Effects of dietary leucine and valine levels on growth performance, glycolipid metabolism and immune response in Tilapia GIFT Oreochromis niloticus.

An 8-week feeding trial was performed to evaluate the effects of dietary leucine (Leu) and valine (Val) levels on growth performance, glycolipid metabolism and immune response in Oreochromis niloticus. Fish (15.23 ± 0.05 g) were randomly fed four diets containing two Leu levels (1.2% and 2.3%) and two Val levels (0.7% and 1.4%) as a 2 × 2 experimental design (LL-LV, LL-HV, HL-LV and HL-HV). Compared with LL-LV group, the growth parameters (final weight, daily growth coefficient (DGC) and growth rate per metabolic body weight (GRMBW)), feed conversion rate (FCR), the activities of intestinal amylase, lipase, creatine kinase (CK) and Na+, K+-ATPase, liver NAD+/NADH ratio, as well as the expression of SIRT1, GK, PK, FBPase, PPARα, CPT IA, ACO and IL10 all increased significantly in the HL-LV group; however, in the high Val group, final weight, DGC, GRMBW, intestinal enzyme activities, as well as the expression of PEPCK, SREBP1, FAS, IL8 and IL10 of the HL-HV group were significantly lower than those of the LL-HV group, while the opposite was true for the remaining indicators. Significant interactions between dietary Leu and Val were observed in final weight, DGC, GRMBW, plasma IL1ß and IL6 levels, intestinal amylase and CK activities, liver NAD+/NADH ratio, as well as the expression of SIRT1, PK, PEPCK, FBPase, SREBP1, FAS, PPARα, CPT IA, ACO, NF-κB1, IL1ß, IL6 and IL10. The highest values of growth parameters, intestinal enzyme activities and expression of SIRT1, FBPase, PPARα, CPT IA and ACO were observed in the HL-LV group, while the opposite was true for the expression of SREBP1, FAS, PPARα, NF-κB1, IL1ß and IL6. Overall, our findings indicated that dietary Leu and Val can effect interactively, and fish fed with diets containing 2.3% Leu with 0.7% Val had the best growth performance and hepatic health status of O. niloticus.

Mitochondrial Fatty Acid ß-Oxidation Inhibition Promotes Glucose Utilization and Protein Deposition through Energy Homeostasis Remodeling in Fish.

BACKGROUND: Fish cannot use carbohydrate efficiently and instead utilize protein for energy supply, thus limiting dietary protein storage. Protein deposition is dependent on protein turnover balance, which correlates tightly with cellular energy homeostasis. Mitochondrial fatty acid ß-oxidation (FAO) plays a crucial role in energy metabolism. However, the effect of remodeled energy homeostasis caused by inhibited mitochondrial FAO on protein deposition in fish has not been intensively studied. OBJECTIVES: This study aimed to identify the regulatory role of mitochondrial FAO in energy homeostasis maintenance and protein deposition by studying lipid, glucose, and protein metabolism in fish. METHODS: Carnitine-depleted male Nile tilapia (initial weight: 4.29 ± 0.12 g; 3 mo old) were established by feeding them with mildronate diets (1000 mg/kg/d) for 6 wk. Zebrafish deficient in the carnitine palmitoyltransferase 1b gene (cpt1b) were produced by using CRISPR/Cas9 gene-editing technology, and their males (154 ± 3.52 mg; 3 mo old) were used for experiments. Normal Nile tilapia and wildtype zebrafish were used as controls. We assessed nutrient metabolism and energy homeostasis-related biochemical and molecular parameters, and performed 14C-labeled nutrient tracking and transcriptomic analyses. RESULTS: The mitochondrial FAO decreased by 33.1-88.9% (liver) and 55.6-68.8% (muscle) in carnitine-depleted Nile tilapia and cpt1b-deficient zebrafish compared with their controls (P < 0.05). Notably, glucose oxidation and muscle protein deposition increased by 20.5-24.4% and 6.40-8.54%, respectively, in the 2 fish models compared with their corresponding controls (P < 0.05). Accordingly, the adenosine 5'-monophosphate-activated protein kinase/protein kinase B-mechanistic target of rapamycin (AMPK/AKT-mTOR) signaling was significantly activated in the 2 fish models with inhibited mitochondrial FAO (P < 0.05). CONCLUSIONS: These data show that inhibited mitochondrial FAO in fish induces energy homeostasis remodeling and enhances glucose utilization and protein deposition. Therefore, fish with inhibited mitochondrial FAO could have high potential to utilize carbohydrate. Our results demonstrate a potentially new approach for increasing protein deposition through energy homeostasis regulation in cultured animals.

