RESUMO
Obesity is a risk factor for many diseases, such as type 2 diabetes and cardiovascular diseases. In line with the need for precision medicine, the search for biomarkers reporting the progression of obesity- and diet-associated disorders is urgent. We used NMR to determine the metabolomics profile of key organs (lung, liver, heart, skeletal muscle, kidney, and brain) and serum from male C57Bl/6J mice (5 weeks old) fed for 6, 10, and 14 weeks on a high-fat and high-sucrose diet (HFHSD) vs. a standard diet (STD). We determined metabolite concentrations in the organs at each time point, which allowed us to discriminate age- and diet-related effects as well as the interactions between both, highlighting the need to evaluate the influence of age as a confounding factor on metabolic signatures. Notably, the analysis revealed the influence of time on metabolite concentrations in the STD condition, probably reflecting the juvenile-to-adult transition. Variations impacted the liver and lung metabolites, revealing the strong influence of the HFHS diet on normal metabolism maturation during youth.
Assuntos
Diabetes Mellitus Tipo 2 , Sacarose , Camundongos , Masculino , Animais , Sacarose/metabolismo , Dieta Hiperlipídica/efeitos adversos , Diabetes Mellitus Tipo 2/complicações , Estudos Transversais , Obesidade/metabolismo , Metabolômica , Fígado/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Ratos , Animais , Humanos , Ácido Pirúvico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ácido Cítrico/farmacologia , Ácido Cítrico/metabolismo , Células Secretoras de Insulina/metabolismo , Glucose/farmacologia , Glucose/metabolismo , Insulina/metabolismoRESUMO
INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown. OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P. METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury. CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.
Assuntos
Epóxido Hidrolases , Traumatismos Cardíacos , Obesidade , Animais , Feminino , Masculino , Ratos , Sistemas CRISPR-Cas , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/patologia , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Resistência à Insulina/genética , Lisofosfolipídeos , Obesidade/genética , Obesidade/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Traumatismo por Reperfusão/genéticaRESUMO
Glucotoxicity-induced ß-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Mitochondria and ER form a network in cells that controls cell function and fate. Mitochondria of the pancreatic ß cell play a central role in the secretion of insulin in response to glucose through their ability to produce ATP. Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. Here, we review MAM functions in the cell and we focus on the crosstalk between the ER and Mitochondria in the context of T2D, highlighting the pivotal role played by MAMs especially in ß cells through inter-organelle calcium exchange and glucotoxicity-associated ß cell dysfunction.
Assuntos
Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias , Animais , Diabetes Mellitus Tipo 2/etiologia , HumanosRESUMO
Cellular metabolomics has become key to elucidate mechanistic aspects in various fields such as cancerology or pharmacology, and is rapidly becoming a standard phenotyping tool accessible to the broad biological community. Acquisition of reliable spectroscopic datasets, such as nuclear magnetic resonance (NMR) spectra, to characterize biological systems depends on the elaboration of robust methods for cellular metabolites extraction. Previous studies have addressed many issues raised by these protocols, however with little pondering on ergonomic and practical aspects of the methods that impact their scalability, reproducibility and hence their suitability to high-throughput studies or their use by non-metabolomics experts. Here, we optimize a fast and ergonomic protocol for extraction of metabolites from adherent mammalian cells for NMR metabolomics studies. The proposed extraction protocol, including cell washing, metabolism quenching and actual extraction of intracellular metabolites, was first optimized on HeLa cells. Efficiency of the protocol, in its globality and for the different individual steps, was assessed by NMR quantification of 27 metabolites from cellular extracts. We show that a single PBS wash provides a seemly compromise between contamination from growth medium and leakage of intracellular metabolites. In HeLa cells, extraction using pure methanol, without cell scraping, recovered a higher amount of intracellular metabolites than the reference methanol/water/chloroform method with cell scraping, with yields varying across metabolite classes. Optimized and reference protocols were further tested on eight cell lines of miscellaneous nature, and inter-operator reproducibility was demonstrated. Our results stress the need for tailored extraction protocols and show that fast protocols minimizing time-consuming steps, without compromising extraction yields, are suitable for high-throughput metabolomics studies. Graphical abstract.
