RESUMEN
Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1â¯mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20⯵M, 4â¯h) prior to exposure to cisplatin (20⯵M, 23â¯h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03â¯mM vs ATX-304 0.02 + 0.01â¯mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20⯵M, 4â¯h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Lesión Renal Aguda , Cisplatino , Ratones Endogámicos C57BL , Animales , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones , Masculino , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Cultivadas , Sustancias Protectoras/farmacología , Fosforilación , Compuestos de Bifenilo , Pironas , TiofenosRESUMEN
OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Insulinas , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Glucosa/metabolismo , Insulinas/metabolismo , Ratones Noqueados , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.
Asunto(s)
Dinaminas , Quinazolinonas , Humanos , Dinaminas/antagonistas & inhibidores , GTP Fosfohidrolasas/metabolismo , Dinámicas Mitocondriales , Quinazolinonas/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Hígado Graso , Animales , Ratones , Fosforilación , Proteínas Quinasas Activadas por AMP/metabolismo , Dieta Alta en Grasa , Procesamiento Proteico-Postraduccional , Obesidad , Ácido Mirístico/metabolismo , Ratones Endogámicos C57BL , Proteína Fosfatasa 2C/metabolismoRESUMEN
The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Diabetes Mellitus Tipo 2 , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Ratones , Músculo Esquelético/metabolismo , FosforilaciónRESUMEN
AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Lisosomas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Secuencia de Aminoácidos , Animales , Activación Enzimática , Femenino , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Células HeLa , Humanos , Isoenzimas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Fosforilación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.
Asunto(s)
Fenómenos Biológicos , Fosfoproteínas , Fosfoproteínas/genética , Fosforilación , Proteómica/métodos , Transducción de Señal/fisiologíaRESUMEN
AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.
Asunto(s)
Dinaminas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidralazina/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Femenino , Células HeLa , Humanos , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de SeñalRESUMEN
The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.
Asunto(s)
Proteínas 14-3-3/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , Proteínas 14-3-3/genética , Animales , Células COS , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática , HumanosRESUMEN
OBJECTIVE: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory ß subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. METHODS: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK ß1 (W100A) or ß2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. RESULTS: While ß1 W100A KI did not affect whole-body metabolism or exercise capacity, ß2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of ß1 W100A and ß2 W98A, respectively, versus WT mice. ß1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in ß1 W100A liver and ß2 W98A skeletal muscle versus WT. CONCLUSIONS: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético/fisiología , Glucógeno/metabolismo , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Femenino , Glucosa/metabolismo , Glucógeno/fisiología , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Fosforilación , Unión ProteicaRESUMEN
Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for ß-oxidation2-6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) ß1-containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the ß-subunit. ß1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK ß1-selective activators and promote LCFA oxidation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acilcoenzima A/fisiología , Regulación Alostérica/fisiología , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Animales , Compuestos de Bifenilo , Dominio Catalítico , Ésteres , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutación/genética , Oxidación-Reducción , Palmitoil Coenzima A/metabolismo , Fosforilación , Pironas/farmacología , Tiofenos/farmacologíaRESUMEN
Meteorin-like (metrnl) is a recently identified adipomyokine that beneficially affects glucose metabolism; however, its underlying mechanism of action is not completely understood. We here show that the level of metrnl increases in vitro under electrical pulse stimulation and in vivo in exercised mice, suggesting that metrnl is secreted during muscle contractions. In addition, metrnl increases glucose uptake via the calcium-dependent AMPKα2 pathway in skeletal muscle cells and increases the phosphorylation of HDAC5, a transcriptional repressor of GLUT4, in an AMPKα2-dependent manner. Phosphorylated HDAC5 interacts with 14-3-3 proteins and sequesters them in the cytoplasm, resulting in the activation of GLUT4 transcription. An intraperitoneal injection of recombinant metrnl improved glucose tolerance in mice with high-fat-diet-induced obesity or type 2 diabetes, but not in AMPK ß1ß2 muscle-specific null mice. Metrnl improves glucose metabolism via AMPKα2 and is a promising therapeutic candidate for glucose-related diseases such as type 2 diabetes.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Diabetes Mellitus Tipo 2/genética , Histona Desacetilasas/genética , Factores de Crecimiento Nervioso/genética , Obesidad/genética , Proteínas 14-3-3/genética , Animales , Línea Celular , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Estimulación Eléctrica , Glucosa/genética , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Humanos , Resistencia a la Insulina/genética , Ratones , Contracción Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Factores de Crecimiento Nervioso/farmacología , Obesidad/tratamiento farmacológico , Obesidad/etiología , Obesidad/patología , Condicionamiento Físico Animal , Proteínas Recombinantes/farmacologíaRESUMEN
Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.
