RESUMO
BACKGROUND: Between 5-10% of patients discontinue statin therapy due to statin-associated adverse reactions, primarily statin associated muscle symptoms (SAMS). The absence of a clear clinical phenotype or of biomarkers poses a challenge for diagnosis and management of SAMS. Similarly, our incomplete understanding of the pathogenesis of SAMS hinders the identification of treatments for SAMS. Metabolomics, the profiling of metabolites in biofluids, cells and tissues is an important tool for biomarker discovery and provides important insight into the origins of symptomatology. In order to better understand the pathophysiology of this common disorder and to identify biomarkers, we undertook comprehensive metabolomic and lipidomic profiling of plasma samples from patients with SAMS who were undergoing statin rechallenge as part of their clinical care. METHODS AND FINDINGS: We report our findings in 67 patients, 28 with SAMS (cases) and 39 statin-tolerant controls. SAMS patients were studied during statin rechallenge and statin tolerant controls were studied while on statin. Plasma samples were analyzed using untargeted LC-MS metabolomics and lipidomics to detect differences between cases and controls. Differences in lipid species in plasma were observed between cases and controls. These included higher levels of linoleic acid containing phospholipids and lower ether lipids and sphingolipids. Reduced levels of acylcarnitines and altered amino acid profile (tryptophan, tyrosine, proline, arginine, and taurine) were observed in cases relative to controls. Pathway analysis identified significant increase of urea cycle metabolites and arginine and proline metabolites among cases along with downregulation of pathways mediating oxidation of branched chain fatty acids, carnitine synthesis, and transfer of acetyl groups into mitochondria. CONCLUSIONS: The plasma metabolome of patients with SAMS exhibited reduced content of long chain fatty acids and increased levels of linoleic acid (18:2) in phospholipids, altered energy production pathways (ß-oxidation, citric acid cycle and urea cycles) as well as reduced levels of carnitine, an essential mediator of mitochondrial energy production. Our findings support the hypothesis that alterations in pro-inflammatory lipids (arachidonic acid pathway) and impaired mitochondrial energy metabolism underlie the muscle symptoms of patients with statin associated muscle symptoms (SAMS).
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Prostaglandinas , Músculos/metabolismo , Carnitina , Ácidos Graxos/metabolismo , Metabolômica/métodos , Prolina , Arginina , Biomarcadores , Ácidos Linoleicos , UreiaRESUMO
Vegetables oils, rich in polyunsaturated fatty acids, are vulnerable to oxidation during manufacturing, processing, and food preparation. Currently, individual oxidation products are not well characterized, and hence, the health impacts of these unique lipid species remain unknown. Here, we introduce an extensive oxidized lipidomics in silico tandem mass spectrometry library and integrate these libraries within a user-friendly software covering a comprehensive redox lipidomics workflow. We apply this workflow to olive, soy, and walnut cooking oil; comparing unheated oil, oil after deep frying potatoes, and oil after oven frying potatoes. We annotated over a thousand oxidized triglycerides across 273 features (many coeluted). This software was validated against traditional chemical assays of oxidation, known oxidized lipids in castor oil, synthesized standards, and an alternate software LPPtiger. Development of these new software programs for redox lipidomics opens the door to characterize health implications of individual oxidation products.
Assuntos
Culinária , Lipidômica/métodos , Óleos de Plantas , Solanum tuberosum/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Oxirredução , Óleos de Plantas/análise , Óleos de Plantas/químicaRESUMO
Exenatide (Exe) is a glucagon-like peptide (GLP)-1 receptor agonist that enhances insulin secretion and is associated with induction of satiety with weight loss. As mitochondrial dysfunction and lipotoxicity are central features of nonalcoholic steatohepatitis (NASH), we tested whether Exe improved mitochondrial function in this setting. We studied C57BL/6J mice fed for 24 weeks either a control- or high-fructose, high-trans-fat (TFD)-diet (i.e., a NASH model previously validated by our laboratory). For the final 8 weeks, mice were treated with Exe (30 µg/kg/day) or vehicle. Mitochondrial metabolism was assessed by infusion of [13C3]propionate, [3,4-13C2]glucose and NMR-based 13C-isotopomer analysis. Exenatide significantly decreased fasting plasma glucose, free fatty acids and triglycerides, as well as adipose tissue insulin resistance. Moreover, Exe reduced 23% hepatic glucose production, 15% tri-carboxylic acid (TCA) cycle flux, 20% anaplerosis and 17% pyruvate cycling resulting in a significant 31% decrease in intrahepatic triglyceride content (P = 0.02). Exenatide improved the lipidomic profile and decreased hepatic lipid byproducts associated with insulin resistance and lipotoxicity, such as diacylglycerols (TFD: 111 ± 13 vs Exe: 64 ± 13 µmol/g protein, P = 0.03) and ceramides (TFD: 1.6 ± 0.1 vs Exe: 1.3 ± 0.1 µmol/g protein, P = 0.03). Exenatide lowered expression of hepatic lipogenic genes (Srebp1C, Cd36) and genes involved in inflammation and fibrosis (Tnfa, Timp1). In conclusion, in a diet-induced mouse model of NASH, Exe ameliorates mitochondrial TCA cycle flux and significantly decreases insulin resistance, steatosis and hepatocyte lipotoxicity. This may have significant clinical implications to the potential mechanism of action of GLP-1 receptor agonists in patients with NASH. Future studies should elucidate the relative contribution of direct vs indirect mechanisms at play.