Assuntos
Adesão Celular , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultura , Ergonomia , Ensaios de Triagem em Larga Escala , Humanos , Mamíferos , Solventes/química , Água/químicaRESUMO
Glucotoxicity-induced ß-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in ß-cells is still largely unknown, and their implication in glucotoxicity-associated ß-cell dysfunction remains to be defined. Here, we report that acute glucose treatment stimulated ER-mitochondria interactions and calcium (Ca2+) exchange in INS-1E cells, whereas disruption of MAMs altered glucose-stimulated insulin secretion (GSIS). Conversely, chronic incubations with high glucose of either INS-1E cells or human pancreatic islets altered GSIS and concomitantly reduced ER Ca2+ store, increased basal mitochondrial Ca2+, and reduced ATP-stimulated ER-mitochondria Ca2+ exchanges, despite an increase of organelle interactions. Furthermore, glucotoxicity-induced perturbations of Ca2+ signaling are associated with ER stress, altered mitochondrial respiration, and mitochondria fragmentation, and these organelle stresses may participate in increased organelle tethering as a protective mechanism. Last, sustained induction of ER-mitochondria interactions using a linker reduced organelle Ca2+ exchange, induced mitochondrial fission, and altered GSIS. Therefore, dynamic organelle coupling participates in GSIS in ß-cells, and over time, disruption of organelle Ca2+ exchange might be a novel mechanism contributing to glucotoxicity-induced ß-cell dysfunction.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , RatosRESUMO
Type 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum-mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 non-diabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering and exchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics as compared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Adulto , Células Cultivadas , Feminino , Imunofluorescência , Humanos , Hibridização In Situ , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Pessoa de Meia-Idade , Membranas Mitocondriais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Background. The use of miRNAs as biomarkers for Type 1 Diabetes (T1D) risk is attractive as T1D is usually diagnosed in front of acute symptoms. As miR-375 is highly expressed in the endocrine pancreas, we postulated that its circulating level might reflect beta cell alterations and might be altered in the blood of T1D patients recently diagnosed. Methods. Sera were obtained from 22 T1D children at onset of the disease, before subcutaneous insulin treatment, and from 10 nondiabetic pediatric controls. MiR-375 seric level was quantified by stem-loop RT-PCR-based assay. MiRNAs regulations in isolated human islets in response to high glucose concentrations were determined by TaqMan Low-Density Array. Results. The abundance of miR-375, among the 410 miRNAs detected in human islets, mirrored its well-established role in rodent islet biology. Upregulated miRNAs targeted genes involved in islet homeostasis and regulation of beta cell mass. Downregulated miRNAs, including miR-375, were involved in pancreas secretion and protein turnover. Seric level of miR-375 was lower in T1D children versus age-matched controls, without any correlations with HbA1c, glycaemia, and number of autoantibodies. Conclusion. Altered circulating level of miR-375 at onset of T1D might be a general biomarker of metabolic alterations and inflammation associated with the disease.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , MicroRNAs/sangue , Adolescente , Criança , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , MasculinoRESUMO
This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice.
Assuntos
Benzoatos/farmacologia , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Cardiopatias/prevenção & controle , Resistência à Insulina , Obesidade/tratamento farmacológico , Ureia/análogos & derivados , Vasodilatação/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Vasos Coronários/enzimologia , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eicosanoides/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Epóxido Hidrolases/metabolismo , Glibureto/farmacologia , Cardiopatias/enzimologia , Cardiopatias/etiologia , Cardiopatias/fisiopatologia , Hipoglicemiantes/farmacologia , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Óxido Nítrico/metabolismo , Obesidade/sangue , Obesidade/complicações , Obesidade/enzimologia , Obesidade/fisiopatologia , Fatores de Tempo , Ureia/farmacologia , Vasodilatadores/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Obesity, through low-grade inflammation, can drive insulin resistance and type 2 diabetes. While infiltration of adipose tissue (AT) with mononuclear cells (MNCs) is well established in obesity, the functional consequences of these interactions are less understood. Herein, we cocultured human adipose-derived stem cells (ASCs) from