infusion or several weeks of high-fat feeding. Recent studies extend findings and show relevance to humans. Muscle cytosolic lipids may accumulate either by increased fatty acid flux into muscle, or by reduced fatty acid oxidation. In some circumstances muscle insulin resistance may be an adaptation to optimize use of fatty acids when they are the predominant available energy fuel. The interactions described here are fundamental to optimizing therapy of insulin resistance based on alterations in muscle lipid metabolism. [source] Metformin action on AMP-activated protein kinase: a translational research approach to understanding a potential new therapeutic targetDIABETIC MEDICINE, Issue 10 2010J. G. Boyle Diabet. Med. 27, 1097,1106 (2010) Abstract Clinical studies in Type 2 diabetes mellitus have shown that the effects of metformin go beyond improving HbA1c and include reductions in cardiovascular endpoints. Metformin therapy has been widely used in the treatment of Type 2 diabetes for many years, yet the precise mode of action remains uncertain. It has recently been proposed that metformin-mediated stimulation of hepatic AMP-activated protein kinase (AMPK) underlies the hypoglycaemic effects of metformin. AMPK is a heterotrimeric enzyme that is expressed in many tissues and plays a central role in the regulation of energy homoeostasis. Furthermore, there is increasing evidence that AMPK is implicated in the pathophysiology of cardiovascular and metabolic diseases. The generation of more specific and potent activators of AMPK, however, could have additional metabolic and vascular benefits for patients with Type 2 diabetes. [source] The mechanisms that underlie glucose sensing during hypoglycaemia in diabetesDIABETIC MEDICINE, Issue 5 2008R. McCrimmon Abstract Hypoglycaemia is a frequent and greatly feared side-effect of insulin therapy, and a major obstacle to achieving near-normal glucose control. This review will focus on the more recent developments in our understanding of the mechanisms that underlie the sensing of hypoglycaemia in both non-diabetic and diabetic individuals, and how this mechanism becomes impaired over time. The research focus of my own laboratory and many others is directed by three principal questions. Where does the body sense a falling glucose? How does the body detect a falling glucose? And why does this mechanism fail in Type 1 diabetes? Hypoglycaemia is sensed by specialized neurons found in the brain and periphery, and of these the ventromedial hypothalamus appears to play a major role. Neurons that react to fluctuations in glucose use mechanisms very similar to those that operate in pancreatic B- and A-cells, in particular in their use of glucokinase and the KATP channel as key steps through which the metabolic signal is translated into altered neuronal firing rates. During hypoglycaemia, glucose-inhibited (GI) neurons may be regulated by the activity of AMP-activated protein kinase. This sensing mechanism is disturbed by recurrent hypoglycaemia, such that counter-regulatory defence responses are triggered at a lower glucose level. Why this should occur is not yet known, but it may involve increased metabolism or fuel delivery to glucose-sensing neurons or alterations in the mechanisms that regulate the stress response. [source] Acute signalling responses to intense endurance training commenced with low or normal muscle glycogenEXPERIMENTAL PHYSIOLOGY, Issue 2 2010Wee Kian Yeo We have previously demonstrated that well-trained subjects who completed a 3 week training programme in which selected high-intensity interval training (HIT) sessions were commenced with low muscle glycogen content increased the maximal activities of several oxidative enzymes that promote endurance adaptations to a greater extent than subjects who began all training sessions with normal glycogen levels. The aim of the present study was to investigate acute skeletal muscle signalling responses to a single bout of HIT commenced with low or normal muscle glycogen stores in an attempt to elucidate potential mechanism(s) that might underlie our previous observations. Six endurance-trained cyclists/triathletes performed a 100 min ride at ,70% peak O2 uptake (AT) on day 1 and HIT (8 × 5 min work bouts at maximal self-selected effort with 1 min rest) 24 h later (HIGH). Another six subjects, matched for fitness and training history, performed AT on day 1 then 1,2 h later, HIT (LOW). Muscle biopsies were taken before and after HIT. Muscle glycogen concentration was higher in HIGH versus LOW before the HIT (390 ± 28 versus 256 ± 67 ,mol (g dry wt),1). After HIT, glycogen levels were reduced in both groups (P < 0.05) but HIGH was elevated compared with LOW (229 ± 29 versus 124 ± 41 ,mol (g dry wt),1; P < 0.05). Phosphorylation of 5,AMP-activated protein kinase (AMPK) increased after HIT, but the magnitude of increase was greater in LOW (P < 0.05). Despite the augmented AMPK response in LOW after HIT, selected downstream AMPK substrates were similar between groups. