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BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.
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Lisofosfolípidos , Monocitos , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Diglicéridos/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipoproteína(a)/metabolismo , Monocitos/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Rationale: The plasma lipidome has the potential to reflect many facets of the host status during severe infection. Previous work is limited to specific lipid groups or was focused on lipids as prognosticators.Objectives: To map the plasma lipidome during sepsis due to community-acquired pneumonia (CAP) and determine the disease specificity and associations with clinical features.Methods: We analyzed 1,833 lipid species across 33 classes in 169 patients admitted to the ICU with sepsis due to CAP, 51 noninfected ICU patients, and 48 outpatient controls. In a paired analysis, we reanalyzed patients still in the ICU 4 days after admission (n = 82).Measurements and Main Results: A total of 58% of plasma lipids were significantly lower in patients with CAP-attributable sepsis compared with outpatient controls (6% higher, 36% not different). We found strong lipid class-specific associations with disease severity, validated across two external cohorts, and inflammatory biomarkers, in which triacylglycerols, cholesterol esters, and lysophospholipids exhibited the strongest associations. A total of 36% of lipids increased over time, and stratification by survival revealed diverging lipid recovery, which was confirmed in an external cohort; specifically, a 10% increase in cholesterol ester levels was related to a lower odds ratio (0.84; P = 0.006) for 30-day mortality (absolute mortality, 18 of 82). Comparison with noninfected ICU patients delineated a substantial common illness response (57.5%) and a distinct lipidomic signal for patients with CAP-attributable sepsis (37%).Conclusions: Patients with sepsis due to CAP exhibit a time-dependent and partially disease-specific shift in their plasma lipidome that correlates with disease severity and systemic inflammation and is associated with higher mortality.
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Infecciones Comunitarias Adquiridas , Neumonía , Sepsis , Humanos , Lipidómica , Neumonía/complicaciones , Sepsis/complicaciones , Lípidos , Índice de Severidad de la Enfermedad , Unidades de Cuidados IntensivosRESUMEN
Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions-retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra-providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD.
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Adrenoleucodistrofia , Espectrometría de Movilidad Iónica , Lipidómica , Adrenoleucodistrofia/metabolismo , Adrenoleucodistrofia/genética , Humanos , Lipidómica/métodos , Lípidos/análisis , Metabolismo de los LípidosRESUMEN
Circadian clock function declines with ageing, which can aggravate ageing-related diseases such as type 2 diabetes and neurodegenerative disorders. Understanding age-related changes in the circadian system at a systemic level can contribute to the development of strategies to promote healthy ageing. The goal of this study was to investigate the impact of ageing on 24-h rhythms in amine metabolites across four tissues in young (2 months of age) and old (22-25 months of age) mice using a targeted metabolomics approach. Liver, plasma, the suprachiasmatic nucleus (SCN; the location of the central circadian clock in the hypothalamus) and the paraventricular nucleus (PVN; a downstream target of the SCN) were collected from young and old mice every 4 h during a 24-h period (n = 6-7 mice per group). Differential rhythmicity analysis revealed that ageing impacts 24-h rhythms in the amine metabolome in a tissue-specific manner. Most profound changes were observed in the liver, in which rhythmicity was lost in 60% of the metabolites in aged mice. Furthermore, we found strong correlations in metabolite levels between the liver and plasma and between the SCN and the PVN in young mice. These correlations were almost completely abolished in old mice. These results indicate that ageing is accompanied by a severe loss of the circadian coordination between tissues and by disturbed rhythmicity of metabolic processes. The tissue-specific impact of ageing may help to differentiate mechanisms of ageing-related disorders in the brain versus peripheral tissues and thereby contribute to the development of potential therapies for these disorders.
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Envejecimiento , Ritmo Circadiano , Hígado , Metaboloma , Núcleo Hipotalámico Paraventricular , Núcleo Supraquiasmático , Animales , Envejecimiento/metabolismo , Envejecimiento/fisiología , Núcleo Supraquiasmático/metabolismo , Ritmo Circadiano/fisiología , Ratones , Hígado/metabolismo , Masculino , Núcleo Hipotalámico Paraventricular/metabolismo , Ratones Endogámicos C57BL , Relojes Circadianos/fisiología , Aminas/metabolismoRESUMEN
PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.
