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BACKGROUND: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SIGNIFICANCE: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.
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Bicarbonatos , Animales , Ratones , Tampones (Química) , Bicarbonatos/química , Lípidos/química , Cromatografía de Fase Inversa/métodos , Propiedades de Superficie , Lipidómica/métodos , Ratones Endogámicos C57BL , Interacciones Hidrofóbicas e Hidrofílicas , Ácidos Fosfatidicos/química , Hígado/químicaRESUMEN
BACKGROUND: Clinical studies demonstrated beneficial effects of sodium-glucose-transporter 2 inhibitors on the risk of cardiovascular death in patients with heart failure with preserved ejection fraction (HFpEF). However, underlying processes for cardioprotection remain unclear. The present study focused on the impact of empagliflozin (Empa) on myocardial function in a rat model with established HFpEF and analyzed underlying molecular mechanisms. METHODS: Obese ZSF1 (Zucker fatty and spontaneously hypertensive) rats were randomized to standard care (HFpEF, n=18) or Empa (HFpEF/Empa, n=18). ZSF1 lean rats (con, n=18) served as healthy controls. Echocardiography was performed at baseline and after 4 and 8 weeks, respectively. After 8 weeks of treatment, hemodynamics were measured invasively, mitochondrial function was assessed and myocardial tissue was collected for either molecular and histological analyses or transmission electron microscopy. RESULTS: In HFpEF Empa significantly improved diastolic function (E/é: con: 17.5±2.8; HFpEF: 24.4±4.6; P<0.001 versus con; HFpEF/Empa: 19.4±3.2; P<0.001 versus HFpEF). This was accompanied by improved hemodynamics and calcium handling and by reduced inflammation, hypertrophy, and fibrosis. Proteomic analysis demonstrated major changes in proteins involved in mitochondrial oxidative phosphorylation. Cardiac mitochondrial respiration was significantly impaired in HFpEF but restored by Empa (Vmax complex IV: con: 0.18±0.07 mmol O2/s/mg; HFpEF: 0.13±0.05 mmol O2/s/mg; P<0.041 versus con; HFpEF/Empa: 0.21±0.05 mmol O2/s/mg; P=0.012 versus HFpEF) without alterations of mitochondrial content. The expression of cardiolipin, an essential stability/functionality-mediating phospholipid of the respiratory chain, was significantly decreased in HFpEF but reverted by Empa (con: 15.9±1.7 nmol/mg protein; HFpEF: 12.5±1.8 nmol/mg protein; P=0.002 versus con; HFpEF/Empa: 14.5±1.8 nmol/mg protein; P=0.03 versus HFpEF). Transmission electron microscopy revealed a reduced size of mitochondria in HFpEF, which was restored by Empa. CONCLUSIONS: The study demonstrates beneficial effects of Empa on diastolic function, hemodynamics, inflammation, and cardiac remodeling in a rat model of HFpEF. These effects were mediated by improved mitochondrial respiratory capacity due to modulated cardiolipin and improved calcium handling.
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Compuestos de Bencidrilo , Diástole , Modelos Animales de Enfermedad , Glucósidos , Insuficiencia Cardíaca , Mitocondrias Cardíacas , Ratas Zucker , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Volumen Sistólico , Animales , Glucósidos/farmacología , Compuestos de Bencidrilo/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Diástole/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Masculino , Función Ventricular Izquierda/efectos de los fármacos , Ratas Endogámicas SHR , Transporte de Electrón/efectos de los fármacos , RatasRESUMEN
Specific biomarker molecules are increasingly being used for detection and quantification in plant and soil samples of arbuscular mycorrhizal (AM) fungi, an important and widespread microbial guild heavily implicated in transfers of nutrients and carbon between plants and soils and in the maintenance of soil physico-chemical properties. Yet, concerns have previously been raised as to the validity of a range of previously used approaches (e.g., microscopy, AM-specific fatty acids, sterols, glomalin-like molecules, ribosomal DNA sequences), justifying further research into novel biomarkers for AM fungal abundance and/or functioning. Here, we focused on complex polar lipids contained in pure biomass of Rhizophagus irregularis and in nonmycorrhizal and mycorrhizal roots of chicory (Cichorium intybus), leek (Allium porrum), and big bluestem (Andropogon gerardii). The lipids were analyzed by shotgun lipidomics using a high-resolution hybrid mass spectrometer. Size range between 1350 and 1550 Da was chosen for the detection of potential biomarkers among cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerols), a specific class of phospholipids. The analysis revealed a variety of molecular species, including cardiolipins containing one or two polyunsaturated fatty acids with 20 carbon atoms each, i.e., arachidonic and/or eicosapentaenoic acids, some of them apparently specific for the mycorrhizal samples. Although further verification using a greater variety of AM fungal species and samples from various soils/ecosystems/environmental conditions is needed, current results suggest the possibility to identify novel biochemical signatures specific for AM fungi within mycorrhizal roots. Whether they could be used for quantification of both root and soil colonization by the AM fungi merits further scrutiny.
