RESUMO
Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.
Assuntos
Adenosina , Astrócitos , Encéfalo , Metabolismo Energético , Neurônios , Transdução de Sinais , Animais , Feminino , Masculino , Camundongos , Ratos , Adenosina/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/citologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Hipocampo/citologia , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Neurônios/metabolismo , Receptor A2B de Adenosina/deficiência , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/metabolismo , Reconhecimento Psicológico/fisiologia , Sono/genética , Sono/fisiologia , Sinapses/metabolismoRESUMO
OBJECTIVE: Gut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health. DESIGN: In 271 middle-aged non-diabetic Danish individuals, who were stratified on habitual dietary intake, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy of urine samples and shotgun-sequencing-based metagenomics of the gut microbiome to explore links between the urine level of hippurate, measures of the gut microbiome, dietary fat and markers of metabolic health. In mechanistic experiments with chronic subcutaneous infusion of hippurate to high-fat-diet-fed obese mice, we tested for causality between hippurate and metabolic phenotypes. RESULTS: In the human study, we showed that urine hippurate positively associates with microbial gene richness and functional modules for microbial benzoate biosynthetic pathways, one of which is less prevalent in the Bacteroides 2 enterotype compared with Ruminococcaceae or Prevotella enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate concentration, independently of gene richness, accounts for links with metabolic health. In the high-fat-fed mice experiments, we demonstrate causality through chronic infusion of hippurate (20 nmol/day) resulting in improved glucose tolerance and enhanced insulin secretion. CONCLUSION: Our human and experimental studies show that a high urine hippurate concentration is a general marker of metabolic health, and in the context of obesity induced by high-fat diets, hippurate contributes to metabolic improvements, highlighting its potential as a mediator of metabolic health.
Assuntos
Biomarcadores/metabolismo , Microbioma Gastrointestinal , Hipuratos/metabolismo , Animais , Biodiversidade , Dinamarca , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metaboloma , Metagenômica , Camundongos , Pessoa de Meia-Idade , FenótipoRESUMO
BACKGROUND: During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. METHODS: We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. RESULTS: Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-ß1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. CONCLUSIONS: Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/efeitos dos fármacos , Piridonas/farmacologia , Pirimidinonas/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Biópsia por Agulha , Células Cultivadas , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , Fibroblastos/efeitos dos fármacos , Fibrose/tratamento farmacológico , Fibrose/patologia , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Terapia de Alvo Molecular/métodos , Distribuição Aleatória , Valores de Referência , Insuficiência Renal Crônica/genética , Transdução de Sinais/genéticaRESUMO
Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11ßHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11ßHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11ßHSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11ßHSD1(-/-) mice and rats treated with a specific 11ßHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11ßHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11ßHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Resistência à Insulina/fisiologia , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/complicações , Uremia/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Análise de Variância , Animais , Glicemia , Carbenoxolona/administração & dosagem , Carbenoxolona/farmacologia , Corticosterona/sangue , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Glucocorticoides/metabolismo , Immunoblotting , Insulina/sangue , Fígado/metabolismo , Camundongos , Camundongos Knockout , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Uremia/etiologiaRESUMO
Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.
Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neovascularização Fisiológica/genética , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/genética , Compostos de Anilina/administração & dosagem , Animais , Endotélio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/genética , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , Neovascularização Fisiológica/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Éteres Fenílicos/administração & dosagem , RNA Interferente Pequeno/genética , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trocador de Sódio e Cálcio/biossíntese , Trocador de Sódio e Cálcio/metabolismo , Trombina/administração & dosagemRESUMO
BACKGROUND: Outcomes after acute myocardial infarction in patients with chronic kidney disease are extremely poor. Ischemic conditioning techniques are among the most powerful cytoprotective strategies discovered to date. However, experimental data suggest that comorbidity may attenuate the protective effects of ischemic conditioning. METHODS AND RESULTS: We conducted investigations into the effects of chronic uremia on myocardial infarct size and the protective effects of ischemic preconditioning (IPC), remote ischemic preconditioning, and ischemic postconditioning in 2 rodent models of chronic uremia. In addition, a limited investigation into the signaling mechanisms involved in cardioprotection after IPC was performed in both uremic and nonuremic animals. Myocardial infarct size was increased in uremic animals, but all 3 conditioning strategies (IPC, remote IPC, ischemic postconditioning) proved highly efficacious in reducing myocardial infarct size (relative reduction, 86%, 39%, and 65% [P<0.005, P<0.05, and P<0.05], respectively). Moreover, some protocols (IPC and ischemic postconditioning) appeared to be more effective in uremic than in sham (nonuremic) animals. Analysis of the signaling mechanisms revealed that components of both the reperfusion injury salvage kinase and survivor activating factor enhancement pathways were similarly upregulated in both uremic and nonuremic animals after an IPC stimulus. CONCLUSION: Conditioning strategies may present the best opportunity to improve outcomes for patients with chronic kidney disease after an acute coronary syndrome.
