Caracterización de la haptoglobina porcina mediante una aproximación Proteómica

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Biochemical and proteomic analyses of the physiological response induced by individual housing in gilts provide new potential stress markers

BACKGROUND: The objective assessment of animal stress and welfare requires proper laboratory biomarkers. In this work, we have analyzed the changes in serum composition in gilts after switching their housing, from pen to individual stalls, which is generally accepted to cause animal discomfort. RESULTS: Blood and saliva samples were collected a day before and up to four days after changing the housing system. Biochemical analyses showed adaptive changes in lipid and protein metabolism after the housing switch, whereas cortisol and muscular markers showed a large variability between animals. 2D-DIGE and iTRAQ proteomic approaches revealed variations in serum protein composition after changing housing and diet of gilts. Both techniques showed alterations in two main homeostatic mechanisms: the innate immune and redox systems. The acute phase proteins haptoglobin, apolipoprotein A-I and α1-antichymotrypsin 3, and the antioxidant enzyme peroxiredoxin 2 were found differentially expressed by 2D-DIGE. Other proteins related to the innate immune system, including lactotransferrin, protegrin 3 and galectin 1 were also identified by iTRAQ, as well as oxidative stress enzymes such as peroxiredoxin 2 and glutathione peroxidase 3. Proteomics also revealed the decrease of apolipoproteins, and the presence of intracellular proteins in serum, which may indicate physical injury to tissues. CONCLUSIONS: Housing of gilts in individual stalls and diet change increase lipid and protein catabolism, oxidative stress, activate the innate immune system and cause a certain degree of tissue damage. We propose that valuable assays for stress assessment in gilts may be based on a score composed by a combination of salivary cortisol, lipid metabolites, innate immunity and oxidative stress markers and intracellular proteins

Making sense of metabolomic data: comprehensive analysis of altered metabolic pathways in diabetes and obesity

Podeu consultar el III Workshop anual INSA-UB complet a: http://hdl.handle.net/2445/118993Sessió 1. Pòster núm.

Evaluation and comparison of bioinformatic tools for the enrichment analysis of metabolomics data

Background: Bioinformatic tools for the enrichment of 'omics' datasets facilitate interpretation and understanding of data. To date few are suitable for metabolomics datasets. The main objective of this work is to give a critical overview, for the first time, of the performance of these tools. To that aim, datasets from metabolomic repositories were selected and enriched data were created. Both types of data were analysed with these tools and outputs were thoroughly examined. Results: An exploratory multivariate analysis of the most used tools for the enrichment of metabolite sets, based on a non-metric multidimensional scaling (NMDS) of Jaccard's distances, was performed and mirrored their diversity. Codes (identifiers) of the metabolites of the datasets were searched in different metabolite databases (HMDB, KEGG, PubChem, ChEBI, BioCyc/HumanCyc, LipidMAPS, ChemSpider, METLIN and Recon2). The databases that presented more identifiers of the metabolites of the dataset were PubChem, followed by METLIN and ChEBI. However, these databases had duplicated entries and might present false positives. The performance of over-representation analysis (ORA) tools, including BioCyc/HumanCyc, ConsensusPathDB, IMPaLA, MBRole, MetaboAnalyst, Metabox, MetExplore, MPEA, PathVisio and Reactome and the mapping tool KEGGREST, was examined. Results were mostly consistent among tools and between real and enriched data despite the variability of the tools. Nevertheless, a few controversial results such as differences in the total number of metabolites were also found. Disease-based enrichment analyses were also assessed, but they were not found to be accurate probably due to the fact that metabolite disease sets are not up-to-date and the difficulty of predicting diseases from a list of metabolites. Conclusions: We have extensively reviewed the state-of-the-art of the available range of tools for metabolomic datasets, the completeness of metabolite databases, the performance of ORA methods and disease-based analyses. Despite the variability of the tools, they provided consistent results independent of their analytic approach. However, more work on the completeness of metabolite and pathway databases is required, which strongly affects the accuracy of enrichment analyses. Improvements will be translated into more accurate and global insights of the metabolome

Metabotypes of response to bariatric surgery independent of the magnitude of weight loss

