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Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.
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Peso Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/metabolismo , Metformina/farmacologia , Administração Oral , Adulto , Idoso , Animais , Glicemia/análise , Glicemia/metabolismo , Dieta Hiperlipídica , Método Duplo-Cego , Ingestão de Energia/efeitos dos fármacos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Feminino , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/antagonistas & inibidores , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/deficiência , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/deficiência , Fator 15 de Diferenciação de Crescimento/genética , Homeostase/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Obesos , Pessoa de Meia-Idade , Redução de Peso/efeitos dos fármacosRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1ß, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.
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Bronquiectasia , Progressão da Doença , Escarro , Humanos , Bronquiectasia/microbiologia , Bronquiectasia/fisiopatologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Escarro/microbiologia , Estudos de Coortes , Elastase de Leucócito/metabolismo , MicrobiotaRESUMO
RATIONALE: The inflammasome is a key regulatory complex of the inflammatory response leading to interleukin-1ß (IL-1ß) release and activation. IL-1ß amplifies inflammatory responses and induces mucus secretion and hyperconcentration in other diseases. The role of IL-1ß in bronchiectasis has not been investigated. OBJECTIVES: To characterise the role of airway IL-1ß in bronchiectasis, including the association with mucus properties, ciliary function, airway inflammation, microbiome and disease severity. METHODS: Stable bronchiectasis patients were enrolled in an international cohort study (n=269). IL-1ß was measured in sputum supernatant. A validation cohort also had sputum rheology and hydration measured (n=53). For analysis, patients were stratified according to the median value of IL-1ß in the population (high versus low) to compare disease severity, airway infection, microbiome (16S rRNA sequencing), inflammation and caspase-1 activity. Primary human nasal epithelial cells grown in air-liquid interface culture were used to study the effect of IL-1ß on cilia function. RESULTS: Patients with high sputum IL-1ß had more severe disease, increased caspase-1 activity and an increased T-helper type 1, T-helper type 2 and neutrophil inflammatory response compared with patients with low IL-1ß. The active-dominant form of IL-1ß was associated with increased disease severity. High IL-1ß was related to higher relative abundance of Proteobacteria in the microbiome and increased mucus solid content and viscoelastic properties. Chronic IL-1ß treatment reduced the functionality of cilia and tight junctions of epithelial cells in vitro. CONCLUSIONS: A subset of stable bronchiectasis patients show increased airway IL-1ß, suggesting pulmonary inflammasome activation is linked with more severe disease, airway infection, mucus dehydration and epithelial dysfunction.
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Bronquiectasia , Interleucina-1beta , Depuração Mucociliar , Índice de Gravidade de Doença , Escarro , Humanos , Interleucina-1beta/metabolismo , Bronquiectasia/fisiopatologia , Bronquiectasia/metabolismo , Bronquiectasia/microbiologia , Feminino , Masculino , Pessoa de Meia-Idade , Escarro/metabolismo , Idoso , Inflamassomos/metabolismo , Muco/metabolismo , Caspase 1/metabolismo , Microbiota , Inflamação , Estudos de Coortes , RNA Ribossômico 16S/genética , Adulto , Cílios/metabolismoRESUMO
Rationale: Although inflammation and infection are key disease drivers in bronchiectasis, few studies have integrated host inflammatory and microbiome data to guide precision medicine. Objectives: To identify clusters among patients with bronchiectasis on the basis of inflammatory markers and to assess the association between inflammatory endotypes, microbiome characteristics, and exacerbation risk. Methods: Patients with stable bronchiectasis were enrolled at three European centers, and cluster analysis was used to stratify the patients according to the levels of 33 sputum and serum inflammatory markers. Clusters were compared in terms of microbiome composition (16S ribosomal RNA sequencing) and exacerbation risk over a 12-month follow-up. Measurements and Main Results: A total of 199 patients were enrolled (109 [54.8%] female; median age, 69 yr). Four clusters of patients were defined according to their inflammatory profiles: cluster 1, milder neutrophilic inflammation; cluster 2, mixed-neutrophilic and type 2; cluster 3, most severe neutrophilic; and cluster 4, mixed-epithelial and type 2. Lower microbiome diversity was associated with more severe inflammatory clusters (P < 0.001), and ß-diversity analysis demonstrated distinct microbiome profiles associated with each inflammatory cluster (P = 0.001). Proteobacteria and Pseudomonas at phylum and genus levels, respectively, were more enriched in clusters 2 and 3 than in clusters 1 and 4. Furthermore, patients in cluster 2 (rate ratio [RR], 1.49; 95% confidence interval [CI], 1.16-1.92) and cluster 3 (RR, 1.61; 95% CI, 1.12-2.32) were at higher risk of exacerbation over a 12-month follow-up compared with cluster 1, even after adjustment for prior exacerbation history. Conclusions: Bronchiectasis inflammatory endotypes are associated with distinct microbiome profiles and future exacerbation risk.
