RESUMEN
CONTEXT: Glucagon-like peptide 1 (GLP-1), an insulinotropic peptide released into the circulation from intestinal enteroendocrine cells, is considered a hormonal mediator of insulin secretion. However, the physiological actions of circulating GLP-1 have been questioned because of the short half-life of the active peptide. Moreover, there is mounting evidence for localized, intra-islet mediation of GLP-1 receptor (GLP-1r) signaling including a role for islet dipeptidyl-peptidase 4 (DPP4). OBJECTIVE: To determine whether GLP-1r signaling contributes to insulin secretion in the absence of enteral stimulation and increased plasma levels, and whether this is affected by DPP4. METHODS: Single-site study conducted at an academic medical center of 20 nondiabetic subjects and 13 subjects with type 2 diabetes. This was a crossover study in which subjects received either a DPP4 inhibitor (DPP4i; sitagliptin) or placebo on 2 separate days. On each day they received a bolus of intravenous (IV) arginine during sequential 60-minute infusions of the GLP-1r blocker exendin[9-39] (Ex-9) and saline. The main outcome measures were arginine-stimulated secretion of C-Peptide (C-PArg) and insulin (InsArg). RESULTS: Plasma GLP-1 remained at fasting levels throughout the experiments and IV arginine stimulated both α- and ß-cell secretion in all subjects. Ex-9 infusion reduced C-PArg in both the diabetic and nondiabetic groups by ~14% (Pâ <â .03 for both groups). Sitagliptin lowered baseline glycemia but did not affect the primary measures of insulin secretion. However, a significant interaction between sitagliptin and Ex-9 suggested more GLP-1r activation with DPP4i treatment in subjects with diabetes. CONCLUSION: GLP-1r activation contributes to ß-cell secretion in diabetic and nondiabetic people during α-cell activation, but in the absence of increased circulating GLP-1. These results are compatible with regulation of ß-cells by paracrine signals from α-cells. This process may be affected by DPP4 inhibition.
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Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , Arginina/uso terapéutico , Estudios Cruzados , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Ayuno , Péptido 1 Similar al Glucagón , Humanos , Insulina/metabolismo , Secreción de Insulina , Fosfato de Sitagliptina/farmacología , Fosfato de Sitagliptina/uso terapéuticoRESUMEN
Exercise is an effective therapy against the metabolic syndrome. However, the molecular pathways underlying the advantageous effects of exercise are elusive. Glucagon receptor signaling is essential for exercise benefits, and recent evidence indicates that a downstream effector of glucagon, fibroblast growth factor 21 (FGF21), is implicated in this response. Therefore, we tested the hypothesis that FGF21 action is necessary in mediating metabolic effects of exercise. We utilized acute exhaustive treadmill exercise in Wistar rats to identify a putative, concomitant increase in plasma glucagon and FGF21 with the increase in glucose and lactate following exercise. To test the necessity of FGF21 action in the exercise response, we exposed FGF21 congenitally deficient mice (Fgf21(-/-)) and their wild-type (Wt) littermates to chronic high-fat (HF) feeding and inoperable (sedentary) or operable (exercise) voluntary running wheels. Physiological tests were performed to assess the role of FGF21 in the beneficial effect of exercise on glucose metabolism. Wt and Fgf21(-/-) littermates exhibited similar running behavior, and exercise was effective in suppressing weight and fat mass gain and dyslipidemia independently of genotype. However, exercise failed to positively affect hepatic triglyceride content and glucose tolerance in HF diet-fed Fgf21(-/-) mice. Furthermore, Fgf21(-/-) mice exhibited an impaired adaptation to exercise training, including reduced AMP-activated protein kinase activity in skeletal muscle. This study demonstrates that FGF21 action is necessary to achieve the full metabolic benefits of exercise during chronic HF feeding.
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Glucemia/metabolismo , Dieta Alta en Grasa , Terapia por Ejercicio/métodos , Factores de Crecimiento de Fibroblastos/metabolismo , Obesidad/metabolismo , Obesidad/terapia , Condicionamiento Físico Animal/métodos , Animales , Peso Corporal , Enfermedad Crónica , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Wistar , Resultado del TratamientoRESUMEN
The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.
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Metabolismo Energético , Ghrelina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Relacionada con Agouti/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa , Metabolismo Energético/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Perfilación de la Expresión Génica , Ghrelina/administración & dosificación , Ghrelina/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Fenotipo , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Receptor de Melanocortina Tipo 3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Ghrelina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.