Objective Bariatric surgery is considered the most efficient treatment for morbid obesity and its related diseases. However, its role as a metabolic modifier is not well understood. We aimed to determine biosignatures of response to bariatric surgery and elucidate short-term metabolic adaptations. Methods We used a LC- and FIA-ESI-MS/MS approach to quantify acylcarnitines, (lyso)phosphatidylcholines, sphingomyelins, amino acids, biogenic amines and hexoses in serum samples of subjects with morbid obesity (n = 39) before and 1, 3 and 6 months after bariatric surgery. K-means cluster analysis allowed to distinguish metabotypes of response to bariatric surgery. Results For the first time, global metabolic changes following bariatric surgery independent of the baseline health status of the subjects have been revealed. We identify two metabolic phenotypes (metabotypes) at the interval 6 months-baseline after surgery, which presented differences in the levels of compounds of urea metabolism, gluconeogenic precursors and (lyso)phospholipid particles. Clinically, metabotypes were different in terms of the degree of improvement in insulin resistance, cholesterol, low-density lipoproteins and uric acid independent of the magnitude of weight loss. Conclusions This study opens new perspectives and new hypotheses on the metabolic benefits of bariatric surgery and understanding of the biology of obesity and its associated diseases

Characterization of metabolomic profile associated with metabolic improvement after bariatric surgery in subjects with morbid obesity

The exact impact of bariatric surgery in metabolically 'healthy' (MH) or 'unhealthy' (MU) phenotypes for the study of the metabolic improvement is still unknown. We applied an untargeted LC-ESI-TripleTOF-MS-driven metabolomics approach in serum samples from 39 patients with morbid obesity (MH and MU) 1, 3, and 6 months after bariatric surgery. Multiple factor analysis, along with correlation and enrichment analyses, was carried out to distinguish those metabolites associated with metabolic improvement. Hydroxypropionic acids, medium-/long-chain hydroxy fatty acids, and bile acid glucuronides were the most discriminative biomarkers of response between MH and MU phenotypes. Hydroxypropionic (hydroxyphenyllactic-related) acids, amino acids, and glycerolipids were the most significant clusters of metabolites altered after bariatric surgery in MU ( p < 0.001). After surgery, MU and MH changed toward a common metabolic state 3 months after surgery. We observed a negative correlation with changes in waist circumference and cholesterol levels with metabolites of lipid metabolism. Glycemic variables were correlated with hexoses, which, in turn, correlated with gluconic acid and amino acid metabolism. Finally, we noted that hydroxyphenyllactic acid was associated with amino acid and lipid metabolism. Microbial metabolism of amino acid and BA glucuronidation pathways may be the key points of metabolic rearrangement after surgery

Biomarkers of Morbid Obesity and Prediabetes by Metabolomic Profiling of Human Discordant Phenotypes

Metabolomic studies aimed to dissect the connection between the development of type 2 diabetes and obesity are still scarce. In the present study, fasting serum from sixty-four adult individuals classified into four sex-matched groups by their BMI [non-obese versus morbid obese] and the increased risk of developing diabetes [prediabetic insulin resistant state versus non-prediabetic non-insulin resistant] was analyzed by LC- and FIA-ESI-MS/MS-driven metabolomic approaches. Altered levels of [lyso]glycerophospholipids was the most specific metabolic trait associated to morbid obesity, particularly lysophosphatidylcholines acylated with margaric, oleic and linoleic acids [lysoPC C17:0: R=-0.56, p=0.0003; lysoPC C18:1: R=-0.61, p=0.0001; lysoPC C18:2 R=-0.64, p<0.0001]. Several amino acids were biomarkers of risk of diabetes onset associated to obesity. For instance, glutamate significantly associated with fasting insulin [R=0.5, p=0.0019] and HOMA-IR [R=0.46, p=0.0072], while glycine showed negative associations [fasting insulin: R=-0.51, p=0.0017; HOMA-IR: R=-0.49, p=0.0033], and the branched chain amino acid valine associated to prediabetes and insulin resistance in a BMI-independent manner [fasting insulin: R=0.37, p=0.0479; HOMA-IR: R=0.37, p=0.0468]. Minority sphingolipids including specific [dihydro]ceramides and sphingomyelins also associated with the prediabetic insulin resistant state, hence deserving attention as potential targets for early diagnosis or therapeutic intervention

