Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.     

Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle.

AMP-activated protein kinase (AMPK) is emerging as an important energy-sensing/signaling system in skeletal muscle. This kinase is activated allosterically by 5'-AMP and inhibited allosterically by creatine phosphate. Phosphorylation of AMPK by an upstream kinase, AMPK kinase (also activated allosterically by 5'-AMP), results in activation. It is activated in both rat and human muscle in response to muscle contraction, the extent of activation depending on work rate and muscle glycogen concentration. AMPK can also be activated chemically in resting muscle with 5-aminoimidazole-4-carboxamide-riboside, which enters the muscle and is phosphorylated to form ZMP, a nucleotide that mimics the effect of 5'-AMP. Once activated, AMPK is hypothesized to phosphorylate proteins involved in triggering fatty acid oxidation and glucose uptake. Evidence is also accumulating for a role of AMPK in inducing some of the adaptations to endurance training, including the increase in muscle GLUT-4, hexokinase, uncoupling protein 3, and some of the mitochondrial oxidative enzymes. It thus appears that AMPK has the capability of monitoring intramuscular energy charge and then acutely stimulating fat oxidation and glucose uptake to counteract the increased rates of ATP utilization during muscle contraction. In addition, this system may have the capability of enhancing capacity for ATP production when the muscle is exposed to endurance training.     

AMP-activated protein kinase and the regulation of glucose transport

The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by acute increases in the cellular [AMP]/[ATP] ratio. In skeletal and/or cardiac muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress. There are many lines of evidence that increasing AMPK activity in skeletal muscle results in increased rates of glucose transport. Although similar to the effects of insulin to increase glucose transport in muscle, it is clear that the underlying mechanisms for AMPK-mediated glucose transport involve proximal signals that are distinct from that of insulin. Here, we discuss the evidence for AMPK regulation of glucose transport in skeletal and cardiac muscle and describe research investigating putative signaling mechanisms mediating this effect. We also discuss evidence that AMPK may play a role in enhancing muscle and whole body insulin sensitivity for glucose transport under conditions such as exercise, as well as the use of the AMPK activator AICAR to reverse insulin-resistant conditions. The identification of AMPK as a novel glucose transport mediator in skeletal muscle is providing important insights for the treatment and prevention of type 2 diabetes.     

Cellular energy sensing and signaling by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy sensing/signaling protein that, when activated, increases ATP production by stimulating glucose uptake and fatty acid oxidation while at the same time inhibiting ATP=consuming processes such as protein synthesis. Chronic activation of AMPK inhibits expression of lipogenic enzymes in the liver and enhances expression of mitochondrial oxidative enzymes in skeletal muscle. Deficiency of muscle LKB1, the upstream kinase of AMPK, results in greater fluctuation in energy charge during muscle contraction and decreased capacity for exercise at higher work rates. Because AMPK enhances both glucose uptake and fatty acid oxidation in skeletal muscle, it has become a target for prevention and treatment of type 2 diabetes and obesity.     

UDP-glucose pyrophosphorylase is upregulated in carriers of the porcine RN- mutation in the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy metabolism in eukaryotic cells acting as a metabolic sensor. In its activated form AMPK inhibits ATP consuming pathways and stimulates ATP generating pathways. A dominant mutation, denoted RN(-), in the porcine PRKAG3 gene, encoding the regulatory gamma3 subunit of AMPK, results in hyperaccumulation of glycogen in glycolytic skeletal muscle cells. To study the effects of this mutation on protein expression patterns in skeletal muscle, comparative proteome analysis of muscle samples from 12 animals (6 rn (+)/rn (+) and 6 RN(-)/rn (+)) was performed. The major finding of the proteome analysis was that the key enzyme in the synthesis of glycogen, UDP-glucose pyrophosphorylase, was significantly up-regulated in RN(-) carriers. This observation was subsequently supported by studies of enzyme activity and Northern blot analysis. Furthermore, the expression patterns of enzymes related to glycolysis and the citric acid cycle were also affected. Our data suggests that hyperaccumulation of glycogen mediated by the RN(-) mutation is due to an increased synthesis of glycogen.     

