RESUMO
A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.
Assuntos
Neoplasias Encefálicas , Encéfalo , Malformações Vasculares do Sistema Nervoso Central , Células Endoteliais , Feto , RNA-Seq , Análise da Expressão Gênica de Célula Única , Feminino , Humanos , Masculino , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Encéfalo/embriologia , Encéfalo/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Comunicação Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/citologia , Feto/irrigação sanguínea , Feto/citologia , Feto/embriologia , Malformações Vasculares do Sistema Nervoso Central/patologia , Antígenos HLA-D/metabolismo , Adulto , SaúdeRESUMO
The CNS critically relies on the formation and proper function of its vasculature during development, adult homeostasis and disease. Angiogenesis - the formation of new blood vessels - is highly active during brain development, enters almost complete quiescence in the healthy adult brain and is reactivated in vascular-dependent brain pathologies such as brain vascular malformations and brain tumours. Despite major advances in the understanding of the cellular and molecular mechanisms driving angiogenesis in peripheral tissues, developmental signalling pathways orchestrating angiogenic processes in the healthy and the diseased CNS remain incompletely understood. Molecular signalling pathways of the 'neurovascular link' defining common mechanisms of nerve and vessel wiring have emerged as crucial regulators of peripheral vascular growth, but their relevance for angiogenesis in brain development and disease remains largely unexplored. Here we review the current knowledge of general and CNS-specific mechanisms of angiogenesis during brain development and in brain vascular malformations and brain tumours, including how key molecular signalling pathways are reactivated in vascular-dependent diseases. We also discuss how these topics can be studied in the single-cell multi-omics era.
Assuntos
Neoplasias Encefálicas , Malformações Vasculares do Sistema Nervoso Central , Humanos , Neovascularização Fisiológica/fisiologia , Encéfalo , Transdução de SinaisRESUMO
Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.
Assuntos
Células Endoteliais/metabolismo , Glicólise , Neovascularização Fisiológica , Fosfofrutoquinase-2/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/citologia , Feminino , Deleção de Genes , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinase-2/genética , Pseudópodes/metabolismo , Peixe-ZebraRESUMO
Glucose is the universal fuel of most mammalian cells, and it is largely replenished through dietary intake. Glucose availability to tissues is paramount for the maintenance of homeostatic energetics and, hence, supply should match demand by the consuming organs. In its journey through the body, glucose encounters cellular barriers for transit at the levels of the absorbing intestinal epithelial wall, the renal epithelium mediating glucose reabsorption, and the tight capillary endothelia (especially in the brain). Glucose transiting through these cellular barriers must escape degradation to ensure optimal glucose delivery to the bloodstream or tissues. The liver, which stores glycogen and generates glucose de novo, must similarly be able to release it intact to the circulation. We present the most up-to-date knowledge on glucose handling by the gut, liver, brain endothelium, and kidney, and discuss underlying molecular mechanisms and open questions. Diseases associated with defects in glucose delivery and homeostasis are also briefly addressed. We propose that the universal problem of sparing glucose from catabolism in favor of translocation across the barriers posed by epithelia and endothelia is resolved through common mechanisms involving glucose transfer to the endoplasmic reticulum, from where glucose exits the cells via unconventional cellular mechanisms.
Assuntos
Encéfalo , Glucose , Animais , Humanos , Glucose/metabolismo , Epitélio/metabolismo , Encéfalo/metabolismo , Transporte Biológico , Intestinos , Mamíferos/metabolismoRESUMO
Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.
Assuntos
Glutamina/metabolismo , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Envelhecimento/metabolismo , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Glutamato Desidrogenase/deficiência , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Macrófagos/enzimologia , Masculino , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Oxirredução , Células Satélites de Músculo Esquelético/citologia , Serina-Treonina Quinases TORRESUMO
Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+ ) levels. The HIF-1α/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD+ metabolism.