Untargeted profiling of concordant/discordant phenotypes of high insulin resistance and obesity to predict the risk of developing diabetes

This study explores the metabolic profiles of concordant/discordant phenotypes of high insulin resistance (IR) and obesity. Through untargeted metabolomics (LC-ESI-QTOF-MS), we analyzed the fasting serum of subjects with high IR and/or obesity ( n = 64). An partial least-squares discriminant analysis with orthogonal signal correction followed by univariate statistics and enrichment analysis allowed exploration of these metabolic profiles. A multivariate regression method (LASSO) was used for variable selection and a predictive biomarker model to identify subjects with high IR regardless of obesity was built. Adrenic acid and a dyglyceride (DG) were shared by high IR and obesity. Uric and margaric acids, 14 DGs, ketocholesterol, and hydroxycorticosterone were unique to high IR, while arachidonic, hydroxyeicosatetraenoic (HETE), palmitoleic, triHETE, and glycocholic acids, HETE lactone, leukotriene B4, and two glutamyl-peptides to obesity. DGs and adrenic acid differed in concordant/discordant phenotypes, thereby revealing protective mechanisms against high IR also in obesity. A biomarker model formed by DGs, uric and adrenic acids presented a high predictive power to identify subjects with high IR [AUC 80.1% (68.9-91.4)]. These findings could become relevant for diabetes risk detection and unveil new potential targets in therapeutic treatments of IR, diabetes, and obesity. An independent validated cohort is needed to confirm these results

Effect of sex and RYR1 gene mutation on the muscle proteomic profile and main physiological biomarkers in pigs at slaughter

Gender and RYR1 gene mutation might have an effect on the muscle metabolic characteristics and on the animal's stress at slaughter, which could influence the process of muscle-to-meat conversion. Forty-eight pigs were distributed in a design including two factors: sex (male/female) and RYR1 genotype (NN/Nn). At slaughter, physiological blood biomarkers and muscle proteome were analyzed and carcass and meat quality traits were registered. Females had higher serum levels of glucose, urea, C-reactive protein "CRP", Pig-MAP and glutation-peroxidase "GPx" and lower levels of lactate, showed faster muscle pH decline and higher meat exudation. RYR1 mutation increased serum creatinine, creatine kinase and CRP and decreased GPx. The proteomic study highlighted significant effects of gender and RYR1 genotype on proteins related to fibre composition, antioxidant defense and post mortem glycolytic pathway, which correlate to differences of meat quality. This study provides interesting information on muscle biomarkers of the ultimate meat quality that are modulated by the animal's individual susceptibility to stress at slaughter.info:eu-repo/semantics/acceptedVersio

Identificación de biomarcadores plasmáticos de bienestar en el ganado porcino mediante la aplicación de técnicas proteómicas