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Proteínas Hierro-Azufre , Pez Cebra , Animales , Humanos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Masculino , Femenino , Fenotipo , Fibroblastos/metabolismo , Fibroblastos/patología , Citosol/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Microcefalia/genética , Microcefalia/patología , Lactante , MetalochaperonasRESUMEN
Altered metabolism is a hallmark of both cell division and cancer. Chronic lymphocytic leukemia (CLL) cells circulate between peripheral blood (PB) and lymph nodes (LNs), where they receive proliferative and prosurvival signals from surrounding cells. However, insight into the metabolism of LN CLL and how this may relate to therapeutic response is lacking. To obtain insight into CLL LN metabolism, we applied a 2-tiered strategy. First, we sampled PB from 8 patients at baseline and after 3-month ibrutinib (IBR) treatment, which forces egress of CLL cells from LNs. Second, we applied in vitro B-cell receptor (BCR) or CD40 stimulation to mimic the LN microenvironment and performed metabolomic and transcriptomic analyses. The combined analyses indicated prominent changes in purine, glucose, and glutamate metabolism occurring in the LNs. CD40 signaling mostly regulated amino acid metabolism, tricarboxylic acid cycle (TCA), and energy production. BCR signaling preferably engaged glucose and glycerol metabolism and several biosynthesis routes. Pathway analyses demonstrated opposite effects of in vitro stimulation vs IBR treatment. In agreement, the metabolic regulator MYC and its target genes were induced after BCR/CD40 stimulation and suppressed by IBR. Next, 13C fluxomics performed on CD40/BCR-stimulated cells confirmed a strong contribution of glutamine as fuel for the TCA cycle, whereas glucose was mainly converted into lactate and ribose-5-phosphate. Finally, inhibition of glutamine import with V9302 attenuated CD40/BCR-induced resistance to venetoclax. Together, these data provide insight into crucial metabolic changes driven by the CLL LN microenvironment. The prominent use of amino acids as fuel for the TCA cycle suggests new therapeutic vulnerabilities.
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Leucemia Linfocítica Crónica de Células B , Antígenos CD40 , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Ganglios Linfáticos/patología , Receptores de Antígenos de Linfocitos B/metabolismo , Microambiente TumoralRESUMEN
STUDY QUESTION: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? SUMMARY ANSWER: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. WHAT IS KNOWN ALREADY: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. STUDY DESIGN, SIZE, DURATION: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. MAIN RESULTS AND THE ROLE OF CHANCE: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. LIMITATIONS, REASONS FOR CAUTION: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. WIDER IMPLICATIONS OF THE FINDINGS: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. TRIAL REGISTRATION NUMBER: N/A.
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NAD , Oocitos , Humanos , Femenino , Masculino , NAD/metabolismo , Oocitos/metabolismo , Estrés Oxidativo , Mitocondrias/metabolismo , EnvejecimientoRESUMEN
Shift-workers show an increased incidence of type 2 diabetes mellitus (T2DM). A possible mechanism is the disruption of the circadian timing of glucose homeostasis. Skeletal muscle mitochondrial function is modulated by the molecular clock. We used time-restricted feeding (TRF) during the inactive phase to investigate how mistimed feeding affects muscle mitochondrial metabolism. Rats on an ad libitum (AL) diet were compared to those that could eat only during the light (inactive) or dark (active) phase. Mitochondrial respiration, metabolic gene expressions, and metabolite concentrations were determined in the soleus muscle. Rats on AL feeding or dark-fed TRF showed a clear daily rhythm in muscle mitochondrial respiration. This rhythm in mitochondrial oxidative phosphorylation capacity was abolished in light-fed TRF animals and overall 24h respiration was lower. The expression of several genes involved in mitochondrial biogenesis and the fission/fusion machinery was altered in light-fed animals. Metabolomics analysis indicated that light-fed animals had lost rhythmic levels of α-ketoglutarate and citric acid. Contrastingly, lipidomics showed that light-fed animals abundantly gained rhythmicity in levels of triglycerides. Furthermore, while the RER shifted entirely with the food intake in the light-fed animals, many measured metabolic parameters (e.g., activity and mitochondrial respiration) did not strictly align with the shifted timing of food intake, resulting in a mismatch between expected metabolic supply/demand (as dictated by the circadian timing system and light/dark-cycle) and the actual metabolic supply/demand (as dictated by the timing of food intake). These data suggest that shift-work impairs mitochondrial metabolism and causes metabolic inflexibility, which can predispose to T2DM.