obese individuals with MNCs and analyzed their reciprocal behavior. Presence of ASCs 1) enhanced interleukin (IL)-17A secretion by Th17 cells, 2) inhibited γ-interferon and tumor necrosis factor α secretion by Th1 cells, and 3) increased monocyte-mediated IL-1ß secretion. IL-17A secretion also occurred in stromal vascular fractions issued from obese but not lean individuals. Th17 polarization mostly depended on physical contacts between ASCs and MNCs-with a contribution of intracellular adhesion molecule-1-and occurred through activation of the inflammasome and phosphatidylinositol 3-kinase pathways. ASCs favored STAT3 over STAT5 transcription factor binding on STAT binding sites within the IL-17A/F gene locus. Finally, conditioned media from activated ASC-MNC cocultures inhibited adipocyte differentiation mRNA markers and impaired insulin-mediated Akt phosphorylation and lipolysis inhibition. In conclusion, we report that obese- but not lean-derived ASCs induce Th17 promotion and monocyte activation. This proinflammatory environment, in turn, inhibits adipogenesis and adipocyte insulin response. The demonstration of an ASC-Th17-monocyte cell axis reveals a novel proinflammatory process taking place in AT during obesity and defines novel putative therapeutic targets.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/citologia , Inflamação/etiologia , Insulina/farmacologia , Monócitos/fisiologia , Obesidade/complicações , Células-Tronco/fisiologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Comunicação Celular , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Fatores de Transcrição STAT/fisiologiaRESUMO
Imeglimin is the first in a new class of oral glucose-lowering agents currently in phase 2b development. Although imeglimin improves insulin sensitivity in humans, the molecular mechanisms are unknown. This study used a model of 16-week high-fat, high-sucrose diet (HFHSD) mice to characterize its antidiabetic effects. Six-week imeglimin treatment significantly decreased glycemia, restored normal glucose tolerance, and improved insulin sensitivity without modifying organs, body weights, and food intake. This was associated with an increase in insulin-stimulated protein kinase B phosphorylation in the liver and muscle. In liver mitochondria, imeglimin redirects substrate flows in favor of complex II, as illustrated by increased respiration with succinate and by the restoration of respiration with glutamate/malate back to control levels. In addition, imeglimin inhibits complex I and restores complex III activities, suggesting an increase in fatty acid oxidation, which is supported by an increase in hepatic 3-hydroxyacetyl-CoA dehydrogenase activity and acylcarnitine profile and the reduction of liver steatosis. Imeglimin also reduces reactive oxygen species production and increases mitochondrial DNA. Finally, imeglimin effects on mitochondrial phospholipid composition could participate in the benefit of imeglimin on mitochondrial function. In conclusion, imeglimin normalizes glucose tolerance and insulin sensitivity by preserving mitochondrial function from oxidative stress and favoring lipid oxidation in liver of HFHSD mice.
Assuntos
Hipoglicemiantes/uso terapêutico , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Triazinas/uso terapêutico , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Autoantibodies against antigens expressed by insulin-producing ß cells are circulating in both healthy individuals and patients at risk of developing Type 1 diabetes. Recent studies suggest that another set of antibodies (anti-idiotypic antibodies) exists in this antibody/antigen interacting network to regulate auto-reactive responses. Anti-idiotypic antibodies may block the antigen-binding site of autoantibodies or inhibit autoantibody expression and secretion. The equilibrium between autoantibodies and anti-idiotypic antibodies plays a critical role in mediating or preventing autoimmunity. In order to investigate the molecular mechanisms underlying such a network in autoimmunity and potentially develop neutralizing reagents to prevent or treat Type 1 diabetes, we need to produce autoantibodies and autoantigens with high quality and purity. Herein, using GAD65/anti-GAD65 autoantibodies as a model system, we aimed to establish reliable approaches for the preparation of highly pure autoantibodies suitable for downstream investigation.
RESUMO
B cells internalize extracellular Ag into endosomes using the Ig component of the BCR. In endosomes, Ag-derived peptides are loaded onto MHC class II proteins. How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC class II peptide loading, coprecipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes colocalize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble DM directly binds the Ig Fab domain and increases levels of free Ag released from immune complexes. Taken together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences.
