RESUMO
N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with â¼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease.
Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Polissacarídeos/metabolismo , Animais , Cromatografia Líquida , Feminino , Glicômica , Masculino , Espectrometria de Massas , Camundongos Endogâmicos C57BL , NanotecnologiaRESUMO
Diarrheal diseases are a leading cause of death in children under the age of 5 years worldwide. Repeated early-life exposures to diarrheal pathogens can result in comorbidities including stunted growth and cognitive deficits, suggesting an impairment in the microbiota-gut-brain (MGB) axis. Neonatal C57BL/6 mice were infected with enteropathogenic Escherichia coli (EPEC) (strain e2348/69; ΔescV [type III secretion system {T3SS} mutant]) or the vehicle (Luria-Bertani [LB] broth) via orogastric gavage at postnatal day 7 (P7). Behavior (novel-object recognition [NOR] task, light/dark [L/D] box, and open-field test [OFT]), intestinal physiology (Ussing chambers), and the gut microbiota (16S Illumina sequencing) were assessed in adulthood (6 to 8 weeks of age). Neonatal infection of mice with EPEC, but not the T3SS mutant, caused ileal inflammation in neonates and impaired recognition memory (NOR task) in adulthood. Cognitive impairments were coupled with increased neurogenesis (Ki67 and doublecortin immunostaining) and neuroinflammation (increased microglia activation [Iba1]) in adulthood. Intestinal pathophysiology in adult mice was characterized by increased secretory state (short-circuit current [Isc]) and permeability (conductance) (fluorescein isothiocyanate [FITC]-dextran flux) in the ileum and colon of neonatally EPEC-infected mice, along with increased expression of proinflammatory cytokines (Tnfα, Il12, and Il6) and pattern recognition receptors (Nod1/2 and Tlr2/4). Finally, neonatal EPEC infection caused significant dysbiosis of the gut microbiota, including decreased Firmicutes, in adulthood. Together, these findings demonstrate that infection in early life can significantly impair the MGB axis in adulthood.
Assuntos
Encéfalo/metabolismo , Escherichia coli Enteropatogênica/fisiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Retroalimentação Fisiológica , Microbioma Gastrointestinal , Intestinos , Animais , Suscetibilidade a Doenças , HumanosRESUMO
Malignant hyperthermia (MH) is characterized by induction of skeletal muscle hyperthermia in response to a dysregulated increase in myoplasmic calcium. Although altered energetics play a central role in MH, MH-susceptible humans and mouse models are often described as having no phenotype until exposure to a triggering agent. The purpose of this study was to determine the influence of the R163C ryanodine receptor 1 mutation, a common MH mutation in humans, on energy expenditure, and voluntary wheel running in mice. Energy expenditure was measured by indirect respiration calorimetry in wild-type (WT) and heterozygous R163C (HET) mice over a range of ambient temperatures. Energy expenditure adjusted for body weight or lean mass was increased (P < .05) in male, but not female, HET mice housed at 22°C or when housed at 28°C with a running wheel. In female mice, voluntary wheel running was decreased (P < .05) in the HET vs WT animals when analyzed across ambient temperatures. The thermoneutral zone was also widened in both male and female HET mice. The results of the study show that the R163C mutations alters energetics even at temperatures that do not typically induce MH.