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Respiración de la Célula/fisiología , Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Ayuno/fisiología , Mitocondrias/fisiología , Músculo Esquelético/fisiología , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Dieta/métodos , Ingestión de Alimentos/fisiología , Metabolismo Energético/fisiología , Conducta Alimentaria/fisiología , Expresión Génica/fisiología , Masculino , Biogénesis de Organelos , Fosforilación Oxidativa , Fotoperiodo , Ratas , Ratas WistarRESUMEN
Circulating nonadherent monocytes can migrate to extravascular sites by a process that involves adherence. Alterations in intracellular metabolism shape the immunological phenotype of phagocytes upon activation. To determine the effect of adherence on their metabolic and functional response human monocytes were stimulated with LPS under nonadherent and adherent conditions. Adherent monocytes (relative to nonadherent monocytes) produced less TNF and IL-1ß (proinflammatory) and more IL-10 (anti-inflammatory) upon LPS stimulation and had an increased capacity to phagocytose and produce reactive oxygen species. RNA sequencing analysis confirmed that adherence modified the LPS-induced response of monocytes, reducing expression of proinflammatory genes involved in TLR signaling and increasing induction of genes involved in pathogen elimination. Adherence resulted in an increased glycolytic response as indicated by lactate release, gene set enrichment, and [13C]-glucose flux analysis. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analogue 2-deoxy-d-glucose (2DG). Although both interventions equally inhibited glycolysis, only 2DG influenced monocyte functions, inhibiting expression of genes involved in TLR signaling and pathogen elimination, as well as cytokine release. 2DG, but not glucose deprivation, reduced expression of genes involved in oxidative phosphorylation. Inhibition of oxidative phosphorylation affected TNF and IL-10 release in a similar way as 2DG. Collectively, these data suggest that adherence may modify the metabolic and immunological profile of monocytes and that inhibition of glycolysis and oxidative phosphorylation, but not inhibition of glycolysis alone, has a profound effect on immune functions of monocytes exposed to LPS.
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Reprogramación Celular , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/toxicidad , Monocitos/inmunología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/inmunología , Humanos , Monocinas/inmunologíaRESUMEN
We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
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Errores Innatos del Metabolismo de los Aminoácidos/genética , Ataxia/genética , Discapacidades del Desarrollo/genética , Glutaminasa/deficiencia , Glutaminasa/genética , Glutamina/metabolismo , Repeticiones de Microsatélite , Mutación , Atrofia/genética , Cerebelo/patología , Preescolar , Femenino , Genotipo , Glutamina/análisis , Humanos , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , Secuenciación Completa del GenomaRESUMEN
Circadian misalignment, as seen in shift work, is associated with an increased risk to develop type 2 diabetes. In an experimental setting, we recently showed that a rapid day-night shift for 3 consecutive nights leads to misalignment of the core molecular clock, induction of the PPAR pathway, and insulin resistance in skeletal muscle of young, healthy men. Here, we investigated if circadian misalignment affects the skeletal muscle lipidome and intramyocellular lipid droplet characteristics, explaining the misalignment-induced insulin resistance. Fourteen healthy men underwent one aligned and one circadian misalignment period, both consisting of ~3.5 days. In the misaligned condition, day and night were rapidly shifted by 12 hours leading to opposite eating, sleep, and activity times compared with the aligned condition. For each condition, two muscle biopsies were taken from the m. vastus lateralis in the morning and evening and subjected to semi-targeted lipidomics and confocal microscopy analysis. We found that only 2% of detected lipids were different between morning and evening in the aligned condition, whereas 12% displayed a morning-evening difference upon misalignment. Triacylglycerols, in particular species of a carbon length ≥55, were the most abundant lipid species changed upon misalignment. Cardiolipins were decreased upon misalignment, whereas phosphatidylcholines consistently followed the same morning-evening pattern, suggesting regulation by the circadian clock. Cholesteryl esters adjusted to the shifted behavior. Lipid droplet characteristics remained unaltered upon misalignment. Together, these findings show that simulated shift work disturbs the skeletal muscle lipidome, which may contribute to misalignment-induced insulin resistance.
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Ritmo Circadiano , Lipidómica/métodos , Lípidos/análisis , Músculo Esquelético/patología , Adulto , Humanos , Masculino , Músculo Esquelético/metabolismo , Adulto JovenRESUMEN
Nicotinamide adenine dinucleotide (NAD+ ) homeostasis is constantly compromised due to degradation by NAD+ -dependent enzymes. NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.
