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Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the ß-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the ß-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.
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Aged individuals with spinal cord injury (SCI) are prevalent with increased mortality and worse outcomes. SCI can cause secondary brain neuroinflammation and neurodegeneration. However, the mechanisms contributing to SCI-induced brain dysfunction are poorly understood. Cell-to-cell signaling through extracellular vesicles (EVs) has emerged as a critical mediator of neuroinflammation, including at a distance through circulation. We have previously shown that SCI in young adult (YA) male mice leads to robust changes in plasma EV count and microRNAs (miRs) content. Here, our goal was to investigate the impact of old age on EVs and brain after SCI. At 24 h post-injury, there was no difference in particle count or size distribution between YA and aged mice. However, aged animals increased expression of EV marker CD63 with SCI. Using the Fireplex® miRs assay, Proteomics, and mass spectrometry-based Lipidomics, circulating EVs analysis identified distinct profiles of miRs, proteins, and lipid components in old and injury animals. In vitro, plasma EVs from aged SCI mice, at a lower concentration comparable to those of YA SCI mice, induced the secretion of pro-inflammatory cytokines and neuronal apoptosis. Systemic administration of plasma EVs from SCI animals was sufficient to impair general physical function and neurological function in intact animals, which is associated with pro-inflammatory changes in the brain. Furthermore, plasma EVs from young animals had rejuvenating effects on naïve aged mice. Collectively, these studies identify the critical changes in circulating EVs cargoes after SCI and in aged animals and support a potential EV-mediated mechanism for SCI-induced brain changes.
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Envejecimiento , Encéfalo , Vesículas Extracelulares , Enfermedades Neuroinflamatorias , Traumatismos de la Médula Espinal , Animales , Vesículas Extracelulares/metabolismo , Masculino , Ratones , Enfermedades Neuroinflamatorias/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Encéfalo/metabolismo , Envejecimiento/metabolismo , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Citocinas/metabolismo , Citocinas/sangre , Neuronas/metabolismo , Inflamación/metabolismoRESUMEN
Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.
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Aldehídos , Química Encefálica , Espectrometría de Masas en Tándem , Animales , Espectrometría de Masas en Tándem/métodos , Aldehídos/análisis , Aldehídos/química , Ratones , Cromatografía Liquida/métodos , Reproducibilidad de los Resultados , Masculino , Modelos Lineales , Encéfalo/metabolismo , Límite de Detección , Ratones Endogámicos C57BLRESUMEN
Transient events during development can exert long-lasting effects on organismal lifespan. Here we demonstrate that exposure of Caenorhabditis elegans to reactive oxygen species during development protects against amyloid-induced proteotoxicity later in life. We show that this protection is initiated by the inactivation of the redox-sensitive H3K4me3-depositing COMPASS complex and conferred by a substantial increase in the heat-shock-independent activity of heat shock factor 1 (HSF-1), a longevity factor known to act predominantly during C. elegans development. We show that depletion of HSF-1 leads to marked rearrangements of the organismal lipid landscape and a significant decrease in mitochondrial ß-oxidation and that both lipid and metabolic changes contribute to the protective effects of HSF-1 against amyloid toxicity. Together, these findings link developmental changes in the histone landscape, HSF-1 activity and lipid metabolism to protection against age-associated amyloid toxicities later in life.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción/genética , Histonas/metabolismo , Metabolismo de los Lípidos/genética , Proteínas Amiloidogénicas/metabolismo , Calidad de Vida , Lípidos/farmacologíaRESUMEN
Emerging and re-emerging zoonotic viral diseases continue to significantly impact public health. Of particular interest are enveloped viruses (e.g., SARS-CoV-2, the causative pathogen of COVID-19), which include emerging pathogens of highest concern. Enveloped viruses contain a viral envelope that encapsulates the genetic material and nucleocapsid, providing structural protection and functional bioactivity. The viral envelope is composed of a coordinated network of glycoproteins and lipids. The lipid composition of the envelope consists of lipids preferentially appropriated from host cell membranes. Subsequently, changes to the host cell lipid metabolism and an accounting of what lipids are changed during viral infection provide an opportunity to fingerprint the host cell's response to the infecting virus. To address this issue, we comprehensively characterized the lipid composition of VeroE6-TMPRSS2 cells infected with SARS-CoV-2. Our approach involved using an innovative solid-phase extraction technique to efficiently extract cellular lipids combined with liquid chromatography coupled to high-resolution tandem mass spectrometry. We identified lipid changes in cells exposed to SARS-CoV-2, of which the ceramide to sphingomyelin ratio was most prominent. The identification of a lipid profile (i.e., lipid fingerprint) that is characteristic of cellular SARS-CoV-2 infection lays the foundation for targeting lipid metabolism pathways to further understand how enveloped viruses infect cells, identifying opportunities to aid antiviral and vaccine development.
