Comparative Study of the Molecular Characterization, Evolution, and Structure Modeling of Digestive Lipase Genes Reveals the Different Evolutionary Selection Between Mammals and Fishes.

Vertebrates need suitable lipases to digest lipids for the requirement of energy and essential nutrients; however, the main digestive lipase genes of fishes have certain controversies. In this study, two types of digestive lipase genes (pancreatic lipase (pl) and bile salt-activated lipase (bsal)) were identified in mammals and fishes. The neighborhood genes and key active sites of the two lipase genes were conserved in mammals and fishes. Three copies of PL genes were found in mammals, but only one copy of the pl gene was found in most of the fish species, and the pl gene was even completely absent in some fish species (e.g., zebrafish, medaka, and common carp). Additionally, the hydrophobic amino acid residues (Ile and Leu) which are important to pancreatic lipase activity were also absent in most of the fish species. The PL was the main digestive lipase gene in mammals, but the pl gene seemed not to be the main digestive lipase gene in fish due to the absence of the pl gene sequence and the important amino acid residues. In contrast, the bsal gene existed in all fish species, even two to five copies of bsal genes were found in most of the fishes, but only one copy of the BSAL gene was found in mammals. The amino acid residues of bile salt-binding sites and the three-dimensional (3D) structure modeling of Bsal proteins were conserved in most of the fish species, so bsal might be the main digestive lipase gene in fish. The phylogenetic analysis also indicated that pl or bsal showed an independent evolution between mammals and fishes. Therefore, we inferred that the evolutionary selection of the main digestive lipase genes diverged into two types between mammals and fishes. These findings will provide valuable evidence for the study of lipid digestion in fish.

Adaptation of AMPK-mTOR-signal pathways and lipid metabolism in response to low- and high-level rapeseed meal diet in Chinese perch (Siniperca chuatsi).

It is well known that carnivorous fish cannot use plant-proteins efficiently. They affect lipid metabolism of fish and cause serious problems to fish health. The reasons for this deficiency of fish metabolism are not known well. Chinese perch, a carnivorous fish, can accept artificial diet after domestication and is also considered as a novel model of fish for nutrition studies. Therefore, the aim of this study was to explore the effect of fish meal replacement by low- or high-rapeseed meal on lipid and glucose metabolism of Chinese perch. Three experimental diets were formulated with 0, 10%, and 30% rapeseed meal, named as Control, RSL, and RSH groups, respectively. After 8-weeks of the feeding trial, the inhibition of growth and fat deposition were observed in Chinese perch fed with rapeseed meal diets compared to the control group. Fish fed with RSL diets showed decreased food intake, serum low density lipoprotein (LDL), phosphorylated Grb10 (P < 0.05), inhibited fatty acid (FA) transport (lipoprotein lipase (LPL)), and glycerol synthesis (phosphoenol pyruvate carboxykinase (PEPCK)) in the liver. In addition, fish fed with RSL diets were also inhibited FA synthesis (fatty acid synthase (FAS), sterol regulatory element binding protein 1 (SREBP1), and Acetyl-CoA carboxylase (ACC1)), lipid uptake (hepatic lipase (HL)), ß-oxidation (carnitine palmitoyltransferase I (CPT1)), and glycerol synthesis (PEPCK) in the visceral adipose tissue. Fish fed with RSH diets showed phosphorylated AMPK, inhibited FA synthesis (SREBP1, ACC1, and FAS), while enhanced lipolysis (hormone-sensitive lipase (HSL)), and then reduced Acetyl-CoA pool. In turn, ß-oxidation (peroxisome proliferator-activated receptor-a (PPARα) and CPT1) was inhibited, while glycolysis (glucose-6-phosphatase (G6PD) and pyruvate carboxylase (PC)) were enhanced, consequently the lipid accumulation was decreased in the liver. Fish were also inhibited lipid uptake (LPL), that caused inhibiting of FA synthesis (SREBP1), ß-oxidation (CPT1), glycerol synthesis (PEPCK), and in turn improved lipolysis (HSL) in the visceral adipose tissue. Our study suggested that both RSL and RSH diets decreased lipid accumulation in Chinese perch; however, the mechanism of lipid metabolism was different. Fish accepted less diet in RSL group, which inhibited lipid metabolism in the liver and in the visceral adipose tissues, while fish in RSH group activated AMPK pathway, inhibited FA synthesis, and enhanced lipolysis, which reduced Acetyl-CoA pool in the liver. Subsequently, lipid uptake and metabolism were inhibited in the visceral adipose tissue of RSH fish.

Metabolic responses of Chinese perch (Siniperca chuatsi) to different levels of dietary carbohydrate.