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Factores de Crecimiento de Fibroblastos/metabolismo , Glucagón/metabolismo , Hepatocitos/metabolismo , Receptores de Glucagón/metabolismo , Adulto , Animales , Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/farmacología , Fármacos Antiobesidad/uso terapéutico , Células Cultivadas , Estudios Cruzados , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Método Doble Ciego , Femenino , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Glucagón/agonistas , Glucagón/farmacología , Células HEK293 , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Resistencia a la Insulina , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Terapia Molecular Dirigida , Obesidad/sangre , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Péptidos/síntesis química , Péptidos/farmacocinética , Péptidos/fisiología , Péptidos/uso terapéutico , Ratas , Receptores de Glucagón/agonistas , Receptores de Glucagón/genética , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismoRESUMEN
The scaffold protein p62 (sequestosome 1; SQSTM1) is an emerging key molecular link among the metabolic, immune, and proliferative processes of the cell. Here, we report that adipocyte-specific, but not CNS-, liver-, muscle-, or myeloid-specific p62-deficient mice are obese and exhibit a decreased metabolic rate caused by impaired nonshivering thermogenesis. Our results show that p62 regulates energy metabolism via control of mitochondrial function in brown adipose tissue (BAT). Accordingly, adipocyte-specific p62 deficiency led to impaired mitochondrial function, causing BAT to become unresponsive to ß-adrenergic stimuli. Ablation of p62 leads to decreased activation of p38 targets, affecting signaling molecules that control mitochondrial function, such as ATF2, CREB, PGC1α, DIO2, NRF1, CYTC, COX2, ATP5ß, and UCP1. p62 ablation in HIB1B and BAT primary cells demonstrated that p62 controls thermogenesis in a cell-autonomous manner, independently of brown adipocyte development or differentiation. Together, our data identify p62 as a novel regulator of mitochondrial function and brown fat thermogenesis.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Termogénesis/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Adipocitos Marrones/citología , Tejido Adiposo Pardo/citología , Animales , Células Cultivadas , Proteínas de Choque Térmico/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Especificidad de Órganos/genética , Proteína Sequestosoma-1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Although obesity rates are rapidly rising, caloric restriction remains one of the few safe therapies. Here we tested the hypothesis that obesity-associated disorders are caused by increased adipose tissue as opposed to excess dietary lipids. Fat mass (FM) of lean C57B6 mice fed a high-fat diet (HFD; FMC mice) was "clamped" to match the FM of mice maintained on a low-fat diet (standard diet [SD] mice). FMC mice displayed improved glucose and insulin tolerance as compared with ad libitum HFD mice (P < 0.001) or SD mice (P < 0.05). These improvements were associated with fewer signs of inflammation, consistent with the less-impaired metabolism. In follow-up studies, diet-induced obese mice were food restricted for 5 weeks to achieve FM levels identical with those of age-matched SD mice. Previously, obese mice exhibited improved glucose and insulin tolerance but showed markedly increased fasting-induced hyperphagia (P < 0.001). When mice were given ad libitum access to the HFD, the hyperphagia of these mice led to accelerated body weight gain as compared with otherwise matched controls without a history of obesity. These results suggest that although caloric restriction on a HFD provides metabolic benefits, maintaining those benefits may require lifelong continuation, at least in individuals with a history of obesity.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Restricción Calórica/efectos adversos , Metabolismo Energético , Adiposidad , Animales , Dieta Alta en Grasa/efectos adversos , Dieta Reductora/efectos adversos , Regulación de la Expresión Génica , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/inmunología , Intolerancia a la Glucosa/metabolismo , Hiperfagia/etiología , Hipotálamo/metabolismo , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/dietoterapia , Obesidad/inmunología , Obesidad/metabolismo , Obesidad/prevención & control , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Distribución Aleatoria , Prevención Secundaria , Aumento de PesoRESUMEN
The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or fibroblast growth factor 21 (FGF21), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF21 monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.
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Dieta/efectos adversos , Factores de Crecimiento de Fibroblastos/farmacología , Leptina/análogos & derivados , Leptina/farmacología , Obesidad/metabolismo , Péptidos/farmacología , Ponzoñas/farmacología , Animales , Peso Corporal , Combinación de Medicamentos , Exenatida , Factores de Crecimiento de Fibroblastos/administración & dosificación , Leptina/administración & dosificación , Leptina/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Obesidad/inducido químicamente , Obesidad/tratamiento farmacológico , Péptidos/administración & dosificación , Polietilenglicoles/química , Ponzoñas/administración & dosificaciónRESUMEN
Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.
