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1.
Rapid Commun Mass Spectrom ; 34(22): e8911, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32738001

RESUMO

RATIONALE: The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. METHODS: Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. RESULTS: Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. CONCLUSIONS: The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.


Assuntos
Lipidômica/métodos , Lipídeos/sangue , Espectrometria de Massas em Tandem/métodos , Idoso , Cromatografia Líquida , Feminino , Humanos , Masculino , Fosfatidilcolinas/sangue , Reprodutibilidade dos Testes , Software , Triglicerídeos/sangue
2.
Int J Mol Sci ; 18(5)2017 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-28531105

RESUMO

We have previously found that cigarette smoke disrupts metabolic function, in part, by increasing muscle ceramide accrual. To further our understanding of this, we sought to determine the role of the cytokine high-mobility group box 1 (HMGB1), which is increased with smoke exposure, in smoke-induced muscle metabolic perturbations. To test this theory, we determined HMGB1 from lungs of human smokers, as well as from lung cells from mice exposed to cigarette smoke. We also treated cells and mice directly with HMGB1, in the presence or absence of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in ceramide biosynthesis. Outcomes included assessments of insulin resistance and muscle mitochondrial function. HMGB1 was significantly increased in both human lungs and rodent alveolar macrophages. Further testing revealed that HMGB1 treatment elicited a widespread increase in ceramide species and reduction in myotube mitochondrial respiration, an increase in reactive oxygen species, and reduced insulin-stimulated Akt phosphorylation. Inhibition of ceramide biosynthesis with myriocin was protective. In mice, by comparing treatments of HMGB1 injections with or without myriocin, we found that HMGB1 injections resulted in increased muscle ceramides, especially C16 and C24, which were necessary for reduced muscle mitochondrial respiration and compromised insulin and glucose tolerance. In conclusion, HMGB1 may be a necessary intermediate in the ceramide-dependent metabolic consequences of cigarette smoke exposure.


Assuntos
Ceramidas/biossíntese , Proteína HMGB1/metabolismo , Pulmão/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Nicotiana/efeitos adversos , Fumaça/efeitos adversos , Fumar/metabolismo , Animais , Respiração Celular , Ceramidas/antagonistas & inibidores , Ceramidas/genética , Ácidos Graxos Monoinsaturados/farmacologia , Proteína HMGB1/sangue , Proteína HMGB1/farmacologia , Humanos , Insulina/metabolismo , Resistência à Insulina , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina C-Palmitoiltransferase/metabolismo
3.
Sci Immunol ; 9(94): eadh2334, 2024 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-38669316

RESUMO

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.


Assuntos
Neoplasias , Linfócitos T , Regulação para Cima , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Fosfolipases A/imunologia , Fosfolipases A/genética , Fosfolipases A2/imunologia , Linfócitos T/imunologia , Regulação para Cima/imunologia
4.
J Chromatogr A ; 1708: 464342, 2023 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-37696124

RESUMO

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.


Assuntos
COVID-19 , Lipidômica , Humanos , Espectrometria de Massas em Tandem , Pandemias , Reprodutibilidade dos Testes , Cromatografia Líquida , Gravidade do Paciente , Lipídeos
5.
Elife ; 122023 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-36951533

RESUMO

Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.


Assuntos
Sarcopenia , Camundongos , Animais , Sarcopenia/patologia , Peróxidos Lipídicos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Músculo Esquelético/metabolismo , Estresse Oxidativo
6.
Front Plant Sci ; 13: 1038161, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36438089

RESUMO

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

7.
J Am Soc Mass Spectrom ; 32(11): 2655-2663, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34637296

RESUMO

Differential mobility spectrometry (DMS) is highly useful for shotgun lipidomic analysis because it overcomes difficulties in measuring isobaric species within a complex lipid sample and allows for acyl tail characterization of phospholipid species. Despite these advantages, the resulting workflow presents technical challenges, including the need to tune the DMS before every batch to update compensative voltages settings within the method. The Sciex Lipidyzer platform uses a Sciex 5500 QTRAP with a DMS (SelexION), an LC system configured for direction infusion experiments, an extensive set of standards designed for quantitative lipidomics, and a software package (Lipidyzer Workflow Manager) that facilitates the workflow and rapidly analyzes the data. Although the Lipidyzer platform remains very useful for DMS-based shotgun lipidomics, the software is no longer updated for current versions of Analyst and Windows. Furthermore, the software is fixed to a single workflow and cannot take advantage of new lipidomics standards or analyze additional lipid species. To address this multitude of issues, we developed Shotgun Lipidomics Assistant (SLA), a Python-based application that facilitates DMS-based lipidomics workflows. SLA provides the user with flexibility in adding and subtracting lipid and standard MRMs. It can report quantitative lipidomics results from raw data in minutes, comparable to the Lipidyzer software. We show that SLA facilitates an expanded lipidomics analysis that measures over 1450 lipid species across 17 (sub)classes. Lastly, we demonstrate that the SLA performs isotope correction, a feature that was absent from the original software.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Lipidômica/métodos , Animais , Análise de Injeção de Fluxo , Lipídeos/análise , Lipídeos/química , Macrófagos , Camundongos , Software , Fluxo de Trabalho
8.
Cell Rep ; 22(7): 1760-1773, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29444429