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Bronquiectasia , Humanos , Feminino , Idoso , Masculino , Bronquiectasia/microbiologia , Biomarcadores , Escarro/microbiologia , Inflamação , Estudos de CoortesRESUMO
AIMS/HYPOTHESIS: Chronic hyperglycaemia and recurrent hypoglycaemia are independently associated with accelerated cognitive decline in type 1 diabetes. Recurrent hypoglycaemia in rodent models of chemically induced (streptozotocin [STZ]) diabetes leads to cognitive impairment in memory-related tasks associated with hippocampal oxidative damage. This study examined the hypothesis that post-hypoglycaemic hyperglycaemia in STZ-diabetes exacerbates hippocampal oxidative stress and explored potential contributory mechanisms. METHODS: The hyperinsulinaemic glucose clamp technique was used to induce equivalent hypoglycaemia and to control post-hypoglycaemic glucose levels in mice with and without STZ-diabetes and Nrf2-/- mice (lacking Nrf2 [also known as Nfe2l2]). Subsequently, quantitative proteomics based on stable isotope labelling by amino acids in cell culture and biochemical approaches were used to assess oxidative damage and explore contributory pathways. RESULTS: Evidence of hippocampal oxidative damage was most marked in mice with STZ-diabetes exposed to post-hypoglycaemic hyperglycaemia; these mice also showed induction of Nrf2 and the Nrf2 transcriptional targets Sod2 and Hmox-1. In this group, hypoglycaemia induced a significant upregulation of proteins involved in alternative fuel provision, reductive biosynthesis and degradation of damaged proteins, and a significant downregulation of proteins mediating the stress response. Key differences emerged between mice with and without STZ-diabetes following recovery from hypoglycaemia in proteins mediating the stress response and reductive biosynthesis. CONCLUSIONS/INTERPRETATION: There is a disruption of the cellular response to a hypoglycaemic challenge in mice with STZ-induced diabetes that is not seen in wild-type non-diabetic animals. The chronic hyperglycaemia of diabetes and post-hypoglycaemic hyperglycaemia act synergistically to induce oxidative stress and damage in the hippocampus, possibly leading to irreversible damage/modification to proteins or synapses between cells. In conclusion, recurrent hypoglycaemia in sub-optimally controlled diabetes may contribute, at least in part, to accelerated cognitive decline through amplifying oxidative damage in key brain regions, such as the hippocampus. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in ProteomeXchange, accession no. 1-20220824-173727 ( www.proteomexchange.org ). Additional datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Hiperglicemia , Hipoglicemia , Camundongos , Animais , Hiperglicemia/metabolismo , Hipoglicemiantes , Diabetes Mellitus Tipo 1/metabolismo , Fator 2 Relacionado a NF-E2/genética , Hipoglicemia/metabolismo , Hipocampo , Estresse Oxidativo , Diabetes Mellitus Experimental/metabolismo , Glicemia/metabolismoRESUMO
Rationale: Bronchiectasis and chronic obstructive pulmonary disease (COPD) are two disease entities with overlapped clinical features, and codiagnosis frequently occurs (termed the "COPD-bronchiectasis association"). Objectives: To investigate the sputum microbiome and proteome in patients with bronchiectasis, COPD, and the COPD-bronchiectasis association with the aim of identifying endotypes that may inform treatment. Methods: Sputum microbiome and protein profiling were carried out using 16S rRNA amplicon sequencing and a label-free proteomics workflow, respectively, in a cohort comprising patients with COPD (n = 43), bronchiectasis (n = 30), and