Assuntos
Linfócitos B/imunologia , Antígenos HLA-D/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Complexo Antígeno-Anticorpo/imunologia , Compartimento Celular/imunologia , Linhagem Celular Tumoral , Endossomos/imunologia , Humanos , Tonsila Palatina/citologia , Tonsila Palatina/imunologiaRESUMO
As shown in a large clinical prospective trial, inhibition of the renin-angiotensin system (RAS) can delay the onset of type 2 diabetes in high-risk individuals. We evaluated the beneficial effects of RAS inhibition on ß-cell function under glucotoxic conditions. Human islets from 13 donors were cultured in 5.5 mM (controls) or 16.7 mM glucose [high glucose (HG)] for 4 d with or without losartan (5 µM), a selective AT1R blocker, and/or U73122 (2 µM), a selective PLC inhibitor, during the last 2 d. HG induced RAS activation with overexpression of AT1R (P<0.05) and angiotensinogen (P<0.001) mRNAs. HG increased endoplasmic reticulum (ER) stress markers (P<0.001) such as GRP78, sXBP1, and ATF4 mRNAs and Grp78 protein levels (P<0.01). HG also decreased reticular calcium concentration (P<0.0001) and modified protein expressions of ER calcium pumps with reduction of SERCA2b (P<0.01) and increase of IP3R2 (P<0.05). Losartan prevented these deleterious effects and was associated with improved insulin secretion despite HG exposure. AT1R activation triggers the PLC-IP3-calcium pathway. Losartan prevented the increase of PLC ß1 and γ1 protein levels induced by HG (P<0.05). U73122 reproduced all the protective effects of losartan. AT1R blockade protects human islets from the deleterious effects of glucose through inhibition of the PLC-IP3-calcium pathway.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Glucose/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Losartan/farmacologia , Fosfolipase C beta/metabolismo , Fosfolipase C gama/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Estrenos/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Fosfolipase C beta/antagonistas & inibidores , Fosfolipase C gama/antagonistas & inibidores , Pirrolidinonas/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
This study aims to investigate gadolinium-based nanoparticles (Gd-HNP) for in vitro labeling of human plasmacytoid dendritic cells (HuPDC) to allow for in vivo tracking and HuPDC quantifying using magnetic resonance imaging (MRI) following parenteral injection. Human plasmacytoid DC were labeled (LabHuPDC) with fluorescent Gd-HNP (Gd-FITC-HNP) and injected via intraperitoneal and intravenous routes in 4-5 NOD-SCID ß2m(-/-)mice (treated mice = TM). Control mice (CM) were similarly injected with unlabeled HuPDC. In vivo 7 T MRI was performed 24 h later and all spleens were removed in order to measure Gd and fluorescence contents and identify HuPDC. Gd-FITC-HNP efficiently labeled HuPDC (0.05 to 0.1 pg per cell), without altering viability and activation properties. The magnetic resonance (MR) signal was exclusively due to HuPDC. The normalized MR splenic intensity for TM was significantly higher than for CM (p < 0.024), and highly correlated with the spleen Gd content (r = 0.97), and the number of HuPDC found in the spleen (r = 0.94). Gd-FITC-HNP allowed for in vivo tracking and HuPDC quantifying by means of MRI following parenteral injection, with very high sensitivity (<3000 cells per mm(3)). The safety of these new nanoparticle types must be confirmed via extensive toxicology tests including in vivo stability and biodistribution studies.
Assuntos
Meios de Contraste/química , Células Dendríticas/citologia , Nanopartículas de Magnetita/química , Animais , Linhagem Celular , Rastreamento de Células , Meios de Contraste/síntese química , Meios de Contraste/farmacocinética , Células Dendríticas/química , Células Dendríticas/transplante , Fluoresceína-5-Isotiocianato/química , Gadolínio/química , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Baço/imunologia , Baço/metabolismo , Distribuição TecidualRESUMO
The identification of disease-specific autoantibodies to the 65-kDa isoform of glutamate decarboxylase (GAD65Ab) epitopes in type 1 diabetes has been hampered by their conformational nature. Here, we compared two methods of GAD65Ab epitope analysis: GAD65/67 fusion proteins and competition assays using GAD65-specific recombinant fraction antigen binding (rFab). Sera from newly diagnosed type 1 diabetes patients (n=61) were studied using both approaches. Competition of GAD65 binding by an rFab to a specific epitope did not correlate with binding to the fusion protein that represented this epitope. Conversely, samples that bound to specific fusion proteins were not necessarily competed with rFab specific to determinants in the same region. We conclude that epitopes of different characteristics are detected by fusion proteins and by competition with rFab. Fusion proteins allow the definition of large epitope regions; however, some conformational GAD65Ab epitopes, especially those residing in the middle region, are destroyed or distorted in the fusion proteins. Competition studies using rFab allow the identification of conformational epitopes. However, monoclonal rFab may only reflect a limited proportion of the epitopes recognized by polyclonal sera. A combined analysis using both approaches may therefore be necessary to gain best understanding of autoantibody characteristics and affinity maturation.
