The relationship between AMP-activated protein kinase activity and AMP concentration in the isolated perfused rat heart.

The objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of beta-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by (31)P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 microm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol.min(-1).microg of protein(-1). The half-maximal AMPK activation (A(0.5)) was 1.8 +/- 0.3 microm AMP. Measurements in vitro have reported similar AMPK A(0.5) at 0.2 mm ATP, but found that A(0.5) increased 10-20-fold at 4 mm ATP. The low A(0.5) of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart.     

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.     

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].     

Activating AMP-activated protein kinase without AMP

Though once believed to be regulated exclusively by the cellular energy state, AMPK has now been shown to be activated by a calcium-dependent signaling pathway.     

α1-肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶

为了验证心脏腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）是否为肾上腺素受体（adrenergic receptor,AR）的下游信号分子,本实验在大鼠心室肌源细胞和大鼠心脏中观察了α-AR对AMPK的激活作用,利用Western blot检测了AMPK-α总蛋白表达量及其172位苏氨酸磷酸化水平。雄性Sprague-Dawley大鼠皮下植入去甲肾上腺素（norepinephrine,NE）,苯肾上腺素（phenylephrine,PE）或者溶剂载体[0．01％（W／V）维生素C]的缓释微泵（osmotic minipump）。NE或PE以每小时0．2 mg／kg的速率持续输注,7 d后用AMPK-α抗体免疫沉淀处理样本并测定AMPK的活性。结果显示,在细胞水平,NE引起的AMPK磷酸化水平增高具有时间依赖和剂量依赖特点, NE处理细胞10 min后AMPK磷酸化水平达到最高峰;NE引起的这种效应对β-AR的拮抗剂普萘洛尔（propranolol）不敏感,但是可以被α1-AR拮抗剂哌唑嗪（prazosin）所阻断。结果提示,α1-AR介导AMPK的磷酸化,但β-AR无此作用。AR激动剂持续灌注7 d后,AMPK的活性在NE（7．4倍）和PE（6．0倍）灌注组较对照组显著增高（P〈0．05,H=6）。PE持续灌注组大鼠与对照组相比无明显的心肌肥厚和组织纤维化变化。本文证明α1-AR激动剂可以增强AMPK的活性,揭示了心脏中α1-AR激动在调控AMPK活性方面的重要作用。深入了解α1-AR介导的AMPK激活可能在心衰治疗中具有重要的临床意义。     

Polyunsaturated fatty acids do not activate AMP-activated protein kinase in mouse tissues

Stearoyl-CoA desaturase 1 (SCD1) deficiency partitions fatty acids away from lipid synthesis towards fatty acid oxidation in liver and skeletal muscle in part due to activation of AMP-activated protein kinase (AMPK) pathway. The mechanism of AMPK activation by SCD1 mutation is unknown, however since SCD1-/- animals have increased relative amounts of polyunsaturated fatty acids (PUFA), we hypothesized that the increased levels of PUFA might be responsible for the activation of AMPK in SCD1 deficient mice. Therefore, the present study was undertaken to analyze the effect of PUFA on AMPK in liver, skeletal muscle, and heart. We fed mice ad libitum for 14 days with diet supplemented with fish oil (5% fat). As expected, fish oil supplementation significantly increased n-3 PUFA content in each of the analyzed tissues. Hepatic mRNA levels of fatty acid synthase and acyl-CoA oxidase decreased by 92% and increased by 60%, respectively, consistent with known PUFA effects. However, after 14 days of PUFA feeding, we did not find any changes in AMPK phosphorylation and protein content in mouse liver, skeletal muscle, and heart. The data suggest that PUFA are not involved in AMPK activation in mouse tissues and that the increased activity of AMPK in SCD1-/- mice is probably PUFA-independent.     

Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.     

Functional role of AMP-activated protein kinase in the heart during exercise

AMP-activated protein kinase (AMPK) plays a critical role in maintaining energy homeostasis and cardiac function during ischemia in the heart. However, the functional role of AMPK in the heart during exercise is unknown. We examined whether acute exercise increases AMPK activity in mouse hearts and determined the significance of these increases by studying transgenic (TG) mice expressing a cardiac-specific dominant-negative (inactivating) AMPKalpha2 subunit. Exercise increased cardiac AMPKalpha2 activity in the wild type mice but not in TG. We found that inactivation of AMPK did not result in abnormal ATP and glycogen consumption during exercise, cardiac function assessed by heart rhythm telemetry and stress echocardiography, or in maximal exercise capacity.     

