ABSTRACT
These days, easy access to commercially available (poly)phenolic compounds has expanded the scope of potential research beyond the field of chemistry, particularly in the area of their bioactivity. However, the quality of these compounds is often overlooked or not even considered. This issue is illustrated in this study through the example of (dihydro)phenanthrenes, a group of natural products present in yams, as AMP-activated protein kinase (AMPK) activators. A study conducted in our group on a series of compounds, fully characterized using a combination of chemical synthesis, NMR and MS techniques, provided evidence that the conclusions of a previous study were erroneous, likely due to the use of a misidentified commercial compound by its supplier. Furthermore, we demonstrated that additional representatives of the (dihydro)phenanthrene phytochemical classes were able to directly activate AMPK, avoiding the risk of misinterpretation of results based on analysis of a single compound alone.
Subject(s)
AMP-Activated Protein Kinases , Phenanthrenes , AMP-Activated Protein Kinases/metabolism , AMP-Activated Protein Kinases/genetics , Phenanthrenes/chemistry , Humans , Biological Products/chemistry , Biological Products/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Molecular StructureABSTRACT
Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.
Subject(s)
Alkaloids , Sarcopenia , Humans , Male , Mice , Animals , Sarcopenia/drug therapy , Sarcopenia/prevention & control , Sarcopenia/metabolism , NAD/metabolism , Caenorhabditis elegans , Aging , Muscle, Skeletal/metabolism , Alkaloids/pharmacology , Alkaloids/therapeutic use , Alkaloids/metabolismABSTRACT
AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK ß subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch" mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKß1 (ß1-G2A). We demonstrate that non-myristoylated AMPKß1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of ß1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the ß1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.
Subject(s)
AMP-Activated Protein Kinases , Fatty Liver , Animals , Mice , Phosphorylation , AMP-Activated Protein Kinases/metabolism , Diet, High-Fat , Protein Processing, Post-Translational , Obesity , Myristic Acid/metabolism , Mice, Inbred C57BL , Protein Phosphatase 2C/metabolismABSTRACT
Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK ß1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKß1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKß1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.
Subject(s)
AMP-Activated Protein Kinases , Fatty Acids , Mice , Animals , Phosphorylation , Fatty Acids/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Mitochondria/metabolism , Homeostasis , Autophagy , Triglycerides/metabolismABSTRACT
AMP-activated protein kinase (AMPK) is a central energy sensor that coordinates the response to energy challenges to maintain cellular ATP levels. AMPK is a potential therapeutic target for treating metabolic disorders, and several direct synthetic activators of AMPK have been developed that show promise in preclinical models of type 2 diabetes. These compounds have been shown to regulate AMPK through binding to a novel allosteric drug and metabolite (ADaM)-binding site on AMPK, and it is possible that other molecules might similarly bind this site. Here, we performed a high-throughput screen with natural plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to other ADaM site activators, Lusianthridin showed preferential activation of AMPKß1-containing complexes in intact cells and was unable to activate an AMPKß1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in de novo lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from ß1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation.
Subject(s)
AMP-Activated Protein Kinases , Hepatocytes , Lipids , Phenanthrenes , AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Allosteric Regulation , Animals , Binding Sites , Diabetes Mellitus, Type 2 , Hepatocytes/drug effects , Hepatocytes/enzymology , Lipid Metabolism , Lipids/biosynthesis , Mice , Phenanthrenes/pharmacology , PhosphorylationABSTRACT
OBJECTIVE: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. METHODS: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and ß subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a ß3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. RESULTS: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and ß2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and ß2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. CONCLUSIONS: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for ß3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Blood Glucose/metabolism , Muscle Fibers, Skeletal/metabolism , AMP-Activated Protein Kinases/genetics , Adipose Tissue, Brown/metabolism , Aminoimidazole Carboxamide/administration & dosage , Aminoimidazole Carboxamide/analogs & derivatives , Animals , Benzimidazoles/administration & dosage , Diet, High-Fat , Female , Glucose Tolerance Test , Insulin/metabolism , Male , Metabolic Clearance Rate/drug effects , Mice , Mice, Knockout , Models, Animal , Pyridines/administration & dosage , Ribonucleotides/administration & dosage , Thermogenesis/drug effectsABSTRACT
AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr172). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the ß subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr172 AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr172 AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr172 phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr172, AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, the basal level of Thr172 phosphorylation is very low (â¼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKß, Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172 AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr172 phosphorylation. However, in a cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , A549 Cells , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases/genetics , Biphenyl Compounds , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Enzyme Activation/drug effects , Enzyme Activation/genetics , HEK293 Cells , Humans , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Serine-Threonine Kinases/genetics , Pyrones/pharmacology , Thiophenes/pharmacologyABSTRACT
Rodents are exquisitely sensitive to light and optogenetic behavioral experiments routinely introduce light-delivery materials into experimental situations, which raises the possibility that light could leak and influence behavioral performance. We examined whether rats respond to a faint diffusion of light, termed caplight, which emanated through the translucent dental acrylic resin used to affix deep-brain optical cannulas in place. Although rats did not display significant changes in locomotion or rearing to caplight in a darkened open field, they did acquire conditional fear via caplight-footshock pairings. These findings highlight the potential confounding influence of extraneous light emanating from light-delivery materials during optogenetic analyses.