Assuntos
Autoanticorpos/imunologia , Mapeamento de Epitopos/métodos , Epitopos/análise , Glutamato Descarboxilase/imunologia , Isoenzimas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Autoanticorpos/química , Diabetes Mellitus Tipo 1/imunologia , Humanos , Proteínas Recombinantes de Fusão/química , Reprodutibilidade dos TestesRESUMO
Autoantibodies to the 65-kDa isoform of GAD (GAD65Abs) are associated with type 1 diabetes development, but the conformational nature of the GAD65Ab epitopes complicates the evaluation of disease risk. Six GAD65-specific recombinant Fabs (rFabs) were cloned from monoclonal antibodies b96.11, DP-C, DP-A, DPD, 144, and 221-442. The binding of GAD65Abs in 61 type 1 diabetic patients to GAD65 was analyzed by competitive radioimmunoassays with the six rFabs to ascertain disease-specific GAD65Ab binding specificities. The median binding was reduced significantly by rFab b96.11 (72%) (P < 0.0001), DP-A (84%) (P < 0.0001), DP-C (84%) (P < 0.0001), 221-442 (79%) (P < 0.0001), and DP-D (80%) (P < 0.0001). The competition pattern in type 1 diabetic patients differed from that in GAD65Ab-positive late autoimmune diabetes in adults (LADA) patients (n = 44), first-degree relatives (n = 38), and healthy individuals (n = 14). Whereas 87 and 72% of the type 1 diabetic sera were competed by rFab b96.11 and DP-C, respectively, only 34 and 26% of LADA patients, 18 and 25% of first-degree relatives, and 7 and 28% of healthy individuals showed competition (P < 0.0001). These findings support the view that type 1 diabetes is associated with disease- and epitope-specific GAD65Abs and supports the notion that the middle epitope is disease associated. These GAD65-specific rFabs should prove useful in predicting type 1 diabetes and in the study of conformational GAD65Ab epitopes.
Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Isoenzimas/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Especificidade de Anticorpos , Criança , Glutamato Descarboxilase/antagonistas & inibidores , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Isoenzimas/antagonistas & inibidores , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Valores de Referência , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
It has been a continuing challenge to try and identify those patients with hyperthyroid Graves' disease likely to remain in remission after an antithyroid drug course or to manage the medical treatment so as to increase the chance of remission. On average, the overall relapse rate is approximately 50% and any significant reduction of this figure would be of practical as well as theoretical value. The numerous controlled prospective studies performed in many parts of the world, with varying iodine intakes, have all confirmed that the main initial features related to the subsequent risk of relapse are: young age, male gender, goiter larger than 40 mL, hypoechogenic and hypervascular gland, high level of anti-thyrotropin receptor antibody (TRAb), detected either with radioreceptor assay (TBII: >40 U/L) or the biologic stimulation assay (thyroid-stimulating antibodies [TSAb]; >300%), severity of hyperthyroidism, and possibly the presence of ophthalmopathy. Alone, each of these has a low predictive value, but together they allow evaluation of the risk of relapse, thus helping treatment choice. As to the modalities of antithyroid drug treatment, dose of the drug or addition of levothyroxine does not affect posttreatment outcome. In contrast, significantly fewer relapses occur for drug courses longer than at least 1 year. Persistence of high levels of TRAb after medical treatment is strongly predictive of relapse but this is of limited value because in most patients, TRAb levels are low or even undetectable at the end of treatment, which does not indicate for further outcome. Smoking is a significant independent risk factor for relapse. In conclusion, reduction of the risk of relapse in patients with medically treated hyperthyroid Graves' disease relies on clinical competence and appropriate management taking into account an array of factors none of which alone has definite predictive value.
Assuntos
Antitireóideos/uso terapêutico , Doença de Graves/tratamento farmacológico , Doença de Graves/epidemiologia , Humanos , Fatores de Risco , Prevenção SecundáriaRESUMO
Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.