Ghrelin protects heart against ERS-induced injury and apoptosis by activating AMP-activated protein kinase

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), is a cardioprotective peptide. In our previous work, we have revealed that ghrelin could protect heart against ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum stress (ERS), which contributes to many heart diseases. In current study, using both in vivo and in vitro models, we investigated how ghrelin inhibits myocardial ERS. In the in vivo rat heart injury model induced by isoproterenol (ISO), we found that exogenous ghrelin could alleviate heart dysfunction, reduce myocardial injury and apoptosis and inhibit the excessive myocardial ERS induced by ISO. More importantly, the activation of AMP-activated protein kinase (AMPK) was observed. To explore the role of AMPK activation in ERS inhibition by ghrelin, we set up two in vitro ERS models by exposing cultured rat cardiomyocytes to tunicamycin(Tm) or dithiothreitol (DTT). In both models, compared with Tm or DTT treatment alone, pre-incubation cardiomyocytes with ghrelin significantly activated AMPK, reversed the upregulation of the ERS markers, C/EBP-homologous protein (CHOP) and cleaved caspase-12, and reduced apoptosis of cardiomyocytes. Further, we found that the ERS inhibitory and anti-apoptotic actions induced by ghrelin were blocked by an AMPK inhibitor. To investigate how ghrelin activates AMPK, selective antagonist of GHS-R1a and inhibitor of Ca²⁺/Calmodulin-dependent protein kinase kinase (CaMKK) were added, respectively, before ghrelin pre-incubation, and we found that AMPK activation was prevented and the ERS inhibitory and anti-apoptotic actions of ghrelin were blocked. In conclusion, ghrelin could protect heart against ERS-induced injury and apoptosis, at least partially through a GHS-R1a/CaMKK/AMPK pathway.     

Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease

Obesity, type 2 diabetes and the metabolic syndrome are disorders of energy balance, which the AMP-activated protein kinase (AMPK) regulates both at the cellular and whole body levels. AMPK switches cells from an anabolic state where nutrients are taken up and stored, to a catabolic state where they are oxidized. Drugs that activate AMPK indirectly (metformin and thiazolidinediones) are now the mainstay of treatment for type 2 diabetes, but more direct AMPK activators may have fewer side effects. However, activating mutations in AMPK can cause heart disease, and it will be important to look for adverse effects in the heart.     

Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart

In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. Because insulin is able to activate Akt and inactivate AMP-activated protein kinase (AMPK) in the heart, we hypothesized that Akt can regulate the activity of AMPK. To address the potential existence of this novel signaling pathway, we used a number of experimental protocols to activate Akt in cardiac myocytes and monitored the activation status of AMPK. Mouse hearts perfused in the presence of insulin demonstrated accelerated glycolysis and glucose oxidation rates as compared with non-insulin-perfused hearts. In addition, insulin caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation at its major regulatory site (threonine 172 of the alpha catalytic subunit). Transgenic mice overexpressing a constitutively active mutant form of Akt1 displayed decreased phosphorylation of cardiac alpha-AMPK. Isolated neonatal cardiac myocytes infected with an adenovirus expressing constitutively active mutant forms of either Akt1 or Akt2 also suppressed AMPK phosphorylation. However, Akt-dependent depression of alpha-AMPK phosphorylation could be overcome in the presence of the AMPK activator, metformin, suggesting that an override mechanism exists that can restore AMPK activity. Taken together, this study suggests that there is cross-talk between the AMPK and Akt pathways and that Akt activation can lead to decreased AMPK activity. In addition, our data suggest that the ability of insulin to inhibit AMPK may be controlled via an Akt-mediated mechanism.     

AMP-activated protein kinase and autophagy

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.     

AMP-activated protein kinase and p38 MAPK activate O-GlcNAcylation of neuronal proteins during glucose deprivation

We have demonstrated previously that a wide array of stress signals induces O-GlcNAc transferase (OGT) expression and increases O-GlcNAcylation of many intracellular proteins, a response that is critical for cell survival. Here, we describe a mechanism by which glucose deprivation induces OGT expression and activity in Neuro-2a neuroblastoma cells. Glucose deprivation increases OGT mRNA and protein expression in an AMP-activated protein kinase-dependent manner, whereas OGT enzymatic activity is regulated in a p38 MAPK-dependent manner. OGT is not phosphorylated by p38, but rather it interacts directly with p38 through its C terminus; this interaction increases with p38 activation during glucose deprivation. Surprisingly, the catalytic activity of OGT, as measured toward peptide substrates, is not altered by glucose deprivation. Instead, p38 regulates OGT activity within the cell by recruiting it to specific targets, including neurofilament H. Neurofilament H is O-GlcNAcylated during glucose deprivation in a p38-dependent manner. Interestingly, neurofilament H solubility is increased by glucose deprivation in an O-GlcNAc-dependent manner, suggesting that O-GlcNAcylation of neurofilament H regulates its disassembly from filaments. Not only do these data help to reveal how OGT is regulated by stress, but these findings also describe a possible mechanism by which defective brain glucose metabolism, as found in aging and ischemia, may directly affect axonal structure.     

AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.     

Functional modulation of AMP-activated protein kinase by cereblon

Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.     

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.     

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.