Subject(s)
Acrylic Resins , Catheters, Indwelling , Fear , Light , Optical Fibers , Optogenetics/instrumentation , Animals , Conditioning, Classical , Electroshock , Freezing Reaction, Cataleptic , Light/adverse effects , Male , Motor Activity , Rats, Long-Evans , Signal Detection, PsychologicalABSTRACT
The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, ß and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that â¼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK ß subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Aging/genetics , Caenorhabditis elegans Proteins/genetics , Insulin-Like Growth Factor I/genetics , Insulin/metabolism , Transcription Factors/genetics , AMP-Activated Protein Kinases/genetics , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/metabolism , Forkhead Transcription Factors , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Longevity/genetics , Protein Isoforms/genetics , Receptor, Insulin/genetics , Signal Transduction/genetics , Transcription Factors/metabolism , Transcriptional Activation/geneticsABSTRACT
AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.
Subject(s)
AMP-Activated Protein Kinases/chemistry , Gene Expression Regulation, Enzymologic , Adenosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Allosteric Site , Binding Sites , Carbohydrates/chemistry , Circular Dichroism , Crystallography, X-Ray , HEK293 Cells , Humans , Interferometry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Threonine/chemistryABSTRACT
The hydrolysis of ATP drives virtually all of the energy-requiring processes in living cells. A prerequisite of living cells is that the concentration of ATP needs to be maintained at sufficiently high levels to sustain essential cellular functions. In eukaryotic cells, the AMPK (AMP-activated protein kinase) cascade is one of the systems that have evolved to ensure that energy homoeostasis is maintained. AMPK is activated in response to a fall in ATP, and recent studies have suggested that ADP plays an important role in regulating AMPK. Once activated, AMPK phosphorylates a broad range of downstream targets, resulting in the overall effect of increasing ATP-producing pathways whilst decreasing ATP-utilizing pathways. Disturbances in energy homoeostasis underlie a number of disease states in humans, e.g. Type 2 diabetes, obesity and cancer. Reflecting its key role in energy metabolism, AMPK has emerged as a potential therapeutic target. In the present review we examine the recent progress aimed at understanding the regulation of AMPK and discuss some of the latest developments that have emerged in key areas of human physiology where AMPK is thought to play an important role.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cardiovascular Diseases/physiopathology , Energy Metabolism , Neoplasms/physiopathology , Obesity/physiopathology , Animals , HumansABSTRACT
Fear conditioning, extinction, and renewal were evaluated in adult (6-month-old) and aging (17-month-old) male C57Bl/6 mice. Mice were subjected to five tone-shock trials and later exposed to 150 tone-alone trials. Thereafter, all mice showed little fear in the extinction context. Adult mice demonstrated return of fear in a distinct context (renewal) but aging mice did not. Aging mice showed normal shock sensitivity, tone fear learning, and extinction. Aging mice thus exhibited a very selective deficit in the contextual gating of extinguished fear. This contextual gating deficit may reflect age-related pathology in the hippocampus and prefrontal cortex.