Mechanisms and metabolic regulation of PPARα activation in Nile tilapia (Oreochromis niloticus).

Although the key metabolic regulatory functions of mammalian peroxisome proliferator-activated receptor α (PPARα) have been thoroughly studied, the molecular mechanisms and metabolic regulation of PPARα activation in fish are less known. In the first part of the present study, Nile tilapia (Nt)PPARα was cloned and identified, and high mRNA expression levels were detected in the brain, liver, and heart. NtPPARα was activated by an agonist (fenofibrate) and by fasting and was verified in primary hepatocytes and living fish by decreased phosphorylation of NtPPARα and/or increased NtPPARα mRNA and protein expression. In the second part of the present work, fenofibrate was fed to fish or fish were fasted for 4weeks to investigate the metabolic regulatory effects of NtPPARα. A transcriptomic study was also performed. The results indicated that fenofibrate decreased hepatic triglyceride and 18C-series fatty acid contents but increased the catabolic rate of intraperitoneally injected [1-(14)C] palmitate in vivo, hepatic mitochondrial ß-oxidation efficiency, the quantity of cytochrome b DNA, and carnitine palmitoyltransferase-1a mRNA expression. Fenofibrate also increased serum glucose, insulin, and lactate concentrations. Fasting had stronger hypolipidemic and gene regulatory effects than those of fenofibrate. Taken together, we conclude that: 1) liver is one of the main target tissues of the metabolic regulation of NtPPARα activation; 2) dephosphorylation is the basal NtPPARα activation mechanism rather than enhanced mRNA and protein expression; 3) activated NtPPARα has a hypolipidemic effect by increasing activity and the number of hepatic mitochondria; and 4) PPARα activation affects carbohydrate metabolism by altering energy homeostasis among nutrients.

Molecular characterization, transcriptional activity and nutritional regulation of peroxisome proliferator activated receptor gamma in Nile tilapia (Oreochromis niloticus).

Peroxisome proliferator activated receptor gamma (PPARγ) is a master regulator in lipid metabolism and widely exists in vertebrates. However, the molecular structure and transcriptional activity of PPARγ in fish are still unclear. This study cloned PPARγ from Nile tilapia (Oreochromis niloticus) referred as NtPPARγ and transfected the NtPPARγ plasmids into HEK-293 cells to explore its mechanism of transcriptional regulation in fish. The expression of NtPPARγ was compared in fed and fasted fish. Two transcripts of NtPPARγ varied at the 5'-untranslated region and the DNA binding domain was highly conserved. Thirty-nine amino acid residues in the ligand binding domain in Nile tilapia were different from those in human. Two transcripts showed different expression profiles in 11 tissues, but both were highly expressed in liver, intestine and kidney. The transcriptional activity assay showed that NtPPARγ collaborates with retinoid X-receptor α (NtRXRα) to regulate the expression of Nile tilapia fatty acid binding protein 4 (FABP4), the compartment of which have been identified as the target gene of PPARγ in human. In the fish fasting trial, the mRNA expression of NtPPARγ1 and NtPPARγ2 in intestine and liver at 3h post-feeding (HPF) was lower than those at 8 HPF, 24 HPF and in fish fasted for 36h, but was relatively stable in kidney among different feeding treatments. In conclusion, the DNA binding domain in PPARγ was highly conserved, while the ligand binding domain was moderately conserved. In Nile tilapia, the PPARγ collaborates with RXRα to perform transcriptional regulation of FABP4 at least in vitro. The plasmid system established in this study along with a cell line from Nile tilapia will be useful tools for the further functional study of PPARγ in fish.

Baicalin promotes antiviral IFNs production and alleviates type I IFN-induced neutrophil inflammation.