the COPD-bronchiectasis association (n = 48). Results were validated in an independent cohort of 91 patients (n = 28-31 each group) using targeted measurements of inflammatory markers, mucins, and bacterial culture. Measurements and Main Results: Principal component analysis of sputum microbiome and protein profiles showed a partial separation between the COPD and the "COPD-bronchiectasis association" group. Further analyses revealed that patients with the "COPD-bronchiectasis association" had a higher abundance of proteobacteria, higher expression of mucin-5AC and proteins from the "neutrophil degranulation" pathway compared to those with COPD. In contrast, patients with COPD had an elevated expression of mucin-5B and several peptidase inhibitors, higher abundance of common commensal taxa, and a greater microbiome diversity. The profiles of "COPD-bronchiectasis association" and bronchiectasis groups were largely overlapping. Five endotypes were proposed with differential inflammatory, mucin, and microbiological features. The key features related to the "COPD-bronchiectasis association" were validated in an independent cohort. Conclusions: Neutrophilic inflammation, differential mucin expression, and Gram-negative infection are dominant traits in patients with the "COPD-bronchiectasis association."
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Bronquiectasia , Microbiota , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/genética , RNA Ribossômico 16S , Escarro/microbiologiaRESUMO
BACKGROUND: The sputum microbiome has a potential role in disease phenotyping and risk stratification in chronic obstructive pulmonary disease (COPD), but few large longitudinal cohort studies exist. OBJECTIVE: Our aim was to investigate the COPD sputum microbiome and its association with inflammatory phenotypes and mortality. METHODS: 16S ribosomal RNA gene sequencing was performed on sputum from 253 clinically stable COPD patients (4-year median follow-up). Samples were classified as Proteobacteria or Firmicutes (phylum level) and Haemophilus or Streptococcus (genus level) dominant. Alpha diversity was measured by using Shannon-Wiener diversity and Berger-Parker dominance indices. Survival was modeled by using Cox proportional hazards regression. A subset of 78 patients had label-free liquid chromatography with tandem mass spectrometry performed, with partial least square discriminant analysis integrating clinical, microbiome, and proteomics data. RESULTS: Proteobacteria dominance and lower diversity was associated with more severe COPD according to the Global Initiative for Chronic Obstructive Lung Disease classification system (P = .0015), more frequent exacerbations (P = .0042), blood eosinophil level less than or equal to 100 cells/µL (P < .0001), and lower FEV1 (P = .026). Blood eosinophil counts showed a positive relationship with percent of Firmicutes and Streptococcus and a negative association with percent Proteobacteria and Haemophilus. Proteobacteria dominance was associated with increased mortality compared with Firmicutes-dominated or balanced microbiome profiles (hazard ratio = 2.58; 95% CI = 1.43-4.66; P = .0017 and hazard ratio = 7.47; 95% CI = 1.02-54.86; P = .048, respectively). Integrated omics analysis showed significant associations between Proteobacteria dominance and the neutrophil activation pathway in sputum. CONCLUSION: The sputum microbiome is associated with clinical and inflammatory phenotypes in COPD. Reduced microbiome diversity, associated with Proteobacteria (predominantly Haemophilus) dominance, is associated with neutrophil-associated protein profiles and an increased risk of mortality.
