Ding-Dong Zhang

Hepatic β-Oxidation and Regulation of Carnitine Palmitoyltransferase (CPT) I in Blunt Snout Bream Megalobrama amblycephala Fed a High Fat Diet

High-fat diets may promote growth, partly through their protein-sparing effects. However, high-fat diets often lead to excessive fat deposition, which may have a negative impact on fish such as poor growth and suppressive immune. Therefore, this study investigated the effects of a fat-rich diet on the mechanisms of fat deposition in the liver. Three-hundred blunt snout bream (Megalobrama amblycephala) juveniles (initial mass 18.00±0.05 g) were fed with one of two diets (5% or 15% fat) for 8 weeks. β-Oxidation capacity and regulation of rate-limiting enzymes were assessed. Large fat droplets were present in hepatocytes of fish fed the high-fat diet. This observation is thought to be largely owing to the reduced capacity for mitochondrial and peroxisomal β-oxidation in the livers of fish fed the high-fat diet, as well as the decreased activities of carnitine palmitoyltransferase (CPT) I and acyl-CoA oxidase (ACO), which are enzymes involved in fatty-acid metabolism. Study of CPT I kinetics showed that CPT I had a low affinity for its substrates and a low catalytic efficiency in fish fed the high-fat diet. Expression of both CPT I and ACO was significantly down-regulated in fish fed the high-fat diet. Moreover, the fatty-acid composition of the mitochondrial membrane varied between the two groups. In conclusion, the attenuated β-oxidation capacity observed in fish fed a high-fat diet is proposed to be owing to decreased activity and/or catalytic efficiency of the rate-limiting enzymes CPT I and ACO, via both genetic and non-genetic mechanisms.

The Effect of Exposure to a High-Fat Diet on MicroRNA Expression in the Liver of Blunt Snout Bream (Megalobrama amblycephala)

Blunt snout bream (Megalobrama amblycephala) are susceptible to hepatic steatosis when maintained in modern intensive culture systems. The aim of this study was to investigate the potential roles of microRNAs (miRNAs) in diet-induced hepatic steatosis in this species. MiRNAs, small non-coding RNAs that regulate gene expression at the posttranscriptional level, are involved in diverse biological processes, including lipid metabolism. Deep sequencing of hepatic small RNA libraries from blunt snout bream fed normal-fat and high-fat diets identified 202 (193 known and 9 novel) miRNAs, of which 12 were differentially expressed between the normal-fat and high-fat diet groups. Quantitative stem-loop reverse transcriptase-polymerase chain reaction analyses confirmed the upregulation of miR-30c and miR-30e-3p and the downregulation of miR-145 and miR-15a-5p in high-fat diet-fed fish. Bioinformatics tools were used to predict the targets of these verified miRNAs and to explore potential downstream gene ontology biological process categories and Kyoto Encyclopedia of Genes and Genomes pathways. Six putative lipid metabolism-related target genes (fetuin-B, Cyp7a1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 2, 3-oxoacid CoA transferase 1b, stearoyl-CoA desaturase, and fatty-acid synthase) were identified as having potential important roles in the development of diet-induced hepatic steatosis in blunt snout bream. The results presented here are a foundation for future studies of miRNA-controlled lipid metabolism regulatory networks in blunt snout bream.

Feeding frequency affects stress, innate immunity and disease resistance of juvenile blunt snout bream Megalobrama amblycephala.

This study aimed to evaluate the effects of feeding frequency on stress, innate immunity and disease resistance of juvenile blunt snout bream Megalobrama amblycephala (average weight: 9.92 ± 0.06 g). Fish were randomly assigned to one of six feeding frequencies (1, 2, 3, 4, 5 and 6 times/day) following the same ration size for 8 weeks. After the feeding trial, fish were challenged by Aeromonas hydrophila and cumulative mortality was recorded for the next 10 days. Daily gain index of fish fed 3-5 times/day was significantly higher than that of the other groups. High feeding frequencies induced significantly elevated plasma levels of both cortisol and lactate. Fish fed 3-4 times/day exhibited relatively low liver catalase and glutathione peroxidase activities as well as malondialdehyde contents, but obtained significantly higher reduced glutathione levels and post-challenged haemato-immunological parameters (include blood leukocyte and erythrocyte counts as well as plasma lysozyme, alternative complement, acid phosphatase and myeloperoxidase activities) compared with that of the other groups. After challenge, the lowest mortality was observed in fish fed 4 times/day. It was significantly lower than that of fish fed 1-3 times/day, but exhibited no statistical difference with that of the other groups. In conclusion, both low and high feeding frequencies could cause oxidative stress of juvenile M. amblycephala, as might consequently lead to the depressed immunity and reduced resistance to A. hydrophila infection. The optimal feeding frequency to enhance growth and boost immunity of this species at juvenile stage is 4 times/day.