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Micorrizas , Cardiolipinas , Ecosistema , Hongos , Plantas , Cebollas , Suelo/química , Carbono , Raíces de Plantas/microbiologíaRESUMEN
Hydrophilic interaction liquid chromatography (HILIC) connected with electrospray high-resolution tandem mass spectrometry (MS) was used for the analysis of unusual amino acid (AA) substituted phosphatidylglycerols (PG) and cardiolipins (CL) in mesophilic and thermophilic bacteria. Individual peaks from the lipid class separation by HILIC were isolated and hydrolyzed to determine the absolute configuration of the aminoacyl side chain. The configuration of the aminoacyl side chain was assigned by indirect liquid chromatography (LC) enantiomer separation after the hydrolysis of the aminoacylated (aminoacyl) lipids using N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester as chiral derivatizing agent and reversed phase LC-MS for analysis. When two chromatographic methods were combined, less common AAs, such as d-allo-Ile and d-allo-Thr, were identified. The taxonomic classification of bacteria showed that bacteria of the family Bacillaceae (Bacillus and Geobacillus) produce branched-chain AAs, that is, d-allo-Ile, d-Ile, and d-Leu. These AAs were present only in the genera Bacillus and Geobacillus and not in Alicyclobacillus acidoterrestris (family Alicyclobacillaceae). On the contrary, hydroxy AAs, that is, l- and d-Thr, and l- and d-allo-Thr, were identified as aminoacyl-PG and aminoacyl-CL in A. acidoterrestris and were not present in the genera Bacillus and Geobacillus. Therefore, the complete analysis made it possible to identify the stereochemistry of AAs in aminoacyl PGs and CLs and use this fact for chemotaxonomy.
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The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to "positive feedback" in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.
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Cardiolipinas , Liposomas , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Cardiolipinas/metabolismo , MutaciónRESUMEN
Parkinson's disease (PD) progresses with the loss of dopaminergic neurons in the substantia nigra pars compacta region of the brain. The superior mechanisms and the cause of this specific localized neurodegeneration is currently unknown. However, experimental evidence indicates a link between PD progression and reactive oxygen species with imbalanced metal homeostasis. Wild-type Caenorhabditis elegans exposed to redox-active metals was used as the model organism to study cellular response to imbalanced metal homeostasis linked to neurodegenerative diseases. Using modern hyphenated techniques such as capillary electrophoresis coupled to inductively coupled plasma mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry, alterations in the lipidome and metallome were determined in vivo. In contrast to iron, most of the absorbed zinc and manganese were loosely bound. We observed changes in the phospholipid composition for acute iron and manganese exposures, as well as chronic zinc exposure. Furthermore, we focused on the mitochondrial membrane alteration due to its importance in neuronal function. However, significant changes in the inner mitochondrial membrane by determination of cardiolipin species could only be observed for acute iron exposure. These results indicate different intracellular sites of local ROS generation, depending on the redox active metal. Our study combines metallomic and lipidomic alterations as the cause and consequence to enlighten intracellular mechanisms in vivo, associated with PD progression. The mass spectrometry raw data have been deposited to the MassIVE database (https://massive.ucsd.edu) with the identifier MSV000090796 and 10.25345/C51J97C8F.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Hierro/metabolismo , Manganeso/metabolismo , Caenorhabditis elegans/genética , Zinc , Lipidómica , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Metales , Neuronas Dopaminérgicas/metabolismoRESUMEN