Assuntos
Precondicionamento Isquêmico Miocárdico/métodos , Infarto do Miocárdio/terapia , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão/prevenção & controle , Uremia/complicações , Síndrome Coronariana Aguda/patologia , Síndrome Coronariana Aguda/terapia , Animais , Doença Crônica , Modelos Animais de Doenças , Masculino , Infarto do Miocárdio/patologia , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Miocárdio/patologia , Ratos , Ratos Wistar , Insuficiência Renal Crônica/complicações , Traumatismo por Reperfusão/patologiaRESUMO
The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied "explainable" machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.
Assuntos
Endocrinologia , Metilaminas , Adulto , Humanos , Causalidade , RimRESUMO
VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time- and dose-dependent manner and attenuated VEGF-induced Ca(2+) transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKCα translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.
Assuntos
Cálcio/metabolismo , Células Endoteliais/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Patológica , Trocador de Sódio e Cálcio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proliferação de Células , Células Cultivadas , Inativação Gênica , Humanos , Íons , Fosforilação , Proteína Quinase C-alfa/metabolismo , Veias Umbilicais/citologiaRESUMO
Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and ß-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCß1, and VGSCß3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.
Assuntos
Células Endoteliais/citologia , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Cálcio/química , Diferenciação Celular , Eletrofisiologia/métodos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Microbiota , Humanos , Estudos Longitudinais , Metaboloma , Pessoa de Meia-IdadeRESUMO
It has been previously demonstrated that the bi-directional transporter Na+/Ca2+ exchanger (NCX) working in the reverse (Ca2+-influx) - mode promotes the activation of ERK1/2 in response to the key pro-angiogenic cytokine VEGF in human endothelial cells (ECs). However, the molecular event(s) that elicit NCX reversal in VEGF-stimulated ECs remain unclear. Here we investigated whether Na+ influx via the diacylglycerol (DAG) - activated non-selective cation channel TRPC3 was functionally associated with NCX and whether its activity was required for VEGF-induced ERK1/2 activation and angiogenesis. We provide evidence that TRPC3 inhibitors and siRNA attenuated ERK1/2 phosphorylation, reduced PKCα activity and partially suppressed Ca2+ transients in response to VEGF. Additionally, TRPC3 inhibitors and siRNA significantly suppressed endothelial tubular differentiation, an in vitro indicator of angiogenesis. We also report that simulating PLCγ activation downstream of VEGF receptor 2 by application of the cell-permeable DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was sufficient to activate ERK1/2 and enhance tubular differentiation. OAG-induced ERK1/2 activation and tubulogenesis were significantly suppressed by TRPC3 and reverse-mode NCX inhibitors and siRNA. Moreover, whilst both reverse-mode NCX and TRPC3 inhibitors attenuated OAG-induced Ca2+ transients, only TRPC3 antagonists blunted Na+ influx in response to OAG. Importantly, when Na+ was increased in ECs by inhibiting the Na+-K+-ATPase, TRPC3 activity was dispensable for OAG-induced ERK1/2 phosphorylation. Collectively, our research suggests that DAG generation downstream of VEGF receptors activatesTRPC3 causing Na+ influx with subsequent reversal of NCX, ERK1/2 activation and ultimately contributes to enhanced angiogenesis. Targeting reverse-mode NCX and its upstream initiator TRPC3 could be clinically relevant in conditions characterised by abnormal VEGF signalling.
Assuntos
Células Endoteliais/metabolismo , Sistema de Sinalização das MAP Quinases , Neovascularização Fisiológica , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Sódio/metabolismoRESUMO
Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions. With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification. This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the well-structured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy. On the other hand, the substitution of Val38Cys, which did not contribute to structural changes, indicates a very possible implication of this amino acid in the protein methylation process. The data reported in this work illustrate the distinctive amino acid dynamics in a thermophilic protein, which, while serving the function common to its counterparts from mesophilic organisms, has had to adapt to the extreme environmental conditions typical of thermophilic organisms.