Inhibiting mitochondrial citrate shuttling induces hepatic triglyceride deposition in Nile tilapia (Oreochromis niloticus) through lipid anabolic remodeling.

The solute carrier family 25 member 1 (Slc25a1)-dependent mitochondrial citrate shuttle is responsible for exporting citrate from the mitochondria to the cytoplasm for supporting lipid biosynthesis and protein acetylation. Previous studies on Slc25a1 concentrated on pathological models. However, the importance of Slc25a1 in maintaining metabolic homeostasis under normal nutritional conditions remains poorly understood. Here, we investigated the mechanism of mitochondrial citrate shuttle in maintaining lipid metabolism homeostasis in male Nile tilapia (Oreochromis niloticus). To achieve the objective, we blocked the mitochondrial citrate shuttle by inhibiting Slc25a1 under normal nutritional conditions. Slc25a1 inhibition was established by feeding Nile tilapia with 250 mg/kg 1,2,3-benzenetricarboxylic acid hydrate for six weeks or intraperitoneal injecting them with dsRNA to knockdown slc25a1b for seven days. The Nile tilapia with Slc25a1 inhibition exhibited an obesity-like phenotype accompanied by fat deposition, liver damage and hyperglycemia. Moreover, Slc25a1 inhibition decreased hepatic citrate-derived acetyl-CoA, but increased hepatic triglyceride levels. Furthermore, Slc25a1 inhibition replenished cytoplasmic acetyl-CoA through enhanced acetate pathway, which led to hepatic triglycerides accumulation. However, acetate-derived acetyl-CoA caused by hepatic Slc25a1 inhibition did not activate de novo lipogenesis, but rather modified protein acetylation. In addition, hepatic Slc25a1 inhibition enhanced fatty acids esterification through acetate-derived acetyl-CoA, which increased Lipin1 acetylation and its protein stability. Collectively, our results illustrate that inhibiting mitochondrial citrate shuttle triggers lipid anabolic remodeling and results in lipid accumulation, indicating the importance of mitochondrial citrate shuttle in maintaining lipid metabolism homeostasis.

Inhibition of mitochondrial fatty acid ß-oxidation activates mTORC1 pathway and protein synthesis via Gcn5-dependent acetylation of Raptor in zebrafish.

Pharmacological inhibition of mitochondrial fatty acid oxidation (FAO) has been clinically used to alleviate certain metabolic diseases by remodeling cellular metabolism. However, mitochondrial FAO inhibition also leads to mechanistic target of rapamycin complex 1 (mTORC1) activation-related protein synthesis and tissue hypertrophy, but the mechanism remains unclear. Here, by using a mitochondrial FAO inhibitor (mildronate or etomoxir) or knocking out carnitine palmitoyltransferase-1, we revealed that mitochondrial FAO inhibition activated the mTORC1 pathway through general control nondepressible 5-dependent Raptor acetylation. Mitochondrial FAO inhibition significantly promoted glucose catabolism and increased intracellular acetyl-CoA levels. In response to the increased intracellular acetyl-CoA, acetyltransferase general control nondepressible 5 activated mTORC1 by catalyzing Raptor acetylation through direct interaction. Further investigation also screened Raptor deacetylase histone deacetylase class II and identified histone deacetylase 7 as a potential regulator of Raptor. These results provide a possible mechanistic explanation for the mTORC1 activation after mitochondrial FAO inhibition and also bring light to reveal the roles of nutrient metabolic remodeling in regulating protein acetylation by affecting acetyl-CoA production.

Hypoxia tolerance in fish depends on catabolic preference between lipids and carbohydrates.

Hypoxia is a common environmental stress factor in aquatic organisms, which varies among fish species. However, the mechanisms underlying the ability of fish species to tolerate hypoxia are not well known. Here, we showed that hypoxia response in different fish species was affected by lipid catabolism and preference for lipid or carbohydrate energy sources. Activation of biochemical lipid catabolism through peroxisome proliferator-activated receptor alpha (Pparα) or increasing mitochondrial fat oxidation in tilapia decreased tolerance to acute hypoxia by increasing oxygen consumption and oxidative damage and reducing carbohydrate catabolism as an energy source. Conversely, lipid catabolism inhibition by suppressing entry of lipids into mitochondria in tilapia or individually knocking out three key genes of lipid catabolism in zebrafish increased tolerance to acute hypoxia by decreasing oxygen consumption and oxidative damage and promoting carbohydrate catabolism. However, anaerobic glycolysis suppression eliminated lipid catabolism inhibition-promoted hypoxia tolerance in adipose triglyceride lipase (atgl) mutant zebrafish. Using 14 fish species with different trophic levels and taxonomic status, the fish preferentially using lipids for energy were more intolerant to acute hypoxia than those preferentially using carbohydrates. Our study shows that hypoxia tolerance in fish depends on catabolic preference for lipids or carbohydrates, which can be modified by regulating lipid catabolism.

