Low phosphorus increases hepatic lipid deposition, oxidative stress and inflammatory response via Acetyl-CoA carboxylase-dependent manner in zebrafish liver cells.

Acetyl-CoA carboxylase (ACC) plays a regulatory role in both fatty acid synthesis and oxidation, controlling the process of lipid deposition in the liver. Given that existing studies have shown a close relationship between low phosphorus (P) and hepatic lipid deposition, this study was conducted to investigate whether ACC plays a crucial role in this relationship. Zebrafish liver cell line (ZFL) was incubated under low P medium (LP, P concentration: 0.77 mg/L) or adequate P medium (AP, P concentration: 35 mg/L) for 240 h. The results showed that, compared with AP-treated cells, LP-treated cells displayed elevated lipid accumulation, and reduced fatty acid ß-oxidation, ATP content, and mitochondrial mass. Furthermore, transcriptomics analysis revealed that LP-treated cells significantly increased lipid synthesis (Acetyl-CoA carboxylases (acc), Stearyl coenzyme A dehydrogenase (scd)) but decreased fatty acid ß-oxidation (Carnitine palmitoyltransferase I (cptI)) and (AMP-activated protein kinase (ampk)) mRNA levels compared to AP-treated cells. The phosphorylation of AMPK and ACC, and the protein expression of CPTI were significantly decreased in LP-treated cells compared with those in AP-treated cells. After 240 h of LP treatment, PF-05175157 (an ACC inhibitor) was supplemented in the LP treatment for an additional 12 h. PF-05175157-treated cells showed higher phosphorylation of ACC, higher protein expression of CPTI, and lower protein expression of FASN, lower TG content, enhanced fatty acid ß-oxidation, increased ATP content, and mitochondrial mass compared with LP-treated cells. PF-05175157 also relieved the LP-induced oxidative stress and inflammatory response. Overall, these findings suggest that ACC is a promising target for treating LP-induced elevation of lipid deposition in ZFL, and can alleviate oxidative stress and inflammatory response.

Comparative analysis of vertebral transcriptome in Japanese seabass (Lateolabrax japonicus) fed diets with varying phosphorus/calcium levels.

Aquaculture jeopardizes the aquatic environment by discharge of the most dietary phosphorus (P) into the water. Reducing the dietary P level is a common approach for decreasing the P discharge but it may result in increased risk of P deficiency leading to vertebral deformities. However, the molecular mechanism of vertebral deformities is poorly understood. We assessed vertebral transcriptome and compared the genes associated with bone metabolism in Japanese seabass (Lateolabrax japonicus) fed three diets containing different P and Ca levels including: diet I (0.4% P, 0.3% Ca), diet II (0.8% P, 0.3% Ca) and diet III (0.8% P, 3% Ca). The results showed that P deficiency reduces the ossification of vertebrae and induces visible vertebral deformities. Moreover, 256 gens were up-regulated and 125 genes were down-regulated in fish fed P deficient diets. Furthermore, administration of the diet with adequate P and Ca excess (diet III) resulted in the significant enhancement in expression of 19 genes and reduced expression of 93 genes. Comparing group II with group III, expression of 109 genes was up-regulated and expression of 1369 genes was down-regulated. Gene ontology enrichment analysis revealed significant alterations in biological functions by P deficiency. In summary, these findings indicated that both dietary P shortage and Ca excess lead to reduced differentiation and proliferation of osteoblast and induce a higher activity of osteoclastogenesis, which could subsequently impair vertebral mineralization and cause skeletal deformities.

Chitooligosaccharide supplementation in low-fish meal diets for Pacific white shrimp (Litopenaeus vannamei): Effects on growth, innate immunity, gut histology, and immune-related genes expression.

This study evaluated the effects of supplementing chitooligosaccharide (COS) in low fish meal (FM) diets on growth, immune response, intestine and hepatopancrease histology, and expression of inflammatory and immune-related genes in Pacific white shrimp (Litopenaeus vannamei). A basal diet was formulated using FM and soybean meal (SM) as primary protein sources and considered as a high FM (HFM) diet, then a low FM (LFM) diet was prepared by substituting 50% of FM with SM and supplemented with 0, 0.3, 0.6, 0.9, 1.2 or 1.5 g COS kg-1 diet (LFM, COS3, COS6, COS9, COS12 and COS15 diets). Each diet was fed to quadruplicate groups of shrimp (0.9 g) to apparent satiation three times daily for eight weeks. At the end of the experiment no significant changes in growth and survival rate were observed among treatments (P > 0.05). FM replacement led to significant (P < 0.05) reduction of serum lysozyme activity and significant improvements were obtained by adding 0.3 or 0.6 g kg-1 COS to the LFM diet. A significant decrease in nitric oxide synthase activity was found in LFM group and no beneficial effects could be achieved by COS application. LFM group showed higher hepatopancrease superoxide dismutase and glutathione peroxidase activities than HFM group and further enhancements were obtained by COS application. Hepatopancrease total antioxidant capacity and alkaline phosphatase activity decreased in LFM group and COS supplementation improved their values. Expression of lysozyme, crustin, Pen3 and proPo genes were significantly up-regulated in hepatopancrease of groups received 0.3-0.9 g COS kg-1 diet. FM substitution enhanced the expression of HSP70 and inflammatory genes such as AIF and TNF in hepatopancrease and intestine, and COS administration at a moderate level down-regulated their expression level. Remarkable enhancement in intestinal fold height was obtained by inclusion of 0.3 or 0.6 g COS kg-1 diet compared to the group received LFM diet. Shrimps fed HFM and COS containing diets exhibited higher number of E-cells within their hepatopancrease tubules than the LFM group. The findings in this study clearly demonstrated that COS could enhance non-specific immune response and antioxidant activity, and ameliorate the negative impacts of high SM diets on gut and hepatopancrease health in pacific white shrimp. The optimum inclusion level of COS seems to be 0.3-0.6 g kg-1 of diet.

