The circadian rhythm regulates branched-chain amino acids metabolism in fast muscle of Chinese perch (Siniperca chuatsi) during short-term fasting by Clock-KLF15-Bcat2 pathway.

As an internal time-keeping mechanism, circadian rhythm plays crucial role in maintaining homoeostasis when in response to nutrition change; meanwhile, branched-chain amino acids (BCAA) in skeletal muscle play an important role in preserving energy homoeostasis during fasting. Previous results from our laboratory suggested that fasting can influence peripheral circadian rhythm and BCAA metabolism in fish, but the relationship between circadian rhythm and BCAA metabolism, and whether circadian rhythm regulates BCAA metabolism to maintain physiological homoeostasis during fasting remains unclear. This study shows that the expression of fifteen core clock genes as well as KLF15 and Bcat2 is highly responsive to short-term fasting in fast muscle of Siniperca chuatsi, and the correlation coefficient between Clock and KLF15 expression is enhanced after fasting treatment. Furthermore, we demonstrate that the transcriptional expression of KLF15 is regulated by Clock, and the transcriptional expression of Bcat2 is regulated by KLF15 by using dual-luciferase reporter gene assay and Vivo-morpholinos-mediated gene knockdown technique. Therefore, fasting imposes a dynamic coordination of transcription between the circadian rhythm and BCAA metabolic pathways. The findings highlight the interaction between circadian rhythm and BCAA metabolism and suggest that fasting induces a switch in KLF15 expression through affecting the rhythmic expression of Clock, and then KLF15 promotes the transcription of Bcat2 to enhance the metabolism of BCAA, thus maintaining energy homoeostasis and providing energy for skeletal muscle as well as other tissues.

Methionine-Mediated Regulation of Intestinal Lipid Transportation Induced by High-Fat Diet in Rice Field Eel (Monopterus Albus).

An eight-week feeding trial explored the mechanism that supplemented methionine (0 g/kg, 4 g/kg, 8 g/kg, and 12 g/kg) in a high-fat diet (120 g/kg fat) on intestinal lipid transportation and gut microbiota of M. Albus (initial weight 25.03 ± 0.13 g) based on the diet (60 g/kg fat), named as Con, HFD+M0, HFD+M4, HFD+M8, and HFD+M12, respectively. Compared with Con, gastric amylase, lipase, trypsin (P < 0.05), and intestinal lipase, amylase, trypsin, Na+/K+ -Adenosinetriphosphatase, depth of gastric fovea, and the number of intestinal villus goblet cells of HFD+M0 were markedly declined (P < 0.05), while intestinal high-density lipoprotein-cholesterol, very low-density lipoprotein-cholesterol and microsomal triglyceride transfer protein of HFD+M0 were markedly enhanced (P < 0.05); compared with HFD+M0, gastric lipase, amylase, trypsin, and intestinal lipase, trypsin, Na+/K+ -Adenosinetriphosphatase, microsomal triglyceride transfer protein, very low-density lipoprotein-cholesterol, and apolipoprotein -A, the height of intestinal villus and the number of intestinal villus goblet cells of HFD+M8 were remarkably enhanced (P < 0.05). Compared with Con, intestinal occ, cl12, cl15, zo-1, zo-2 of HFD + M0 were markedly down-regulated (P <0.05), while intestinal vldlr, npc1l1, cd36, fatp1, fatp2, fatp6, fatp7, apo, apoa, apob, apof, apoo, mct1, mct2, mct4, mct7, mct12, lpl, mttp, moat2, dgat2 of HFD M0 were remarkably upregulated (P < 0.05); compared with HFD+M0, intestinal gcn2 and eif2α of HFD+M8 were remarkably downregulated (P < 0.05), intestinal occ, cl12, cl15, zo-1, zo-2, hdlbp, ldlrap, vldlr, cd36, fatp1, fatp2, fatp6, apo, apoa, apob, apof, apoo, mct1, mct2, mct8, mct12, lpl, mttp, moat2, and dgat2 were remarkably upregulated (P < 0.05). Compared with Con, the diversity of gut microbiota of HFD+M0 was significantly declined (P < 0.05), while the diversity of gut microbiota in HFD+M8 was significantly higher than that in HFD+M0 (P < 0.05). In conclusion, a high-fat methionine deficiency diet destroyed the intestinal barrier, reduced the capacity of intestinal digestion and absorption, and disrupted the balance of gut microbiota; supplemented methionine promoted the digestion and absorption of lipids, and also improved the balance of gut microbiota.