AMP-activated protein kinase: balancing the scales

AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a key role in the regulation of energy control. AMPK is activated in response to an increase in the ratio of AMP:ATP within the cell. Activation requires phosphorylation of threonine 172 within the catalytic subunit of AMPK by an upstream kinase. The identity of the upstream kinase in the cascade remained frustratingly elusive for many years, but was recently identified as LKB1, a kinase that is inactivated in a rare hereditary form of cancer called Peutz-Jeghers syndrome. Once activated, AMPK initiates a series of responses that are aimed at restoring the energy balance within the cell. ATP-consuming, anabolic pathways, such as fatty acid synthesis and protein synthesis are switched-off, whereas ATP-generating, catabolic pathways, such as fatty acid oxidation and glycolysis, are switched-on. More recent studies have indicated, that AMPK plays an important role in the regulation of whole body energy metabolism. The adipocyte-derived hormones, leptin and adiponectin, activate AMPK in peripheral tissues, including skeletal muscle and liver, increasing energy expenditure. In the hypothalamus, AMPK is inhibited by leptin and insulin, hormones which suppress feeding, whilst ghrelin, a hormone that increases food intake, activates AMPK. Furthermore, direct pharmacological activation of AMPK in the hypothalamus by 5-aminoimidazole-4-carboxamide ribose increases food intake in rats, demonstrating that AMPK plays a direct role in the regulation of feeding. Taken together these findings indicate that AMPK has a pivotal role in regulating pathways that control both energy expenditure and energy intake.     

AMP-activated protein kinase: the energy charge hypothesis revisited.

The AMP-activated protein kinase cascade is a sensor of cellular energy charge, and its existence provides strong support for the energy charge hypothesis first proposed by Daniel Atkinson in the 1960s. The system is activated in an ultrasensitive manner by cellular stresses that deplete ATP (and consequently elevate AMP), either by inhibiting ATP production (e.g., hypoxia), or by accelerating ATP consumption (e.g., exercise in muscle). Once activated, it switches on catabolic pathways, both acutely by phosphorylation of metabolic enzymes and chronically by effects on gene expression, and switches off many ATP-consuming processes. Recent work suggests that activation of AMPK is responsible for many of the effects of physical exercise, both the rapid metabolic effects and the adaptations that occur during training. Dominant mutations in regulatory subunit isoforms (gamma2 and gamma3) of AMPK, which appear to increase the basal activity in the absence of AMP, lead to hypertrophy of cardiac and skeletal muscle respectively.     

腺苷-磷酸激活的蛋白激酶在脂类营养代谢中的研究及应用

腺苷-磷酸激活的蛋白激酶（AMP-activated protein kinase,AMPK）是公认的重要能量感受酶。其作用与多个代谢途径有关,尤其在脂类营养代谢过程中发挥着关键的调控作用。AMPK对脂质代谢的调控通过多个信号通路进行,涉及到骨骼肌、肝脏、乳腺等多个组织。对AMPK调控脂类营养代谢机理的研究为2型糖尿病、脂肪肝、肥胖症、癌症等多种疾病的治疗提供了靶点,但AMPK在奶牛乳腺组织的研究较少,其在提高奶牛生产性能方面潜能巨大。     

Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells

Myostatin (Mstn) is a secreted growth factor predominately expressed in skeletal muscle that negatively regulates skeletal muscle mass. Recent studies have indicated that loss function of myostatin not only increases muscle mass but also improves insulin sensitivity in vivo. In the present report, we demonstrated that myostatin regulates glucose metabolism by promoting glucose consumption and glucose uptake, increasing glycolysis, and inhibiting glycogen synthesis in skeletal muscle cells. Microarray analysis revealed that myostatin upregulates several genes involved in regulating glucose metabolism such as Glut1, Glut4, Hk2, and IL-6. Further investigation of the molecular basis of these phenomena revealed that AMP-activated protein kinase (AMPK), a key component for maintaining energy homeostasis, was activated by myostatin for promotion of glycolysis. Taken together, these findings provide the first experimental evidence that myostatin regulates glucose metabolism through the AMPK signal pathway in muscle cells. Importantly, our findings highlight that continued investigation of the metabolic function of myostatin is necessary for a comprehensive understanding of its active role in the regulation of skeletal muscle energy metabolism.     