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Bioquímica/métodos , Péptidos/metabolismo , Proteínas/química , Proteínas/metabolismo , Análisis de Secuencia/métodos , Acilación , Colorimetría , Formiatos/metabolismo , Hidrolasas/metabolismo , Isotiocianatos/metabolismo , Fragmentos de Péptidos/metabolismo , Péptido Hidrolasas/metabolismo , Piroglutamil-Peptidasa I/metabolismo , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/aislamiento & purificación , Soluciones , Anhídridos Succínicos/metabolismoRESUMEN
UNLABELLED: Genetic control of root development in rice is complex and the underlying mechanisms (constitutive and adaptive) are poorly understood. Lowland and upland varieties of indica and japonica rice with contrasting root development characteristics have been crossed, mapping populations developed and a number of QTLs in different chromosomes were identified. As these studies have used different sets of markers and many of the QTLs identified are long, it is difficult to exploit the varietal difference for improved root traits by marker assisted selection and for identification of concerned alleles. Intensive data mining of literature resulted in the identification 861 root development QTLs and associated microsatellite markers located on different chromosomes. The QTL and marker data generated and the genome sequence of rice were used for construction of a relational database, Rootbrowse, using MySQL relational database management system and Bio::DB::GFF schema. The data is viewed using GBrowse visualization tool. It graphically displays a section of the genome and all features annotated on it including the QTLs. The QTLs can be displayed along with SSR markers, protein coding genes and/or known root development genes for prediction of probable candidate genes. AVAILABILITY: Rootbrowse is freely available at http://www.ricebrowse.org.
RESUMEN
Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn(268) or Asn(268) and Asn(302) in the alpha-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 +/- 0.15 vs. 0.24 +/- 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 +/- 0.29) than the 34-kDa form (1.08 +/- 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC(50), 0.68 nM) than the 34-kDa isoform (IC(50), 8.2 nM) at displacing [(125)I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn(302) of the alpha-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.
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Inhibinas/metabolismo , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Hormona Folículo Estimulante/metabolismo , Glicosilación , Humanos , Técnicas In Vitro , Inhibinas/química , Inhibinas/aislamiento & purificación , Isomerismo , Hipófisis/citología , Unión Proteica , Proteoglicanos/genética , Ratas , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
OBJECTIVES: Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking. DESIGN AND METHODS: Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile. RESULTS: Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p<0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1. CONCLUSIONS: The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.
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Ensayo de Inmunoadsorción Enzimática/métodos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Fosfoserina/sangre , Adolescente , Adulto , Líquido Amniótico/química , Anticuerpos/inmunología , Femenino , Humanos , Persona de Mediana Edad , Embarazo , Isoformas de Proteínas/sangre , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: Determination of insulin-like growth factor (IGF)-I is now a routine adjunct to multiple research and clinical investigations. Evidence has associated higher IGF-I levels with various human pathologies, but the reported associations have not been invariably confirmed. We examined the potential for post-sampling proteolysis and evaluated the impact of such events on IGF-I immunoassays. DESIGN AND METHODS: We compared IGF-I in different sets of fresh and frozen old samples using four different and commonly used immunoassays. The potential for post-sampling proteolysis was further examined by assaying fresh samples stored for 4 weeks at various temperatures in the absence or presence of protease inhibitors. RESULTS: IGF-I levels in fresh serum samples from adult males, females, and pregnant subjects by all methods were similar and were highly correlated (r=0.85-0.97). The same was true for levels in frozen ( approximately 2 years at --80 degrees C) samples from diabetic patients, which are reportedly associated with enhanced proteolytic activity. In contrast, in another set of frozen adult male and female samples ( approximately 8 years at --20 degrees C), the inter-method median IGF-I levels varied by approximately 3- to 4-fold and the values poorly correlated. Similar variability in the inter-method response was also observed when IGF-I in the replicates of fresh samples stored at 4 degrees C for 4 weeks was measured. However, the 4 degrees C storage effect could be completely blocked by the addition of protease inhibitors, allowing for all assays to detect 92--101% of the expected mean levels. CONCLUSIONS: The data indicate susceptibility of IGF-I to significant post-sampling proteolysis and suggest the importance of immunoassays for the intact molecule. Immunoassays that lack specificity for intact IGF-I may mask the potential pathophysiological effects of proteolysis and generate misleading results, particularly in studies involving inappropriately stored and/or proteolyzed samples. In such cases, underestimation of the in vivo levels by the intact assays would occur, but the findings of low IGF-I levels may be indicative of questionable sample quality.
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Ensayo de Inmunoadsorción Enzimática , Factor I del Crecimiento Similar a la Insulina/análisis , Adulto , Anciano , Aprotinina/farmacología , Diabetes Mellitus Tipo 2/sangre , Endopeptidasas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Congelación , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Persona de Mediana Edad , Fluoruro de Fenilmetilsulfonilo/farmacología , Embarazo , Inhibidores de Proteasas/farmacología , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
OBJECTIVE: Inhibin circulates in various molecular weight forms. Alpha (alpha)-subunit-directed total inhibin immunoassays, which detect all forms of alpha subunits plus the alpha/beta inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. DESIGN AND METHODS: Method development involved a pair of well-characterized inhibin alpha subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. RESULTS: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5-500 ng/l, and intra- and inter-assay imprecision of 2.3-4.6% and 3.3-5.1% at total inhibin concentrations of approximately 60-400 ng/l, respectively. The mean (+/-SD) recovery from spiked serum samples averaged 109 +/- 14% and recovery in response to serial sample dilution was 99 +/- 10%. Serum values by the direct method (n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS (r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8-250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. CONCLUSION: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.