Subject(s)
Aging/psychology , Fear/psychology , Aging/physiology , Animals , Behavior, Animal , Conditioning, Psychological , Extinction, Psychological , Learning , Male , Memory , Mice , Mice, Inbred C57BL , Perception , Psychomotor PerformanceABSTRACT
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
Subject(s)
Adenosine Diphosphate/metabolism , Enzyme Activation/genetics , Glucose/metabolism , Protein Phosphatase 1/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Signal Transduction , Adenosine Diphosphate/chemistry , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Amino Acid Sequence , Catalytic Domain/genetics , Conserved Sequence , Gene Expression Regulation, Fungal/physiology , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Protein Interaction Domains and Motifs , Protein Phosphatase 1/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity , Threonine/metabolismABSTRACT
Maintaining sufficient levels of ATP (the immediate source of cellular energy) is essential for the proper functioning of all living cells. As a consequence, cells require mechanisms to balance energy demand with supply. In eukaryotic cells the AMP-activated protein kinase (AMPK) cascade has an important role in this homeostasis. AMPK is activated by a fall in ATP (concomitant with a rise in ADP and AMP), which leads to the activation of catabolic pathways and the inhibition of anabolic pathways. Here we review the role of AMPK as an energy sensor and consider the recent finding that ADP, as well as AMP, causes activation of mammalian AMPK. We also review recent progress in structural studies on phosphorylated AMPK that provides a mechanism for the regulation of AMPK in which AMP and ADP protect it against dephosphorylation. Finally, we briefly survey some of the outstanding questions concerning the regulation of AMPK.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adenine Nucleotides/physiology , Energy Metabolism/physiology , Gene Expression Regulation, Enzymologic/physiology , AMP-Activated Protein Kinases/genetics , Signal Transduction/physiology , Species SpecificityABSTRACT
The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
Subject(s)
AMP-Activated Protein Kinases/chemistry , AMP-Activated Protein Kinases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , AMP-Activated Protein Kinases/genetics , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Animals , Binding Sites , Crystallography, X-Ray , Enzyme Activation/drug effects , Enzyme Activation/genetics , Kinetics , Magnesium/metabolism , Mammals , Models, Molecular , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Binding , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , ThermodynamicsABSTRACT
In three experiments, chronic stress enhancement of subsequent fear learning was investigated in C57Bl/6 mice. The first experiment focused on the influence of stressor type on subsequent Pavlovian fear learning. Male mice were subjected to 7d of either repeated restraint stress or chronic variable stress before undergoing a fear conditioning procedure with three tone-shock trials. Subsequent tests were conducted of contextual and tone fear, through measures of the freezing response. Repeated restraint altered pre-training activity and the unconditional response to shock, but was ineffective in influencing conditional fear. Chronic variable stress significantly inflated contextual fear without altering tone fear. In a second experiment, investigating potential sex differences in the fear-enhancing effects of stress, female mice were subjected to the very same procedures. Among females, chronic variable stress selectively altered tone fear, rather than contextual fear. A final experiment investigated the potential role of ovarian hormones by subjecting female mice to either ovariectomy or sham surgery before the stress procedures. Ovariectomy had no significant effect on the ability of stress to enhance fear in females. In sum, the experiments indicate that stressor type significantly influences subsequent fear learning, that males and females are differentially sensitive to fear enhancement by stress, and that the mechanisms mediating these sex differences lie outside of the immediate influence of ovarian hormones. The findings should allow for refinement of animal models of human psychiatric disorders and for further investigations into the genetic and molecular substrates of significant gender differences in fear and anxiety.