Effect of starvation and refeeding on reactive oxygen species, autophagy and oxidative stress in Chinese perch (Siniperca chuatsi) muscle growth.

In skeletal muscle, autophagy regulates the development and growth of muscle fibres and maintains the normal muscle metabolism. Under starvation and refeeding conditions, the effect of reactive oxygen species (ROS) levels on skeletal muscle autophagy is still unclear, although the excessive accumulation of ROS has been shown to increase autophagy in cells. The purpose of this study was to explore the effects of starvation and diet after starvation on the autophagy of adult Chinese perch muscle, and to determine the level of ROS in the muscle. We performed zero (Normal control), three and seven starvation treatments on adult Chinese perch, and returned to normal feeding for 3 days after starvation for 7 days. In the muscles of the adult Chinese perch muscle after 3 days of starvation, the autophagy marker protein LC3 and the number of autophagosomes remained basically the same as in the normal feeding situation. However, on starvation for 7 days, the mitochondrial autophagy was sensitive and the number of autophagosomes increased, but the antioxidant-related molecules (malondialdehyde, catalase, glutathione S-transferase, glutathione and anti-superoxide anion) decreased and the accumulation of ROS was obvious. In addition, the extended starvation time also increased the level of LC3 protein. However, by refeeding after starvation this nutritional stress resulted in a decrease in ROS levels and a partial restoration of antioxidant enzyme activity. Our data show that in the adult Chinese perch muscle, starvation could reduce the antioxidant activity through the accumulation of ROS, and that the number of autophagosomes continues to increase. Refeeding after starvation could effectively compensate for the level of ROS, and restore the mRNA abundance of antioxidant genes and the activity of antioxidant enzymes to reduce autophagy and improve feed efficiency. Further research should optimize starvation conditions to reduce autophagy in muscles and maintain normal muscle metabolism.

The miRNA expression profile directly reflects the energy metabolic differences between slow and fast muscle with nutritional regulation of the Chinese perch (Siniperca chuatsi).

Fish skeletal muscles are composed of two distinct types, slow and fast muscles, and they play important roles in maintaining the body's movement and energy metabolism. The two types of muscle are easy to separate, so they are often used as the model system for studies on their physiological and functional characteristics. In this study, we revealed that the carbohydrate and lipid metabolic KEGG pathways are different between slow and fast muscles of Chinese perch with transcriptome analysis. In fast muscle, glucose metabolism was catabolic with higher glycolysis capacity, while in slow muscle, glucose metabolism was anabolic with more glycogen synthesis. In addition, oxidative metabolism in slow muscle was stronger than that in fast muscle. By analyzing the expression levels of 40 miRNAs involved in metabolism in the muscles of Chinese perch, 18 miRNAs were significantly upregulated and 7 were significantly downregulated in slow muscle compared with fast muscle. Based on functional enrichment analysis of their target genes, the differential expression levels of 17 miRNAs in slow and fast muscles were reflected in their carbohydrate and lipid metabolism. Among these, 15 miRNAs were associated with carbohydrate metabolism, and 6 miRNAs were associated with lipid metabolism. After 3 days of starvation, the expression levels of 15 miRNAs involved in glucose metabolism in fast and slow muscles increased. However, after 7 days of starvation, the mRNA levels of miR-22a, miR-23a, miR-133a-3p, miR-139, miR-143, miR-144, miR-181a and miR-206 decreased to basal levels. Our data suggest that the possible reason for the difference in glucose and lipid metabolism is that more miRNAs inhibit the expression of target genes in slow muscle.