Assuntos
Metabolismo Energético/fisiologia , Hipertermia/patologia , Hipertermia Maligna/patologia , Atividade Motora/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Feminino , Heterozigoto , Hipertermia/metabolismo , Masculino , Hipertermia Maligna/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Myelination in the peripheral and central nervous systems is critical in regulating motor, sensory, and cognitive functions. As myelination occurs rapidly during early life, neonatal gut dysbiosis during early colonization can potentially alter proper myelination by dysregulating immune responses and neuronal differentiation. Despite common usage of antibiotics (Abx) in children, the impact of neonatal Abx-induced dysbiosis on the development of microbiota, gut, brain (MGB) axis, including myelination and behavior, is unknown. We hypothesized that neonatal Abx-induced dysbiosis dysregulates host-microbe interactions, impairing myelination in the brain, and altering the MGB axis. Neonatal C57BL/6 mice were orally gavaged daily with an Abx cocktail (neomycin, vancomycin, ampicillin) or water (vehicle) from postnatal day 7 (P7) until weaning (P23) to induce gut dysbiosis. Behavior (cognition; anxiety-like behavior), microbiota sequencing, and qPCR (ileum, colon, hippocampus and pre-frontal cortex [PFC]) were performed in adult mice (6-8 weeks). Neonatal Abx administration led to intestinal dysbiosis in adulthood, impaired intestinal physiology, coupled with perturbations of bacterial metabolites and behavioral alterations (cognitive deficits and anxiolytic behavior). Expression of myelin-related genes (Mag, Mog, Mbp, Mobp, Plp) and transcription factors (Sox10, Myrf) important for oligodendrocytes were significantly increased in the PFC region of Abx-treated mice. Increased myelination was confirmed by immunofluorescence imaging and western blot analysis, demonstrating increased expression of MBP, SOX10 and MYRF in neonatally Abx-treated mice compared to sham controls in adulthood. Finally, administration of the short chain fatty acid butyrate following completion of the Abx treatment restored intestinal physiology, behavior, and myelination impairments, suggesting a critical role for the gut microbiota in mediating these effects. Taken together, we identified a long-lasting impact of neonatal Abx administration on the MGB axis, specifically on myelin regulation in the PFC region, potentially contributing to impaired cognitive function and bacterial metabolites are effective in reversing this altered phenotype.
Assuntos
Microbioma Gastrointestinal , Microbiota , Animais , Antibacterianos , Encéfalo , Camundongos , Camundongos Endogâmicos C57BL , Bainha de MielinaRESUMO
Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Atividade Motora/genética , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Feminino , Regulação da Expressão Gênica/genética , Heterozigoto , Humanos , Masculino , Camundongos , Atividade Motora/fisiologia , Mutação , Fenótipo , Isoformas de Proteínas/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologiaRESUMO
Inflammatory bowel diseases (IBDs) are chronic intestinal diseases, frequently associated with comorbid psychological and cognitive deficits. These neuropsychiatric effects include anxiety, depression, and memory impairments that can be seen both during active disease and following remission and are more frequently seen in pediatric patients. The mechanism(s) through which these extraintestinal deficits develop remain unknown, and the study of these phenomenon is hampered by a lack of murine pediatric IBD models. Herein we describe microbiota-gut-brain (MGB) axis deficits following induction of colitis in a pediatric setting. Acute colitis was induced by administration of 2% dextran sodium sulfate (DSS) for 5 days starting at weaning [postnatal day (P)21] causing reduced weight gain, colonic shortening, and colonic inflammation by 8 days post-DSS (P29), which were mostly resolved in adult (P56) mice. Despite resolution of acute disease, cognitive deficits (novel object recognition task) and anxiety-like behavior (light/dark box) were identified in the absence of changes in exploratory behavior (open field test) in P56 mice previously treated with DSS at weaning. Behavioral deficits were found in conjunction with neuroinflammation, decreased neurogenesis, and altered expression of pattern recognition receptor genes in the hippocampus. Additionally, persistent alterations in the gut microbiota composition were observed at P56, including reduced butyrate-producing species. Taken together, these results describe for the first time the presence of MGB axis deficits following induction of colitis at weaning, which persist in adulthood.NEW & NOTEWORTHY Here we describe long-lasting impacts on the microbiota-gut-brain (MGB) axis following administration of low-dose dextran sodium sulfate (DSS) to weaning mice (P21), including gut dysbiosis, colonic inflammation, and brain/behavioral deficits in adulthood (P56). Early-life DSS leads to acute colonic inflammation, similar to adult mice; however, it results in long-lasting deficits in the MGB axis in adulthood (P56), in contrast to the transient deficits seen in adult DSS. This model highlights the unique features of pediatric inflammatory bowel disease.