Double-edged effect of sodium citrate in Nile tilapia (Oreochromis niloticus): Promoting lipid and protein deposition vs. causing hyperglycemia and insulin resistance.

Citrate is an essential substrate for energy metabolism that plays critical roles in regulating glucose and lipid metabolic homeostasis. However, the action of citrate in regulating nutrient metabolism in fish remains poorly understood. Here, we investigated the effects of dietary sodium citrate on growth performance and systematic energy metabolism in juvenile Nile tilapia (Oreochromis niloticus). A total of 270 Nile tilapia (2.81 ± 0.01 g) were randomly divided into three groups (3 replicates per group, 30 fish per replicate) and fed with control diet (35% protein and 6% lipid), 2% and 4% sodium citrate diets, respectively, for 8 weeks. The results showed that sodium citrate exhibited no effect on growth performance (P > 0.05). The whole-body crude protein, serum triglyceride and hepatic glycogen contents were significantly increased in the 4% sodium citrate group (P < 0.05), but not in the 2% sodium citrate group (P > 0.05). The 4% sodium citrate treatment significantly increased the serum glucose and insulin levels at the end of feeding trial and also in the glucose tolerance test (P < 0.05). The 4% sodium citrate significantly enhanced the hepatic phosphofructokinase activity and inhibited the expression of pyruvate dehydrogenase kinase isozyme 2 and phosphor-pyruvate dehydrogenase E1 component subunit alpha proteins (P < 0.05). Additionally, the 4% sodium citrate significantly increased hepatic triglyceride and acetyl-CoA levels, while the expressions of carnitine palmitoyl transferase 1a protein were significantly down-regulated by the 4% sodium citrate (P < 0.05). Besides, the 4% sodium citrate induced crude protein deposition in muscle by activating mTOR signaling and inhibiting AMPK signaling (P < 0.05). Furthermore, the 4% sodium citrate significantly suppressed serum aspartate aminotransferase and alanine aminotransferase activities, along with the lowered expression of pro-inflammatory genes, such as nfκb, tnfα and il8 (P < 0.05). Although the 4% sodium citrate significantly increased phosphor-nuclear factor-kB p65 protein expression (P < 0.05), no significant tissue damage or inflammation occurred. Taken together, dietary supplementation of sodium citrate could exhibit a double-edged effect in Nile tilapia, with the positive aspect in promoting nutrient deposition and the negative aspect in causing hyperglycemia and insulin resistance.

High cholesterol intake remodels cholesterol turnover and energy homeostasis in Nile tilapia (Oreochromis niloticus).

The roles of dietary cholesterol in fish physiology are currently contradictory. The issue reflects the limited studies on the metabolic consequences of cholesterol intake in fish. The present study investigated the metabolic responses to high cholesterol intake in Nile tilapia (Oreochromis niloticus), which were fed with four cholesterol-contained diets (0.8, 1.6, 2.4 and 3.2%) and a control diet for eight weeks. All fish-fed cholesterol diets showed increased body weight, but accumulated cholesterol (the peak level was in the 1.6% cholesterol group). Then, we selected 1.6% cholesterol and control diets for further analysis. The high cholesterol diet impaired liver function and reduced mitochondria number in fish. Furthermore, high cholesterol intake triggered protective adaptation via (1) inhibiting endogenous cholesterol synthesis, (2) elevating the expression of genes related to cholesterol esterification and efflux, and (3) promoting chenodeoxycholic acid synthesis and efflux. Accordingly, high cholesterol intake reshaped the fish gut microbiome by increasing the abundance of Lactobacillus spp. and Mycobacterium spp., both of which are involved in cholesterol and/or bile acids catabolism. Moreover, high cholesterol intake inhibited lipid catabolic activities through mitochondrial ß-oxidation, and lysosome-mediated lipophagy, and depressed insulin signaling sensitivity. Protein catabolism was elevated as a compulsory response to maintain energy homeostasis. Therefore, although high cholesterol intake promoted growth, it led to metabolic disorders in fish. For the first time, this study provides evidence for the systemic metabolic response to high cholesterol intake in fish. This knowledge contributes to an understanding of the metabolic syndromes caused by high cholesterol intake or deposition in fish. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00158-7.