A Study on How Methionine Restriction Decreases the Body's Hepatic and Lipid Deposition in Rice Field Eel (Monopterus albus).

Methionine restriction reduces animal lipid deposition. However, the molecular mechanism underlying how the body reacts to the condition and regulates lipid metabolism remains unknown. In this study, a feeding trial was performed on rice field eel Monopterus albus with six isonitrogenous and isoenergetic feeds that included different levels of methionine (0, 2, 4, 6, 8, and 10 g/kg). Compared with M0 (0 g/kg), the crude lipid and crude protein of M. albus increased markedly in M8 (8 g/kg) (p < 0.05), serum (total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and non-esterified free fatty acids), and hepatic contents (hepatic lipase, apolipoprotein-A, fatty acid synthetase, total cholesterol, triglyceride, and lipoprteinlipase). However, in the serum, very-low-density lipoprotein and hepatic contents (hormone-sensitive triglyceride lipase, Acetyl CoA carboxylase, carnitine palmitoyltransterase, and mirosomal triglygeride transfer protein) decreased markedly in M8 (p < 0.05). The contents of hepatic C18:2n-6, C22:6n-3, and n-3PUFA in the M8 group were significantly higher than those in M0 (p < 0.05), and the contents of lipid droplets in M8 were higher than those in M0. Compared with M0, the hepatic gcn2, eif2α, hsl, mttp, ldlrap, pparα, cpt1, and cpt2 were remarkably downregulated in M8, while srebf2, lpl, moat2, dgat2, hdlbp, srebf1, fas, fads2, me1, pfae, and icdh were markedly upregulated in M8. Moreover, hepatic SREBP1 and FAS protein expression were upregulated significantly in M8 (p < 0.01). In short, methionine restriction decreased the lipid deposition of M. albus, especially for hepatic lipid deposition, and mainly downregulated hepatic fatty acid metabolism. Besides, gcn2 could be activated under methionine restriction.

Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Two smyd1 paralogues, smyd1a and smyd1b, have been identified in zebrafish. Although Smyd1b function has been reported in fast muscle, its function in slow muscle and the function of Smyd1a, in general, are uncertain. In this study, we generated 2 smyd1a mutant alleles and analyzed the muscle defects in smyd1a and smyd1b single and double mutants in zebrafish. We demonstrated that knockout of smyd1a alone had no visible effect on muscle development and fish survival. This was in contrast to the smyd1b mutant, which exhibited skeletal and cardiac muscle defects, leading to early embryonic lethality. The smyd1a and smyd1b double mutants, however, showed a stronger muscle defect compared with smyd1a or smyd1b mutation alone, namely, the complete disruption of sarcomere organization in slow and fast muscles. Immunostaining revealed that smyd1a; smyd1b double mutations had no effect on myosin gene expression but resulted in a dramatic reduction of myosin protein levels in muscle cells of zebrafish embryos. This was accompanied by the up-regulation of hsp40 and hsp90-α1 gene expression. Together, our studies indicate that both Smyd1a and Smyd1b partake in slow and fast muscle development although Smyd1b plays a dominant role compared with Smyd1a.-Cai, M., Han, L., Liu, L., He, F., Chu, W., Zhang, J., Tian, Z., Du, S. Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Exposure to waterborne cadmium induce oxidative stress, autophagy and mitochondrial dysfunction in the liver of Procypris merus.

