Frontiers in Marine Science (Jun 2014)
Metabolic regulation in meagre, <i>Argyrosomus regius </i>(Asso, 1801): Study of gene-diet interactions on lipid metabolism
Abstract
Fish oil is the most important source of n-3 highly unsaturated fatty acids (HUFAs) for humans. With the stagnation of world marine fisheries, the role of aquaculture stocks increased rapidly as source of n-3 HUFA (FAO, 2012), but not sufficiently. To reduce the dependence as well as find ways to use of marine sources more economically and efficiently, the use of vegetable oils (VO) have been widely investigated (Estévez et al., 2011). Vegetable oils are rich in C18 PUFA, but devoided of the docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), essential fatty acids involved in maintaining cell membrane structure, among other essential functions. The capacity of fish to thrive on diets containing only the C18 PUFA, 18 : 2n − 6 and 18 : 3n – 3 varies among species (Tocher, 2003; Leaver et al., 2008; Castro et al., 2012). It is well documented that teleosts have different enzyme capacity to desaturate-elongate 18C fatty acids into 20-22C LC-PUFAs (Cook & McMaster, 2004; Leaver et al., 2008). Generally, freshwater fish species can convert 18:2 n−6 and 18:3 n−3 to HUFA more efficiently than marine species, especially carnivorous which prey on organisms rich in HUFA (Tocher, 2003). Thus, the modulation of expression and translation of elongases and desaturases is of great importance to achieve independence of (carnivorous) marine fish aquaculture from fishmeal and fish oil. There is currently considerable interest in the HUFA biosynthetic pathway in fish aiming to determine the effectiveness of vegetable oils as a total replacement of fish oil in the aquaculture of carnivore fish species (Vagner & Santigosa, 2011). Selenium is a structural component for several enzymes, including glutathione peroxidase and thioredoxine (Perottoni et al., 2004). These enzymes have physiological antioxidant properties and thus, protect the tissues of lipid peroxidation products (Orun et al., 2008). The current study aimed to evaluate the effects of dietary lipid profile on fads2 (fatty acyl desaturase gene) and elovl5 (fatty acyl elongase gene) expression in liver and brain of meagre (Argyrosomus regius). The four isoproteic and isolipidic diets (50% protein and 12% lipid, dry matter basis) were formulated with fish oil (FO) or a blend of vegetable oils (VO, rapeseed, linseed and soybean), each with selenium (S, 1mg/kg diet) or without selenium (NS). Fish were fed ad libitum for 60 days under a controlled rearing conditions (temperature = 20,7 ± 0,7 ºC; pH = 8; O2 = > 6 ppm). Ribonucleic acid (RNA) was extracted from two tissues: liver and brain using RNAspin Mini RNAIsolation Kit (GE Healthcare), with includes a step with DNase I to remove the presence of DNA. The integrity of the extracted RNA was verified by agarose electrophoresis. The RNA concentrations were obtained by nanodrop. From the total RNA, 0.5µg were transcribed to complementary DNA (cDNA) using iScript Reverse Transcription Supermix for RT-PCR (BioRad) following the manufacturer protocol. Gene expression from fads2 and elovl5 were quantified by analysis of RT-PCR. The β-actine was used as a reference gene. On VO diet hepatic fads2 expression was significantly higher, but not elovl5 expression. In brain of VO fed fish, fads2 and elovl5 expression was not significantly different when compared with FO fed fish (Table 1). With selenium supplementation hepatic fads2 expression was lower in FO (P 0.05). The same trend was evident for hepatic elovl5 expression (P> 0.05) (Table1). Recently, Monroig et al. (2013) described the first functional characterization of meagre Fads2 and Elovl5. This work concluded that, unlike most teleosts, the Fads2 pocesses ∆6 and ∆8 activity. However, conversion rates of meagre Fads2 were low when compared to Salmo salar ∆6 Fads_c (Monroig et al., 2011). On the other hand, meagre Elovl5 showed high activity towards C18 and C20. Thus, ∆6/∆8 activity might be ineffective in converting C18 PUFA and therefore an increase of dietary C18 PUFA may stimulates the expression of the fads2. In contrast, Elovl5 is very efficient to convert the desaturated products of ∆6/∆8 and so Elovl5 expression is not enhanced, since the activity of this enzyme is already higher. These findings may explain the differences in the expression of two genes, between hepatic fads2 and elovl5. Furthermore, increased hepatic fads2 expression between FO-S and VO-S treatments was 114 fold. Such induction was much greater than observed in Salmo salad (Zheng et al., 2005). However, Salmo salad has separate and distinct genes for ∆6 and ∆5 desaturases (Zheng et al., 2005) and, due to genome duplication, it has two genes with ∆6 activity (Monroig et al., 2010). Moreover, lipid peroxidation in liver increases with the number of fatty acid (FA) double bonds (Haggag, Elsanhoty & Ramadan, 2014). D'Aquino et al. (1991) observed that rats fed diets with fish oil had increased lipid peroxidation. Our results indicate that, in FO-S, selenium may have protected FA from peroxidation, thus dietary HUFA seemed to have been sufficient to maintain the phospholipid turnover and induction of FA metabolism genes did not occur. In FO-NS diet membranes were not protected efficiently from lipid peroxidation, and therefore a higher expression of FA metabolism genes was necessary to offset the damage, consequently, biosynthesis of HUFA was more stimulated. ROS-induced oxidative stress has been associated with expression and protein levels of transcription factors (Okuno et al., 2012). A reduction of ROS (Reactive Oxygen Species) has been observed in fish fed VO with selenium, when compared to VO without selenium (data not presented). It is plausible to infer that a stimulation of expression and level of protein SREBP-1 by a reduction of ROS. SREBP-1 play a role on the regulation of genes involved in biosynthesis of HUFA, as fads2 and elovl5 (Jump, Tripathy & Depner, 2013). In conclusion, our results showed that vegetable oils have an effect on expression level of genes involved in HUFAs biosynthesis in meagre, mainly fads2, which seems to be the rate limiting enzyme in this pathway. In addition, dietary selenium seems to favor the expression of genes involved in the biosynthesis of HUFAs, when meagre is fed on VO-based diet.
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