Type I and III interferons (IFNs) both serve as pivotal components of the host antiviral innate immune system. Although they exert similar antiviral effects, type I IFNs can also activate neutrophil inflammation, a function not born by type III IFNs. Baicalin, the main bioactive component of Scutellariae radix, has been shown to exert therapeutic effects on viral diseases due to its anti-viral, anti-inflammatory and immunomulatory activities. There is uncertainty, however, on the association between the antiviral effects of baicalin and the modulation of anti-viral IFNs production and the immunological effects of type I IFNs. Here, a Poly (I:C)-stimulated A549 cell line was established to mimic a viral infection model. Our results demonstrated that baicalin could elevate the expression of type I and III IFNs and their receptors in Poly (I:C)-stimulated A549 cells. Moreover, the potential regulation effects of baicalin for type I IFN-induced neutrophil inflammation was further explored. Results showed that baicalin diminished the production of the pro-inflammatory cytokines (IL-1ß, IL-6, IL-17 and TNF-α), ROS, and neutrophil extracellular traps and suppressed chemotaxis. Collectively, all these data indicated that baicalin had a dual role on IFNs production and effects: (1) Baicalin was able to elevate the expression of type I and III IFNs and their receptors, (2) and it alleviated type I IFN-mediated neutrophil inflammatory response. This meant that baicalin has the potential to act as an eximious immunomodulator, exerting antiviral effects and reducing inflammation.

DHA alleviates high glucose-induced mitochondrial dysfunction in Oreochromis niloticus by inhibiting DRP1-mediated mitochondrial fission.

Dynamin-related protein 1 (DRP1) is a key regulator in the maintenance of mammalian glucose homeostasis, but the relevant information remains poorly understood on aquatic animals. In the study, DRP1 is formally described for the first time in Oreochromis niloticus. DRP1 encodes a peptide of 673 amino acid residues that contained three conserved domains: a GTPase domain, a dynamin middle domain and a dynamin GTPase effector domain. DRP1 transcripts are widely distributed in all of the detected seven organs/tissues, and the highest mRNA levels in brain. High-carbohydrate (45 %) fed fish showed a significant upregulation of liver DRP1 expression than that of control (30 %) group. Glucose administration upregulated liver DRP1 expression, with peak values observed at 1 h; then its expression returned to the basal value at 12 h. In the in vitro study, DRP1 over-expression significantly decreased mitochondrial abundance in hepatocytes. DHA significantly increased mitochondrial abundance, transcriptions of mitochondrial transcription factor A (TFAM) and mitofusin 1 and 2 (MFN1 and MFN2) and complex II and III activities of high glucose-treated hepatocyte, whereas the opposite was true for DRP1, mitochondrial fission factor (MFF) and fission (FIS) expression. Together, these findings illustrated that O. niloticus DRP1 is highly conserved, and it participated in glucose control of fish. DHA could alleviate high glucose-induced mitochondrial dysfunction of fish by inhibiting DRP1-mediated mitochondrial fission.

Microbe-based management for colorectal cancer.

ABSTRACT: Colorectal cancer (CRC) is one of the most prevalent, most lethal cancers in the world. Increasing evidence suggests that the intestinal microbiota is closely related to the pathogenesis and prognosis of CRC. The normal microbiota plays an essential role in maintaining gut barrier function and the immune microenvironment. Recent studies have identified carcinogenic bacteria such as enterotoxigenic Bacteroides fragilis (ETBF) and Streptococcus gallolyticus (S. gallolyticus), as well as protective bacterial such as Akkermansia muciniphila (A. muciniphila), as potential targets of CRC treatment. Gut microbiota modulation aims to restore gut dysbiosis, regulate the intestinal immune system and prevent from pathogen invasion, all of which are beneficial for CRC prevention and prognosis. The utility of probiotics, prebiotics, postbiotics, fecal microbiota transplantation and dietary inventions to treat CRC makes them novel microbe-based management tools. In this review, we describe the mechanisms involved in bacteria-derived colorectal carcinogenesis and summarized novel bacteria-related therapies for CRC. In summary, we hope to facilitate clinical applications of intestinal bacteria for preventing and treating CRC.

Dietary oils modify lipid molecules and nutritional value of fillet in Nile tilapia: A deep lipidomics analysis.