Assuntos
Encéfalo/fisiopatologia , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/fisiopatologia , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Vias Neurais/fisiopatologia , Animais , Ansiedade/psicologia , Comportamento Animal , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/psicologia , Colite/induzido quimicamente , Colite/microbiologia , Colite/fisiopatologia , Sulfato de Dextrana , Modelos Animais de Doenças , Disbiose , Feminino , Hipocampo/metabolismo , Humanos , Doenças Inflamatórias Intestinais/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese , Aumento de PesoRESUMO
Surveys report that 25-57 % of cats are overweight or obese. The most evinced cause is neutering. Weight loss often fails; thus, new strategies are needed. Obesity has been associated with altered gut bacterial populations and increases in microbial dietary energy extraction, body weight and adiposity. This study aimed to determine whether alterations in intestinal bacteria were associated with obesity, energy restriction and neutering by characterising faecal microbiota using 16S rRNA gene sequencing in eight lean intact, eight lean neutered and eight obese neutered cats before and after 6 weeks of energy restriction. Lean neutered cats had a bacterial profile similar to obese rodents and humans, with a greater abundance (P<0·05) of Firmicutes and lower abundance (P<0·05) of Bacteroidetes compared with the other groups. The greater abundance of Firmicutes in lean neutered cats was due to a bloom in Peptostreptococcaceae. Obese cats had an 18 % reduction in fat mass after energy restriction (P<0·05). Energy reduction was concurrent with significant shifts in two low-abundance bacterial genera and trends in four additional genera. The greatest change was a reduction in the Firmicutes genus, Sarcina, from 4·54 to 0·65 % abundance after energy restriction. The short duration of energy restriction may explain why few bacterial changes were observed in the obese cats. Additional work is needed to understand how neutering, obesity and weight loss are related to changes in feline microbiota and how these microbial shifts affect host physiology.
Assuntos
Restrição Calórica , Castração , Fezes/microbiologia , Microbioma Gastrointestinal , Obesidade/veterinária , Animais , Bacteroidetes/isolamento & purificação , Composição Corporal , Peso Corporal , Gatos , DNA Bacteriano/isolamento & purificação , Dieta/veterinária , Feminino , Bactérias Gram-Positivas/isolamento & purificação , Masculino , Análise Multivariada , Obesidade/microbiologia , RNA Ribossômico 16S/isolamento & purificação , Análise de Sequência de RNARESUMO
Insulin resistance may be linked to incomplete fatty acid ß-oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid ß-oxidation, oxidative stress, inflammation, and insulin-resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20-30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitate-induced insulin resistance concomitant with an â¼25% decrease in short-chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2-3 times by short- or long-chain acylcarnitines. Acylcarnitine-induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid ß-oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.
Assuntos
Carnitina/análogos & derivados , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Adulto , Animais , Antioxidantes/farmacologia , Carnitina/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Ácidos Graxos/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Camundongos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Obesidade/metabolismo , Oxirredução , Estresse OxidativoRESUMO
Blood and urine acylcarnitine profiles are commonly used to diagnose long-chain fatty acid oxidation disorders (FAOD: i.e., long-chain hydroxy-acyl-CoA dehydrogenase [LCHAD] and carnitine palmitoyltransferase 2 [CPT2] deficiency), but the global metabolic impact of long-chain FAOD has not been reported. We utilized untargeted metabolomics to characterize plasma metabolites in 12 overnight-fasted individuals with FAOD (10 LCHAD, two CPT2) and 11 healthy age-, sex-, and body mass index (BMI)-matched controls, with the caveat that individuals with FAOD consume a low-fat diet supplemented with medium-chain triglycerides (MCT) while matched controls consume a typical American diet. In plasma 832 metabolites were identified, and partial least squared-discriminant analysis (PLS-DA) identified 114 non-acylcarnitine variables that discriminated FAOD subjects and controls. FAOD individuals had significantly higher triglycerides and lower specific phosphatidylethanolamines, ceramides, and sphingomyelins. Differences in phosphatidylcholines were also found but the directionality differed by metabolite species. Further, there were few differences in non-lipid metabolites, indicating the metabolic impact of FAOD specifically on lipid pathways. This analysis provides evidence that LCHAD/CPT2 deficiency significantly alters complex lipid pathway flux. This metabolic signature may provide new clinical tools capable of confirming or diagnosing FAOD, even in subjects with a mild phenotype, and may provide clues regarding the biochemical and metabolic impact of FAOD that is relevant to the etiology of FAOD symptoms.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Carnitina O-Palmitoiltransferase/deficiência , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo/metabolismo , Plasma/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Adolescente , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Estudos de Casos e Controles , Ceramidas/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos , Masculino , Redes e Vias Metabólicas/fisiologia , Oxirredução , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Esfingomielinas/metabolismo , Triglicerídeos/metabolismoRESUMO
Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 µM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 µM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 µM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.