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was unchanged for both groups before and after the HIT training sessions. We conclude that despite a greater activation AMPK phosphorylation when HIT was commenced with low compared with normal muscle glycogen availability, the localization and phosphorylation state of selected downstream targets of AMPK were similar in response to the two interventions. [source] Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistanceEXPERIMENTAL PHYSIOLOGY, Issue 1 2010T. Fuentes To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean ±s.d. age, 31 ± 5 years; height, 184 ± 9 cm; weight, 91 ± 13 kg; and percentage body fat, 24.8 ± 5.8%) and 10 obese (age, 30 ± 7 years; height, 184 ± 8 cm; weight, 115 ± 8 kg; and percentage body fat, 34.9 ± 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPK, and ACC, phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPK, and ACC, phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles. [source] Role of exercise-induced brain-derived neurotrophic factor production in the regulation of energy homeostasis in mammalsEXPERIMENTAL PHYSIOLOGY, Issue 12 2009Bente K. Pedersen Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal development and plasticity and plays a role in learning and memory. Moreover, it is well established that BDNF plays a role in the hypothalamic pathway that controls body weight and energy homeostasis. Recent evidence identifies BDNF as a player not only in central metabolism, but also in regulating energy metabolism in peripheral organs. Low levels of BDNF are found in patients with neurodegenerative diseases, including Alzheimer's disease and major depression. In addition, BDNF levels are low in obesity and independently so in patients with type 2 diabetes. Brain-derived neurotrophic factor is expressed in non-neurogenic tissues, including skeletal muscle, and exercise increases BDNF levels not only in the brain and in plasma, but in skeletal muscle as well. Brain-derived neurotrophic factor mRNA and protein expression was increased in muscle cells that were electrically stimulated, and BDNF increased phosphorylation of AMP-activated protein kinase (AMPK) and acetyl coenzyme A carboxylase-beta (ACC,) and enhanced fatty oxidation both in vitro and ex vivo. These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing lipid oxidation in skeletal muscle via activation of AMPK. Thus, BDNF appears to play a role both in neurobiology and in central as well as peripheral metabolism. The finding of low BDNF levels both in neurodegenerative diseases and in type 2 diabetes may explain the clustering of these diseases. Brain-derived neurotrophic factor is likely to mediate some of the beneficial effects of exercise with regard to protection against dementia and type 2 diabetes. [source] Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5,AMP-activated protein kinase (AMPK)FEBS JOURNAL, Issue 13 2004AMPK by adenoviral gene transfer technique, Studies using H9c2 cells overexpressing MCD Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria. [source] Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytesFEBS JOURNAL, Issue 15 2002Ulrike Krause Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways. [source] Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A-related phosphatase inhibitorGENES TO CELLS, Issue 5 2009Yuichiro Hanyu Calcium/calmodulin-dependent protein kinase (CaMK) is required for diverse cellular functions, and similar kinases exist in fungi. Although mammalian CaMK kinase (CaMKK) activates CaMK and also evolutionarily-conserved AMP-activated protein kinase (AMPK), CaMKK is yet to be established in yeast. We here report that the fission yeast Schizosaccharomyces pombe Ssp1 kinase, which controls G2/M transition and response to stress, is the putative CaMKK. Ssp1 has a CaM binding domain (CBD) and associates with 14-3-3 proteins as mammalian CaMKK does. Temperature-sensitive ssp1 mutants isolated are defective in the tolerance to limited glucose, and this tolerance requires the conserved stretch present between the kinase domain and CBD. Sds23, multi-copy suppressor for mutants defective in type 1 phosphatase and APC/cyclosome, also suppresses the ssp1 phenotype, and is required for the tolerance to limited glucose. We demonstrate that Sds23 binds to type 2A protein phosphatases (PP2A) and PP2A-related phosphatase