Role of AMP-activated protein kinase in autophagy and proteasome function

In this work, we have examined the possible role of AMP-activated protein kinase (a key energy sensor) in regulating intracellular protein degradation. We have found that AICAR, a known activator of AMPK, has a dual effect. On one hand, it inhibits autophagy by a mechanism independent of AMPK activity; AICAR decreases class III PI3-kinase binding to beclin-1 and this effect counteracts and reverses the known positive effect of AMPK activity on autophagy. On the other hand, AICAR inhibits the proteasomal degradation of proteins by an AMPK-dependent mechanism. This is a novel function of AMPK that allows the regulation of proteasomal activity under conditions of energy demand.     

A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle

Eukaryotic cells possess systems for sensing nutritional stress and inducing compensatory mechanisms that minimize the consumption of ATP while utilizing alternative energy sources. Such stress can also be imposed by increased energy needs, such as in skeletal muscle of exercising animals. In these studies, we consider the role of the metabolic sensor, AMP-activated protein kinase (AMPK), in the regulation of glucose transport in skeletal muscle. Expression in mouse muscle of a dominant inhibitory mutant of AMPK completely blocked the ability of hypoxia or AICAR to activate hexose uptake, while only partially reducing contraction-stimulated hexose uptake. These data indicate that AMPK transmits a portion of the signal by which muscle contraction increases glucose uptake, but other AMPK-independent pathways also contribute to the response.     

The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis

The AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status switch regulating several systems including glucose and lipid metabolism. Recently, AMPK has been implicated in the control of skeletal muscle mass by decreasing mTORC1 activity and increasing protein degradation through regulation of ubiquitin-proteasome and autophagy pathways. In this review, we give an overview of the central role of AMPK in the control of skeletal muscle plasticity. We detail particularly its implication in the control of the hypertrophic and atrophic signaling pathways. In the light of these cumulative and attractive results, AMPK appears as a key player in regulating muscle homeostasis and the modulation of its activity may constitute a therapeutic potential in treating muscle wasting syndromes in humans.     

Regulation of nutrient uptake by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that is a key regulator of energy balance at both the cellular and whole-body level. AMPK acts to stimulate ATP production and reduce ATP consumption when cellular ATP levels fall, thereby normalizing energy balance. Given the central role of AMPK in cellular carbohydrate and lipid metabolism, AMPK activation has been proposed to be a therapeutic target for conditions associated with dysfunctional nutrient metabolism including obesity, type 2 diabetes, hepatic steatosis, cardiovascular diseases and cancer. One way by which increased ATP production can be achieved is by increasing the supply of nutrient substrates. In the 1990s, AMPK activation was demonstrated to stimulate glucose uptake in striated muscle, thereby improving substrate supply for ATP production. Subsequently AMPK activation was postulated to underlie the increase in glucose uptake that occurs during muscle contraction. More recently, however, several lines of evidence have demonstrated that AMPK activation is unlikely to be required for contraction-mediated glucose uptake. Furthermore, despite the importance of AMPK in cellular and whole-body metabolism, far fewer studies have investigated either the role of AMPK in glucose uptake by non-muscle tissues or whether AMPK regulates the uptake of fatty acids. In the present review, we discuss the role of AMPK in nutrient uptake by tissues, focusing on glucose uptake out with muscle and fatty acid uptake.     

Isozymes of creatine kinase in mammalian cell culures

Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine-phosphocreatine–creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts. Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats. The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived. Furthermore, the addition of creatine to culture media stimulates its synthesis. The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line. Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine-phosphocreatine–creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present. This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally.     

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.     

Role of AMP-activated protein kinase in the regulation of gene transcription

AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism in response to changes in the energy status of the cells. AMPK was known to shut down energy-consuming pathways in response to a fall in the ATP/AMP ratio by phosphorylating key enzymes of intermediate metabolism. Here we will discuss the recent evidence implicating AMPK in the regulation of gene expression in mammals, mainly in the liver and in the pancreatic beta-cells.