The present study was performed to determine the effect of waterborne cadmium (Cd) exposure on oxidative stress, autophagy and mitochondrial dysfunction, and to explore the mechanism of Cd-induced liver damage in freshwater teleost Procypris merus. To this end, P. merus were exposed to waterborne 0, 0.25 and 0.5 mg/L Cd for 30 days (equal to 0, 2.22 and 4.45 µmol Cd/l). The waterborne Cd exposure significantly increased hepatic Cd accumulation and impaired histological structure of the liver of P. merus. both low and high-dose waterborne Cd exposure induced oxidative stress in the liver of P. merus, through increases Malondialdehyde (MDA) and reactive oxide species (ROS) accumulation in the liver. The Cd-induced oxidative stress in liver may result from reduction of enzyme activities (superoxide dismutases (SOD), catalases (CAT), GSH-S-transferases (GST)) and transcriptional expression of antioxidant related genes (gpx1, gpx2, cata, gsta1, sod1). Furthermore, the present study showed that waterborne Cd exposure decreased the transcriptional factor (nrf2) expression, which might lead to the down-regulation of antioxidant gene expression. Transmission electron microscopy (TEM) observations demonstrated that waterborne Cd exposure induced autophagy in the liver of P. merus. Gene expression analysis showed that waterborne Cd exposure also induced mRNA expression of a set of genes (beclin1, ulk1, atg5, lc3a, atg4b, atg9a, and p62) involved in the autophagy process, indicating that the influence of Cd on autophagy involved transcription regulation of autophagy gene expression. Waterborne Cd exposure induced a sharp decrease in ATP content in the liver of P. merus. In addition, the expression of mitochondrial function genes (sdha, cox4i1, cox1, atp5f1, and mt-cyb) are significantly decreased in the liver of P. merus in Cd treated groups, manifesting the suppression of Cd on mitochondrial energy metabolism. Taken together, our experiments demonstrate that waterborne Cd exposure induced oxidative stress, autophagy and mitochondrial dysfunction in the liver of P. merus. These results may contribute to the understanding of mechanisms that hepatotoxicity of Cd in teleost.

Nr1d1 affects autophagy in the skeletal muscles of juvenile Nile tilapia by regulating the rhythmic expression of autophagy-related genes.

Autophagy is an important evolutionary conserved process in eukaryotic organisms for the turnover of intracellular substances. Recent studies revealed that autophagy displays circadian rhythms in mice and zebrafish. To date, there is no report focused on the rhythmic changes of autophagy in fish skeletal muscles upon nutritional deprivation. In this study, we examined the circadian rhythms of 158 functional genes in tilapia muscle in response to starvation. We found that 12 genes were involved in autophagy changed their rhythm after starvation. Among these genes, Atg4c, Bnip3la, Lc3a, Lc3b, Lc3c, and Ulk1a exhibited a daily rhythmicity in tilapia muscle, and Atg4b, becn1, bnip3la, bnip3lb, Lc3a, and ulk1b were significantly upregulated in response to starvation. The number of autophagosomes was dramatically increased in fasted fish, indicating that nutritional signals affect not only the muscular clock system but also its autophagy behavior. Administration of GSK4112, an activator of Nr1d1, altered rhythmic expression of both circadian clock genes and autophagy genes in tilapia muscle. Taken together, these findings provide evidence that nutritional deficiency affects both circadian regulation and autophagy activities in skeletal muscle.

Dietary Ginkgo biloba leaf extract alters immune-related gene expression and disease resistance to Aeromonas hydrophila in common carp Cyprinus carpio.