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NAD/metabolismo , Mononucleótido de Nicotinamida/metabolismo , Animales , Línea Celular , Supervivencia Celular , Células Epiteliales/efectos de los fármacos , Homeostasis , Humanos , Túbulos Renales , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , NAD/genética , Mononucleótido de Nicotinamida/química , Daño por ReperfusiónRESUMEN
RATIONALE: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state. OBJECTIVE: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism. METHODS AND RESULTS: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration. CONCLUSIONS: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease.
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Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Glucólisis , Leucocitos/metabolismo , Lipoproteína(a)/metabolismo , Migración Transendotelial y Transepitelial , Anciano , Anciano de 80 o más Años , Animales , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/terapia , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Humanos , Mediadores de Inflamación , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/patología , Lipoproteína(a)/genética , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Mutación , Oligonucleótidos Antisentido/uso terapéutico , Fosfofructoquinasa-2/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.
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Galactosemias , Humanos , Galactosemias/genética , Galactosa/metabolismo , Redes y Vías Metabólicas , Biomarcadores/metabolismo , Fosfatos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
IgG Abs are crucial for various immune functions, including neutralization, phagocytosis, and Ab-dependent cellular cytotoxicity. In this study, we identified another function of IgG by showing that IgG immune complexes elicit distinct cytokine profiles by human myeloid immune cells, which are dependent on FcγR activation by the different IgG subclasses. Using monoclonal IgG subclasses with identical Ag specificity, our data demonstrate that the production of Th17-inducing cytokines, such as TNF, IL-1ß, and IL-23, is particularly dependent on IgG2, whereas type I IFN responses are controlled by IgG3, and IgG1 is able to regulate both. In addition, we identified that subclass-specific cytokine production is orchestrated at the posttranscriptional level through distinct glycolytic reprogramming of human myeloid immune cells. Combined, these data identify that IgG subclasses provide pathogen- and cell type-specific immunity through differential metabolic reprogramming by FcγRs. These findings may be relevant for future design of Ab-related therapies in the context of infectious diseases, chronic inflammation, and cancer.
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Citocinas/inmunología , Inmunoglobulina G/inmunología , Células Mieloides/inmunología , Receptores de IgG/inmunología , Humanos , Células Mieloides/citologíaRESUMEN
Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.
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Anemia de Diamond-Blackfan/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Niño , Células Eritroides , Femenino , Humanos , Masculino , Mutación/genética , Precursores del ARN/genética , ARN Mensajero/genética , Secuenciación del ExomaRESUMEN
BACKGROUND: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology. OBJECTIVE: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty. METHODS: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7). RESULTS: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways. CONCLUSION: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty.
Asunto(s)
Asma/metabolismo , Bronquios/patología , Termoplastia Bronquial , Mucosa Respiratoria/metabolismo , Adolescente , Adulto , Anciano , Asma/patología , Asma/terapia , Femenino , Perfilación de la Expresión Génica , Voluntarios Sanos , Humanos , Metabolismo de los Lípidos/genética , Masculino , Persona de Mediana Edad , Fosforilación Oxidativa , Mucosa Respiratoria/patología , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors belonging to the nuclear receptor superfamily. Since most target genes of PPARs are implicated in lipid and glucose metabolism, regulation by PPARs could be used as a screening tool to identify novel genes involved in lipid or glucose metabolism. Here, we identify Adtrp, a serine hydrolase enzyme that was reported to catalyze the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs), as a novel PPAR-regulated gene. Adtrp was significantly upregulated by PPARα activation in mouse primary hepatocytes, liver slices, and whole liver. In addition, Adtrp was upregulated by PPARγ activation in 3L3-L1 adipocytes and in white adipose tissue. ChIP-SEQ identified a strong PPAR-binding site in the immediate upstream promoter of the Adtrp gene. Adenoviral-mediated hepatic overexpression of Adtrp in diet-induced obese mice caused a modest increase in plasma nonesterified fatty acids but did not influence diet-induced obesity, liver triglyceride levels, liver lipidomic profiles, liver transcriptomic profiles, plasma cholesterol, triglyceride, glycerol, and glucose levels. Moreover, hepatic Adtrp overexpression did not lead to significant changes in FAHFA levels in plasma or liver and did not influence glucose and insulin tolerance. Finally, hepatic overexpression of Adtrp did not influence liver triglycerides and levels of plasma metabolites after a 24-h fast. Taken together, our data suggest that despite being a PPAR-regulated gene, hepatic Adtrp does not seem to play a major role in lipid and glucose metabolism and does not regulate FAHFA levels.