Effects of dietary fructooligosaccharide levels and feeding modes on growth, immune responses, antioxidant capability and disease resistance of blunt snout bream (Megalobrama amblycephala)

This study aimed to determine the effects of fructooligosaccharide (FOS) levels and its feeding modes on growth, immune response, antioxidant capability and disease resistance of blunt snout bream (Megalobrama amblycephala). Fish (12.5 ± 0.5 g) were subjected to three FOS levels (0, 0.4% and 0.8%) and two feeding modes (supplementing FOS continuously and supplementing FOS two days interval 5 days) according to a 3 × 2 factorial design. At the end of 8-week feeding trial, fish were challenged by Aeromonas hydrophila with concentration of 1 × 10(5) CFU mL(-1) and mortality was recorded for the next 96 h. Fish fed 0.4% FOS continuously (D2) and fish fed the basal diet for 5 days followed by 0.8% FOS for 2 days (D5) showed admirable growth performance. The highest plasma lysozyme, acid phosphatase and myeloperoxidase activities as well as complement component 3, total protein and immunoglobulin M (IgM) levels were all observed in fish fed D5. They were significantly higher (P < 0.05) than those of the control group and/or fish fed 0.8% FOS continuously, but exhibited no statistical difference (P > 0.05) with that of fish fed D2. A similar trend was also observed in antioxidant capability as well as the expression of Leap-I and Leap-Ⅱ. Mortality showed an opposite trend with the immune response with the lowest rate observed in fish fed D5. The results indicated that diet supplementing FOS in appropriate levels and feeding modes could improve the growth, immune response and antioxidant capability of fish, as might consequently lead to enhanced disease resistance. It can be speculated that the basal diet for 5 days followed by 0.8% FOS for 2 days was most suitable for blunt snout bream.

Effects of light intensity on growth, immune responses, antioxidant capability and disease resistance of juvenile blunt snout bream Megalobrama amblycephala.

Light is necessary for many fish species to develop and grow normally since most fishes are visual feeders. However, too intense light may be stressful or even lethal. Thus, this study was conducted to evaluate the effect of light intensity on growth, immune response, antioxidant capability and disease resistance of juvenile blunt snout bream Megalobrama amblycephala. Fish (18.04 ± 0.22 g) randomly divided into 5 groups were exposed to a range of light intensities (100, 200, 400, 800 and 1600 lx) in cultures for 8 weeks. After the feeding trial, fish were challenged by Aeromonas hydrophila and cumulative mortality was recorded for the next 96 h. The results demonstrated that fish subjected to 400 lx showed the greatest weight gain (125.70 ± 5.29%). Plasma levels of glucose and lactate increased with light intensity rising from 100 lx to 1600 lx while the lowest plasma levels of cortisol was observed at 400 lx group. Post-challenged haemato-immunological parameters (including plasma lysozyme and alternative complement activities, as well as plasma nitric oxide level and globulin contents) improved with light intensity increasing from 100 lx to 400 lx, and then decreased with further increasing light intensity. However, antioxidant biomarkers such as liver catalase and malondialdehyde showed an opposite trend with immune response with the lowest values observed at 400 lx groups. The application of light intensity at 1600 lx significantly lowered liver glutathione activity to 76.78 ± 6.91 μmol g(-1). Within a range of light intensity from 100 to 400 lx, no differences were observed in liver total superoxide dismutase and glutathione peroxidase activities while they were significantly higher at 800 and 1600 lx. After challenge, the lowest mortality was observed in fish exposed to 400 lx. It was significantly lower than that of fish exposed to 100 and 1600 lx. The results of the present study indicated that high light intensities (more than 800 lx) not only produced poor growth, but also led to stress response, as might consequently result in the elevated liver oxidation rates and depress immunity of this species. Although no stress response was observed, fish subjected to low light intensities (lower than 400 lx) also showed oxidative stress, immunosuppression and reduced disease resistance. Taken together, the optimal light intensity to enhance growth and boost immunity of this species at juvenile stage is 400 lx.