The presence of thrombotic events in COVID-19 patients has been described since the beginning of the pandemic. This association has been confirmed in most of the reported studies. Autopsy reports have shown that most thromboses are located in the lung, although they have also been observed in other organs such as the skin and kidneys. SARS-CoV2 infection induces a generalized prothrombotic state, which is attributed to a combination of factors such as hypoxia, excess cellular apoptosis, and mainly to overactivation of the immune system. Among immune-mediated prothrombotic situations, antiphospholipid syndrome (APS) stands out. Recurrent thrombotic events are observed in APS in the presence of antiphospholipid antibodies (aPL). There are numerous studies that report high prevalence of aPL in patients with COVID-19 infection. However, the results show discrepancies in the data on the prevalence of aPL, and its role in the pathogenesis of thrombosis in these patients. This could be due to the heterogeneity of the detection procedures for aPL or to transient elevations of non-pathogenic aPL levels in the context of infection. In this review we try to clarify the role of aPL in COVID-19 infection, and attempt to answer the question of whether it is a coagulopathy of its own, or secondary to APS.
La presencia de eventos trombóticos en los pacientes con COVID-19 se describió desde el inicio de la pandemia, asociación que ha sido confirmada en la mayoría de los estudios reportados. Los informes de necropsias han puesto de manifiesto que la mayoría de las trombosis se localiza en el pulmón, aunque también se han observado en otros órganos, como la piel y los riñones. La infección por SARS-CoV-2 induce un estado protrombótico generalizado que se atribuye a una conjunción de factores como la hipoxia, el exceso de apoptosis celular y, sobre todo, una hiperactivación del sistema inmune. Entre las situaciones protrombóticas inmunomediadas destaca el síndrome antifosfolipídico, en el cual se observan eventos trombóticos de repetición en presencia de anticuerpos antifosfolipídicos (AAF). Existen numerosos estudios que reportan una elevada prevalencia de AAF en los pacientes con infección por la COVID-19; sin embargo, los resultados muestran discordancias en los datos de prevalencia de AAF y su rol en la patogenia sobre la trombosis en estos pacientes, lo que que podría deberse a la heterogeneidad de los procedimientos de detección de los AAF o a elevaciones transitorias de los niveles de AAF no patogénicos en el contexto de la infección. En esta revisión se busca aclarar el papel de los AAF en la infección por COVID-19, intentando responder a la pregunta de si se trata de una coagulopatía propia o es secundaria a un síndrome antifosfolipídico.
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Humanos , Fosfatidilgliceroles , Enfermedades Autoinmunes , Cardiolipinas , Síndrome Antifosfolípido , Enfermedades del Sistema Inmune , Lípidos , Lípidos de la MembranaRESUMEN
Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid-lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase-UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn-around-time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.
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Síndrome de Barth/diagnóstico , Cardiolipinas/sangre , Linfocitos/metabolismo , Lisofosfolípidos/sangre , Adolescente , Adulto , Síndrome de Barth/sangre , Niño , Preescolar , Femenino , Humanos , Modelos Lineales , Linfocitos/química , Masculino , Espectrometría de Masas , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.
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BACKGROUND: The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects. METHODS: To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs. RESULTS: Cardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected. CONCLUSION: Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.
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Cardiolipinas , Cardiopatías , Animales , Femenino , Fibrosis , Masculino , Ratones , Ratones Noqueados , MitocondriasAsunto(s)
Cardiolipinas/genética , Corazón/embriología , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Morfogénesis/genética , Organogénesis/genética , Fosfohidrolasa PTEN/genética , Animales , Cardiolipinas/biosíntesis , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , RatonesRESUMEN
Untargeted lipidomics profiling by liquid chromatography -mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection . Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.