Asunto(s)
Esterasas/biosíntesis , Glucosa/metabolismo , Hepatocitos/enzimología , Metabolismo de los Lípidos , Lípidos/sangre , Proteínas de la Membrana/biosíntesis , Células 3T3-L1 , Adipocitos/enzimología , Animales , Modelos Animales de Enfermedad , Inducción Enzimática , Esterasas/genética , Ayuno/metabolismo , Femenino , Lipidómica , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Obesidad/enzimología , Obesidad/genética , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gamma/metabolismoRESUMEN
AIMS/HYPOTHESIS: In our current society sedentary behaviour predominates in most people and is associated with the risk of developing type 2 diabetes. It has been suggested that replacing sitting time by standing and walking could be beneficial for individuals with type 2 diabetes but the underlying mechanisms are unknown and direct comparisons with exercise are lacking. Our objective was to directly compare metabolic responses of either sitting less or exercising, relative to being sedentary. METHODS: We performed a randomised, crossover intervention study in 12 overweight women who performed three well-controlled 4 day activity regimens: (1) sitting regimen (sitting 14 h/day); (2) exercise regimen (sitting 13 h/day, exercise 1 h/day); and (3) sitting less regimen (sitting 9 h/day, standing 4 h/day and walking 3 h/day). The primary outcome was insulin sensitivity measured by a two-step hyperinsulinaemic-euglycaemic clamp. We additionally performed metabolomics on muscle biopsies taken before the clamp to identify changes at the molecular level. RESULTS: Replacing sitting time by standing and walking over 4 days resulted in improved peripheral insulin sensitivity, comparable with the improvement achieved by moderate-to-vigorous exercise. Specifically, we report a significant improvement in peripheral insulin sensitivity in the sitting less (~13%) and the exercise regimen (~20%), compared with the sitting regimen. Furthermore, sitting less shifted the underlying muscle metabolome towards that seen with moderate-to-vigorous exercise, compared with the sitting regimen. CONCLUSIONS/INTERPRETATIONS: Replacing sitting time by standing and walking is an attractive alternative to moderate-to-vigorous exercise for improving metabolic health. TRIAL REGISTRATION: ClinicalTrials.gov NCT03912922.
Asunto(s)
Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Femenino , Humanos , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Posmenopausia , Sedestación , Caminata/fisiologíaRESUMEN
BACKGROUND AND AIMS: Glycogen storage disease (GSD) type 1a is an inborn error of metabolism caused by defective glucose-6-phosphatase catalytic subunit (G6PC) activity. Patients with GSD 1a exhibit severe hepatomegaly due to glycogen and triglyceride (TG) accumulation in the liver. We have shown that the activity of carbohydrate response element binding protein (ChREBP), a key regulator of glycolysis and de novo lipogenesis, is increased in GSD 1a. In the current study, we assessed the contribution of ChREBP to nonalcoholic fatty liver disease (NAFLD) development in a mouse model for hepatic GSD 1a. APPROACH AND RESULTS: Liver-specific G6pc-knockout (L-G6pc-/- ) mice were treated with adeno-associated viruses (AAVs) 2 or 8 directed against short hairpin ChREBP to normalize hepatic ChREBP activity to levels observed in wild-type mice receiving AAV8-scrambled short hairpin RNA (shSCR). Hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L-G6pc-/- mice. This was associated with hepatic accumulation of G6P, glycogen, and lipids, whereas the expression of glycolytic and lipogenic genes was reduced. Enzyme activities, flux measurements, hepatic metabolite analysis and very low density lipoprotein (VLDL)-TG secretion assays revealed that hepatic ChREBP knockdown reduced downstream glycolysis and de novo lipogenesis but also strongly suppressed hepatic VLDL lipidation, hence promoting the storage of "old fat." Interestingly, enhanced VLDL-TG secretion in shSCR-treated L-G6pc-/- mice associated with a ChREBP-dependent induction of the VLDL lipidation proteins microsomal TG transfer protein and transmembrane 6 superfamily member 2 (TM6SF2), the latter being confirmed by ChIP-qPCR. CONCLUSIONS: Attenuation of hepatic ChREBP induction in GSD 1a liver aggravates hepatomegaly because of further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL-TG secretion. TM6SF2, critical for VLDL formation, was identified as a ChREBP target in mouse liver. Altogether, our data show that enhanced ChREBP activity limits NAFLD development in GSD 1a by balancing hepatic TG production and secretion.