Assuntos
Cálcio/farmacologia , Carnitina/análogos & derivados , Fibras Musculares Esqueléticas/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Animais , Carnitina/química , Carnitina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: It has been suggested that perturbations in branched-chain amino acid (BCAA) catabolism are associated with insulin resistance and contribute to elevated systemic BCAAs. Evidence in rodents suggests dietary protein rich in BCAAs can increase BCAA catabolism, but there is limited evidence in humans. OBJECTIVE: We hypothesize that a diet rich in BCAAs will increase BCAA catabolism, which will manifest in a reduction of fasting plasma BCAA concentrations. METHODS: The metabolome of 27 obese women with metabolic syndrome before and after weight loss was investigated to identify changes in BCAA metabolism using GC-time-of-flight mass spectrometry. Subjects were enrolled in an 8-wk weight-loss study including either a 20-g/d whey (whey group, n = 16) or gelatin (gelatin group, n = 11) protein supplement. When matched for total protein by weight, whey protein has 3 times the amount of BCAAs compared with gelatin protein. RESULTS: Postintervention plasma abundances of Ile (gelatin group: 637 ± 18, quantifier ion peak height ÷ 100; whey group: 744 ± 65), Leu (gelatin group: 1210 ± 33; whey group: 1380 ± 79), and Val (gelatin group: 2080 ± 59; whey group: 2510 ± 230) did not differ between treatment groups. BCAAs were significantly correlated with homeostasis model assessment of insulin resistance at baseline (r = 0.52, 0.43, and 0.49 for Leu, Ile, and Val, respectively; all, P < 0.05), but correlations were no longer significant at postintervention. Pro- and Cys-related pathways were found discriminant of whey protein vs. gelatin protein supplementation in multivariate statistical analyses. CONCLUSIONS: These findings suggest that BCAA metabolism is, at best, only modestly affected at a whey protein supplementation dose of 20 g/d. Furthermore, the loss of an association between postintervention BCAA and homeostasis model assessment suggests that factors associated with calorie restriction or protein intake affect how plasma BCAAs relate to insulin sensitivity. This trial was registered at clinicaltrials.gov as NCT00739479.
Assuntos
Aminoácidos de Cadeia Ramificada/sangue , Suplementos Nutricionais , Metaboloma , Proteínas do Leite/administração & dosagem , Obesidade/dietoterapia , Redução de Peso , Adulto , Aminoácidos de Cadeia Ramificada/administração & dosagem , Glicemia , Índice de Massa Corporal , Restrição Calórica , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Gelatina/administração & dosagem , Homeostase , Humanos , Insulina/sangue , Pessoa de Meia-Idade , Análise Multivariada , Obesidade/sangue , Circunferência da Cintura , Proteínas do Soro do LeiteRESUMO
Incomplete ß-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete ß-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 µM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.
Assuntos
Carnitina/análogos & derivados , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , Macrófagos/imunologia , Receptores de Reconhecimento de Padrão/agonistas , Animais , Carnitina/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Indução Enzimática , Inativação Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/agonistas , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Ácidos Mirísticos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPß), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPß-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPß-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPß, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPß and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Fígado Gorduroso/virologia , Genoma Viral , Hepacivirus/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Proteínas não Estruturais Virais/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/virologia , Indução Enzimática , Fígado Gorduroso/enzimologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genes Reporter , Gluconeogênese/genética , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Hepacivirus/fisiologia , Humanos , Insulina/fisiologia , Metabolismo dos Lipídeos/genética , Luciferases/biossíntese , Luciferases/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas , Ratos , Vesículas Secretórias/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
Insulin resistance occurs in 20%-25% of the human population, and the condition is a chief component of type 2 diabetes mellitus and a risk factor for cardiovascular disease and certain forms of cancer. Herein, we demonstrate that the sphingolipid ceramide is a common molecular intermediate linking several different pathological metabolic stresses (i.e., glucocorticoids and saturated fats, but not unsaturated fats) to the induction of insulin resistance. Moreover, inhibition of ceramide synthesis markedly improves glucose tolerance and prevents the onset of frank diabetes in obese rodents. Collectively, these data have two important implications. First, they indicate that different fatty acids induce insulin resistance by distinct mechanisms discerned by their reliance on sphingolipid synthesis. Second, they identify enzymes required for ceramide synthesis as therapeutic targets for combating insulin resistance caused by nutrient excess or glucocorticoid therapy.