There are great differences in metabolic responses to different levels of carbohydrate among different carnivorous fish species. To explore metabolic responses of Chinese perch to moderate and high level of dietary carbohydrates, three diets containing 7.3% (LC), 17.5% (MC), and 27.5% (HC) of carbohydrates were provided to Chinese perch for 56 days. The results showed that MC and HC groups exhibited an increase in weight gain (WG) and hepatic glycogen content, and a decrease in feed conversion efficiency, compared with the LC group. The MC and HC groups also showed the increase in mRNA levels of phosphofructokinase and citrate synthase related to the aerobic oxidation pathway, which might be responsible for the increase in WG. Moreover, compared with the LC group, the HC group exhibited high levels of plasma indices (glucose, pyruvic acid, lactic acid, total triglyceride, total cholesterol, and low-density lipoprotein) and liver lipid resulting from the increased mRNA levels of fatty acid synthesis-related genes (ATP citrate lyase, acetyl-CoA carboxylase α, and fatty acid synthase), low level of crude protein caused by inhibition of TOR pathway, and liver damage induced by low antioxidant capacity and infiltration of inflammatory cells, but the MC group did not. The above results indicated that 17.5% dietary carbohydrate might be utilized effectively in Chinese perch and part carbohydrates were converted into glycogen to maintain glucose homeostasis; 27.5% dietary carbohydrate could not be fully utilized. The 27.5% carbohydrate diet induced the up-regulation of aerobic oxidation, glycogen synthesis, and fat synthesis pathways which might not be sufficient to maintain glucose homeostasis.

Effects of High Carbohydrate Diet-Modulated Microbiota on Gut Health in Chinese Perch.

High carbohydrate diet-induced damage in gut is linked to changes in gut permeability and microbiota. However, the mechanisms of action are not clear, especially in non-mammals. We performed the gut microbiota profiling in Chinese perch fed with different content of starch diets (0, 10, and 20%) by 16S rRNA sequencing. The gut permeability, metabolites, histological analysis, and inflammatory infiltration were evaluated. We found that gut microbial diversity, beneficial bacteria quantity, and lactic acid content were higher in C10 group than in the other groups. The lower level of gut microbial diversity was observed in C20 group, and mycoplasma was the overwhelmingly dominant species, but the butyric acid-producing bacteria and butyric acid level were significantly reduced. The gut permeability in C20 group was also increased due to the decreased mRNA expression levels of tight junction proteins caused by the butyric acid deficiency and gut lipid droplets accumulation. Then a large amount of LPS penetrated into the plasma, resulting in inflammation. These results suggested that high carbohydrate diet-induced damage in gut could be attributed to the endotoxemia, permeability, and gut microbiota, especially the role of mycoplasma and butyric acid-producing bacteria. In addition, predictive functional profiling of microbial communities by PICRUSt showed that C10 group enriched pathway related to membrane transport and down-regulated the pathways related to energy, coenzyme factor and vitamin metabolism, while C20 group exhibited reversed results. These data showed that the high-carbohydrate diet reversed the beneficial changes in gut microbial metabolism resulted from the medium-carbohydrate diet, and further demonstrated that microbiota played a key role in the gut damage caused by the high-carbohydrate diet. Our findings provide a reference for the targeted regulation of gut microbiota to mitigate the damage caused by the increase in starch content in fish feed (cost saving).

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

Growth and Metabolic Response of Chinese Perch to Different Dietary Protein-to-Energy Ratios in Artificial Diets.

The effect of dietary nutrients on novel farm species has always garnered wide research and economic interest. Chinese perch, an economically important carnivorous fish, accepts an artificial diet after taming, so it is essential to evaluate and optimize the nutritional and metabolic demands of this species. However, little is known about the effect of an artificial diet on the growth and metabolism of Chinese perch. Therefore, the present study evaluated the growth and metabolic responses of Chinese perch to experimental diets with different dietary protein/energy (P/E) ratios. Five isoenergetic diets (18 kJ/g) with graded levels of P/E ratios of 30.58, 33.22, 35.90, 38.6, and 41.35 mg/kJ (named A, B, C, D, and E) were formulated. A total of 225 Chinese perch (64.89 ± 0.28 g) were divided into five groups (triplicate tanks for each group), distributed into 15 (350 L) fiberglass tanks, and fed twice a day at 4% of fish wet body weight with the respective P/E ratio diets for 10 weeks. Compared with the other groups, Chinese perch in Group C showed significantly improved growth performance, weight gain (WG), specific growth rate (SGR), viscerosomatic index (VSI), hepatosomatic index (HSI), intraperitoneal fat (IPF), feed utilization, feed intake (FI), feed conversion ratio (FCR), protein efficiency ratio (PER), protein retention efficiency (PRE), energy retention efficiency (ERE), and feed efficiency (FE) as well as whole-body, muscle, and liver composition. Chinese perch in Group A, on the other hand, had the lowest growth performance, feed utilization, and body composition compared with the other groups. The activities of nitrogen metabolism-related enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST) glutamate dehydrogenase (GDH), and adenosine 5'-monophosphate deaminase (AMPD)) as well as the mRNA expression of the GDH and AMPD genes were significantly lower than those in the other groups. Similarly, the expression of NPY and AgRp were significantly higher in Group C compared with the other groups. However, the gene expression of CART and POMC was not affected by the dietary P/E ratios. In Group A, the expression of mTOR, S6K, and 4EBP1 was significantly lower and that of AMPK, LKB1, and eEF2 was significantly higher when compared with the other groups. Biochemical analysis of blood showed that ALT, AST, total protein (TP), alkaline phosphatase (ALP), glucose (GLU), blood urea nitrogen (BUN), and triglyceride (TG) levels were also affected by the dietary P/E ratio. From our results, we concluded that Chinese perch growth performance and nutrient metabolism were significantly affected by the P/E ratio of the artificial diet. Second-order polynomial regression analysis revealed that Chinese perch growth performance was optimal at a P/E ratio of 37.98 in the artificial diet.