Effects of Dietary Pantothenic Acid on Growth, Intestinal Function, Anti-Oxidative Status and Fatty Acids Synthesis of Juvenile Blunt Snout Bream Megalobrama amblycephala

Four groups of juvenile Megalobrama amblycephala were fed three times daily with six semi-purified diets containing 3.39 (PA unsupplied diet), 10.54, 19.28, 31.04, 48.38 and 59.72 mg kg-1 calcium D-pantothenate. The results showed that survival rate, final weight, specific growth rate, protein efficiency ratio and nitrogen retention efficiency all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 19.28 mg kg-1, whereas the opposite was true for feed conversion ratio. Whole-body crude protein increased as dietary PA levels increased, while the opposite pattern was found for the crude lipid content. Intestinal α-amylase, lipase, protease, Na+-K+-ATPase, alkaline phosphatase and gamma-glutamyl transferase activities were all elevated in fish fed PA-supplemented diets. Hepatic catalase activities improved with increases in dietary PA, while the opposite was true for malondialdehyde contents. The liver PA concentration and coenzyme A content rose significantly (P<0.01), up to 31.04 mg kg-1, with increasing dietary PA levels and then plateaued. The percentage of hepatic saturated fatty acids increased significantly (P<0.01) as dietary PA levels increased, while the percentages of monounsaturated fatty acids and polyunsaturated fatty acid (PUFA) decreased as dietary PA increased. Fish fed diets containing 19.28 and 31.04 mg kg-1 PA exhibited higher (P<0.01) docosahexaenoic acid and PUFA percentages in muscle than those fed with other diets. The expression of the gene encoding pantothenate kinase was significantly up-regulated (P<0.01) in fish fed PA-supplemented diets. Hepatic Acetyl-CoA carboxylase α, fatty acid synthetase, stearoyl regulatory element-binding protein 1 and X receptor α genes all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 31.04 mg kg-1. Based on broken-line regression analyses of weight gain, liver CoA concentrations and PA contents against dietary PA levels, the optimal dietary PA requirements of juvenile blunt snout bream were estimated to be 24.08 mg kg-1.

Identification and differential expression of hepatopancreas microRNAs in red swamp crayfish fed with emodin diet

Using high-throughput Illumina Solexa system, the differential miRNA expressions from hepatopancreas in red swamp crayfish (Procambarus clarkii) fed with diets containing 0 (control) and 75 mg emodin kg(-1) (trial) were identified, respectively. As a result, 13,335,928 raw reads from the control sample and 14,938,951 raw reads from the trial sample were obtained while 13,053,344 (98.77%) and 14,517,522 (98.34%) small RNA were identified, respectively. 106 mature miRNAs (belonging to 68 miRNA gene families) were identified. 35 miRNAs displayed significantly differential expressions between two libraries. Of these, comparing to the control library, 6 miRNAs were significantly up-regulated and 29 miRNAs were significantly down-regulated. Moreover, 5 novel miRNAs (2 from control sample, 3 from trial sample) and target genes were predicted. GO analysis suggested that these miRNAs might be involved in innate immune response, growth, metabolism, cellular process, biological regulation and stimulus response. Our knowledge from this study could contribute to a better understanding of the miRNAs roles in regulating innate immune response and the study of miRNA function in crayfish.

Molecular characterization of carnitine palmitoyltransferase IA in Megalobrama amblycephala and effects on its expression of feeding status and dietary lipid and berberine.