Assuntos
Ceramidas/metabolismo , Ácidos Graxos/metabolismo , Glucocorticoides/metabolismo , Resistência à Insulina , Obesidade/metabolismo , Animais , Ceramidas/biossíntese , Diabetes Mellitus Tipo 2/metabolismo , Gorduras Insaturadas/metabolismo , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Oxirredutases/genética , Ratos , Ratos Sprague-Dawley , Esfingolipídeos/metabolismoRESUMO
The gut microbiota and associated metabolites have emerged as potential modulators of pathophysiological changes in obesity and related metabolic disorders. Butyrate, a product of bacterial fermentation, has been shown to have beneficial effects in obesity and rodent models of diet-induced obesity. Here, we aimed to determine the beneficial effects of butyrate (as glycerol ester of butyrate monobutyrin, MB) supplementation on metabolic phenotype, intestinal permeability and inflammation, feeding behavior, and the gut microbiota in low-fat (LF)- and high-fat (HF)-fed mice. Two cohorts (separated by 2 weeks) of male C57BL/6J mice (n = 24 in each cohort, 6/group/cohort; 6 weeks old) were separated into four weight-matched groups and fed either a LF (10 % fat/kcal) or HF (45% fat/kcal) with or without supplementation of MB (LF/MB or HF/MB) at 0.25% (w/v) in drinking water for 6 weeks. Metabolic phenotypes (body weight and adiposity), intestinal inflammation, feeding behavior, and fecal microbiome and metabolites were measured. Despite identical genetic and experimental conditions, we found marked differences between cohorts in the response (body weight gain, adiposity, and intestinal permeability) to HF-diet and MB. Notably, the composition of the gut microbiota was significantly different between cohorts, characterized by lower species richness and differential abundance of a large number of taxa, including subtaxa from five phyla, including increased abundance of the genera Bacteroides, Proteobacteria, and Parasutterella in cohort 2 compared to cohort 1. These differences may have contributed to the differential response in intestinal permeability to the HF diet in cohort 2. MB supplementation had no significant effect on metabolic phenotype, but there was a trend to protect from HF-induced impairments in intestinal barrier function in cohort 1 and in sensitivity to cholecystokinin (CCK) in both cohorts. These data support the concept that microbiota composition may have a crucial effect on metabolic responses of a host to dietary interventions and highlight the importance of taking steps to ensure reproducibility in rodent studies.
Assuntos
Butiratos/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Inflamação/induzido quimicamente , Obesidade/induzido quimicamente , Ração Animal/análise , Animais , Peso Corporal , Dieta/veterinária , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Suplementos Nutricionais , Fezes/microbiologia , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológicoRESUMO
The NCoR corepressor plays critical roles in mediating transcriptional repression by both nuclear receptors and non-receptor transcription factors. Alternative mRNA splicing of NCoR produces a series of variants with differing molecular and biological properties. The NCoRω splice-variant inhibits adipogenesis whereas the NCoRδ splice-variant promotes it, and mice bearing a splice-specific knockout of NCoRω display enhanced hepatic steatosis and overall weight gain on a high fat diet as well as a greatly increased resistance to diet-induced glucose intolerance. We report here that the reciprocal NCoRδ splice-specific knock-out mice display the contrary phenotypes of reduced hepatic steatosis and reduced weight gain relative to the NCoRω-/- mice. The NCoRδ-/- mice also fail to demonstrate the strong resistance to diet-induced glucose intolerance exhibited by the NCoRω-/- animals. The NCoR δ and ω variants possess both unique and shared transcriptional targets, with expression of certain hepatic genes affected in opposite directions in the two mutants, others altered in one but not the other genotype, and yet others changed in parallel in both NCoRδ-/- and NCoRω-/- animals versus WT. Gene set expression analysis (GSEA) identified a series of lipid, carbohydrate, and amino acid metabolic pathways that are likely to contribute to their distinct steatosis and glucose tolerance phenotypes. We conclude that alternative-splicing of the NCoR corepressor plays a key role in the regulation of hepatic energy storage and utilization, with the NCoRδ and NCoRω variants exerting both opposing and shared functions in many aspects of this phenomenon and in the organism as a whole.