Ppe1, and that Sds23 inhibits Ppe1 phosphatase activity. Ssp1 and Ppe1 thus seem to antagonize in utilizing limited glucose. We also show that Ppk9 and Ssp2 are the catalytic subunits of AMPK and AMPK-related kinases, respectively, which bind to common ,-(Amk2) and ,-(Cbs2) subunits. [source] Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell lineINTERNATIONAL ENDODONTIC JOURNAL, Issue 3 2008Y. Fukuyama Abstract Aim, To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP-activated protein kinase (AMPK) in rat dental pulp cell line RPC-C2A. Methodology, RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 °C, in a humidified atmosphere at 5% CO2. Cells were cultured in the presence or absence of H2O2 for up to 60 min at concentrations of from 0.1 to 3.0 mmol L,1. Cell viability was analysed by WST-1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic ,1 and regulatory ,1 and ,1 subunit isoforms. The effect of silencing AMPK,1 on cell viability was determined using siRNA. Results, Exposure to H2O2 decreased cell viability in a time- and dose-dependent manner. The catalytic AMPK,1 subunit and its activated form, phospho-AMPK,, increased with exposure to H2O2 in a time- and dose-dependent manner, whereas the regulatory ,1 and ,1 subunits showed no change. Downregulation of AMPK,1 resulted in a reduction in cell viability in H2O2 -treated cells at a concentration of 0.1 mmol L,1 for 30 min incubation, indicating an increased sensitivity to H2O2. Conclusions, Reactive oxygen induced energy fuel gauge enzyme AMPK, expression and its activation by phosphorylation in RPC-C2A cells, suggesting that AMPK is essential for protection against H2O2 -induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine,pulp complex injured by reactive oxygen. [source] Molecular Genetic Analysis of PRKAG2 in Sporadic Wolff-Parkinson-White SyndromeJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 3 2003CARL J. VAUGHAN Introduction: Mutations in the PRKAG2 gene that encodes the gamma2 regulatory subunit of AMP-activated protein kinase have been shown to cause autosomal dominant Wolff-Parkinson-White (WPW) syndrome associated with hypertrophic cardiomyopathy. Prior studies focused on familial WPW syndrome associated with other heart disease such as hypertrophic cardiomyopathy. However, such disease accounts for only a small fraction of WPW cases, and the contribution of PRKAG2 mutations to sporadic isolated WPW syndrome is unknown. Methods and Results: Subjects presented for clinical electrophysiologic evaluation of suspected WPW syndrome. WPW syndrome was diagnosed by ECG findings and/or by clinically indicated electrophysiologic study prior to enrollment. Echocardiography excluded hypertrophic cardiomyopathy. Denaturing high-performance liquid chromatography and automated sequencing were used to search for PRKAG2 mutations. Twenty-six patients without a family history of WPW syndrome were studied. No subject had cardiac hypertrophy, and only one patient had associated congenital heart disease. Accessory pathways were detected at diverse locations within the heart. Two polymorphisms in PRKAG2 were detected. [inv6+36insA] occurred in intron 6 in 4 WPW patients and [inv10+10delT] in intron 10 in 1 WPW patient. Both occurred in normal unrelated chromosomes. No PRKAG2 mutations were detected. Conclusion: This study shows that, unlike familial WPW syndrome, constitutional mutation of PRKAG2 is not commonly associated with sporadic WPW syndrome. Although polymorphisms within the PRKAG2 introns were identified, there is no evidence that these polymorphisms predispose to accessory pathway formation because their frequency is similarly high in both WPW patients and normal individuals. Further studies are warranted to identify the molecular basis of common sporadic WPW syndrome.(J Cardiovasc Electrophysiol, Vol. 14, pp. 263-268, March 2003) [source] AMP-activated protein kinase enhances the expression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt signaling in C2C12 myotubesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jun F. Tong Abstract Two muscle-specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor-1 (IGF-1) pathway inhibits muscle UL expression through Akt-mediated inhibition of FoxO transcription factors, while AMP-activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF-1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0,mM 5-aminoimidazole-4-carboxamide-1-,- D -ribofuranoside (AICAR) in the presence or absence of 50,ng/ml IGF-1. IGF-1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0,mM AICAR synergized IGF-1-induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF-1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK. J. Cell. Biochem. 