Dietary docosahexaenoic acid reduces fat deposition and alleviates liver damage induced by D-galactosamine and lipopolysaccharides in Nile tilapia (Oreochromis niloticus).

Liver health is important to maintain survival and growth of fish. Currently, the role of dietary docosahexaenoic acid (DHA) in improving fish liver health is largely unknown. This study investigated the role of DHA supplementation in fat deposition and liver damage caused by D-galactosamine (D-GalN) and lipopolysaccharides (LPS) in Nile tilapia (Oreochromis niloticus). Four diets were formulated as control diet (Con), Con supplemented with 1 % DHA, 2 % DHA and 4 % DHA diets, respectively. The diets were fed to 25 Nile tilapia (2.0 ± 0.1 g, average initial weight) in triplicates for four weeks. After the four weeks, 20 fish in each treatment were randomly selected and injected with a mixture of 500 mg D-GalN and 10 µL LPS per mL to induce acute liver injury. The results showed that the Nile tilapia fed on DHA diets decreased visceral somatic index, liver lipid content and serum and liver triglyceride concentrations than those fed on the Con diet. Moreover, after D-GalN/LPS injection, the fish fed on DHA diets decreased alanine aminotransferase and aspartate transaminase activities in the serum. The results of liver qPCR and transcriptomics assays together showed that the DHA diets feeding improved liver health by downregulating the expression of the genes related to toll-like receptor 4 (TLR4) signaling pathway, inflammation and apoptosis. This study indicates that DHA supplementation in Nile tilapia alleviates the liver damage caused by D-GalN/LPS through increasing lipid catabolism, decreasing lipogenesis, TLR4 signaling pathway, inflammation, and apoptosis. Our study provides novel knowledge on the role of DHA in improving liver health in cultured aquatic animals for sustainable aquaculture.

Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary.

Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.

High dissolved oxygen exacerbates ammonia toxicity with sex-dependent manner in zebrafish.

Ammonia nitrogen is one of the important environmental factors, and causes negative effects for fish health in ecosystem and aquaculture. The toxic effects and mechanisms of ammonia in fish deserve further investigation. In the present study, we exposed female and male zebrafish (Danio rerio) to ammonia (50 mg/L NH4Cl) with oxygenated (7.5-7.8 mg/L) or non­oxygenated (3.8-4.5 mg/L) water, to identify the combined effects of dissolved oxygen and ammonia on fish with gender difference. The results showed that oxygenated ammonia exposure increased fish mortality, gill secondary lamellas damage and gill tissue spaces, gene expressions of proinflammatory interleukin 1 beta (il-1ß) and apoptotic caspase8 as compared with non­oxygenated ammonia. Besides, oxygenated ammonia elevated plasma ammonia contents, and decreased the discharge of body ammonia through gills by depressing the enzyme activity of Na+/K+-ATPase. Notably, when zebrafish were subjected to ammonia stress, more severe mortality, gill damage and tissue inflammatory response were observed in males than females. This is the first study to clarify the gender-dependent impacts of ammonia toxicity, and the adverse effects of oxygenation on ammonia resistance in zebrafish.

Atorvastatin remodels lipid distribution between liver and adipose tissues through blocking lipoprotein efflux in fish.

The regulation of cholesterol metabolism in fish is still unclear. Statins play important roles in promoting cholesterol metabolism development in mammals. However, studies on the role of statins in cholesterol metabolism in fish are currently limited. The present study evaluated the effects of statins on cholesterol metabolism in fish. Nile tilapia (Oreochromis niloticus) were fed on control diets supplemented with three atorvastatin levels (0, 12, and 24 mg/kg diet, ATV0, ATV12, and ATV24, respectively) for 4 wk. Intriguingly, the results showed that both atorvastatin treatments increased hepatic cholesterol and triglyceride contents mainly through inhibiting bile acid synthesis and efflux, and compensatorily enhancing cholesterol synthesis in fish liver (P < 0.05). Moreover, atorvastatin treatment significantly inhibited hepatic very-low-density lipoprotein (VLDL) assembly and thus decreased serum VLDL content (P < 0.05). However, fish treated with atorvastatin significantly reduced cholesterol and triglycerides contents in adipose tissue (P < 0.05). Further molecular analysis showed that atorvastatin treatment promoted cholesterol synthesis and lipogenesis pathways, but inhibited lipid catabolism and low-density lipoprotein (LDL) uptake in the adipose tissue of fish (P < 0.05). In general, atorvastatin induced the remodeling of lipid distribution between liver and adipose tissues through blocking VLDL efflux from the liver to adipose tissue of fish. Our results provide a novel regulatory pattern of cholesterol metabolism response caused by atorvastatin in fish, which is distinct from mammals: cholesterol inhibition by atorvastatin activates hepatic cholesterol synthesis and inhibits its efflux to maintain cholesterol homeostasis, consequently reduces cholesterol storage in fish adipose tissue.