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COVID-19 , Humanos , SARS-CoV-2 , LípidosRESUMEN
Lipid peroxidation is a key component in the pathogenesis of numerous disease states, where the oxidative damage of lipids frequently leads to membrane dysfunction and subsequent cellular death. Glycerophosphoethanolamine (PE) is the second most abundant phospholipid found in cellular membranes and, when oxidized, has been identified as an executor of ferroptotic cell death. PE commonly exists in the plasmalogen form, where the presence of the vinyl ether bond and its enrichment in polyunsaturated fatty acids make it especially susceptible to oxidative degradation. This results in a multitude of oxidized products complicating identification and often requiring several analytical techniques for interpretation. In the present study, we outline an analytical approach for the structural characterization of intact oxidized products of arachidonate-containing diacyl and plasmalogen PE. Intact oxidized PE structures, including structural and positional isomers, were identified using complementary liquid chromatography techniques, drift tube ion mobility, and high-resolution tandem mass spectrometry. This work establishes a comprehensive method for the analysis of intact lipid peroxidation products and provides an important pathway to investigate how lipid peroxidation initially impacts glycerophospholipids and their role in redox biology.
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Plasmalógenos , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Plasmalógenos/química , Plasmalógenos/metabolismo , Oxidación-Reducción , GlicerofosfolípidosRESUMEN
Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.
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Cobre , Síndrome del Pelo Ensortijado , Ratones , Animales , ATPasas Transportadoras de Cobre , Cobre/metabolismo , Plexo Coroideo/metabolismo , Síndrome del Pelo Ensortijado/metabolismo , Encéfalo/metabolismoRESUMEN
BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Hígado Graso , Animales , Ratones , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Etanol/farmacología , Hígado Graso/metabolismo , Hígado/metabolismo , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.
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Neoplasias de la Mama , Proteínas Celulares de Unión al Retinol/metabolismo , Tretinoina , Neoplasias de la Mama/metabolismo , Proliferación Celular , Colágeno/metabolismo , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Humanos , Retinoides/metabolismo , Proteínas Celulares de Unión al Retinol/genética , Tretinoina/metabolismo , Microambiente Tumoral , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1 loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222 metabolite and metabolite combinations were analyzed, with 33 having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1 loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1 loss and reduced RA in the adult heart.
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Retinoides , Vitamina A , Animales , Homeostasis , Ratones , Proteómica , Retinoides/metabolismo , Proteínas de Unión al Retinol , Proteínas Celulares de Unión al Retinol/genética , Proteínas Celulares de Unión al Retinol/metabolismo , Tretinoina/metabolismo , Vitamina A/metabolismoRESUMEN
Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.
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Oligonucleótidos , Espectroscopía de Resonancia Magnética , Oligonucleótidos/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genéticaRESUMEN
Changes in plasmalogen glycerophosphoethanolamine (PE-P) composition (structure and abundance) are a key indicator of altered lipid metabolism. Differential changes in the levels of PE-P have been reported in different disease states, including neurodegenerative diseases. Of particular interest, traumatic brain injury (TBI) has resulted in altered expression of glycerophospholipid profiles, including PE-P. To date, most analytical assays assessing PE-P have focused on general lipidomic workflows to evaluate the relative, semi-quantitative abundance of PE-P during disease progression. This approach provides a broad evaluation of PE-P, yet often lacks specificity and sensitivity for individual PE-P structures which is a necessity for robust quantitative data. The present study highlights the development of a targeted, quantitative method using a HILIC separation and selective reaction monitoring mass spectrometry for the confident identification and accurate quantitation of PE-P. Our innovative method incorporates both the sn-1 alkyl vinyl ether and sn-2 acyl chain as product ion transitions, for specific and sensitive quantitation of 100 PE-P structures. Our method also uniquely allowed for the unambiguous assignment and quantitation of di-unsaturated sn-1 PE-P structures, which to date have not been conclusively quantified. Application of this assay to a TBI mouse model resulted in distinct temporal profiles for plasma PE-P up to 28 days post injury. Plasma PE-P were significantly increased 24 h after induced TBI, followed by a gradual reduction to sham concentrations by day 28. Overall, we established a structure-specific, quantitative assay for identification and quantitation of a comprehensive set of PE-P structures with demonstrated relevance to brain injury.