Carnitine palmitoyltransferase I (CPT I, EC 2.3.1.21) controls the main regulatory step of fatty acid oxidation, and hence studies of its molecular characterization are useful to understand lipid metabolism in cultured fish. Here, a full-length cDNA coding CPT I was cloned from liver of blunt snout bream Megalobrama amblycephala. This cDNA obtained covered 2499bp with an open reading frame of 2181bp encoding 726 amino acids. This CPT I mRNA predominantly expressed in heart and white muscle, while little in eye and spleen. The phylogenetic tree constructed on the basis of sequence alignments among several vertebrate species suggests that this blunt snout bream CPT I sequence belongs to the CPT IA family. In order to investigate the characterization of CPT IA mRNA expression, post-prandial experiment and feeding trial were conducted. The results showed that CPT IA mRNA expression was unchanged from 2 to 12h, and then significantly increased at 24h post-feeding in liver and heart. Berberine, an alkaloid, was identified as a promising lipid-lowering drug. In order to elucidate the effect of berberine on CPT I expression, fish were fed for 8 weeks with three diets (low-fat diet (LFD, 5% fat), high-fat diet (HFD, 15% fat), and berberine-supplemented diet (BSD, 15% fat). The results showed that HFD could decrease the expression of CPT IA and PPARα, while BSD increased those expressions.

Feeding rates affect stress and non-specific immune responses of juvenile blunt snout bream Megalobrama amblycephala subjected to hypoxia

This study aimed to investigate the effects of feeding rates on stress response, innate immunity and hypoxia resistance of juvenile blunt snout bream Megalobrama amblycephala. Fish were randomly assigned to one of six feeding rates (2, 3, 4, 5, 6 and 7% of body weight/day) for 60 days. Then, fish were subjected to hypoxic conditions and haemato-immunological parameters were analyzed pre- and post-challenge. Low feed ration resulted in decreased liver superoxide dismutase and catalase activities and reduced glutathione contents. Inadequate feeding also adversely affected the immune functions of fish, as was characterized by the relatively low haemato-immunological parameters (including alternative complement (ACH50), myeloperoxidase (MPO), plasma protein profiles and transferring) and high hypoxia-induced mortality. High feed ration did not lead to the improvement in antioxidant capability, immune responses and survival. In addition, plasma cortisol, glucose and transferrin levels as well as lysozyme activities all increased significantly after hypoxia challenge, whereas the opposite was true for plasma ACH50 and MPO activities as well as protein profiles in terms of hypoxia challenge. An interaction between feeding rate and hypoxia was also observed in plasma cortisol, glucose and protein profiles. In conclusion, a feeding rate of 4-5% of body weight/day is optimal to boost the innate immunity of juvenile blunt snout bream. Low ration resulted in decreased antioxidant capability, compromised immune functions and reduced hypoxia resistance, while over feeding did not benefit the health status.

Molecular characterization of AMP-activated protein kinase α2 from herbivorous fish Megalobrama amblycephala and responsiveness to glucose loading and dietary carbohydrate levels

This study aimed to characterize the full-length cDNA of AMPKα2 in Megalobrama amblycephala, and evaluate its potential role in glucose homeostasis and carbohydrate metabolism. The cDNA obtained covered 1942bp with an open reading frame of 1635bp encoding 545 amino acids. Multiple alignments and phylogenetic analysis revealed a high homology (91-100%) among most fish and higher vertebrates. This AMPKα2 mRNA predominantly expressed in muscle, liver and brain, while little in gill and intestine. Then, the AMPKα2 expressions were determined in the muscle, liver and brain of fish subjected to a glucose load (injected intraperitoneally with 0, 1.67 and 3.34g glucose per kg body weight) and after a 12-week feeding trial (fed two dietary carbohydrate levels: 30% and 43%), respectively. After the glucose load, plasma glycemia peaked at 1h in fish. Thereafter, it decreased significantly to the basal level at 8h. However, AMPKα2 expression in muscle, liver and brain all decreased significantly during the first 2h, then returned to the basal value at 24h. Unlikely, tissue AMPKα2 expression of fish receiving saline solution increased significantly during the whole sampling period. Additionally, high-carbohydrate diet enhanced its expression in liver and muscle, but not that in brain. These findings indicated that the AMPKα2 gene shared a high degree of conservation with that of the other vertebrates. Muscle, liver and brain AMPKα2 expressions were highly induced by glucose administration. Furthermore, high dietary carbohydrate modified its expressions in these tissues.