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1.
Nature ; 632(8023): 157-165, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39020175

RESUMEN

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7. Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people.


Asunto(s)
Envejecimiento , Interleucina-11 , Longevidad , Transducción de Señal , Animales , Femenino , Masculino , Ratones , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Fragilidad/genética , Fragilidad/metabolismo , Fragilidad/prevención & control , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Interleucina-11/antagonistas & inhibidores , Interleucina-11/deficiencia , Interleucina-11/genética , Interleucina-11/metabolismo , Subunidad alfa del Receptor de Interleucina-11/metabolismo , Subunidad alfa del Receptor de Interleucina-11/deficiencia , Longevidad/efectos de los fármacos , Longevidad/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Humanos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología
3.
Biochem J ; 481(5): 345-362, 2024 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-38314646

RESUMEN

Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Adipogénesis , Animales , Ratones , Células 3T3-L1 , Adipocitos/metabolismo , Adipogénesis/genética , Tejido Adiposo/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Leptina/genética , Leptina/farmacología , Leptina/metabolismo
4.
Br J Cancer ; 128(12): 2326-2337, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37076563

RESUMEN

BACKGROUND: Current strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC. METHODS: The antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide. RESULTS: CT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo. CONCLUSIONS: This study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.


Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Nitrilos/uso terapéutico , Quinasas Ciclina-Dependientes/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Proliferación Celular
5.
Diabetologia ; 65(6): 997-1011, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35294578

RESUMEN

AIMS/HYPOTHESIS: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. METHODS: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. RESULTS: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). CONCLUSIONS/INTERPRETATION: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones
6.
Circ Res ; 127(7): 928-944, 2020 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-32611235

RESUMEN

RATIONALE: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; HMOX1) and lipid homeostasis genes. OBJECTIVE: We asked whether this pathway had an in vivo role in mice. METHODS AND RESULTS: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene Spic. This response was lost in bone marrow-derived macrophages from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent. CONCLUSIONS: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Factor de Transcripción Activador 1/metabolismo , Hematoma/metabolismo , Macrófagos/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Factor de Transcripción Activador 1/genética , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Femenino , Hematoma/genética , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hierro/metabolismo , Metabolismo de los Lípidos , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Factores de Tiempo
7.
Clin Sci (Lond) ; 135(20): 2393-2408, 2021 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-34622923

RESUMEN

AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Riñón/enzimología , Enfermedades Renales Poliquísticas/enzimología , Proteínas Quinasas Activadas por AMP/genética , Adulto , Factores de Edad , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , AMP Cíclico/metabolismo , Metabolismo Energético , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Hexoquinasa/metabolismo , Humanos , Riñón/patología , Masculino , Ratones Transgénicos , Fenotipo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Riñón Poliquístico Autosómico Dominante/enzimología , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Transducción de Señal
8.
Biochem J ; 477(11): 2071-2093, 2020 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-32539124

RESUMEN

Metabolic inflexibility, defined as the inability to respond or adapt to metabolic demand, is now recognised as a driving factor behind many pathologies associated with obesity and the metabolic syndrome. Adipose tissue plays a pivotal role in the ability of an organism to sense, adapt to and counteract environmental changes. It provides a buffer in times of nutrient excess, a fuel reserve during starvation and the ability to resist cold-stress through non-shivering thermogenesis. Recent advances in single-cell RNA sequencing combined with lineage tracing, transcriptomic and proteomic analyses have identified novel adipocyte progenitors that give rise to specialised adipocytes with diverse functions, some of which have the potential to be exploited therapeutically. This review will highlight the common and distinct functions of well-known adipocyte populations with respect to their lineage and plasticity, as well as introducing the most recent members of the adipocyte family and their roles in whole organism energy homeostasis. Finally, this article will outline some of the more preliminary findings from large data sets generated by single-cell transcriptomics of mouse and human adipose tissue and their implications for the field, both for discovery and for therapy.


Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Termogénesis/fisiología , Adipocitos/citología , Tejido Adiposo Pardo/citología , Animales , Humanos , Ratones
9.
Biochem J ; 477(17): 3453-3469, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32869834

RESUMEN

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Colforsina/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Serina/metabolismo
10.
J Biol Chem ; 293(44): 17208-17217, 2018 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-30232152

RESUMEN

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide-dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2 In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Activación Enzimática , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Mitocondrias/genética , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Oxidación-Reducción
12.
Biochem J ; 475(18): 2969-2983, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30135087

RESUMEN

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237 Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Mutación Missense , Obesidad/enzimología , Proteínas Quinasas Activadas por AMP/genética , Sustitución de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animales , Femenino , Proteínas Activadoras de GTPasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Obesidad/genética , Fosforilación , Ribonucleótidos/genética , Ribonucleótidos/metabolismo , Caracteres Sexuales
13.
Proc Natl Acad Sci U S A ; 113(26): E3706-15, 2016 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-27303042