Assuntos
NAD , Triptofano , Camundongos , Animais , Triptofano/metabolismo , Células Matadoras Naturais , Glicólise/genética , Hipóxia/metabolismo , Hipóxia Celular , Oxigênio/metabolismo , Homeostase , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.
Assuntos
Células Endoteliais , Neovascularização Fisiológica , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Hipóxia/metabolismo , Isquemia/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Proteínas Imediatamente Precoces/metabolismoRESUMO
RATIONALE: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. OBJECTIVE: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. METHODS AND RESULTS: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. CONCLUSIONS: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.
Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Transportador de Glucose Tipo 1/fisiologia , Neovascularização Fisiológica , Vasos Retinianos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Encéfalo/citologia , Movimento Celular , Proliferação de Células , Células Endoteliais/fisiologia , Endotélio , Endotélio Vascular/fisiologia , Metabolismo Energético , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Glicólise , Humanos , Camundongos , Retina/citologiaRESUMO
Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8Y389C knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain.
Assuntos
Doenças Cerebelares , Creatina , Animais , Cerebelo/metabolismo , Guanidinoacetato N-Metiltransferase/genética , Humanos , Proteínas de Membrana Transportadoras , Músculos/metabolismo , Atrofia Muscular , Ratos , SíndromeRESUMO
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.
Assuntos
Carbono/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Nucleotídeos/biossíntese , Ácido Acético/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo do Ácido Cítrico , DNA/biossíntese , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Inativação Gênica , Glucose/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Nucleotídeos/química , Nucleotídeos/farmacologia , Oxirredução/efeitos dos fármacos , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal neurodegenerative disorder that primarily affects motor neurons. Despite our increased understanding of the genetic factors contributing to ALS, no effective treatment is available. A growing body of evidence shows disturbances in energy metabolism in ALS. Moreover, the remarkable vulnerability of motor neurons to ATP depletion has become increasingly clear. Here, we review metabolic alterations present in ALS patients and models, discuss the selective vulnerability of motor neurons to energetic stress, and provide an overview of tested and emerging metabolic approaches to treat ALS. We believe that a further understanding of the metabolic biology of ALS can lead to the identification of novel therapeutic targets.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Metabolismo Energético , Esclerose Lateral Amiotrófica/terapia , Animais , HumanosRESUMO
The aim of this exploratory study was to investigate how sedentary behavior (SB) and physical activity (PA) influence DNA methylation at a global, gene-specific, and health-related pathway level. SB, light PA (LPA), and moderate-to-vigorous PA (MVPA) were assessed objectively for 41 Flemish men using the SenseWear Pro 3 Armband. CpG site-specific methylation in leukocytes was determined using the Illumina HumanMethylation 450 BeadChip. Correlations were calculated between time spent on the three PA intensity levels and global DNA methylation, using a z-score-based method to determine global DNA methylation levels. To determine whether CpG site-specific methylation can be predicted by these three PA intensity levels, linear regression analyses were performed. Based on the significantly associated CpG sites at α = 0.005, lists were created including all genes with a promoter region overlapping these CpG sites. A biological pathway analysis determined to what extent these genes are overrepresented within several pathways. No significant associations were observed between global DNA methylation and SB (r = 0.084), LPA (r = -0.168), or MVPA (r = -0.125), although the direction of the correlation coefficients is opposite to what is generally reported in literature. SB has a different impact on global and gene-specific methylation than PA, but also LPA and MVPA affect separate genes and pathways. Furthermore, the function of a pathway seems to determine its association with SB, LPA, or MVPA. Multiple PA intensity levels, including SB, should be taken into account in future studies investigating the effect of physical (in)activity on human health through epigenetic mechanisms.