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Fosfatidiletanolaminas , Plasmalógenos , Animales , Lipidómica , Espectrometría de Masas , RatonesRESUMEN
ABSTRACT: Radiation sequelae is complex and characterized by multiple pathologies, which occur over time and nonuniformly throughout different organs. The study of the mesenteric lymph node (MLN) due to its importance in the gastrointestinal system is of particular interest. Other studies have shown an immediate post-irradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations, but the molecular and functional mechanisms that lead to these cellular alterations remain limited. In this work, we show the use of lipidomic, proteomic, and mass spectrometry imaging in the characterization of the effects of acute radiation exposure on the MLN at different time points after ionizing radiation (IR) from 4 d to 21 d after 12 Gy partial body irradiation with 2.5% bone marrow sparing. The combined analyses showed a dysregulation of the lipid and protein composition in the MLN after IR. Protein expression was affected in numerous pathways, including pathways regulating lipids such as LXR/RXR activation and acute phase response. Lipid distribution and abundance was also affected by IR in the MLN, including an accumulation of triacylglycerides, a decrease in polyunsaturated glycerophospholipids, and changes in polyunsaturated fatty acids. Those changes were observed as early as 4 d after IR and were more pronounced for lipids with a higher concentration in the nodules and the medulla of the MLN. These results provide molecular insight into the MLN that can inform on injury mechanism in a non-human primate model of the acute radiation syndrome of the gastrointestinal tract. Those findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.
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Médula Ósea , Traumatismos Experimentales por Radiación , Animales , Médula Ósea/efectos de la radiación , Lipidómica , Ganglios Linfáticos/patología , Macaca mulatta , Espectrometría de Masas , Proteómica , Traumatismos Experimentales por Radiación/patologíaRESUMEN
Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass.
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Glutamina , Sulfuro de Hidrógeno , NAD , Metabolismo Energético , Lipogénesis , Mitocondrias/metabolismo , Transducción de SeñalRESUMEN
There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry. AP-MALDI combined with an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone spiked with lithium salt, resulted in a robust and high-throughput lipid detection platform, specifically geared to sphingolipid detection. Application of the developed workflow included the structural characterization of prominent sphingolipids and detection of over 130 lipid structures from Influenza A virions. Overall, we demonstrate a high-throughput workflow for the detection and structural characterization of total lipid extracts from enveloped viruses using AP-MALDI HRMS and lithium adduct consolidation.
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Litio/química , Lípidos de la Membrana/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Lípidos de la Membrana/química , Esfingolípidos/análisis , Esfingolípidos/químicaRESUMEN
Although low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we showed that proteomic analyses of human and guinea pig heart failure (HF) were consistent with a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry revealed 32% and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2 mg/kg/d) was sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Although cardiac ATRA declined in guinea pig HF and human IDCM, levels of certain retinoid metabolic enzymes diverged. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, blunted the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.