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Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Inhibinas/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Líquido Folicular/química , Humanos , Inhibinas/química , Inhibinas/inmunología , Masculino , Análisis por Apareamiento , Persona de Mediana Edad , Neoplasias Ováricas/sangre , Posmenopausia/sangre , Sensibilidad y EspecificidadRESUMEN
The IGF-binding protein (IGFBP)-related proteins (rPs) are a group of recently described cysteine-rich proteins that share significant amino-terminal structural similarity with the conventional IGFBPs. IGFBP-rP1 (also known as MAC25/angiomodulin/prostacyclin-stimulating factor and T1A12), regulates cellular proliferation, adhesion, and angiogenesis and stimulates prostacyclin synthesis. We characterized new monoclonal antibodies generated against IGFBP-rP1 and have used them to study the distribution of IGFBP-rP1 in human biological fluids and tissues. Additionally, we have developed a noncompetitive sandwich-type immunoassay to quantitate the concentrations of IGFBP-rP1 in human serum. IGFBP-rP1 was readily detectable in serum, urine, amniotic fluid, and cerebrospinal fluid by immunoblot analysis. Evaluation of the newly developed immunoassay demonstrated acceptable analytical performance, with a detection limit of 0.7 micro g/liter, a dynamic range of 3.1-100 micro g/liter, and intra- and interassay coefficients of variation of 2.5-6.8% and 3.1-6.4% at approximately 24-85 ng/ml IGFBP-rP-1, respectively. No significant cross-reactivity with IGFBP-1-6 was observed. In random normal human adult sera (n = 37), the median IGFBP-rP1 was 21.0 micro g/liter, and values did not correlate with levels of IGF-I (r = 0.085, P = 0.61), IGF-II (r = 0.051, P = 0.75), or IGFBP-3 (r = 0.061, P = 0.74). The monoclonal anti-IGFBP-rP1 antibodies also readily detected IGFBP-rP1 expression in human tissue sections, with preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. In summary, using newly developed IGFBP-rP1 monoclonal antibodies, we confirm the presence of IGFBP-rP1 in the major human body fluids, provide quantitative normative data on the concentrations of IGFBP-rP1 in human serum, and show preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. The use of these novel IGFBP-rP1 detection tools should prove useful in the elucidation of the biological role(s) of this protein.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Adulto , Anciano , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Líquidos Corporales/química , Femenino , Humanos , Hibridomas , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , EmbarazoRESUMEN
OBJECTIVES: Markers of myocardial injury have been vital in the assessment of patients with coronary heart disease. Pregnancy associated plasma protein A (PAPP)-A is an insulin-like growth factor (IGF) binding protein (IGFBP)-4 protease and a potential early indicator of unstable angina. We developed an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for PAPP-A and measured serum PAPP-A in patients with biochemical evidence of acute coronary syndrome. DESIGN AND METHODS: Method development was based on pair-wise evaluation of a panel of antibodies and determination of PAPP-A specificity and sensitivity relative to those of a conventional method. Association of PAPP-A with myocardial damage was assessed in serum samples classified based on serum creatine kinase (CK)-MB or cardiac troponin-T levels. RESULTS: Serum PAPP-A was significantly higher in samples with elevated CK-MB or troponin-T than in samples with normal CK-MB (p < 0.001). Marker-association studies showed strong correlation between PAPP-A and troponin-T (r = 0.59, p < 0.001) in a subset of troponin-T positive samples. Indications for both parallel as well as divergence in the expression of PAPP-A and troponin-T was also evident when serial timed samples available from a number of patients were analyzed. CONCLUSIONS: The data are consistent with the conclusion that expression of PAPP-A is enhanced in patients with biochemical evidence of acute coronary syndrome and suggest strongly that demonstration of PAPP-A association with other cardiac markers might be influenced by their relative release dynamics (timing and duration). The availability of the ultrasensitive PAPP-A ELISA should facilitate systematic investigations of PAPP-A expression in this and other pathophysiological conditions that might involve altered expression of the IGF/PAPP-A system.
Asunto(s)
Enfermedad Coronaria/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Proteína Plasmática A Asociada al Embarazo/metabolismo , Adulto , Anciano , Análisis de Varianza , Anticuerpos/inmunología , Especificidad de Anticuerpos , Biomarcadores/sangre , Enfermedad Coronaria/diagnóstico , Creatina Quinasa/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Proteína Plasmática A Asociada al Embarazo/inmunología , Análisis de Regresión , Sensibilidad y Especificidad , Troponina T/sangreRESUMEN
A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.