||The present study cloned full-length genes of peroxisome proliferators activated receptors (PPARs) of the cobia Rachycentron canadum and investigated their expressions in association with cobias’ body adiposity and lipid-metabolism related physiological parameters. In addition to gene cloning, several studies evaluating the roles of PPARs were carried out, including: a time-series study on cage-farmed cobias from week 5 to week 52 post-hatching, a study comparing fish groups with contrasting growth performance and a study elucidating the effects of dietary fatty acids.|
Three isotypes, PPAR α, PPARβ and PPARγ, that were cloned from cobia’s cDNA contained 2046 bp, 2702 bp and 1943 bp, respectively. Their open reading frames encode 476, 510 and 531 amino acids, respectively. The identity in amino acid sequences between the PPARs are 52% (between PPARα and PPARβ), 52% (between PPARα and PPARγ), and 44% (between PPARβ and PPARγ), respectively. RT-PCR and real-time PCR (qPCR) analyses showed that expression of PPARα mRNA predominated in red muscle, heart and liver, and at a lower level in the head kidney and dorsal muscle. PPARβ transcripts were particularly abundant in the heart, liver, brain, and pyloric caeca. In contrast, PPARγ mRNA was detected primarily in the adipose tissues, liver, and pyloric caeca.
In the time-series study, the PPARs expression was related to the body adiposity and lipid-metabolism related physiological parameters of the cobias that were raised for one year to approximately 4.5 Kg in a commercial cage-culture farm. Ten samplings were conducted on weeks 5, 7, 9, 14, 18, 23, 29, 34, 41, and 52 post-hatching. The cobias were raised in an outdoor nursery to 88 g before being transferred to an offshore cage on week 9. The adipocytes in the liver and ventral muscle showed a hypertrophic (increase in cell size) increase towards the end of the nursery phase. Their cell size decreased significantly after the cage transfer and was maintained afterwards a size spectrum dominated by small cells until week 34. The cobias grew rapidly after the offshore transfer and reached 330 g on week 14. They showed a concurrent increase in fat deposition in the liver and ventral muscle and a concurrent hyperplasia increase in density of adipocytes in the ventral and dorsal muscle. Adipocyte hypertrophy was obvious on week 41 and regressed afterwards. As the fish grew, serum phospholipids concentration increased significantly from approximately 380 to 750 mg/dL. Time-series pattern for the specific activity of two NADPH-generating enzymes, malic enzyme and glucose-6- phosphate dehydrogenase, were reciprocal and compensatory. The expression of liver PPARα mRNA was negatively correlated to fat deposition and adiposity. There was a significant increase in body lipid deposition and hepatic PPARγ expression as the fish grew. Hepatic PPARγ expression could be a sufficient parameter describing its expression in whole body. These results showed that PPARγ and PPARα played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia.
In the study comparing differential fish growth, two groups of cobias were selected based on their growth performance from a same batch of fish raised in a nursery. The large-size group that was regarded as superior grower was 54.1 cm in total length and 1,287 g in weight; while the small-size fish (inferior grower) was 36.8 cm and 386 g. Compared to large cobias, small cobias showed a similar hepatosomatic index and viscerasomatic index, but a significantly (p ＜ 0.05) smaller mesenteric fat index (MFI).The levels of crude lipid in the liver (35% vs. 26%) and the proportions of neutral lipids in lipid were higher in large cobias than in small cobias. Concentrations of serum phospholipids, free fatty acids and total protein of large cobias were significantly higher than those of small cobias. Adipocyte density of liver and ventral muscle was increased with increasing fish size. The PPARα mRNA expression in the liver of small cobias was significantly higher (p ＜ 0.05) than large cobias, ascribing to possible stress effect from their inferior growth. The growth superiority obviously affected PPARα mRNA expression and fat deposition in the liver. In general, the expression of liver PPARα mRNA was negatively correlated to body weight, body length, MFI, and serum NEFA, as well as lipid concentration, adiposity (adipocyte density and adipocyte size), G6PDH enzyme activity in the liver. The PPARγ mRNA expression in the liver was positively correlated to size of the adipocytes size.
The effects of dietary fatty acids on PPARs expression were evaluated in a 10-week growth trial, in which cobias with an initial weight of 80 g were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were fish oil (rich in EPA and DHA), perilla oil (C18:3n-3), safflower oil (C18:2n-6), olive oil (C18:1n-9) or palm oil (C16:0). Significant difference was detected in PPARs mRNA expression among dietary treatments and among tissues. In the liver, among the dietary treatments, significantly higher expression levels of PPARα mRNA were detected in perilla oil and olive oil group, PPARβ mRNA in palm oil group and PPARγ mRNA in fish oil group. Linear regression analysis showed that liver PPARα mRNA expression was positively (p ＜ 0.05) correlated with dietary C18:3n-3 levels and negatively with dietary C18:0 levels. Liver PPARβ mRNA expression was positively correlated to C16:0 or C18:0 levels in diets. The PPARγ expression was positively (p ＜ 0.001) correlated to dietary levels of C20:1n-9, C20:5n-3 and C22:6n-3.
In summary, the mRNA expression pattern of PPARs was tissue or organ-specific with the expression of PPARα occurred predominantly in the liver and PPARγ in the adipose tissues. The expressions of PPARs in the liver were more related to their physiological roles than in other tissues or organs studied in the present study. The expression of PPARα in the liver was shown correlated negatively to body fat deposition; and reciprocally, expression of PPARγ was positively correlated to fat deposition. PPARs mRNA expression was also associated with major dietary fatty acids. Increased dietary C18:0 levels down-regulated PPARα and up-regulated PPARβ. Up-regulation of PPARγ was significantly related to increased levels of highly (C>20) unsaturated fatty acid in diets. Dietary C16–C18 fatty acids on the other hand were more related to expressions of PPARα and PPARβ. These results suggest that fish oil could be partially replaced by plant oils as the lipid source in the diet of the cobia. In addition to highly unsaturated fatty acids, reduction in dietary C18:3n-3 and increase in C18:0 lead to increased fat deposition, implicating a possible strategy to modulate body lipid contents of the cobia through dietary manipulation.