Ginkgo biloba leaf is widely used in traditional medicine in China. The present study aimed to illustrate the effects of dietary Ginkgo biloba leaf extract (GBLE) on growth performance and immune responses in common carp infected by Aeromonas hydrophila. Six different diets either not treated (control) or treated with 0.5, 1, 2, 5 and 10 g/kg of GBLE were designed to feed the fishes for 8 weeks. The results indicated that, compared to the control groups, 10 g/kg dietary GBLE significantly increased body growth and feed utilization. In GBLE dietary groups, red blood cell levels, white blood cells, hematocrit, hemoglobin, total protein, albumin and globulin were significantly increased relative to the control groups. Dietary supplementation with 5 g/kg GBLE increased the phagocytic ratio, and phagocytic indexes increased in the 2, 5 and 10 g/kg groups relative to the control groups. Moreover, 2, 5 and 10 g/kg GBLE diets increased O2- production compared to the control groups. Additionally, GBLE diets stimulated lysozyme activity (in 10 g/kg group) and inhibited bactericidal activity (in 0.5, 2, 5 and 10 g/kg group). Quantitative real-time PCR showed that IL1ß, IL8, TNF-α, IL10, TGFß, and inducible enzyme genes were prone to decrease while SAA, hepcidin and GPX1 were increased due to the GBLE diet in the intestine. In the head-kidney, the GBLE treatment decreased IL1ß, IL8, TNF-α, IL10, TGFß, INOS and arginase gene expressions, whereas SOD upregulation was found in the GBLE condition. The mRNA expressions of IL1ß, IL8, TNF-α, IL10 and INOS were decreased, but SAA, hepcidin, GPX1 and SOD mRNA levels were increased in the spleen in the GBLE diet compared to the control. Additionally, diet supplemented with GBLE improved the survival rate infected with A. hydrophila. Our observations suggest that GBLE effectively enhanced growth performance, modulated immune-related gene expression. It improved survival rate of common carp after A. hydrophila infection and the optimum concentration we recommend is 10 g/kg of GBLE.

Expression analysis of the heat shock protein genes and cellular reaction in dojo loach (Misgurnus anguillicaudatus) under the different pathogenic invasion.

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90ß, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90ß were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach.

Gene structure, recombinant expression and function characterization of Siniperca chuatsi Fsrp-3.

A full-length complementary (c)DNA sequence encoding follistatin-related protein 3 (fsrp-3) was determined from skeletal muscle in Chinese mandarin fish Siniperca chuatsi, its molecular structure was characterised and its function suggested. The putative structure of S. chuatsi Fsrp-3 contains an N-terminal domain and two follistatin domains. Quantitative reverse-transcription (qRT)-PCR assays revealed that fsrp-3 messenger (m)RNA was differentially expressed among assayed tissues and was highly expressed in heart and intestine. fsrp-3 mRNA exhibited increasing expression from the larval to the juvenile stage (500 g). To investigate the potential function of S. chuatsi fsrp-3 in muscle growth, we constructed a Fsrp-3 prokaryotic expression system and injected the purified Fsrp-3 fusion protein into the dorsal muscle. Fsrp-3 administration significantly influenced cross-section area, satellite cell activation frequency and nuclear density of S. chuatsi muscle fibres. Following Fsrp-3 treatment, the expression of myogenic regulatory factors was up-regulated and decline in the expression of myostatin was observed. The study revealed that Fsrp-3 may affect muscle growth by regulating myogenic regulatory factor expression and antagonizing myostatin function to initiate satellite cell activation and differentiation in S. chuatsi.

Daily rhythmicity of clock gene transcript levels in fast and slow muscle fibers from Chinese perch (Siniperca chuatsi).

BACKGROUND: Clock genes are considered to be the molecular core of biological clock in vertebrates and they are directly involved in the regulation of daily rhythms in vertebrate tissues such as skeletal muscles. Fish myotomes are composed of anatomically segregated fast and slow muscle fibers that possess different metabolic and contractile properties. To date, there is no report on the characterization of the circadian clock system components of slow muscles in fish. RESULTS: In the present study, the molecular clock components (clock, arntl1/2, cry1/2/3, cry-dash, npas2, nr1d1/2, per1/2/3, rorα and tim genes) and their daily transcription levels were characterized in slow and fast muscles of Chinese perch (Siniperca chuatsi). Among the 15 clock genes, nrld2 and per3 had no daily rhythmicity in slow muscles, and cry2/3 and tim displayed no daily rhythmicity in fast muscles of the adult fish. In the slow muscles, the highest expression of the most clock paralogs occurred at the dark period except arntl1, nr1d1, nr1d2 and tim. With the exception of nr1d2 and tim, the other clock genes had an acrophase at the light period in fast muscles. The circadian expression of the myogenic regulatory factors (mrf4 and myf5), mstn and pnca showed either a positive or a negative correlation with the transcription pattern of the clock genes in both types of muscles. CONCLUSIONS: It was the first report to unravel the molecular clock components of the slow and fast muscles in vertebrates. The expressional pattern differences of the clock genes between the two types of muscle fibers suggest that the clock system may play key roles on muscle type-specific tissue maintenance and function.