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Lipidómica/métodos , Lisofosfatidilcolinas/análisis , Mitocondrias Hepáticas/química , Animales , Cromatografía Liquida , Técnicas de Inactivación de Genes , Ratones , Ratas , Flujo de Trabajo , alfa-Sinucleína/genéticaRESUMEN
Cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) (CLs) are widespread in many organisms, from bacteria to higher green plants and mammals. CLs were observed in Gram-positive bacterium of the genus Kocuria, brewer's yeast Saccharomyces, the green alga Chlamydomonas, spinach and beef heart. A mixture of molecular species of CLs was obtained from total lipids by hydrophilic interaction liquid chromatography (HILIC), and these were further separated and identified by reversed phase LC/MS with negative tandem electrospray ionization. The majority of CLs molecular species from each organism were cleaved using phospholipase C from Bacillus cereus. This phospholipase cleaves CLs into 1,2-diglycerols and phosphatidylglycerol 3-phosphates, which were then separated. After CLs cleavage, diacylglycerols such as sn-1,2-diacyl-3-acetyl-glycerols (i.e., triacylglycerols) were separated and identified by chiral chromatography/MS-positive tandem ESI. Significant differences in the composition of the molecular species between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of CLs were found in all organisms tested. Molecular species of CLs that contained four different fatty acids were identified in all five samples, and CLs containing very long chain fatty acids were identified in yeast. In addition, CLs containing both enantiomers (at the sn-2 carbon) were present in the bacterium tested. These findings were further supported by data already published in GenBank where, in the same family - Micrococcaceae - both enzymes responsible for chirality in the sn-2 position, glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases, were present.
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Cardiolipinas/química , Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bovinos , Fraccionamiento Químico , Chlamydomonas reinhardtii/química , Ácidos Grasos/análisis , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Estereoisomerismo , Triglicéridos/químicaRESUMEN
BACKGROUND/AIMS: The use of novel cryo-additive agents to increase cell viability post-cryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryo-preserved in DMSO alone and with 300 µM Nigerose (Nig), 200 µM Salidroside (Sal) or a combination of Nig (150 µM) and Sal (100 µM). METHODS: HL-60 cells were pre-incubated with Nig/Sal prior, during and post cryopreservation, and subjected to global lipidomic analysis. Malondialdeyhde (MDA), released lactate dehydrogenase (LDH) and reactive oxygen scavenger (ROS) measurements were also carried out to evaluate levels of lipid peroxidation and cytotoxicity. RESULTS: Cryopreserving HL-60 cells in DMSO with Nig and Sal provided optimal protection against unsaturated fatty acid oxidation. Post-thaw, cellular phospholipids and mitochondrial cardiolipins were increased by Nig/Sal as the ratio of unsaturated to saturated fatty acids 2.08 +/- 0.03 and 0.95 +/- 0.09 folds respectively in comparison to cells cryopreserved in DMSO alone (0.49 +/- 0.05 and 0.86 +/- 0.10 folds). HL-60 lipid peroxidation levels in the presence of DMSO + Nig and Sal combined were significantly reduced relative to pre-cryopreservation levels (10.91 +/- 2.13 nmole) compared to DMSO (17.1 +/- 3.96 nmole). DMSO + Nig/Sal combined also significantly reduced cell cytotoxicity post-thaw (0.0128 +/- 0.00182 mU/mL) in comparison to DMSO (0.0164 +/- 0.00126 mU/mL). The combination of Nig/Sal also reduced significantly ROS levels to the levels of prior cryopreservation of HL-60. CONCLUSION: Overall, the establishment of the cryopreserved HL-60 cells lipidomic and the corresponding biological profiles showed an improved cryo-formulation in the presence of DMSO with the Nig/Sal combination by protecting the, mitochondrial inner membrane, unsaturated fatty acid components (i. e. Cardiolipins) and total phospholipids.