Subject(s)
Association Learning/physiology , Conditioning, Classical/physiology , Fear , Gonadal Steroid Hormones/physiology , Stress, Psychological/psychology , Animals , Female , Male , Mice , Mice, Inbred C57BL , Ovariectomy , Restraint, Physical , Sex Factors , Stress, Psychological/classificationABSTRACT
AMP-activated protein kinase (AMPK) plays a key role in maintaining energy homeostasis. Activation of AMPK in peripheral tissues has been shown to alleviate the symptoms of metabolic diseases, such as type 2 diabetes, and consequently AMPK is a target for treatment of these diseases. Recently, a small molecule activator (A-769662) of AMPK was identified that had beneficial effects on metabolism in ob/ob mice. Here we show that A-769662 activates AMPK both allosterically and by inhibiting dephosphorylation of AMPK on Thr-172, similar to the effects of AMP. A-769662 activates AMPK harboring a mutation in the gamma subunit that abolishes activation by AMP. An AMPK complex lacking the glycogen binding domain of the beta subunit abolishes the allosteric effect of A-769662 but not the allosteric activation by AMP. Moreover, mutation of serine 108 to alanine, an autophosphorylation site within the glycogen binding domain of the beta1 subunit, almost completely abolishes activation of AMPK by A-769662 in cells and in vitro, while only partially reducing activation by AMP. Based on our results we propose a model for activation of AMPK by A-769662. Importantly, this model may provide clues for understanding the mechanism by which AMP leads to activation of AMPK, which in turn may help in the identification of other AMPK activators.
Subject(s)
Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Pyridines/pharmacology , Pyrones/pharmacology , Thiophenes/pharmacology , AMP-Activated Protein Kinases , Biphenyl Compounds , Cell Line , Enzyme Activation/drug effects , Humans , Multienzyme Complexes/genetics , Mutation/genetics , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Pyridines/classification , Pyrones/classification , Thiophenes/classification , Threonine/genetics , Threonine/metabolismABSTRACT
AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr172 within the catalytic alpha subunit. Here we show that mutations in the regulatory gamma subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr172 by PP (protein phosphatase)2Calpha. Moreover, a mutation in the gamma subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr172 phosphorylation by either LKB1 or CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase beta) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Calpha in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Calpha, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr172 from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKKbeta. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKKbeta and an AMP-dependent pathway, mediated by LKB1.
Subject(s)
Adenosine Monophosphate/metabolism , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases , Animals , Cloning, Molecular , Enzyme Activation , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Liver/enzymology , Models, Biological , Multienzyme Complexes/genetics , Phosphorylation , Phosphothreonine/metabolism , Protein Serine-Threonine Kinases/genetics , Rats , Recombinant Proteins/metabolismABSTRACT
Traumatic brain injury (TBI) is a principal cause of long-term physical, cognitive, behavioral, and social deficits in young adults, which frequently coexist with a high incidence of substance abuse disorders. However, few studies have examined the long-term effects of TBI on the neuroendocrine-immune system. TBI was induced in adult male rats under isoflurane anesthesia by cortical contusion injury with a pneumatic piston positioned stereotaxically over the left parietal cortex. Controls underwent sham surgery without injury. At 4 weeks post-injury, the plasma corticosterone response to 30-min restraint stress was significantly blunted in TBI rats compared to the sham controls. One week later, transmitters were implanted for continuous biotelemetric recording of body temperature and spontaneous locomotor activity. At 6 weeks post-injury, the febrile response to i.p. injection of the bacterial endotoxin, lipopolysaccharide (LPS; 50 microg/kg), was significantly lower in TBI than in sham rats. At 8 weeks, swimming in the forced swim test was significantly less in TBI than sham rats. At 9 weeks, rats were rendered ethanol (EtOH) dependent by feeding an EtOH-containing liquid diet for 14 days. Cosine rhythmometry analysis of circadian body temperature Midline Estimating Statistic of Rhythm (MESOR), amplitudes, and acrophases indicated differential effects of EtOH and withdrawal in the two groups. Light- and dark-phase activity analysis indicated that TBI rats were significantly more active than the sham group, and that EtOH and withdrawal differentially affected their activity. Given the extensive interactions of the neuroendocrine-immune systems, these results demonstrate that TBI produces lasting dysregulation amidst the central substrates for allostasis and circadian rhythmicity.
Subject(s)
Allostasis/physiology , Brain Injuries/physiopathology , Immune System/physiopathology , Neurosecretory Systems/physiopathology , Alcohol-Related Disorders/etiology , Animals , Body Temperature Regulation/physiology , Brain Injuries/blood , Brain Injuries/psychology , Circadian Rhythm/physiology , Corticosterone/blood , Male , Motor Activity/physiology , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
Signaling by the Ca(2+)/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase alpha (CaMKKalpha) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKalpha mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKalpha mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKalpha in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.