108: 458,468, 2009. © 2009 Wiley-Liss, Inc. [source] Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathwayAGING CELL, Issue 6 2009David S. Williams Summary Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF-16, and the energy sensor, AMP-activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health-related benefits of dietary restriction. [source] Muscle mitochondrial activity increases rapidly after an endotoxin challenge in human volunteersACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 3 2009K. FREDRIKSSON Background: Mitochondrial derangements in muscle of patients suffering from sepsis have been established in several studies and have been related to muscle dysfunction and organ failure. It is not possible to study the early phase of sepsis in patients; therefore, we used a human endotoxaemia model to study the effect of early sepsis on muscle mitochondria. Methods: Seven healthy male volunteers received a standardised endotoxin challenge. Muscle biopsies were obtained immediately before the challenge, and at 2 and 4 h following the endotoxin challenge. The muscle biopsies were analysed for maximal activities of citrate synthase and complexes I and IV of the respiratory chain. In addition, total and mitochondrial superoxide dismutase (SOD) activities were analysed. The concentrations of ATP, creatine phosphate and lactate were analysed to assess the cellular energy status. Total and phosphorylated AMP-activated protein kinase (AMPK-P), a key regulator in intracellular energy metabolism, was measured. Results: Activities of citrate synthase and complex I were significantly increased 2 h after the endotoxin challenge. SOD activities were unaffected by the endotoxin challenge. No changes in ATP, creatine phosphate or lactate were observed. Neither total nor AMPK-P changed. Conclusions: An endotoxin challenge given to healthy volunteers rapidly increases mitochondrial enzyme activity in skeletal muscle. The results of this human model indicate that possibly early during sepsis, mitochondrial activity might be increased in contrast to what has been shown in the later phases of sepsis. It is possible that this early activation leads to exhaustion of the mitochondria and a decreased function later during sepsis. [source] Comparative study between the effect of the peroxisome proliferator activated receptor-, ligands fenofibrate and n-3 polyunsaturated fatty acids on activation of 5,-AMP-activated protein kinase-,1 in high-fat fed ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2009Tarek M. Kamal Motawi Abstract Objectives Obesity is a risk factor for type 2 diabetes mellitus. It results from an energy imbalance in which energy intake exceeds energy expenditure. The cellular fuel gauge 5,-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of one catalytic subunit (,) and two non-catalytic subunits (, and ,), and approximately equal levels of ,1 and ,2 complexes are present in the liver. AMPK regulates metabolic pathways in response to metabolic stress and in particular ATP depletion to switch on energy-producing catabolic pathways such as ,-oxidation of fatty acids and switch off energy-depleting processes such as synthesis of fatty acid and cholesterol. A high-fat diet alters AMPK-,1 gene expression in the liver and skeletal muscle of rats and results in body weight gain and hyperglycaemia. The aim of this study was to investigate and compare the potential effects of peroxisome proliferator-activated receptor (PPAR)-, agonists fenofibrate and n-3 polyunsaturated fatty acids (PUFAs) in modulation of AMPK-,1 activity in liver and skeletal muscle of high-fat diet fed rats. Methods Reverse transcription,polymerase chain reaction was used for determination of AMPK-,1 in liver and soleus muscle and both PPAR-, and CPT-1 in hepatic tissues. Serum, total cholesterol, triacylglycerol, fatty acid and fasting blood glucose were determined colorimetrically. Key findings Both PPAR-, agonists, fenofibrate and n-3 PUFA, increased the mRNA expression of AMPK-,1 activity in liver and skeletal muscle of obese diabetic rats. Fenofibrate was superior in its activation of hepatic mRNA expression of AMPK-, 1 to exert more lipolytic effect and body weight reduction, as estimated through the decrease of triacylglycerol output and serum levels of fatty acid on the one hand and the increase in CPT-1 mRNA expression, the key enzyme in ,-oxidation of fatty acid, on the other hand. n-3 PUFA activated AMPK-,1 mRNA expression in skeletal muscle much more than fenofibrate to reveal more hypoglycaemic effect. Conclusions The PPAR-, agonists fenofibrate and n-3 PUFA could efficiently activate AMPK-,1 mRNA expression in liver and