RESUMEN

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Linfocitos B/citología , Cardiomiopatías/metabolismo , Proteínas Portadoras/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Linfocitos B/enzimología , Linfocitos B/metabolismo , Cardiomiopatías/genética , Proteínas Portadoras/metabolismo , Recuento de Células , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética
14.
Biochem J ; 474(10): 1741-1754, 2017 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-28302767

RESUMEN

AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. AMPK activation results in a wide range of downstream responses, many of which are associated with improved metabolic outcome, making AMPK an attractive target for the treatment of metabolic diseases. AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (ß and γ). The γ-subunit harbours the nucleotide-binding sites and plays an important role in AMPK regulation in response to cellular energy levels. In mammals, there are three isoforms of the γ-subunit and these respond differently to regulation by nucleotides, but there is limited information regarding their role in activation by small molecules. Here, we determined the effect of different γ-isoforms on AMPK by a direct activator, 991. In cells, 991 led to a greater activation of γ2-containing AMPK complexes compared with either γ1 or γ3. This effect was dependent on the long N-terminal region of the γ2-isoform. We were able to rule out an effect of Ser108 phosphorylation, since mutation of Ser108 to alanine in the ß2-isoform had no effect on activation of AMPK by 991 in either γ1- or γ2-complexes. The rate of dephosphorylation of Thr172 was slower for γ2- compared with γ1-complexes, both in the absence and presence of 991. Our studies show that activation of AMPK by 991 depends on the nature of the γ-isoform. This finding may have implications for the design of isoform-selective AMPK activators.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Regulación Alostérica/efectos de los fármacos , Sustitución de Aminoácidos , Aminopiridinas/farmacología , Bencimidazoles/farmacología , Benzoatos/farmacología , Sitios de Unión , Sistemas CRISPR-Cas , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Células HEK293 , Humanos , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ligandos , Mutación , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Treonina/metabolismo
15.
Biochem J ; 474(17): 3059-3073, 2017 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-28694351

RESUMEN

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 (∼1%), 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.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células A549 , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/genética , Compuestos de Bifenilo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Células HEK293 , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Pironas/farmacología , Tiofenos/farmacología
16.
Nature ; 472(7342): 230-3, 2011 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-21399626

RESUMEN

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.


Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Proteínas Quinasas Activadas por AMP/genética , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Cinética , Magnesio/metabolismo , Mamíferos , Modelos Moleculares , Fosforilación/efectos de los fármacos , Fosforilación/genética , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Termodinámica
17.
Sensors (Basel) ; 16(8)2016 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-27548185

RESUMEN

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.


Asunto(s)
Técnicas Biosensibles/métodos , Técnicas de Cultivo de Célula , Imagen Molecular/métodos , Proteínas Quinasas/aislamiento & purificación , Quinasas de la Proteína-Quinasa Activada por el AMP , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Humanos , Imagen Óptica/métodos , Esferoides Celulares/citología
18.
Trends Biochem Sci ; 36(9): 470-7, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21782450

RESUMEN

AMPK is a ubiquitous sensor of cellular energy status in eukaryotic cells. It is activated by stresses causing ATP depletion and, once activated, maintains energy homeostasis by phosphorylating targets that activate catabolism and inhibit energy-consuming processes. Evidence derived from non-mammalian orthologs suggests that its ancestral role was in the response to starvation for a carbon source. We review recent findings showing that AMPK is activated by ADP as well as AMP, and discuss the mechanism by which binding of these nucleotides prevent its dephosphorylation and inactivation. We also discuss the role of the carbohydrate-binding module on the ß subunit and the mechanisms by which it is activated by drugs and xenobiotics such as metformin and resveratrol.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Transporte de Electrón/efectos de los fármacos , Metabolismo Energético , Activación Enzimática , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Glucógeno/metabolismo , Humanos , Metformina/farmacología , Fosforilación , Conformación Proteica , Resveratrol , Estilbenos/farmacología , Transcripción Genética , Levaduras/metabolismo
19.
J Neurochem ; 133(2): 242-52, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25598140

RESUMEN

Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKß-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipoxia-Isquemia Encefálica/enzimología , Hipoxia-Isquemia Encefálica/patología , Animales , Animales Recién Nacidos , Bencimidazoles/farmacología , Muerte Celular , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Glucosa/deficiencia , Hipoxia/patología , Ionomicina/farmacología , L-Lactato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Naftalimidas/farmacología , Neuronas/metabolismo , Transducción de Señal/fisiología , Factores de Tiempo
20.
EMBO J ; 29(2): 376-86, 2010 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-19927127

RESUMEN

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.


Asunto(s)
Senescencia Celular , Fibroblastos/citología , Ploidias , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Línea Celular , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética
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