Assuntos
Processamento Alternativo/genética , Fígado/metabolismo , Correpressor 1 de Receptor Nuclear/genética , Animais , Dieta , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Comportamento Alimentar , Regulação da Expressão Gênica , Intolerância à Glucose/complicações , Resistência à Insulina , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Aumento de PesoRESUMO
BACKGROUND: Consumption of high-fat diet (HF) leads to hyperphagia and increased body weight in male rodents. Female rodents are relatively resistant to hyperphagia and weight gain in response to HF, in part via effects of estrogen that suppresses food intake and increases energy expenditure. However, sex differences in energy expenditure and activity levels with HF challenge have not been systemically described. We hypothesized that, in response to short-term HF feeding, female mice will have a higher energy expenditure and be more resistant to HF-induced hyperphagia than male mice. METHODS: Six-week-old male and female C57BL/6 J mice were fed either low fat (LF, 10% fat) or moderate HF (45% fat) for 5 weeks, and energy expenditure, activity and meal pattern measured using comprehensive laboratory animal monitoring system (CLAMS). RESULTS: After 5 weeks, HF-fed male mice had a significant increase in body weight and fat mass, compared with LF-fed male mice. HF-fed female had a significant increase in body weight compared with LF-fed female mice, but there was no significant difference in fat mass. HF-fed male mice had lower energy expenditure compared to HF-fed female mice, likely due in part to reduced physical activity in the light phase. HF-fed male mice also had increased energy intake in the dark phase compared to LF-fed male mice and a reduced response to exogenous cholecystokinin-induced inhibition of food intake. In contrast, there was no difference in energy intake between LF-fed and HF-fed female mice. CONCLUSIONS: The data show that female mice are generally protected from short-term HF-induced alterations in energy balance, possibly by maintaining higher energy expenditure and an absence of hyperphagia. However, HF-feeding in male mice induced weight and fat mass gain and hyperphagia. These findings suggest that there is a sex difference in the response to short-term HF-feeding in terms of both energy expenditure and control of food intake.
Assuntos
Dieta Hiperlipídica , Caracteres Sexuais , Animais , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta , Ingestão de Energia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Excessive cellular accumulation or exposure to lipids such as long-chain acylcarnitines (LCACs), ceramides, and others is implicated in cell stress and inflammation. Such a situation might manifest when there is a significant mismatch between long-chain fatty acid (LCFA) availability versus storage and oxidative utilization; for example, in cardiac ischemia, increased LCACs may contribute to tissue cell stress and infarct damage. Perturbed LCFAß-oxidation is also seen in fatty acid oxidation disorders (FAODs). FAODs typically manifest with fasting- or stress-induced symptoms, and patients can manage many symptoms through control of diet and physical activity. However, episodic clinical events involving cardiac and skeletal muscle myopathies are common and can present without an obvious molecular trigger. We have speculated that systemic or tissue-specific lipotoxicity and activation of inflammation pathways contribute to long-chain FAOD pathophysiology. With this in mind, we characterized inflammatory phenotype (14 blood plasma cytokines) in resting, overnight-fasted (~10 h), or exercise-challenged subjects with clinically well-controlled long-chain FAODs (n = 12; 10 long-chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD]; 2 carnitine palmitoyltransferase 2 [CPT2]) compared to healthy controls (n = 12). Across experimental conditions, concentrations of three cytokines were modestly but significantly increased in FAOD (IFNγ, IL-8, and MDC), and plasma levels of IL-10 (considered an inflammation-dampening cytokine) were significantly decreased. These novel results indicate that while asymptomatic FAOD patients do not display gross body-wide inflammation even after moderate exercise, ß-oxidation deficiencies might be associated with chronic and subtle activation of "sterile inflammation." Further studies are warranted to determine if inflammation is more apparent in poorly controlled long-chain FAOD or when long-chain FAOD-associated symptoms are present.