Selection of reference genes for microRNA quantitative expression analysis in Chinese perch, Siniperca chuatsi.

Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most effective and sensitive techniques in gene expression assay, for which selection of reference genes is a prerequisite. In teleost species, such as Chinese perch, the expression profiling of miRNAs as reference genes for RT-qPCR has not been intensively studied. In the present study, the expression profiles of six miRNAs (miR-101a, miR-146a, miR-22a, miR-23a, miR-26a and let-7a) and one small nuclear RNA (U6) were assayed with RT-qPCR in different adult tissues, developmental stages and growth conditions of Chinese perch, Siniperca chuatsi. The analyses revealed that embryonic developmental stage is an important variability factor in the expression stability of miRNAs. All six miRNAs exhibited better expression consistency than U6 in most of the conditions examined, and therefore, they may be more suitable as a reference gene for miRNA quantification. When different tissues and developmental stages were considered, miR-22a demonstrated the most consistent expression pattern, and the best combination of reference genes was miR-22a and miR-23a. Our study offers useful data for selecting miRNAs as reference genes for RT-qPCR analysis of miRNAs in teleost fishes under different conditions.

Circadian Rhythm Alteration of the Core Clock Genes and the Lipid Metabolism Genes Induced by High-Fat Diet (HFD) in the Liver Tissue of the Chinese Soft-Shelled Turtle (Trionyx sinensis).

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/ß/γ ß and γ (Rorß/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, ß and γ (Pparα/ß/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorß/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country.

Ontogenic expression of the amino acid transporter b(0,+)AT in suckling Huanjiang piglets: effect of intra-uterine growth restriction.

Intestinal amino acid (AA) transport is critical for the supply of AA to other tissues. Few studies regarding AA intestinal transport systems during the period from postnatal intense development of piglets until weaning are available. In the present study, we measured the intestinal expression of b(0,+)AT according to developmental stage using the suckling Huanjiang piglet model, and documented the effect of intra-uterine growth restriction (IUGR) on such expression using real-time PCR and Western blot analysis. Suckling piglets that recovered after IUGR and those with normal body weights (NBW) were used after birth or at 7, 14 and 21 d of age. Blood samples were used for the measurement of plasma AA concentrations, and the jejunum was collected for the measurement of b(0,+)AT expression. In NBW piglets, b(0,+)AT expression was markedly decreased from days 0 to 21 (P< 0.01) and remained at a low level during all the suckling periods. In IUGR piglets, there was a marked decrease in b(0,+)AT expression at birth, which remained lower, when compared with NBW piglets, during the suckling period. These results coincided with decreased plasma arginine concentration at birth and decreased lysine concentration in 21-d-old piglets (P< 0.05). It is concluded that the high expression of b(0,+)AT at birth decreases during the suckling period, and that IUGR is associated with decreased expression of this apical AA transporter. The possible causal relationship between decreased b(0,+)AT expression and lower body weight of IUGR piglets in the suckling period is discussed.

Method Validation and Measurement Uncertainty (MU) Evaluation on Enrofloxacin and Ciprofloxacin in the Aquatic Products.

This study aimed to investigate a detection method of enrofloxacin and ciprofloxacin to be avail for strictly supervising the quality and safety of aquatic products. The results displayed that the optimal extraction conditions for enrofloxacin and ciprofloxacin were the following five aspects: 15 g dosages of Na2SO4 to dehydrate, 8‰ of acetonitrile and 50% hydrochloric acid to deproteinization, 2 mL dosages of n-hexane to degrease, 10 min of ultrasonic time, and 20 min of extraction (stand) time. Meanwhile, it was also obtained for the optimal detection performance indexes of the recovery, precision, and accuracy from the tests of shrimp, grass carp, and tilapia. In particular, the expanded uncertainties were 2.8601 and 0.8613, and the factors of both the calibration curves (Urel(C)) and the analysis of the experiment (Urel(E)) were the two MU main contributors for enrofloxacin and ciprofloxacin together with the results above 40%. Consequently, the developed novel method was suited for the determination of the enrofloxacin and ciprofloxacin residues in aquatic products and would contribute to reinforce in supervision and inspection of the quality and safety of aquatic products.