skeletal muscle to exert body weight reduction and hypoglycaemic effect, respectively. [source] Molecular Mechanisms of Alcoholic Fatty LiverALCOHOLISM, Issue 2 2009Vishnudutt Purohit Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD+ ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-, (PPAR-,) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-, (TNF-,) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-, agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-, and insulin signaling pathways while down-regulating SREBP-1 activity and suppressing TNF-, production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal ,-oxidation of fatty acids could contribute to fat accumulation in the liver. [source] Snf1-independent, glucose-resistant transcription of Adr1-dependent genes in a mediator mutant of Saccharomyces cerevisiaeMOLECULAR MICROBIOLOGY, Issue 2 2009Elton T. Young Summary Glucose represses transcription of a network of co-regulated genes in Saccharomyces cerevisiae, ensuring that it is utilized before poorer carbon sources are metabolized. Adr1 is a glucose-regulated transcription factor whose promoter binding and activity require Snf1, the yeast homologue of the AMP-activated protein kinase in higher eukaryotes. In this study we found that a temperature-sensitive allele of MED14, a Mediator middle subunit that tethers the tail to the body, allowed a low level of Adr1-independent ADH2 expression that can be enhanced by Adr1 in a dose-dependent manner. A low level of TATA-independent ADH2 expression was observed in the med14 -truncated strain and transcription of ADH2 and other Adr1-dependent genes occurred in the absence of Snf1 and chromatin remodeling coactivators. Loss of ADH2 promoter nucleosomes had occurred in the med14 strain in repressing conditions and did not require ADR1. A global analysis of transcription revealed that loss of Med14 function was associated with both up- and down- regulation of several groups of co-regulated genes, with ADR1 -dependent genes being the most highly represented in the upregulated class. Expression of most genes was not significantly affected by the loss of Med14 function. [source] Epigallocatechin gallate (EGCG) attenuates high glucose-induced insulin signaling blockade in human hepG2 hepatoma cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 8 2008Chih-Li Lin Abstract Insulin resistance is the primary characteristic of type 2 diabetes which as a result of insulin signaling defects. It has been suggested that the tea polyphenol (,)-epigallocatechin-3-gallate (EGCG) displays some antidiabetic effects, but the mechanism for EGCG insulin-enhancing effects is incompletely understood. In the present study, the investigations of EGCG on insulin signaling are performed in insulin-responsive human HepG2 cells cotreated with high glucose. We found that the high glucose condition causes significant increasing Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1), leading to reduce insulin-stimulated phosphorylation of Akt. As the results, the insulin metabolic effects of glycogen synthesis and glucose uptake are inhibited by high glucose. However, the treatment of EGCG improves insulin-stimulated downsignaling by reducing IRS-1 Ser307 phosphorylation. Furthermore, we also demonstrated these EGCG effects are essential depends on the 5,-AMP-activated protein kinase (AMPK) activation. Together, our data suggest a putative link between high glucose and insulin resistance in HepG2 cells, and the EGCG treatment attenuates insulin signaling blockade by reducing IRS-1 Ser307 phosphorylation through the AMPK activation pathway. [source] Fatty acid oxidation and meiotic resumption in mouse oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2009Stephen M. Downs We have examined the potential role of fatty acid oxidation (FAO) in AMP-activated protein kinase (AMPK)-induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase-1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP-arrested cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75-induced maturation but was ineffective in cerulenin-treated oocytes, suggesting that the meiosis-inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO. Mol. Reprod. Dev. 76: 844,853, 2009. © 2009 Wiley-Liss, Inc. [source] Differential expression of skeletal muscle proteins in high-fat diet-fed rats in response to capsaicin feedingPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2010Dong Hyun Kim Abstract In this study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague,Dawley rats fed with a high-fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8,wk. After HFD feeding, capsaicin-treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2-DE