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Microbiota , Proteobactérias/classificação , Doença Pulmonar Obstrutiva Crônica , Escarro/microbiologia , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Inflamação , Estudos Longitudinais , Masculino , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/mortalidade , Taxa de SobrevidaRESUMO
Rationale: PZP (pregnancy zone protein) is a broad-spectrum immunosuppressive protein believed to suppress T-cell function during pregnancy to prevent fetal rejection. It has not previously been reported in the airway.Objectives: To characterize PZP in the bronchiectasis airway, including its relationship with disease severity.Methods: Label-free liquid chromatography/mass spectrometry was performed for sputum protein profiling of patients with bronchiectasis confirmed by high-resolution computed tomography. Results for patients with and without Pseudomonas aeruginosa infection were compared. Sputum and serum PZP was measured by validated ELISA. Airway infection status was established by culture and 16S ribosomal RNA sequencing. Immunofluorescence, ELISA, and electron microscopy were used to identify the cellular source of PZP in neutrophils treated with multiple stimuli.Measurements and Main Results: Elevated PZP was identified by label-free liquid chromatography/mass spectrometry as being associated with P. aeruginosa infection. In a validation study of 124 patients, sputum but not serum concentrations of PZP were significantly associated with the Bronchiectasis Severity Index, the frequency of exacerbations, and symptoms. Airway infection with Proteobacteria such as P. aeruginosa was associated with higher concentrations of PZP. PZP in sputum was directly related to airway bacterial load. Neutrophils induced to form neutrophil extracellular traps (NETs) with phorbol myristate acetate released high concentrations of PZP in vitro, and fluorescence microscopy confirmed the presence of PZP in NETs, whereas fluorescence and electron microscopy localized PZP to the cytoplasm and nuclei of neutrophils. Effective antibiotic therapy reduced sputum PZP.Conclusions: PZP is released into NETs. We report a novel link between airway infection, NET formation, and disease severity in bronchiectasis during chronic airway inflammation.
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Bronquiectasia/etiologia , Bronquiectasia/fisiopatologia , Armadilhas Extracelulares/metabolismo , Proteínas da Gravidez/efeitos adversos , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/fisiopatologia , Infecções Respiratórias/etiologia , Infecções Respiratórias/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Gravidez , Proteínas da Gravidez/sangueRESUMO
Aberrant extracellular matrix (ECM) remodelling in muscle, liver and adipose tissue is a key characteristic of obesity and insulin resistance. Despite its emerging importance, the effective ECM targets remain largely undefined due to limitations of current approaches. Here, we developed a novel ECM-specific mass spectrometry-based proteomics technique to characterise the global view of the ECM changes in the skeletal muscle and liver of mice after high fat (HF) diet feeding. We identified distinct signatures of HF-induced protein changes between skeletal muscle and liver where the ECM remodelling was more prominent in the muscle than liver. In particular, most muscle collagen isoforms were increased by HF diet feeding whereas the liver collagens were differentially but moderately affected highlighting a different role of the ECM remodelling in different tissues of obesity. Moreover, we identified a novel association between collagen 24α1 and insulin resistance in the skeletal muscle. Using quantitative gene expression analysis, we extended this association to the white adipose tissue. Importantly, collagen 24α1 mRNA was increased in the visceral adipose tissue, but not the subcutaneous adipose tissue of obese diabetic subjects compared to lean controls, implying a potential pathogenic role of collagen 24α1 in obesity and type 2 diabetes.
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Colágeno/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Animais , Colágeno/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Expressão Gênica , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , RNA Mensageiro/genéticaRESUMO
Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal excitability in a nongenomic manner. Dysregulation of their synthesis or metabolism has been implicated in many pathological conditions. Here, we describe an isotope dilution based targeted and nontargeted (ID-TNT) profiling of carbonyl neurosteroids/steroids. The method combines stable isotope dilution, hydroxylamine derivatization, high-resolution MS scanning, and data-dependent MS/MS analysis, allowing absolute quantification of pregnenolone, progesterone, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone, and 3ß,5α-tetrahydroprogesterone, and relative quantification of other carbonyl containing steroids. The utility and validity of this approach was tested in an acute stress mouse model and via pharmacological manipulation of the steroid metabolic pathway with finasteride. We report that brain levels of 3α,5α-tetrahydroprogesterone, a potent enhancer of GABAA receptor (GABAAR-mediated inhibitory function, from control mice is in the 5-40 pmol/g range, a value greater than previously reported. The approach allows the use of data from targeted analysis to guide the normalization strategy for nontargeted data. Furthermore, novel findings, including a striking increase of brain pregnenolone following finasteride administration were discovered in this study. Collectively, our results indicate that this approach has distinct advantages for examining targeted and nontargeted neurosteroid/steroid pathways in animal models and could facilitate a better understanding of the physiological and pathological roles of neurosteroids as modulators of brain excitability.