The nutritional value of fish fillet can be largely affected by dietary oils. However, little is known about how dietary oils modify lipid molecules in fish fillets. Through biochemical and lipidomics assays, this study demonstrated the molecular characteristics of fillet lipids in Nile tilapia fed with different oils for six weeks. High 18:2n-6 and low 18:3n-3 deposition in phosphoglycerides resulted high 18:2n-6/18:3n-3 ratio in tilapia. Dietary n-3 VLCUFAs intake increased its deposition at sn-1/3 of triglycerides and at sn-2 of phosphatidylcholines. Irrespective of dietary oil, 16:0 was distributed preferentially at the outer positions of glycerol backbone. High 18:2n-6 accumulated at sn-2 position for fish fed with n-3 PUFA-enriched oils. High 18:3n-3 deposited at sn-1/3 in TG, sn-1 in phosphatidylethanolamines, while at sn-2 in phosphatidylcholines. Together, dietary oils change the composition and positional distribution of fatty acids on the glycerol backbone, and change nutritional value of fish for human health.

Leptin Selectively Regulates Nutrients Metabolism in Nile Tilapia Fed on High Carbohydrate or High Fat Diet.

Leptin is known to inhibit appetite and promote energy metabolism in vertebrates. Leptin resistance (LR) commonly occurs in diet-induced obesity (DIO) in mammals. However, the roles of leptin in the energy homeostasis in DIO animals with LR remain unclear. Here we first verified the high expression of leptin in subcutaneous adipose tissue (SCAT) as in liver in Nile tilapia. Furthermore, we produced two types of DIO Nile tilapia by using a high-carbohydrate diet (HCD) or a high-fat diet (HFD), and confirmed the existence of LR in both models. Notably, we found that HCD-DIO fish retained leptin action in the activation of lipid metabolism and showed LR in glucose metabolism regulation, while this selective leptin action between lipid and glucose metabolism was reversed in HFD-DIO fish. Fasting the fish for 1 week completely recovered leptin actions in the regulation of lipid and glucose metabolism. Therefore, leptin may retain more of its activities in animals with LR than previously believed. Evolutionally, this selective regulation of leptin in nutrients metabolism could be an adaptive mechanism in animals to store surplus calories when different types of food are abundant.

Nutritional background changes the hypolipidemic effects of fenofibrate in Nile tilapia (Oreochromis niloticus).

Peroxisome proliferation activated receptor α (PPARα) is an important transcriptional regulator of lipid metabolism and is activated by high-fat diet (HFD) and fibrates in mammals. However, whether nutritional background affects PPARα activation and the hypolipidemic effects of PPARα ligands have not been investigated in fish. In the present two-phase study of Nile tilapia (Oreochromis niloticus), fish were first fed a HFD (13% fat) or low-fat diet (LFD; 1% fat) diet for 10 weeks, and then fish from the first phase were fed the HFD or LFD supplemented with 200 mg/kg body weight fenofibrate for 4 weeks. The results indicated that the HFD did not activate PPARα or other lipid catabolism-related genes. Hepatic fatty acid ß-oxidation increased significantly in the HFD and LFD groups after the fenofibrate treatment, when exogenous substrates were sufficiently provided. Only in the HFD group, fenofibrate significantly increased hepatic PPARα mRNA and protein expression, and decreased liver and plasma triglyceride concentrations. This is the first study to show that body fat deposition and dietary lipid content affects PPARα activation and the hypolipidemic effects of fenofibrate in fish, and this could be due to differences in substrate availability for lipid catabolism in fish fed with different diets.

Systemic regulation of L-carnitine in nutritional metabolism in zebrafish, Danio rerio.