El benestar animal és un problema de gran importància en el món de la ramaderia actual a causa de la seva relació amb la seguretat i qualitat alimentària, amb implicacions legislatives i econòmiques. No obstant, els criteris actuals per avaluar el grau de benestar d’un animal encara estan lluny de ser suficientment objectius i específics. El descobriment de nous marcadors o d'un perfil de marcadors relacionats amb el benestar permetria millorar la prevenció i el tractament de les malalties, i optimitzar el maneig i la productivitat dels animals de granja. L'objectiu principal d'aquesta tesi ha estat la cerca de biomarcadors plasmàtics de benestar en tres situacions a les que estan exposats habitualment els porcs durant la seva etapa productiva a les granges d'explotació porcina: l'elevada densitat d'estabulació, l'allotjament en gàbies individuals amb fins reproductius (pràctica prohibida a Espanya des del gener de 2013) i la infecció pel circovirus porcí 2 (PCV2). A conseqüència de la innovació i el desenvolupament de les tecnologies -òmiques, que permeten fer un anàlisi massiu de les molècules presents en una cèl·lula, teixit o fluid en un moment determinat, la cerca de nous biomarcadors ha pres un fort impuls i s’ha convertit en un dels objectius de més interés per als investigadors. El suport bibliogràfic i l'anàlisi diferencial mitjançant 2-DE DIGE i iTRAQ del proteoma sanguini dels animals sotmesos a les dues primeres situacions de pobre benestar ha permès proposar un perfil de benestar porcí de laboratori basat en la quantificació de: 1) cortisol en sèrum i saliva; 2) paràmetres relacionats amb el metabolisme dels lípids (colesterol i les seves partícules transportadores, àcids grassos i triglicèrids); 3) indicadors de l'estat de salut de l'animal (marcadors musculars, hepàtics, renals i immunoglobulines); 4) proteïnes de fase aguda (haptoglobina, Pig-MAP i apolipoproteïna A-I) i altres components del sistema immune innat (pèptids antimicrobians); 5) marcadors d'estrès oxidatiu (glutatió peroxidasa, superòxid dismutasa, glutatió total, peroxiredoxina 2 i grups carbonil de les proteïnes oxidades), 6) proteïnes estructurals del citosquelet; i 7) proteïnes de xoc tèrmic. A més, s'han caracteritzat estructuralment les isoformes proteiques de l'haptoglobina i l'apolipoproteïna A-I mitjançant espectrometria de masses en tàndem. Per una banda, es van detectar cadenes glucídiques de tipus N- i O- a les quatres isoformes principals de la cadena β de l'haptoglobina, mentre que les tres isoformes de l'apolipoproteïna A-I estaven oxidades. Així mateix, es va observar un comportament diferent entre les tres isoformes de l'apolipoproteïna A-I en els porcs infectats pel virus PCV2: la isoforma que correspon a la preproteïna va augmentar i les altres dos van disminuir. En canvi, la freqüència relativa de les isoformes de la cadena β i α de l'haptoglobina no va variar. Per una altra banda, s'ha iniciat la posta a punt d'una tècnica quantitativa basada en espectrometria de masses, el SRM, per a la mesura d'algunes proteïnes de fase aguda porcines com ara l'haptoglobina, la Pig-MAP, l'apolipoproteïna A-I i la glicoproteïna α2-Heremans Schmid. Aquesta tecnologia s’està erigint com una bona alternativa a l’ús dels reactius comercials, que no solen presentar reactivitat contra les proteïnes d'origen porcí.El bienestar animal es un problema de gran importancia en el mundo de la ganadería actual debido a su relación con la seguridad y calidad alimentaria, con implicaciones legislativas y económicas. Sin embargo, los criterios actuales para evaluar el grado de bienestar aún están lejos de ser lo suficientemente objetivos y específicos. El descubrimiento de nuevos marcadores o de un perfil de marcadores relacionados con el bienestar permitiría mejorar la prevención y el tratamiento de las enfermedades, y optimizar el manejo y la productividad de los animales de granja. El objetivo principal de esta tesis ha sido la búsqueda de biomarcadores plasmáticos de bienestar en tres situaciones a las que están expuestos habitualmente los cerdos durante su etapa productiva en las granjas de explotación porcina: la elevada densidad de estabulación, el alojamiento en jaulas individuales con fines reproductivos (práctica prohibida en España desde enero del 2013) y la infección por el circovirus porcino 2 (PCV2). A consecuencia de la innovación y el desarrollo de las tecnologías –ómicas, que permiten realizar un análisis masivo de las moléculas presentes en una célula, tejido o fluido en un momento determinado, la búsqueda de nuevos biomarcadores ha tomado un fuerte impulso y se ha convertido en uno de los objetivos de mayor interés para los investigadores. El soporte bibliográfico y el análisis diferencial mediante 2-DE DIGE e iTRAQ del proteoma sanguíneo de los animales sometidos a las dos primeras situaciones