Paper de leptina i grelina sobre el teixit adipós i estudi de la proteolisi muscular en peixos = Role of leptin and ghrelin on adipose tissue and study of muscle proteolysis in fish

Abstract

[cat] Els estudis que composen aquesta tesi tenen com a objectiu optimitzar el creixement muscular i reduir l'acumulació de greix en dues espècies d'aqüicultura, l’orada i la truita irisada. Primerament, es van identificar i caracteritzar en orada, alguns membres clau dels diferents sistemes proteolítics cel•lulars relacionats amb la degradació muscular com les calpaïnes (capn), catepsines (CTS) i via ubiquitina-proteasoma (UbP). La seva transcripció gènica muscular es va estudiar en orades: en tres etapes de la vida (aleví, juvenil i adult), sota dos estats nutricionals (dejuni i realimentació) i en resposta a dietes amb diferent nivell de proteïna i carbohidrat. En segon lloc, en truita irisada es va investigar in vivo l'efecte de l'adipositat sobre la leptina en plasma, i in vitro, el paper de certes hormones i nutrients en l’adipogènesi i en el metabolisme lipídic dels adipòcits. Finalment, es va establir per primera vegada, un cultiu primari de pre-adipòcits a partir de teixit adipós visceral en l'orada, i es van analitzar els efectes d'algunes hormones sobre la proliferació i la diferenciació cel•lular. Aquests estudis han demostrat que: a) l'expressió de les calpaïnes és modulada per l'estat nutricional i la composició de la dieta en l'orada, i que l'expressió de diversos membres del sistema de les calpaïnes es correlacionen amb la textura muscular, el que indica el seu potencial ús com a marcadors moleculars de la qualitat del filet d’orada, b) les SaCTSs i els membres del sistema SaUbP són reguladors del recanvi de les proteïnes musculars, l'expressió dels quals canvia durant la vida i en relació amb l'estat nutricional dels peixos, c) els nivells circulants de leptina poden transmetre informació sobre l'estat energètic del cos, i la regulació de la secreció de leptina dels adipòcits és modulada per la història nutricional dels peixos i per la influència d’hormones i nutrients, d) la grelina sembla ser una potenciadora del metabolisme lipídic en el teixit adipós de truita irisada, mentre que la leptina sembla regular-lo negativament. Aquests estudis han ajudat a entendre millor la proteòlisi muscular així com l’adipogènesi i el metabolisme lipídic d'aquestes espècies d'aqüicultura per millorar la seva qualitat.

[eng] The studies included in this thesis aim to gain knowledge on skeletal muscle protein turnover and adipose tissue development and lipid metabolism regulation in two species of farmed fish to optimize its muscle growth and to reduce fat accumulation. To do this, we identified and characterized in skeletal muscle of gilthead sea bream (sa), some key members of the different cellular proteolytic systems: calpains (capn), cathepsins (CTS) and ubiquitin-proteasome pathway (UbP) related to muscle degradation. Then, muscle gene transcription was studied in gilthead sea bream at: three life stages (fingerlings, juveniles and adults), under two nutritional conditions (fasting and re-feeding) and in response to diets containing different levels of protein and carbohydrates. The most significant results were: a) the identified proteases showed the structural domains and the specific catalytic motifs to be functional as their orthologues in mammals, b) the reduction of the expression of saCTSs and saUbP members together with PCNA and MHC, indicated that muscle protein turnover is reduced with age; SaUb increased again in adults suggesting an increase in ubiquitination in this stage, c) fasting increased sacapns1b expression, whereas re-feeding decreased the expression of almost all proteases analyzed, indicating a negative regulation of proteolysis under anabolic conditions, d) the expression of sacapn1 and sacapns1a was modulated by diet composition, and correlated negatively with the texture of the fillet, pointing out to these genes as potential molecular markers of flesh quality in gilthead sea bream and, e) SaCTSDb showed higher expression in muscle and a distinct pattern of transcriptional regulation compared with SaCTSDa, supporting a more important role for SaCTSDb in muscle proteolysis. Furthermore, it was also investigated in vivo the effect of adiposity on plasma leptin, and in vitro, the role of certain hormones (leptin, ghrelin and insulin) and nutrients (leucine and EPA) on adipogenesis and lipid metabolism in rainbow trout. Finally, it was first established a primary culture of preadipocytes from visceral adipose tissue of gilthead sea bream and, the effects of some hormones (insulin, IGF -I and GH) on proliferation and differentiation were analyzed. The most relevant findings were: a) nutritional restriction decreased adipose tissue and liver weights but increased plasma leptin levels, which were negatively correlated, suggesting that this hormone can act as a nutritional signal regulating appetite, b) adipocytes and preadipocytes express and secrete leptin, contributing to the levels of plasma leptin; insulin and ghrelin increased leptin secretion in a dose-dependent manner in adipocytes of rainbow trout fed with a regular diet, c) rainbow trout adipocytes of fish nutritionally restricted secreted more leptin than those of fish fed to satiety. Besides, only adipocytes from feed restricted fish respond to leucine, a signal that could be interpreted as greater food availability, reducing the secretion of leptin, thus contributing to the stimulation of appetite, d) ghrelin activated lipid metabolism at different levels, increasing lipogenesis and oxidation as well as lipolysis. On the other hand, leptin was anti-lipogenic, as it increased lipolysis and inhibited the expression of a fatty acid transporter, e) leptin and ghrelin did not influence in vitro rainbow trout adipogenesis; however, ghrelin and its receptor were identified in mature adipocytes, indicating other possible endocrine or local effects of this hormone in this species, f) insulin, IGF-I and GH stimulated proliferation in preadipocytes of gilthead sea bream and, the presence of an adipogenic medium supplemented with lipids, as well as IGF -I stimulated differentiation. These studies have helped to better understand muscle proteolysis as well as adipogenesis and lipid metabolism in these aquaculture species to improve its quality