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RATIONALE: Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis. METHODS: This was a single-center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high-resolution computed tomography-confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity-based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross-sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over 3 years. MEASUREMENT AND MAIN RESULTS: Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV1% predicted (r = -0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3-year follow-up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV1 decline (ß coefficient, -0.139; P = 0.001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval [CI], 0.72-0.79) and all-cause mortality (area under the curve, 0.70; 95% CI, 0.67-0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42-5.29; P = 0.003) but not lung function decline. CONCLUSIONS: Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.
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Bronquiectasia/metabolismo , Bronquiectasia/fisiopatologia , Elastase de Leucócito/metabolismo , Pulmão/fisiopatologia , Idoso , Biomarcadores/metabolismo , Estudos de Coortes , Desmosina/metabolismo , Progressão da Doença , Feminino , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Estudos Prospectivos , Sistema de Registros , Índice de Gravidade de Doença , Escarro/metabolismo , Reino UnidoRESUMO
Ponatinib, a pan-BCR-ABL tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia (CML), causes severe side effects including vascular occlusions, pancreatitis, and liver toxicity, although the underlying mechanisms remain unclear. Modifications of critical proteins through reactive metabolites are thought to be responsible for a number of adverse drug reactions. In vitro metabolite screening of ponatinib with human liver microsomes and glutathione revealed unambiguous signals of ponatinib-glutathione (P-GSH) adducts. Further profiling of human cytochrome P450 (P450) indicated that CYP1A1 was the predominant P450 enzyme driving this reaction. P-GSH conjugate formation paralleled the disappearance of hydroxylated ponatinib metabolites, suggesting the initial reaction was epoxide generation. Mouse glutathione S-transferase p1 (mGstp1) further enhanced P-GSH adduct formation in vitro. Ponatinib pharmacokinetics were determined in vivo in wild-type (WT) mice and mice humanized for CYP1A1/2 and treated with the CYP1A1 inducers 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrene. Ponatinib exposure was significantly decreased in treated mice compared with controls (7.7- and 2.2-fold for WT and humanized CYP1A1/2, respectively). Interestingly, the P-GSH conjugate was only found in the feces of CYP1A1-induced mice, but not in control animals. Protein adducts were also identified by liquid chromatography-tandem mass spectrometry analysis of mGstp1 tryptic digests. These results indicate that not only could CYP1A1 be involved in ponatinib disposition, which has not been previously reported, but also that electrophilic intermediates resulting from CYP1A1 metabolism in normal tissues may contribute to ponatinib toxicity. These data are consistent with a recent report that CML patients who smoke are at greater risk of disease progression and premature death.
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Biocatálise , Citocromo P-450 CYP1A1/metabolismo , Imidazóis/metabolismo , Piridazinas/metabolismo , Animais , Glutationa/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Imidazóis/toxicidade , Masculino , Camundongos , Piridazinas/toxicidade , Proteínas Recombinantes/metabolismoRESUMO
Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.
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Aminoácidos/metabolismo , Enzimas/isolamento & purificação , Marcação por Isótopo/métodos , Fígado/enzimologia , Modelos Biológicos , Animais , Técnicas de Cultura de Células , Cromatografia Líquida , Grupo dos Citocromos a/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH-Ferri-Hemoproteína Redutase/genética , Espectrometria de Massas em TandemRESUMO
Elastin degradation is a key feature of emphysema and may have a role in the pathogenesis of atherosclerosis associated with chronic obstructive pulmonary disease (COPD). Circulating desmosine is a specific biomarker of elastin degradation. We investigated the association between plasma desmosine (pDES) and emphysema severity/progression, coronary artery calcium score (CACS) and mortality.pDES was measured in 1177 COPD patients and 110 healthy control subjects from two independent cohorts. Emphysema was assessed on chest computed tomography scans. Aortic arterial stiffness was measured as the aortic-femoral pulse wave velocity.pDES was elevated in patients with cardiovascular disease (p<0.005) and correlated with age (rho=0.39, p<0.0005), CACS (rho=0.19, p<0.0005) modified Medical Research Council dyspnoea score (rho=0.15, p<0.0005), 6-min walking distance (rho=-0.17, p<0.0005) and body mass index, airflow obstruction, dyspnoea, exercise capacity index (rho=0.10, p<0.01), but not with emphysema, emphysema progression or forced expiratory volume in 1â s decline. pDES predicted all-cause mortality independently of several confounding factors (p<0.005). In an independent cohort of 186 patients with COPD and 110 control subjects, pDES levels were higher in COPD patients with cardiovascular disease and correlated with arterial stiffness (p<0.05).In COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. pDES is a good biomarker of cardiovascular risk and mortality in COPD.