Effects of organic acids and essential oils blend on growth, gut microbiota, immune response and disease resistance of Pacific white shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus.

An 8-week feeding trial was undertaken to evaluate supplemental effects of AviPlus® (AP), a blend of organic acids [citric acid, 25%; sorbic acid, 16.7%] and essential oils [thymol, 1.7%; vanillin, 1.0%], on growth, gut microbiota, innate immunity and disease resistance of Pacific white shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus. A basal experimental diet was formulated and supplemented with 0, 0.3, 0.6, 0.9 and 1.2 g kg-1 AP to produce five test diets (Con, AP0.3, AP0.6, AP0.9 and AP1.2). Each diet was fed to triplicate groups of shrimp (0.2 ± 0.01 g, mean ± SE) to apparent satiation three times daily. Growth performance and survival rate were not significantly influenced by AP supplementation (P > 0.05). Significantly (P < 0.05) higher serum total protein was found in groups fed ≥ 0.6 g kg-1 AP compared to control. Serum alkaline phosphatase and phenoloxidase activities were significantly increased in AP0.9 and AP1.2 groups. Also, the group received AP0.6 diet showed significantly higher glutathione peroxidase activity than control. Expression of gut pro-inflammatory genes including TNF-α, LITAF and RAB6A were down-regulated by AP administration. Gut microbiota analysis showed the significant enhancement of the operational taxonomic unit (OTU) diversity and richness indices by AP application. AP supplementation led to increased abundance of Firmicutes and a reduction in abundance of Proteobacteria. Also, dietary inclusion of 1.2 g kg-1 AP led to a significant increase in the abundance of Lactobacillus in shrimp gut. The group offered AP0.3 diet showed significantly higher disease resistance than control group. Furthermore, AP application significantly enhanced relative expression of immune related genes including lysozyme, penaeidin and catalase at 48 h post challenge. In conclusion, these findings show that the tested organic acids and essential oils mixture beneficially affects intestinal microflora and improves immune response and disease resistance of L. vannamei.

De novo assembly and characterization of seabass Lateolabrax japonicus transcriptome and expression of hepatic genes following different dietary phosphorus/calcium levels.

Fish farming seriously influences the aquatic environment because most dietary phosphorus (P) is excreted in the effluent. To increase the P utilization in fish, molecular techniques should be explored given the remarkable development of these techniques. Thus, to identify the candidate genes related to P utilization and molecular alterations following administration of a P-deficient diet in seabass Lateolabrax japonicus, we assessed the de novo pituitary, gill, intestine, liver, kidney, scales and vertebra transcriptomes, and we compared the expression of hepatic genes with three diets varying in P and Ca levels: diet I (0.4% P, 0.3% Ca), diet II (0.8% P, 0.3% Ca), and diet III (0.8% P, 3% Ca). In total, we identified 99,392 unigenes, and 37,086 (37.31%) unigenes were annotated. The results showed that 48 unigenes were significantly (P<0.05) up-regulated, while 55 genes were significantly down-regulated in the liver of group I compared with group II. Offering the P-sufficient and high Ca diet, diet III significantly up-regulated 24 unigenes and down-regulated 46 genes in the liver. There were significant differences in the regulation of 8 unigenes (3 up-regulated and 5 down-regulated) between groups II and III. Gene ontology (GO) functional enrichment and KEGG pathway analysis of differently expressed genes were performed for each pair of groups. The GO analysis showed that a large number of biological processes were significantly altered between P-deficient and P-sufficient treatments (I vs II and I vs III). Comparing group I and group II, seven KEGG terms were enriched significantly: glycine, serine and threonine metabolism, one carbon pool by folate, arginine and proline metabolism, the biosynthesis of unsaturated fatty acids, fatty acid elongation, drug metabolism-cytochrome P450, and fatty acid metabolism. There was no significantly enriched KEGG pathway between groups II and III. In conclusion, our study revealed that a P-deficient diet could increase catabolism and decrease anabolism of protein, as highlighted by low protein efficiency in fish fed the P-deficient diet. Furthermore, P-deficiency could motivate the biosynthesis of fatty acids. However, the dietary Ca level had no significant effect on the growth and expression of hepatic genes in L. japonicus.

Berberine attenuates oxidative stress and hepatocytes apoptosis via protecting mitochondria in blunt snout bream Megalobrama amblycephala fed high-fat diets.

High-fat diets may have favorable effects on growth and cost, but high-fat diets often induce excessive fat deposition, resulting in liver damage. This study aimed to identify the hepatoprotective of a Chinese herb (berberine) for blunt snout bream (Megalobrama amblycephala). Fish were fed with a normal diet (LFD, 5 % fat), high-fat diet (HFD, 15 % fat) or berberine-supplemented diets (BSD, 15 % fat with berberine 50 or 100 mg/kg level) for 8 weeks. After the feeding, histology, oxidative status and mitochondrial function of liver were assessed. The results showed that HFD caused fat accumulation, oxidative stress and apoptosis in hepatocytes of fish. Hepatocytes in HFD group appeared to be hypertrophied, with larger liver cells diameter than these of LFD group. Berberine-supplemented diets could attenuate oxidative stress and hepatocytes apoptosis. HFD induced the decreasing mitochondrial complexes activities and bulk density and surface area density. Berberine improved function of mitochondrial respiratory chain via increasing the complex activities. Moreover, the histological results showed that berberine has the potential to repair mitochondrial ultrastructural damage and elevate the density in cells. In conclusion, our study demonstrated that berberine has attenuated liver damage induced by the high fat mainly via the protection for mitochondria.

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.