for detection of HFD-associated markers. Proteomic analysis using 2-DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for the determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased uncoupling protein 3 (UCP3) protein expression in HFD-fed rats, whereas contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMP-activated protein kinase (AMPIC) CP3 and acetyl-CoA carboxylase (ACC). To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK-ACC-malonyl-CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet-induced alterations of protein expression that are essential for energy expenditure in rat muscle. [source] AMP-activated protein kinase signalling pathways are down regulated and skeletal muscle development impaired in fetuses of obese, over-nourished sheepTHE JOURNAL OF PHYSIOLOGY, Issue 10 2008Mei J. Zhu Maternal obesity and over-nutrition give rise to both obstetric problems and neonatal morbidity. The objective of this study was to evaluate effects of maternal obesity and over-nutrition on signalling of the AMP-activated protein kinase (AMPK) pathway in fetal skeletal muscle in an obese pregnant sheep model. Non-pregnant ewes were assigned to a control group (Con, fed 100% of NRC nutrient recommendations, n= 7) or obesogenic group (OB, fed 150% of National Research Council (NRC) recommendations, n= 7) diet from 60 days before to 75 days after conception (term 150 days) when fetal semitendinosus skeletal muscle (St) was sampled. OB mothers developed severe obesity accompanied by higher maternal and fetal plasma glucose and insulin levels. In fetal St, activity of phosphoinositide-3 kinase (PI3K) associated with insulin receptor substrate-1 (IRS-1) was attenuated (P < 0.05), in agreement with the increased phophorylation of IRS-1 at serine 1011. Phosphorylation of AMP-activated protein kinase (AMPK) at Thr 172, acetyl-CoA carboxylase at Ser 79, tuberous sclerosis 2 at Thr 1462 and eukaryotic translation initiation factor 4E-binding protein 1 at Thr 37/46 were reduced in OB compared to Con fetal St. No difference in energy status (AMP/ATP ratio) was observed. The expression of protein phosphatase 2C was increased in OB compared to Con fetal St. Plasma tumour necrosis factor , (TNF,) was increased in OB fetuses indicating an increased inflammatory state. Expression of peroxisome proliferator-activated receptor , (PPAR,) was higher in OB St, indicating enhanced adipogenesis. The glutathione: glutathione disulphide ratio was also lower, showing increased oxidative stress in OB fetal St. In summary, we have demonstrated decreased signalling of the AMPK system in skeletal muscle of fetuses of OB mothers, which may play a role in altered muscle development and development of insulin resistance in the offspring. [source] Acute physical exercise reverses S -nitrosation of the insulin receptor, insulin receptor substrate 1 and protein kinase B/Akt in diet-induced obese Wistar ratsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2008José R. Pauli Early evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance. Here, we investigated whether this insulin resistance, mediated by S -nitrosation of proteins involved in early steps of the insulin signal transduction pathway, could be reversed by acute physical exercise. Rats on a high-fat diet were subjected to swimming for two 3 h-long bouts, separated by a 45 min rest period. Two or 16 h after the exercise protocol the rats were killed and proteins from the insulin signalling pathway were analysed by immunoprecipitation and immunoblotting. We demonstrated that a high-fat diet led to an increase in the iNOS protein level and S -nitrosation of insulin receptor , (IR,), insulin receptor substrate 1 (IRS1) and Akt. Interestingly, an acute bout of exercise reduced iNOS expression and S -nitrosation of proteins involved in the early steps of insulin action, and improved insulin sensitivity in diet-induced obesity rats. Furthermore, administration of GSNO (NO donor) prevents this improvement in insulin action and the use of an inhibitor of iNOS (l- N6 -(1-iminoethyl)lysine; l -NIL) simulates the effects of exercise on insulin action, insulin signalling and S -nitrosation of IR,, IRS1 and Akt. In summary, a single bout of exercise reverses insulin sensitivity in diet-induced obese rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S -nitrosation of IR/IRS1/Akt. The decrease in iNOS protein expression in the muscle of diet-induced obese rats after an acute bout of exercise was accompanied by an increase in AMP-activated protein kinase (AMPK) activity. These results provide new insights into the mechanism by which exercise restores insulin sensitivity. [source] | ||||||||||||||||||||||||||||