RESUMO

The antidiabetic potential of glucagon receptor antagonism presents an opportunity for use in an insulin-centric clinical environment. To investigate the metabolic effects of glucagon receptor antagonism in type 2 diabetes, we treated Leprdb/db and Lepob/ob mice with REMD 2.59, a human monoclonal antibody and competitive antagonist of the glucagon receptor. As expected, REMD 2.59 suppresses hepatic glucose production and improves glycemia. Surprisingly, it also enhances insulin action in both liver and skeletal muscle, coinciding with an increase in AMP-activated protein kinase (AMPK)-mediated lipid oxidation. Furthermore, weekly REMD 2.59 treatment over a period of months protects against diabetic cardiomyopathy. These functional improvements are not derived simply from correcting the systemic milieu; nondiabetic mice with cardiac-specific overexpression of lipoprotein lipase also show improvements in contractile function after REMD 2.59 treatment. These observations suggest that hyperglucagonemia enables lipotoxic conditions, allowing the development of insulin resistance and cardiac dysfunction during disease progression.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Glucose/metabolismo , Coração/fisiopatologia , Receptores de Glucagon/antagonistas & inibidores , Adenilato Quinase/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Cardiomiopatias Diabéticas/complicações , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Teste de Tolerância a Glucose , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/toxicidade , Fígado/metabolismo , Camundongos , Receptores de Glucagon/metabolismo
9.
Elife ; 62017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28722653

RESUMO

Visceral adiposity confers significant risk for developing metabolic disease in obesity whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective thermogenic phenotype characterized by the accumulation of Ucp1+ beige/BRITE adipocytes (termed 'browning'). In this study, we investigated the physiological consequences of browning murine visceral WAT by selective genetic ablation of Zfp423, a transcriptional suppressor of the adipocyte thermogenic program. Zfp423 deletion in fetal visceral adipose precursors (Zfp423loxP/loxP; Wt1-Cre), or adult visceral white adipose precursors (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP), results in the accumulation of beige-like thermogenic adipocytes within multiple visceral adipose depots. Thermogenic visceral WAT improves cold tolerance and prevents and reverses insulin resistance in obesity. These data indicate that beneficial visceral WAT browning can be engineered by directing visceral white adipocyte precursors to a thermogenic adipocyte fate, and suggest a novel strategy to combat insulin resistance in obesity.


Assuntos
Adipócitos Brancos/fisiologia , Diferenciação Celular , Resistência à Insulina , Camundongos Obesos , Células-Tronco/fisiologia , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/fisiologia , Animais , Proteínas de Ligação a DNA/deficiência , Termogênese , Fatores de Transcrição/deficiência
10.
Mol Metab ; 6(3): 267-275, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28271033

RESUMO

OBJECTIVE: Adiponectin and the signaling induced by its cognate receptors, AdipoR1 and AdipoR2, have garnered attention for their ability to promote insulin sensitivity and oppose steatosis. Activation of these receptors promotes the deacylation of ceramide, a lipid metabolite that appears to play a causal role in impairing insulin signaling. METHODS: Here, we have developed transgenic mice that overexpress AdipoR1 or AdipoR2 under the inducible control of a tetracycline response element. These represent the first inducible genetic models that acutely manipulate adiponectin receptor signaling in adult mouse tissues, which allows us to directly assess AdipoR signaling on glucose and lipid metabolism. RESULTS: Overexpression of either adiponectin receptor isoform in the adipocyte or hepatocyte is sufficient to enhance ceramidase activity, whole body glucose metabolism, and hepatic insulin sensitivity, while opposing hepatic steatosis. Importantly, metabolic improvements fail to occur in an adiponectin knockout background. When challenged with a leptin-deficient genetic model of type 2 diabetes, AdipoR2 expression in adipose or liver is sufficient to reverse hyperglycemia and glucose intolerance. CONCLUSION: These observations reveal that adiponectin is critical for AdipoR-induced ceramidase activation which enhances hepatic glucose and lipid metabolism via rapidly acting "cross-talk" between liver and adipose tissue sphingolipids.


Assuntos
Ceramidases/metabolismo , Receptores de Adiponectina/fisiologia , Adipócitos/metabolismo , Adiponectina/genética , Tecido Adiposo/metabolismo , Animais , Ceramidases/genética , Ceramidases/fisiologia , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Glucose/fisiologia , Hepatócitos/metabolismo , Homeostase/genética , Insulina/metabolismo , Resistência à Insulina/genética , Leptina/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Piperidinas/metabolismo , Receptores de Adiponectina/antagonistas & inibidores , Receptores de Adiponectina/genética
11.
Cell Metab ; 22(2): 266-278, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26190650

RESUMO

Sphingolipids have garnered attention for their role in insulin resistance and lipotoxic cell death. We have developed transgenic mice inducibly expressing acid ceramidase that display a reduction in ceramides in adult mouse tissues. Hepatic overexpression of acid ceramidase prevents hepatic steatosis and prompts improvements in insulin action in liver and adipose tissue upon exposure to high-fat diet. Conversely, overexpression of acid ceramidase within adipose tissue also prevents hepatic steatosis and systemic insulin resistance. Induction of ceramidase activity in either tissue promotes a lowering of hepatic ceramides and reduced activation of the ceramide-activated protein kinase C isoform PKCζ, though the induction of ceramidase activity in the adipocyte prompts more rapid resolution of hepatic steatosis than overexpression of the enzyme directly in the liver. Collectively, our observations suggest the existence of a rapidly acting "cross-talk" between liver and adipose tissue sphingolipids, critically regulating glucose metabolism and hepatic lipid uptake.


Assuntos
Ceramidase Ácida/biossíntese , Ceramidas/metabolismo , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Ceramidase Ácida/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Ceramidas/genética , Indução Enzimática , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/patologia , Camundongos , Camundongos Transgênicos , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo
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