de pobre bienestar ha permitido proponer un perfil de bienestar porcino de laboratorio basado en la cuantificación de: 1) cortisol en suero y saliva; 2) parámetros relacionados con el metabolismo de los lípidos (colesterol y sus partículas transportadoras, ácidos grasos y triglicéridos); 3) indicadores del estado de salud del animal (marcadores musculares, hepáticos, renales e inmunoglobulinas); 4) proteínas de fase aguda (haptoglobina, Pig-MAP y apolipoproteína A-I) y otros componentes del sistema inmune innato (péptidos antimicrobianos); 5) marcadores de estrés oxidativo (glutatión peroxidasa, superóxido dismutasa, glutatión total, peroxiredoxina 2 y grupos carbonilo de las proteínas oxidadas), 6) proteínas estructurales del citosqueleto; y 7) proteínas de choque térmico. Además, se han caracterizado estructuralmente las isoformas proteicas de la haptoglobina y la apolipoproteína A-I mediante espectrometría de masas en tándem. Por un lado, se detectaron cadenas glucídicas de tipo N- y O- en las cuatro isoformas principales de la cadena β de la haptoglobina, mientras que las tres isoformas de la apolipoproteína A-I estaban oxidadas. Asimismo, se observó un comportamiento diferente entre las tres isoformas de la apolipoproteína A-I en los cerdos infectados por el virus PCV2: la isoforma que corresponde a la preproteína aumentó y las otras dos disminuyeron. En cambio, la frecuencia relativa de las isoformas de la cadena β y α de la haptoglobina no variaron. Por otro lado, se ha iniciado la puesta a punto de una técnica cuantitativa basada en espectrometría de masas, el SRM, para la determinación de algunas proteínas de fase aguda porcinas como la haptoglobina, la Pig-MAP, la apolipoproteína A-I y la glicoproteína α2-Heremans Schmid. Esta tecnología se está erigiendo como una buena alternativa al uso de reactivos comerciales, que no suelen presentar reactividad contra las proteínas de origen porcino.Animal welfare is an issue of great importance due to its relationship with food safety and quality, and its legislative, economic, ethical and public opinion implications. However, the current criteria for assessing the degree of animal welfare are still far from being sufficiently objective and specific. The discovery of new markers or combination of markers associated to welfare would improve the prevention and treatment of diseases, and optimize the management and productivity of farm animals. The main aim of this thesis was the search for new serum biomarkers in three common situations in the pig farm which adversely affect the well-being of the animals: the high density housing, the accommodation in individual cages for breeding purposes (practice banned in Spain since January 2013) and the infection with porcine's circovirus 2 (PCV2). As a result of the innovation and development of the -omics technologies, which allow a massive analysis of the molecules present in a cell, tissue or fluid at a given time, the search for new biomarkers has taken a strong run and has become one of the most common goals among researchers. Bibliographical support and experimental analysis of differential serum proteome of animals subjected to these two first situations of poor welfare with 2-DE DIGE and iTRAQ has led to propose a profile of laboratory-based parameters based on 1) cortisol in serum and saliva; 2) lipid metabolism parameters (cholesterol and its transporting particles, fatty acids and triglycerides), 3) indicators of the health of the animal (muscle, liver and kidney markers and immunoglobulins), 4) acute phase proteins (haptoglobin, Pig-MAP and apolipoprotein AI) and other components of the innate immune system (antimicrobial peptides), 5) oxidative stress markers (glutathione peroxidase and total glutathione, superoxide dismutase, peroxiredoxin 2 and carbonyl groups of the oxidized proteins) 6) filamentous cytoskeletal proteins; and 7) heat shock proteins. Moreover, we characterized the haptoglobin and apolipoprotein A-I protein isoforms with tandem mass spectrometry. On one hand, we detected N- and O-glycan chains in the four main isoforms of the haptoglobin β chain, while the three isoforms of apolipoprotein A-I were oxidized. Furthermore, we observed a different behaviour between the three isoforms of apolipoprotein A-I in pigs infected with PCV2: the isoform corresponding to the preprotein increased and whereas the other two decreased. However, the relative frequency of the β and α chain haptoglobin isoforms did not change. On the other hand, we optimized a quantitative method based on mass spectrometry to measure several acute phase proteins as haptoglobin, Pig-MAP, apolipoprotein A-I and α2-Heremans Schmid glycoprotein. SRM is being set up as a good alternative to the use of commercial reagents, which many times have a poor reactivity against proteins of porcine origin