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Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Tretinoina/metabolismo , Vitamina A/metabolismo , Adulto , Anciano , Animales , Animales Recién Nacidos , Benzotiazoles/farmacología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Familia 26 del Citocromo P450/antagonistas & inhibidores , Femenino , Regulación de la Expresión Génica , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Ratas , Tretinoina/farmacología , Triazoles/farmacología , Remodelación Ventricular/efectos de los fármacos , Adulto JovenRESUMEN
Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, the gene that encodes lysosomal ß-glucocerebrosidase (GCase). Mild mutations in GBA1 cause type 1 non-neuronopathic GD, whereas severe mutations cause types 2 and 3 neuronopathic GD (nGD). GCase deficiency results in the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). GlcSph is formed by deacylation of GlcCer by the lysosomal enzyme acid ceramidase. Brains from patients with nGD have high levels of GlcSph, a lipid believed to play an important role in nGD, but the mechanisms involved remain unclear. To identify these mechanisms, we used human induced pluripotent stem cell-derived neurons from nGD patients. We found that elevated levels of GlcSph activate mammalian target of rapamycin (mTOR) complex 1 (mTORC1), interfering with lysosomal biogenesis and autophagy, which were restored by incubation of nGD neurons with mTOR inhibitors. We also found that inhibition of acid ceramidase prevented both, mTOR hyperactivity and lysosomal dysfunction, suggesting that these alterations were caused by GlcSph accumulation in the mutant neurons. To directly determine whether GlcSph can cause mTOR hyperactivation, we incubated wild-type neurons with exogenous GlcSph. Remarkably, GlcSph treatment recapitulated the mTOR hyperactivation and lysosomal abnormalities in mutant neurons, which were prevented by coincubation of GlcSph with mTOR inhibitors. We conclude that elevated GlcSph activates an mTORC1-dependent pathogenic mechanism that is responsible for the lysosomal abnormalities of nGD neurons. We also identify acid ceramidase as essential to the pathogenesis of nGD, providing a new therapeutic target for treating GBA1-associated neurodegeneration.
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Enfermedad de Gaucher , Células Madre Pluripotentes Inducidas , Diana Mecanicista del Complejo 1 de la Rapamicina , Neuronas , Psicosina/análogos & derivados , Ceramidasa Ácida/antagonistas & inhibidores , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Lisosomas , Inhibidores mTOR , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/citología , Psicosina/sangreRESUMEN
Sphingolipids have diverse structural and bioactive functions that play important roles in many key biological processes. Factors such as low relative abundance, varied structures, and a dynamic concentration range provide a difficult analytical challenge for sphingolipid detection. To further improve mass-spectrometry-based sphingolipid analysis, lithium adduct consolidation was implemented to decrease spectral complexity and combine signal intensities, leading to increased specificity and sensitivity. We report the use of lithium hydroxide as a base in a routine hydrolysis procedure in order to effectively remove common ionization suppressants (such as glycolipids and glycerophospholipids) and introduce a source of lithium into the sample. In conjunction, an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone (THAP) is used to facilitate lithium adduct consolidation during the MALDI process. The result is a robust and high-throughput sphingolipid detection scheme, particularly of low-abundance ceramides. Application of our developed workflow includes the detection of differentially expressed liver sphingolipid profiles from a high-fat-induced obesity mouse model. We also demonstrate the method's effectiveness in detecting various sphingolipids in brain and plasma matrices. These results were corroborated with data from UHPLC HR MS/MS and MALDI FT-ICR, verifying the efficacy of the method application. Overall, we demonstrate a high-throughput workflow for sphingolipid analysis in various biological matrices by the use of MALDI TOF and lithium adduct consolidation.
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Compuestos de Litio/química , Hígado/química , Obesidad/etiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Esfingolípidos/análisis , Acetofenonas/química , Animales , Cromatografía Líquida de Alta Presión , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Hidrólisis , Hígado/metabolismo , Ratones Endogámicos C57BL , Esfingolípidos/química , Flujo de TrabajoRESUMEN
Drug-induced liver injury (DILI) has a substantial impact on human health and is a major monetary burden on the drug development process. Presently, there is a lack of robust and analytically validated markers for predicting and early diagnosis of DILI. Sphingolipid metabolism and subsequent disruption of sphingolipid homeostasis has been documented to play a key role contributing to hepatocellular death and subsequent liver injury. A more comprehensive understanding of sphingolipid metabolism in response to liver toxicity has great potential to gain mechanistic insight into hepatotoxicity and define molecular markers that are responsible for hepatocyte dysfunction. Here, we present an analytical platform that provides multidimensional mass spectrometry-based datasets for comprehensive structure characterization of sphingolipids extracted from human primary hepatocytes (HPH) exposed to toxic levels of acetaminophen (APAP). Sphingolipid metabolism as measured by characterization of individual sphingolipid structure was sensitive to APAP toxicity displaying a concentration-dependent response. A number of sphingolipid structures were differentially expressed across varying APAP exposures highlighting the unique role sphingolipid metabolism has in response to hepatotoxicity and its potential use as a molecular marker in DILI.