Assuntos
Exenatida/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tecido Adiposo/metabolismo , Animais , Glicemia/análise , Ciclo do Ácido Cítrico , Dieta Hiperlipídica , Ácidos Graxos não Esterificados/metabolismo , Fibrose , Perfilação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Inflamação , Resistência à Insulina , Lipidômica , Lipídeos/química , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Triglicerídeos/metabolismoRESUMO
Pioglitazone is effective in improving insulin resistance and liver histology in patients with nonalcoholic steatohepatitis (NASH). Because dysfunctional mitochondrial metabolism is a central feature of NASH, we hypothesized that an important target of pioglitazone would be alleviating mitochondrial oxidative dysfunction. To this end, we studied hepatic mitochondrial metabolism in mice fed high-fructose high-transfat diet (TFD) supplemented with pioglitazone for 20 wk, using nuclear magnetic resonance-based 13C isotopomer analysis. Pioglitazone improved whole body and adipose insulin sensitivity in TFD-fed mice. Furthermore, pioglitazone reduced intrahepatic triglyceride content and fed plasma ketones and hepatic TCA cycle flux, anaplerosis, and pyruvate cycling in mice with NASH. This was associated with a marked reduction in most intrahepatic diacylglycerol classes and, to a lesser extent, some ceramide species (C22:1, C23:0). Considering the cross-talk between mitochondrial function and branched-chain amino acid (BCAA) metabolism, pioglitazone's impact on plasma BCAA profile was determined in a cohort of human subjects. Pioglitazone improved the plasma BCAA concentration profile in patients with NASH. This appeared to be related to an improvement in BCAA degradation in multiple tissues. These results provide evidence that pioglitazone-induced changes in NASH are related to improvements in hepatic mitochondrial oxidative dysfunction and changes in whole body BCAA metabolism.
Assuntos
Hipoglicemiantes/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Pioglitazona/farmacologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Dieta , Feminino , Frutose/toxicidade , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Cetonas/sangue , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pioglitazona/uso terapêutico , Ácido Pirúvico/metabolismoRESUMO
Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders.
Assuntos
Citoesqueleto/metabolismo , Leucócitos Mononucleares/imunologia , Proteínas da Gravidez/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Biologia Computacional , Feminino , Humanos , Imunidade Humoral , Imunomodulação , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Gravidez , Proteínas da Gravidez/genética , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Vimentina/metabolismoRESUMO
BACKGROUND: Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. METHODS: FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. RESULTS: PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase ß-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. CONCLUSION: Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.
Assuntos
Embrião de Mamíferos/metabolismo , Peptídeos/fisiologia , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Desenvolvimento Embrionário , Cavalos/embriologia , Camundongos , Modelos Moleculares , Estresse Oxidativo , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Tiorredoxinas/metabolismoRESUMO
The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.
Assuntos
Autoanticorpos , Lesões Encefálicas/sangue , Lesões Encefálicas/imunologia , Proteína Glial Fibrilar Ácida/imunologia , Imunoglobulina G , Adulto , Animais , Astrócitos/imunologia , Astrócitos/metabolismo , Astrócitos/patologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Lesões Encefálicas/patologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Several common degenerative mechanisms and mediators underlying the neuronal injury pathways characterize several neurodegenerative diseases including Alzheimer's, Parkinson's, and Huntington's disease, as well as brain neurotrauma. Such common ground invites the emergence of new approaches and tools to study the altered pathways involved in neural injury alongside with neuritogenesis, an intricate process that commences with neuronal differentiation. Achieving a greater understanding of the impaired pathways of neuritogenesis would significantly help in uncovering detailed mechanisms of axonal regeneration. Among the several agents involved in neuritogenesis are the Rho and Rho kinases (ROCKs), which constitute key integral points in the Rho/ROCK pathway that is known to be disrupted in multiple neuropathologies such as spinal cord injury, traumatic brain injury, and Alzheimer's disease. This in turn renders ROCK inhibition as a promising candidate for therapeutic targets for treatment of neurodegenerative diseases. Among the novel tools to investigate the mechanisms involved in a specific disorder is the use of neuroproteomics/systems biology approach, a growing subfield of bioinformatics aiming to study and establishing a global assessment of the entire neuronal proteome, addressing the dynamic protein changes and interactions. This review aims to examine recent updates regarding how neuroproteomics aids in the understanding of molecular mechanisms of activation and inhibition in the area of neurogenesis and how Rho/ROCK pathway/ROCK inhibitors, primarily Y-27632 and Fasudil compounds, are applied in biological settings, promoting neuronal survival and neuroprotection that has direct future implications in neurotrauma.