Molecular and endocrine mechanisms that regulate skeletal muscle growth in the fine flounder (Paralichthys adspersus)

Tesis (Doctor en Biotecnología)The aquaculture industry represents one of the largest and most important businesses for Chile's economy; however, the centralization and focus of this industry in cultivating is almost exclusively dedicated to salmonids and is bringing several negative consequences. This strongly suggests that diversification of the industry is a necessity. The fine flounder is one of the endemic fish that is a potential candidate to achieve this diversification; however, its farming potential is limited by its slow growth rate, with skeletal muscle being the main tissue responsible for this impairment. In this context, knowledge regarding the physiology and underlying mechanisms that control muscle growth and mass in this species could deliver a decisive advantage for optimizing farming protocols and improving growth rates in this species. The control of growth in vertebrates is modulated by the endocrine system via the GHIIGF-I axis and received nutrients; nevertheless, the molecular and endocrine mechanisms that modulate muscle mass in fish are not known, nor are the contributions of the local autocrine-paracrine actions of these molecules. Therefore, the following question arose: "Are the local andlor svstemic comuonents of the GHIIGF svstem, together with the nutritional status, responsible modulators of muscle mass and subsequent slow arowth in the fine flounder?" This question, and previously exposed antecedents, made it conceivable to formulate the following central hypothesis, which points out that: The nutritional status requlates muscle mass via the svstemic and local components of the GHIIGF svstem in the fine flounder (Paralichthvs ads~ersusla, nd these com Donents are res~onsiblefo r the slow arowth ~erformanceo f this s~ecies." The central hypothesis was tested by pursuing the following general aim: "Evaluate the dvnamics of al1 the comuonents of the GHIIGF-I svstem in the fine flounder in vivo under different nutritional statuses." In order to achieve the general aim, different methodological approaches were carried out. First, compensatory growth trials were implemented as a model with the purpose of manipulating growth in the fine flounder. Therefore, different nutritional conditions were tested, including feeding (basal), fasting, and refeeding periods. Growth performance was evaluated by measuring body weight, length, condition factor, and specific growth rates. Through radioimmunoassay (RIA), plasma GH and IGF-l levels were measured. Contents of the GHR and IGF-IR were evaluated by Western blot and qPCR. By using Western blotting, the GH activated signaling pathways (JAK21STAT5) were assessed in addition to the main signal transductions activated by IGF-1, the RafIMeklERK and the P13K/AktTTOR. Other components of the IGF system, such as the IGFBPs, IGF-IR, and IGF-II, were analyzed by qPCR. A second experimental approach that involved the blockage of the two previously mentioned main signaling pathways activated by IGF-I was carried out. In this approach, fish were subjected to 3 wk of fasting, and this was followed by 2 wk of refeeding with the administration of rapamicyn (TOR blockage) and PD98058 (MEK blockage). The whole IGF system was evaluated by qPCR and Western blot, moreover muscle cellularity was analyzed by histological analyzes and growth performance was assessed. In the present thesis, it was found that the fine flounder possess higher plasma GH levels than of IGF-1, a higher contents of truncated than full length GHR in the skeletal muscle, and an impairment in the JAK2lSTAT5 signaling pathway. All of this triggers the fine flounder to produce low levels of musclederived IGF-l. Moreover, these components are significantly affected by different nutritional statuses. Fish undergoing long-term fasting had an increased GH resistance, a state that is rapidly reversed when the fish are fed again, thus reducing the GH resistance in this tissue and promoting growth compensation. These changes during fasting and refeeding were concomitant with alterations in al1 of the molecules of the IGF system, including the IGFBPs, IGF-IR, and IGF-II. Altogether these results display the molecular and endocrine basis of slow growth in the fine flounder and elucidate the mechanisms modulating muscle mass atrophy and hypertrophy as regulated by nutrients in this species; therefore, validating the central hypothesis. These findings lay the foundation of knowledge for the molecular and endocrine mechanisms controlling muscle mass in the fine flounder and fish in general. The basic knowledge produced in this thesis shows an enormous potential for future applied research in the field of fish growth endocrinology. In particular, the present thesis shows key molecules involved in fish muscle growth, and manipulation of these molecules may be crucial towards improving growth performance in the fine flounder and other fish species