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Doenças Cardiovasculares/sangue , Desmosina/sangue , Enfisema/sangue , Doença Pulmonar Obstrutiva Crônica/sangue , Adulto , Idoso , Biomarcadores/sangue , Composição Corporal , Broncodilatadores/farmacologia , Calcinose , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/mortalidade , Estudos de Casos e Controles , Vasos Coronários/patologia , Progressão da Doença , Elastina/sangue , Elastina/metabolismo , Enfisema/complicações , Enfisema/mortalidade , Feminino , Volume Expiratório Forçado , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/mortalidade , Enfisema Pulmonar/fisiopatologia , Análise de Onda de Pulso , Testes de Função Respiratória , Fatores de Risco , Fumar/metabolismo , Rigidez VascularAssuntos
Biomarcadores/sangue , Bronquiectasia/sangue , Bronquiectasia/mortalidade , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/mortalidade , Desmosina/sangue , Idoso , Bronquiectasia/fisiopatologia , Doenças Cardiovasculares/fisiopatologia , Causas de Morte , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.
Assuntos
Anormalidades do Olho/genética , Proteínas dos Microfilamentos/genética , Microftalmia/genética , Mutação/efeitos da radiação , Animais , Inversão Cromossômica/genética , Cromossomos Humanos Par 18/genética , Éxons , Olho/crescimento & desenvolvimento , Olho/fisiopatologia , Anormalidades do Olho/fisiopatologia , Fibrilina-2 , Fibrilinas , Mutação da Fase de Leitura , Humanos , Camundongos , Microftalmia/fisiopatologia , Fenótipo , Sindactilia/genética , Sindactilia/fisiopatologia , Via de Sinalização Wnt/genéticaRESUMO
The acetylenic tricyclic bis(cyanoenone) TBE-31 is a highly potent cysteine targeting compound with a reversible covalent mode of action; its best-characterized target being Kelch-like ECH-associated protein-1 (Keap1), the cellular sensor for oxidants and electrophiles. TBE-31 reacts with cysteines of Keap1, impairing its ability to target nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) for degradation. Consequently, Nrf2 accumulates and orchestrates cytoprotective gene expression. In this study we investigated the pharmacokinetic and pharmacodynamic properties of TBE-31 in C57BL/6 mice. After a single oral dose of 10 µmol/kg (â¼200 nmol/animal), the concentration of TBE-31 in blood exhibited two peaks, at 22.3 nM and at 15.5 nM, 40 min and 4 h after dosing, respectively, as determined by a quantitative stable isotope dilution LC-MS/MS method. The AUC0-24h was 195.5 h/nmol/l, the terminal elimination half-life was 10.2 h, and the kel was 0.068 h(-1). To assess the pharmacodynamics of Nrf2 activation by TBE-31, we determined the enzyme activity of its prototypic target, NAD(P)H: quinone oxidoreductase 1 (NQO1) and found it elevated by 2.4- and 1.5-fold in liver and heart, respectively. Continuous feeding for 18 days with diet delivering the same daily doses of TBE-31 under conditions of concurrent treatment with the immunosuppressive agent azathioprine had a similar effect on Nrf2 activation without any indications of toxicity. Together with previous reports showing the cytoprotective effects of TBE-31 in animal models of carcinogenesis, our results demonstrate the high potency, efficacy and suitability for chronic administration of cysteine targeting reversible covalent drugs.