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Disacáridos/química , Leucemia/metabolismo , Mitocondrias/metabolismo , Cardiolipinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Criopreservación , Dimetilsulfóxido/farmacología , Disacáridos/farmacología , Glucósidos/farmacología , Células HL-60 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Fenoles/farmacologíaRESUMEN
AIMS/HYPOTHESIS: We sought to determine putative relationships among improved mitochondrial respiration, insulin sensitivity and altered skeletal muscle lipids and metabolite signature in response to combined aerobic and resistance training in women with obesity. METHODS: This study reports a secondary analysis of a randomised controlled trial including additional measures of mitochondrial respiration, skeletal muscle lipidomics, metabolomics and protein content. Women with obesity were randomised into 12 weeks of combined aerobic and resistance exercise training (n = 20) or control (n = 15) groups. Pre- and post-intervention testing included peak oxygen consumption, whole-body insulin sensitivity (intravenous glucose tolerance test), skeletal muscle mitochondrial respiration (high-resolution respirometry), lipidomics and metabolomics (mass spectrometry) and lipid content (magnetic resonance imaging and spectroscopy). Proteins involved in glucose transport (i.e. GLUT4) and lipid turnover (i.e. sphingomyelin synthase 1 and 2) were assessed by western blotting. RESULTS: The original randomised controlled trial showed that exercise training increased insulin sensitivity (median [IQR]; 3.4 [2.0-4.6] to 3.6 [2.4-6.2] x10-5 pmol l-1 min-1), peak oxygen consumption (mean ± SD; 24.9 ± 2.4 to 27.6 ± 3.4 ml kg-1 min-1), and decreased body weight (84.1 ± 8.7 to 83.3 ± 9.7 kg), with an increase in weight (pre intervention, 87.8± 10.9 to post intervention 88.8 ± 11.0 kg) in the control group (interaction p < 0.05). The current study shows an increase in mitochondrial respiration and content in response to exercise training (interaction p < 0.05). The metabolite and lipid signature at baseline were significantly associated with mitochondrial respiratory capacity (p < 0.05) but were not associated with whole-body insulin sensitivity or GLUT4 protein content. Exercise training significantly altered the skeletal muscle lipid profile, increasing specific diacylglycerol(32:2) and ceramide(d18:1/24:0) levels, without changes in other intermediates or total content of diacylglycerol and ceramide. The total content of cardiolipin, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased with exercise training with a decrease in the PC:PE ratios containing 22:5 and 20:4 fatty acids. These changes were associated with content-driven increases in mitochondrial respiration (p < 0.05), but not with the increase in whole-body insulin sensitivity or GLUT4 protein content. Exercise training increased sphingomyelin synthase 1 (p < 0.05), with no change in plasma-membrane-located sphingomyelin synthase 2. CONCLUSIONS/INTERPRETATION: The major findings of our study were that exercise training altered specific intramuscular lipid intermediates, associated with content-driven increases in mitochondrial respiration but not whole-body insulin sensitivity. This highlights the benefits of exercise training and presents putative target pathways for preventing lipotoxicity in skeletal muscle, which is typically associated with the development of type 2 diabetes.
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Ejercicio Físico/fisiología , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidad , Fosfolípidos/metabolismo , Adulto , Respiración de la Célula , Femenino , Estudios de Seguimiento , Prueba de Tolerancia a la Glucosa , Humanos , Resistencia a la Insulina/fisiología , Masculino , Obesidad/metabolismo , Obesidad/patología , Obesidad/terapia , Adulto JovenRESUMEN
Branched-chain amino acids (BCAA) reverse malnutrition and l-carnitine leads to the reduction of hyperammonemia and muscle cramps in cirrhotic patients. BCAA and l-carnitine are involved in glucose and fatty acid metabolism, however their mechanistic activity in cirrhotic liver is not fully understood. We aim to define the molecular mechanism(s) and combined effects of BCAA and l-carnitine using a cirrhotic rat model. Rats were administered carbon tetrachloride for 10 weeks to induce cirrhosis. During the last 6 weeks of administration, cirrhotic rats received BCAA, l-carnitine or a combination of BCAA and l-carnitine daily via gavage. We found that BCAA and l-carnitine treatments significantly improved hepatocellular function associated with reduced triglyceride level, lipid deposition and adipophilin expression, in cirrhotic liver. Lipidomic analysis revealed dynamic changes in hepatic lipid composition by BCAA and l-carnitine administrations. BCAA and l-carnitine globally increased molecular species of phosphatidylcholine. Liver triacylglycerol and phosphatidylcholine hydroperoxides were significantly decreased by BCAA and l-carnitine. Furthermore, serum and liver ATP levels were significantly increased in all treatments, which were attributed to the elevation of mature cardiolipins and mitochondrial component gene expressions. Finally, BCAA and l-carnitine dramatically reduced hepatocellular death. In conclusion, BCAA and l-carnitine treatments attenuate hepatocellular damage through the reduction of lipid peroxides and the overall maintenance of mitochondrial integrity within the cirrhotic liver. These effectiveness of BCAA and l-carnitine support the therapeutic strategies in human chronic liver diseases.
