Identificación mediante proteómica de nuevas adipoquinas y mioquinas implicadas en la obesidad

La obesidad y la ausencia de ejercicio son los dos factores de riesgo más importantes para el desarrollo de enfermedades metabólicas como la diabetes tipo 2, hipertensión, ateroesclerosis o incluso varios tipos de cáncer. En los últimos años se ha avanzado en el estudio del tejido adiposo y el tejido muscular esquelético desde una perspectiva endocrinológica como tejidos implicados en la producción y secreción a la circulación de diversos factores con propiedades paracrinas/endocrinas capaces de regular la homeostasis energética del organismo. Estos factores reciben el nombre de adipoquinas y mioquinas respectivamente. A pesar de que cada vez se identifican más miembros de esta clase de proteínas, el conocimiento que existe sobre estos factores y sus efectos sobre el organismo todavía no ha aclarado la etiología de muchas de las enfermedades relacionadas con la obesidad o la ausencia de ejercicio. Así, la identificación de nuevas adipoquinas y mioquinas y la caracterización de las mismas es un paso de gran importancia para aclarar los mecanismos de regulación en los que se encuentran implicados. Los modelos animales han sido durante muchas décadas muy importantes en el estudio endocrino de diversas enfermedades metabólicas, dada su capacidad de emular diversas condiciones observadas en humanos como la obesidad o la anorexia; por otro lado, la proteómica se ha destacado en los últimos años como una herramienta fundamental para el estudio e identificación de un gran número de proteínas en una muestra biológica en un relativo corto espacio de tiempo. Por lo tanto, la combinación del uso de modelos animales y de herramientas proteómicas se definen de gran utilidad en el estudio endocrino de la secreción tanto del tejido adiposo como del tejido muscular esquelético. En esta tesis doctoral se han establecido una serie de modelos animales tanto fisiológicos como patológicos para su uso en la caracterización de proteínas secretadas por el tejido adiposo y muscular, se ha optimizado la técnica de obtención de secretomas de explantes de tejido adiposo, y desarrollado una nueva técnica para la obtención del secretoma de explantes de tejido muscular esquelético. De esta forma, se han identificado por proteómica nuevas adipoquinas en los depósitos más relevantes de tejido adiposo con potencial importancia en estudios endocrinos futuros como la fetuina A, caracterizada en este trabajo, que juega un papel importante en el desarrollo de enfermedades como la diabetes tipo II. En cuanto al tejido muscular esquelético, se han identificado nuevas mioquinas y se ha demostrado que existen diferencias en la secreción y expresión de estas proteínas según el tipo de fibras que presente el músculo. Finalmente se ha caracterizado en los modelos animales establecidos la nueva mioquina FNDC5/irisina, descubriendo por primera vez que esta proteína también es producida y secretada por el tejido adiposo jugando un papel importante sobretodo en condiciones de índice de masa corporal elevado

Aplicación de Proteómica cuantitativa y tecnología cHiPLC al estudio de secretomas

Comunicaciones a congreso

Obesidómica: caracterización del secretoma del tejido adiposo de diferentes localizaciones anatómicas

Comunicaciones a congreso

Nutrición y secreción gástrica: un nuevo enfoque proteómico

Comunicación a congreso

Caracterización por Proteómica del tejido muscular esquelético como órgano endocrino

Comunicaciones a congreso

Effect of excess body adiposity on the expression of genes involved in early steps of mammary carcinogenesis on diet-induced obese female rats

Introduction: Obesity is increasing worldwide and is associated with higher risk for some cancers. However, the mechanisms underlying this association are unclear. Because the obesity microenvironment could promote the onset of carcinogenesis, the aim of this study was to evaluate the association between excess body adiposity and the expression of genes related to the activation of early steps of tumor promotion on the mammary gland. Methods: Three weeks-old female Sprague-Dawley rats were fed a high fat diet (DIO: 60% Kcal/g fat, n = 14) or standard chow (LEAN: 3% Kcal/g fat, n = 15) for 10 weeks. Body weight and food intake were measured weekly. After sacrifice, retroperitoneal fat tissue was weighed and mammary tissue was extracted for qRT-PCR analysis. Genes associated with cell proliferation (Survivin/BIRC5 and MYC), DNA repair (TP53), and antioxidant protection (GSTM2, ALDH3A1) were quantified. Results: The DIO group showed a body weight 14.1% higher than LEAN group (p < 0.001). These differences were reflected on higher retroperitoneal fat content on DIO (3.22 ± 0.89g) vs. LEAN group (2.33 ± 0.52g; p = 0.012). Interestingly, DIO rats showed a higher gene expression for Survivin (∆68.2%), MYC (∆50.1%), TP53 (∆40.5%), ALDH3A1 (∆74.1%), and GSTM2 (∆25.7%) with respect to LEAN group. Conclusion: These data show that obesity is associated with changes potentially involved in early steps of tumor promotion, as shown by an increase in cellular proliferation and DNA damage related genes, even before detecting histological changes on the mammary tissue of obese female individuals. Further studies are needed to elucidate weather reducing body weight might be a therapeutic strategy to prevent this process

Rat adipose tissue secretome differential analysis from different anatomical locations

Comunicaciones a congreso

Obestatin as a regulator of adipocyte metabolism and adipogenesis

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3alpha/beta, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPalpha, C/EBPbeta, C/EBPdelta and PPARgamma promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome

CILAIR-Based Secretome Analysis of Obese Visceral and Subcutaneous Adipose Tissues Reveals Distinctive ECM Remodeling and Inflammation Mediators

In the context of obesity, strong evidences support a distinctive pathological contribution of adipose tissue depending on its anatomical site of accumulation. Therefore, subcutaneous adipose tissue (SAT) has been lately considered metabolically benign compared to visceral fat (VAT), whose location is associated to the risk of developing cardiovascular disease, insulin resistance, and other associated comorbidities. Under the above situation, the chronic local inflammation that characterizes obese adipose tissue, has acquired a major role on the pathogenesis of obesity. In this work, we have analyzed for the first time human obese VAT and SAT secretomes using an improved quantitative proteomic approach for the study of tissue secretomes, Comparison of Isotope-Labeled Amino acid Incorporation Rates (CILAIR). The use of double isotope-labeling-CILAIR approach to analyze VAT and SAT secretomes allowed the identification of location-specific secreted proteins and its differential secretion. Additionally to the very high percentage of identified proteins previously implicated in obesity or in its comorbidities, this approach was revealed as a useful tool for the study of the obese adipose tissue microenvironment including extracellular matrix (ECM) remodeling and inflammatory status. The results herein presented reinforce the fact that VAT and SAT depots have distinct features and contribute differentially to metabolic disease

Comparative secretome analysis of rat stomach under different nutritional status

The fact that gastric surgery is at the moment the most effective treatment to fight against obesity highlights the relevance of gastric derived proteins as potential targets to treat this pathology. Taking advantage of a previously established gastric explant model for endocrine studies, the proteomic analysis of gastric secretome was performed. To validate this gastric explant system for proteomic analysis, the identification of ghrelin, a classical gastric derived peptide, was performed by MS. In addition, the differential analysis of gastric secretomes under differential nutritional status (control feeding vs fasting vs re-feeding) was performed. The MS identified proteins are showed in the present manuscript. The data supplied in this article is related to the research article entitled "Comparative secretome analysis of rat stomach under different nutritional status" (L.L. Senin, A. Roca-Rivada, C. Castelao, J. Alonso, C. Folgueira, F.F. Casanueva, M. Pardo, L.M. Seoane Comparative secretome analysis of rat stomach under different nutritional status J. Proteomics (2015))