Assuntos
Citocinas/sangue , Ácidos Graxos/metabolismo , Mediadores da Inflamação/sangue , Erros Inatos do Metabolismo Lipídico/sangue , Adolescente , Adulto , Biomarcadores/sangue , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Estudos de Casos e Controles , Criança , Exercício Físico , Feminino , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-8/sangue , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/imunologia , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/deficiência , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/genética , Masculino , Oxirredução , Fenótipo , Período Pós-Prandial , Fatores de Tempo , Adulto JovemRESUMO
Recently, we characterized tumor suppressor candidate 5 (Tusc5) as an adipocyte-neuron PPARgamma target gene. Our objective herein was to identify additional genes that display distinctly high expression in fat and neurons, because such a pattern could signal previously uncharacterized functional pathways shared in these disparate tissues. gamma-Synuclein, a marker of peripheral and select central nervous system neurons, was strongly expressed in white adipose tissue (WAT) and peripheral nervous system ganglia using bioinformatics and quantitative PCR approaches. Gamma-synuclein expression was determined during adipogenesis and in subcutaneous (SC) and visceral adipose tissue (VAT) from obese and nonobese humans. Gamma-synuclein mRNA increased from trace levels in preadipocytes to high levels in mature 3T3-L1 adipocytes and decreased approximately 50% following treatment with the PPARgamma agonist GW1929 (P < 0.01). Because gamma-synuclein limits growth arrest and is implicated in cancer progression in nonadipocytes, we suspected that expression would be increased in situations where WAT plasticity/adipocyte turnover are engaged. Consistent with this postulate, human WAT gamma-synuclein mRNA levels consistently increased in obesity and were higher in SC than in VAT; i.e. they increased approximately 1.7-fold in obese Pima Indian adipocytes (P = 0.003) and approximately 2-fold in SC and VAT of other obese cohorts relative to nonobese subjects. Expression correlated with leptin transcript levels in human SC and VAT (r = 0.887; P < 0.0001; n = 44). Gamma-synuclein protein was observed in rodent and human WAT but not in negative control liver. These results are consistent with the hypothesis that gamma-synuclein plays an important role in adipocyte physiology.
Assuntos
Tecido Adiposo/química , Expressão Gênica , Leptina/genética , Obesidade/metabolismo , gama-Sinucleína/genética , Células 3T3-L1 , Adipócitos/química , Adipócitos/citologia , Animais , Benzofenonas/farmacologia , Western Blotting , Diferenciação Celular , Feminino , Humanos , Imuno-Histoquímica , Indígenas Norte-Americanos , Camundongos , PPAR gama/agonistas , Sistema Nervoso Periférico/química , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Tirosina/análogos & derivados , Tirosina/farmacologia , gama-Sinucleína/análiseRESUMO
The Amargosa vole is a highly endangered rodent endemic to a small stretch of the Amargosa River basin in Inyo County, California. It specializes on a single, nutritionally marginal food source in nature. As part of a conservation effort to preserve the species, a captive breeding population was established to serve as an insurance colony and a source of individuals to release into the wild as restored habitat becomes available. The colony has successfully been maintained on commercial diets for multiple generations, but there are concerns that colony animals could lose gut microbes necessary to digest a wild diet. We analyzed feces from colony-reared and recently captured wild-born voles on various diets, and foregut contents from colony and wild voles. Unexpectedly, fecal microbial composition did not greatly differ despite drastically different diets and differences observed were mostly in low-abundance microbes. In contrast, colony vole foregut microbiomes were dominated by Allobaculum sp. while wild foreguts were dominated by Lactobacillus sp. If these bacterial community differences result in beneficial functional differences in digestion, then captive-reared Amargosa voles should be prepared prior to release into the wild to minimize or eliminate those differences to maximize their chance of success.