Asunto(s)
Adenilato Quinasa/antagonistas & inhibidores , Proliferación Celular/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Nutrientes/metabolismo , Estrés Fisiológico , Adenilato Quinasa/química , Adenilato Quinasa/metabolismo , Dominio Catalítico , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Activación Enzimática , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Neoplasias/metabolismo , Fosforilación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de Señal/fisiologíaRESUMEN
ATPase inhibitory factor 1 (IF1) is an ATP synthase-interacting protein that suppresses the hydrolysis activity of ATP synthase. In this study, we observed that the expression of IF1 was up-regulated in response to electrical pulse stimulation of skeletal muscle cells and in exercized mice and healthy men. IF1 stimulates glucose uptake via AMPK in skeletal muscle cells and primary cultured myoblasts. Reactive oxygen species and Rac family small GTPase 1 (Rac1) function in the upstream and downstream of AMPK, respectively, in IF1-mediated glucose uptake. In diabetic animal models, the administration of recombinant IF1 improved glucose tolerance and down-regulated blood glucose level. In addition, IF1 inhibits ATP hydrolysis by ß-F1-ATPase in plasma membrane, thereby increasing extracellular ATP and activating the protein kinase B (Akt) pathway, ultimately leading to glucose uptake. Thus, we suggest that IF1 is a novel myokine and propose a mechanism by which AMPK and Akt contribute independently to IF1-mediated improvement of glucose tolerance impairment. These results demonstrate the importance of IF1 as a potential antidiabetic agent.-Lee, H. J., Moon, J., Chung, I., Chung, J. H., Park, C., Lee, J. O., Han, J. A., Kang, M. J., Yoo, E. H., Kwak, S.-Y., Jo, G., Park, W., Park, J., Kim, K. M., Lim, S., Ngoei, K. R. W., Ling, N. X. Y., Oakhill, J. S., Galic, S., Murray-Segal, L., Kemp, B. E., Mantzoros, C. S., Krauss, R. M., Shin, M.-J., Kim, H. S. ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways.
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Glucosa/metabolismo , Mioblastos/metabolismo , Proteínas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenosina Trifosfato/metabolismo , Adulto , Animales , Línea Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas/metabolismo , Proteínas/genética , Proteínas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/uso terapéutico , Proteína Inhibidora ATPasaRESUMEN
Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Benzamidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Benzamidas/química , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Pirimidinas/químicaRESUMEN
The AMP-activated protein kinase (AMPK) αßγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2ß2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated α2ß2γ1 and α2ß1γ1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and ß2-Asp111, which provide a design paradigm for ß2-AMPK therapeutics. The α2ß2γ1/SC4 structure reveals an interaction between a ß2 N-terminal α helix and the α2 autoinhibitory domain. Our results provide a structure-function guide to accelerate development of potent, but importantly tissue-specific, ß2-AMPK therapeutics.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Benzoatos/farmacología , Glucosa/metabolismo , Músculo Esquelético/efectos de los fármacos , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Benzoatos/síntesis química , Benzoatos/química , Células COS , Línea Celular , Chlorocebus aethiops , Cristalografía por Rayos X , Activación Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Músculo Esquelético/metabolismo , Piridinas/síntesis química , Piridinas/química , Ratas , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.
RESUMEN
AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the ß1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a ß1-Ser108 kinase in cells. Cellular ß1-Ser108 phosphorylation by ULK1 was dependent on AMPK ß-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates ß1-Ser108 on the regulatory ß1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/fisiología , Estrés Fisiológico , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Compuestos de Bifenilo , Células COS , Chlorocebus aethiops , Células HEK293 , Homeostasis , Humanos , Modelos Biológicos , Fosforilación , Pironas/farmacología , Salicilatos/farmacología , Tiofenos/farmacologíaRESUMEN
The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Regulación de la Expresión Génica , Variación Genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , FosforilaciónRESUMEN
The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.