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Aminoácidos de Cadena Ramificada/farmacología , Carnitina/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hígado Graso/prevención & control , Hepatocitos/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Animales , Tetracloruro de Carbono , Muerte Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Ratas WistarRESUMEN
Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Ácidos Grasos/biosíntesis , Animales , Humanos , RatonesRESUMEN
The occurrence of 3-methylglutaconic aciduria (3-MGA) is a well understood phenomenon in leucine oxidation and ketogenesis disorders (primary 3-MGAs). In contrast, its genesis in non-canonical (secondary) 3-MGAs, a growing-up group of disorders encompassing more than a dozen of inherited metabolic diseases, is a mystery still remaining unresolved for three decades. To puzzle out this anthologic problem of metabolism, three clues were considered: (i) the variety of disorders suggests a common cellular target at the cross-road of metabolic and signaling pathways, (ii) the response to leucine loading test only discriminative for primary but not secondary 3-MGAs suggests these latter are disorders of extramitochondrial HMG-CoA metabolism as also attested by their failure to increase 3-hydroxyisovalerate, a mitochondrial metabolite accumulating only in primary 3-MGAs, (iii) the peroxisome is an extramitochondrial site possessing its own pool and displaying metabolism of HMG-CoA, suggesting its possible involvement in producing extramitochondrial 3-methylglutaconate (3-MG). Following these clues provides a unifying common basis to non-canonical 3-MGAs: constitutive mitochondrial dysfunction induces AMPK activation which, by inhibiting early steps in cholesterol and fatty acid syntheses, pipelines cytoplasmic acetyl-CoA to peroxisomes where a rise in HMG-CoA followed by local dehydration and hydrolysis may lead to 3-MGA yield. Additional contributors are considered, notably for 3-MGAs associated with hyperammonemia, and to a lesser extent in CLPB deficiency. Metabolic and signaling itineraries followed by the proposed scenario are essentially sketched, being provided with compelling evidence from the literature coming in their support.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Errores Innatos del Metabolismo/metabolismo , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Acetilcoenzima A/metabolismo , Animales , HumanosRESUMEN
Collision cross section (CCS) values are descriptors of the 3D structure of ions which can be determined by ion mobility spectrometry (IMS). Currently, most lipidomic studies involving CCS value determination concern eukaryote samples (e.g. human, bovine) and to a lower extent prokaryote samples (e.g. bacteria). Here, we report CCS values obtained from traveling wave ion mobility spectrometry (TWCCSN2) measurements from the bacterial membrane of Pseudomonas aeruginosa-a bacterium ranked as priority 1 for the R&D of new antibiotics by the World Health Organization. In order to cover the lack of reference compounds which could cover the m/z and CCS ranges of the membrane lipids of P. aeruginosa, three calibrants (polyalanine, dextran and phospholipids) were used for the TWCCSN2 calibration. A shift from the published lipid CCS values was systematically observed (ΔCCS% up to 9%); thus, we proposed a CCS correction strategy. This correction strategy allowed a reduction in the shift (ΔCCS%) between our measurements and published values to less than 2%. This correction was then applied to determine the CCS values of Pseudomonas aeruginosa lipids which have not been published yet. As a result, 32 TWCCSN2 values for [M+H]+ ions and 24 TWCCSN2 values for [M-H]- ions were obtained for four classes of phospholipids (phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycerols (PG) and diphosphatidylglycerols-known as cardiolipins (CL)). Graphical abstract.
Asunto(s)
Cardiolipinas/análisis , Espectrometría de Movilidad Iónica/métodos , Espectrometría de Masas/métodos , Fosfolípidos/análisis , CalibraciónRESUMEN
Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.