Excess fat accumulation has been observed widely in farmed fish; therefore, efficient lipid-lowering factors have obtained high attention in the current fish nutrition studies. Dietary L-carnitine can increase fatty acid ß-oxidation in mammals, but has produced contradictory results in different fish species. To date, the mechanisms of metabolic regulation of L-carnitine in fish have not been fully determined. The present study used zebrafish to investigate the systemic regulation of nutrient metabolism by dietary L-carnitine supplementation. L-carnitine significantly decreased the lipid content in liver and muscle, accompanied by increased concentrations of total and free carnitine in tissues. Meanwhile, L-carnitine enhanced mitochondrial ß-oxidation activities and the expression of carnitine palmitoyltransferase 1 mRNA significantly, whereas it depressed the mRNA expression of adipogenesis-related genes. In addition, L-carnitine caused higher glycogen deposition in the fasting state, and increased and decreased the mRNA expressions of gluconeogenesis-related and glycolysis-related genes, respectively. L-carnitine also increased the hepatic expression of mTOR in the feeding state. Taken together, dietary L-carnitine supplementation decreased lipid deposition by increasing mitochondrial fatty acid ß-oxidation, and is likely to promote protein synthesis. However, the L-carnitine-enhanced lipid catabolism would cause a decrease in glucose utilization. Therefore, L-carnitine has comprehensive effects on nutrient metabolism in fish.

Systemic adaptation of lipid metabolism in response to low- and high-fat diet in Nile tilapia (Oreochromis niloticus).

Natural selection endows animals with the abilities to store lipid when food is abundant and to synthesize lipid when it is limited. However, the relevant adaptive strategy of lipid metabolism has not been clearly elucidated in fish. This study examined the systemic metabolic strategies of Nile tilapia to maintain lipid homeostasis when fed with low- or high-fat diets. Three diets with different lipid contents (1%, 7%, and 13%) were formulated and fed to tilapias for 10 weeks. At the end of the feeding trial, the growth rate, hepatic somatic index, and the triglyceride (TG) contents of serum, liver, muscle, and adipose tissue were comparable among three groups, whereas the total body lipid contents and the mass of adipose tissue increased with the increased dietary lipid levels. Overall quantitative PCR, western blotting and transcriptomic assays indicated that the liver was the primary responding organ to low-fat (LF) diet feeding, and the elevated glycolysis and accelerated biosynthesis of fatty acids (FA) in the liver is likely to be the main strategies of tilapia toward LF intake. In contrast, excess ingested lipid was preferentially stored in adipose tissue through increasing the capability of FA uptake and TG synthesis. Increasing numbers, but not enlarging size, of adipocytes may be the main strategy of Nile tilapia responding to continuous high-fat (HF) diet feeding. This is the first study illuminating the systemic adaptation of lipid metabolism responding to LF or HF diet in fish, and our results shed new light on fish physiology.

Identification, characterization and nutritional regulation of two isoforms of acyl-coenzyme A oxidase 1 gene in Nile tilapia (Oreochromis niloticus).

In peroxisome, acyl-coenzyme A oxidase 1 (ACOX1) is the first rate-limiting enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. Two isoforms of acyl-coenzyme A oxidase 1 were firstly identified in Nile tilapia (Oreochromis niloticus) in this study. ACOX1 isoform1 (ACOX1i1) and ACOX1 isoform2 (ACOX1i2) were encoded by the single gene with 661 amino acids in length. The coding region of both isoforms consisted of 14 exons. The residues from 89 to 193 in ACOX1i1 were encoded by exon 3b, while in ACOX1i2 they were encoded by exon 3a. Homologous alignment analysis indicated that the varied region (the residues from 89 to 193) of ACOX1i1 was more conserved than ACOX1i2 in vertebrates (Mammalia, Aves, Amphibia and Pisces). The mRNA expression level of ACOX1i1 and ACOX1i2 was detected separately in eleven tissues and the results indicated that ACOXi1 expression was the highest in liver followed by kidney and brain, while the expression of ACOXi2 was the highest in kidney followed by liver. The normalized levels of both transcript variants were comparable in most tissues, however the level of ACOX1i2 was significantly higher than that of ACOX1i1 in white muscle and kidney (5.1 fold and 3.1 fold), and ACOX1i1 was significantly higher than ACOX1i2 in gill and brain (4.8 fold and 1.9 fold). In different nutritional states, the expression levels of both isoforms in liver were comparable between fasting and most of post-feeding time points, except that the expression at 3h post-feeding was significantly lower than others. The expression of ACOX1i1 in the kidney also showed the similar pattern, indicating the lowest expression at 8h post-feeding, however, no significant change was seen in ACOX2i2 among all nutritional states. These results suggested that ACOX1i1 and i2 may play different roles in tissues, and their expression levels were differently modulated by nutritional stage.