Characterization of myosin heavy chain (MYH) genes and their differential expression in white and red muscles of Chinese perch, Siniperca chuatsi.

Fish skeletal muscle is composed of two anatomically and functionally different fiber layers, white or fast and red or slow muscles. Myosin, the major structural protein of fish skeletal muscle, contains multiple myosin heavy chain (MYH) isoforms involved in the high plasticity of muscle in response to varying functional demands and/or environmental changes. In this study, we comparatively assayed the cellular and ultrastructural feature of white and red skeletal muscles. Then, a total of 28 class II myosin heavy chain genes were identified in by searching the Chinese perch genome database. Among them, 14 genes code for the fast-muscle-type myosin heavy chain, and 7 genes code for the slow-muscle-type myosin heavy chain. Further, the different isoform gene structures, function domains, phylogenetic relations, and muscle-fiber type-specific expression were characterized. This is the first systematic work on the molecular characterization of class II myosin heavy chain isoforms and the differential analysis of their expression in red and white muscle tissues in Chinese perch Siniperca chuatsi. Our work provided valuable information for a better understanding of myh genes and their molecular characteristics, and the correlations of multiple myosin isoforms with potential functions in response to varying functional demands and/or environmental changes.

Dysbiosis of Gut Microbiota and Lipidomics of Content Induced by Dietary Methionine Restriction in Rice Field Eel (Monopterus albus).

An 8-week feeding trial was conducted using the rice field eel (Monopterus albus) with six isonitrogenous and isoenergetic experimental diets of basic feed supplemented with different levels of methionine (0, 2, 4, 6, 8, or 10 g/kg). This study built upon previous research findings that showed dietary methionine restriction (M0, 0 g/kg) inhibited hepatic fatty acid metabolism and intestinal fatty acid transportation, but both are improved by dietary supplementation with a suitable level of methionine (M8, 8 g/kg). Hence, M0 and M8 were selected to investigate how methionine regulates the gut microbiota and lipidomics of M. albus. Compared with M0, values for gut bacterial Sobs, Shannon, ACE, and Chao1 indices of M8 were remarkably increased (p < 0.05), with Fusobacteria, Firmicutes, and Proteobacteria the dominant phyla and Cetobacterium, Plesiomonas, and Bacillus the main genera in the community under the M0 vs. M8 treatments. However, compared with M0, the proportion of phyla consisting of Fusobacteria decreased in M8, as did the Cetobacterium and Lactococcus at the genus level; conversely, the proportions corresponding to Firmicutes, Proteobacteria, and Chioroflexi phyla increased in M8, as did the Clostridium and Streptococcus genera. Many edges appeared in the circus and networks, demonstrating the interspecies interactions among different operational taxonomic units (OTUs). In addition, various OTUs within the same phylum were clustered within one module. Cooperative interactions were predominant in the two networks, while competitive interactions were prevalent in their submodules. Gut microbiota mainly played roles in nutrition (lipid, amino acid, and carbohydrate) transport and metabolism under the M0 vs. M8 treatments. The PLS-DA scores indicated a significant difference in the main lipidomic components between the M0 and M8 treatment groups. Namely, the TG(26:0/16:0/17:0), TG(28:0/16:0/16:0), TG(26:0/16:0/16:0), and TG(30:0/16:0/16:0)-among others-comprising the gut content were reduced under the M8 treatment (p < 0.001). The genus Clostridium was positively correlated with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(18:0/18:1/20:3), and other compounds, yet negatively correlated with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), and TG(16:0/16:0/24:0), among others as well. According to the lipidomics analysis, the predicted KEGG pathways mainly included lipid and glycan biosynthesis and metabolism, and digestive, sensory, and immune systems. In conclusion, methionine restriction disturbed the microbial community balance and induced microbial dysfunctions, whereas methionine supplementation improved the homeostasis of gut microbiota and lipid metabolism of the rice eel.

Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates.

The objective of this study was to determine developmental changes in mRNA and protein levels for N-acetylglutamate synthase (NAGS; a key enzyme in synthesis of citrulline and arginine from glutamine/glutamate and proline) in the small intestine of suckling piglets. The porcine NAGS gene was cloned using the real-time polymerase-chain reaction (RT-PCR) method. The porcine NAGS gene encoded 368 amino acid residues and had a high degree of sequence similarity to the "conserved domain" of human and mouse NAGS genes. The porcine NAGS gene was expressed in E. coli BL21 and a polyclonal antibody against the porcine NAGS protein was developed. Real-time RT-PCR and western-blot analyses were performed to quantify NAGS mRNA and protein, respectively, in the jejunum and ileum of 1- to 28-day-old pigs. Results indicated that intestinal NAGS mRNA levels were lower in 7- to 28-day-old than in 1-day-old pigs. Immunochemical analysis revealed that NAGS protein was localized in enterocytes of the gut. Notably, intestinal NAGS protein abundance declined progressively during the 28-day suckling period. The postnatal decrease in NAGS protein levels was consistent with the previous report of reduced NAGS enzymatic activity as well as reduced synthesis of citrulline and arginine in the small intestine of 7- to 28-day-old pigs. Collectively, these results suggest that intestinal NAGS expression is regulated primarily at the post-transcriptional level. The findings also provide a new molecular basis to explain that endogenous synthesis of arginine is impaired in sow-reared piglets and arginine is a nutritionally essential amino acid for the neonates.

Genome-wide DNA methylation profiles provide insight into epigenetic regulation of red and white muscle development in Chinese perch Siniperca chuatsi.

Fish skeletal muscles are composed of spatially well-separated fiber types, namely, red and white muscles with different physiological functions and metabolism. To compare the DNA methylation profiles of the two types of muscle tissues and identify potential candidate genes for the muscle growth and development under epigenetic regulation, genome-wide DNA methylation of the red and white muscle in Chinese perch Siniperca chuatsi were comparatively analyzed using bisulfate sequencing methods. An average of 0.9 billion 150-bp paired-end reads were obtained, of which 86% were uniquely mapped to the genome. Methylation mostly occurred at CG sites at a ratio of 94.43% in the red muscle and 93.16% in the white muscle. The mean methylation levels at C-sites were 5.95% in red muscle and 5.83% in white muscle, whereas the mean methylation levels of CG, CHG, and CHH were 73.23%, 0.62%, and 0.67% in red muscle, and 71.01%, 0.62%, and 0.67% in white muscle, respectively. A total of 4192 differentially methylated genes (DMGs) were identified significantly enriched in cell signaling pathways related to skeletal muscle differentiation and growth. Various muscle-related genes, including myosin gene isoforms and regulatory factors, are differentially methylated in the promoter region between the red and white muscles. Further analysis of the transcriptional expression of these genes showed that the muscle regulatory factors (myf5, myog, pax3, pax7, and twitst2) and myosin genes (myh10, myh16, myo18a, myo7a, myo9a, and myl3) were differentially expressed between the two kinds of muscles, consistent with the DNA methylation analysis results. ELISA assays confirmed that the level of 5mC in red muscle was significantly higher than in white muscle (P < 0.05). The RT-qPCR assays revealed that the expression levels of the three DNA methylation transferase (dnmt) subtypes, dnmt1, dnmt3ab, and dnmt3bb1, were significantly higher in red muscle than in white muscle. The higher DNA methylation levels in the red muscle may result from higher DNA methylation transferase expression in the red muscles. Thus, this study might provide a theoretical foundation to better understand epigenetic regulation in the growth and development of red and white muscles in animals, at least in Chinese perch fish.