Assuntos
Ilhas de CpG , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Exercício Físico/fisiologia , Leucócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The tight interrelationship between peroxisomes and mitochondria is illustrated by their cooperation in lipid metabolism, antiviral innate immunity and shared use of proteins executing organellar fission. In addition, we previously reported that disruption of peroxisome biogenesis in hepatocytes severely impacts on mitochondrial integrity, primarily damaging the inner membrane. Here we investigated the molecular impairments of the dysfunctional mitochondria in hepatocyte selective Pex5 knockout mice. First, by using blue native electrophoresis and in-gel activity stainings we showed that the respiratory complexes were differentially affected with reduction of complexes I and III and incomplete assembly of complex V, whereas complexes II and IV were normally active. This resulted in impaired oxygen consumption in cultured Pex5(-/-) hepatocytes. Second, mitochondrial DNA was depleted causing an imbalance in the expression of mitochondrial- and nuclear-encoded subunits of the respiratory chain complexes. Third, mitochondrial membranes showed increased permeability and fluidity despite reduced content of the polyunsaturated fatty acid docosahexaenoic acid. Fourth, the affected mitochondria in peroxisome deficient hepatocytes displayed increased oxidative stress. Acute deletion of PEX5 in vivo using adeno-Cre virus phenocopied these effects, indicating that mitochondrial perturbations closely follow the loss of functional peroxisomes in time. Likely to compensate for the functional impairments, the volume of the mitochondrial compartment was increased several folds. This was not driven by PGC-1α but mediated by activation of PPARα, possibly through c-myc overexpression. In conclusion, loss of peroxisomal metabolism in hepatocytes perturbs the mitochondrial inner membrane, depletes mitochondrial DNA and causes mitochondrial biogenesis independent of PGC-1α.
Assuntos
DNA Mitocondrial/metabolismo , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Compartimento Celular , Proliferação de Células , Respiração Celular , Transporte de Elétrons , Deleção de Genes , Hepatócitos/ultraestrutura , Lipídeos/química , Fluidez de Membrana , Camundongos Knockout , Mitocôndrias/ultraestrutura , Oxirredução , Fosforilação Oxidativa , Estresse Oxidativo , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptor 1 de Sinal de Orientação para Peroxissomos , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-ß ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.
Assuntos
Linhagem da Célula , Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/fisiologia , Miostatina/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Envelhecimento/metabolismo , Animais , Proliferação de Células , Deleção de Genes , Marcação de Genes , Hipertrofia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Miostatina/metabolismo , Transdução de SinaisRESUMO
OBJECTIVES: We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury. METHODS: To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45+CD11b+F4/80+CD64+ macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively. RESULTS: We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes. CONCLUSIONS: Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration.
Assuntos
Histonas , Isquemia , Macrófagos , Camundongos Endogâmicos C57BL , Músculo Esquelético , Regeneração , Animais , Macrófagos/metabolismo , Camundongos , Histonas/metabolismo , Músculo Esquelético/metabolismo , Isquemia/metabolismo , Masculino , Expressão Gênica/genéticaRESUMO
Dietary restriction of the sulfur-containing amino acids methionine and cysteine (SAAR) improves body composition, enhances insulin sensitivity, and extends lifespan; benefits seen also with endurance exercise. Yet, the impact of SAAR on skeletal muscle remains largely unexplored. Here we demonstrate that one week of SAAR in sedentary, young, male mice increases endurance exercise capacity. Indirect calorimetry showed that SAAR increased lipid oxidation at rest and delayed the onset of carbohydrate utilization during exercise. Transcriptomic analysis revealed increased expression of genes involved in fatty acid catabolism especially in glycolytic muscle following SAAR. These findings were functionally supported by increased fatty acid circulatory turnover flux and muscle ß-oxidation. Reducing lipid uptake from circulation through endothelial cell (EC)-specific CD36 deletion attenuated the running phenotype. Mechanistically, VEGF-signaling inhibition prevented exercise increases following SAAR, without affecting angiogenesis, implicating noncanonical VEGF signaling and EC CD36-dependent fatty acid transport in regulating exercise capacity by influencing muscle substrate availability.
RESUMO
Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.
RESUMO
Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.