Bacillus amyloliquefaciens protects Nile tilapia against Aeromonas hydrophila infection and alleviates liver inflammation induced by high-carbohydrate diet.

Carbohydrates are widely distributed in nature as an important nutritional substance and energy source. However, the utilization efficiency of carbohydrates is very poor in fish. Over consumption of carbohydrates will cause excessive inflammatory response and result in lower pathogen resistance in fish. Probiotics have been widely used to prevent inflammation, but the underlying mechanism still needs more exploration. In this study, three diets, including a control diet (CD), a high-carbohydrate diet (HD) and the HD supplemented with Bacillus amyloliquefaciens SS1 (HDB) were used to feed Nile tilapia for 10 weeks. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila (A. hydrophila) for 7 days. The data showed that the addition of Bacillus amyloliquefaciens SS1 (B. amyloliquefaciens SS1) significantly increased the survival rate and enhanced the respiratory burst activity of head kidney leukocytes in Nile tilapia. B. amyloliquefaciens SS1 treatment significantly elevated the anti-oxidative capability, which was evidenced by higher activities of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), and higher content of reduced glutathione (GSH) in the serum. Administration with B. amyloliquefaciens SS1 effectively suppressed inflammatory response in the liver by inhibiting nuclear factor kappa-B (NF-κB)/interleukin-1 beta (IL-1ß) inflammatory signaling pathway. In vitro analysis suggested that intestinal bacteria derived-acetate has the antioxidant capability, which may account for the alleviation of inflammation. Overall, this study demonstrated that dietary supplementation with B. amyloliquefaciens SS1 protected Nile Tilapia against A. hydrophila infection and suppressed liver inflammation by enhancing antioxidant capability.

Lactobacillus plantarum Ameliorates High-Carbohydrate Diet-Induced Hepatic Lipid Accumulation and Oxidative Stress by Upregulating Uridine Synthesis.

The overconsumption of carbohydrates induces oxidative stress and lipid accumulation in the liver, which can be alleviated by modulation of intestinal microbiota; however, the underlying mechanism remains unclear. Here, we demonstrated that a strain affiliated with Lactobacillus plantarum (designed as MR1) efficiently attenuated lipid deposition, oxidative stress, as well as inflammatory response, which are caused by high-carbohydrate diet (HC) in fish with poor utilization ability of carbohydrates. Serum untargeted metabolome analysis indicated that pyrimidine metabolism was the significantly changed pathway among the groups. In addition, the content of serum uridine was significantly decreased in the HC group compared with the control group, while it increased by supplementation with L. plantarum MR1. Further analysis showed that addition of L. plantarum MR1 reshaped the composition of gut microbiota and increased the content of intestinal acetate. In vitro experiment showed that sodium acetate could induce the synthesis of uridine in hepatocytes. Furthermore, we proved that uridine could directly ameliorate oxidative stress and decrease liver lipid accumulation in the hepatocytes. In conclusion, this study indicated that probiotic L. plantarum MR1 ameliorated high-carbohydrate diet-induced hepatic lipid accumulation and oxidative stress by increasing the circulating uridine, suggesting that intestinal microbiota can regulate the metabolism of nucleotides to maintain host physiological homeostasis.

Peroxisome proliferator-activated receptor gamma is essential for stress adaptation by maintaining lipid homeostasis in female fish.

Reduction of lipid synthesis often causes free fatty acid (FFA) overload, resulting consequential oxidative stress and health damage. Environmental stresses also induce cellular oxidative stress in organisms. The functional peroxisome proliferator-activated receptor gamma (pparg) gene is essential for lipid synthesis and homeostatic lipid maintenance. However, the relationship between the pparg-mediated lipid synthesis and environmental stress adaptation awaits full elucidation. Here, we generated a pparg-knockout zebrafish model. The conversion of free fatty acids into triglycerides in the female pparg mutants was hampered by reduced esterification efficiency, thus induced lipotoxicity, as evidenced by high oxidative stress and damaged health in these mutants, which led to reduced resistance to cold, heat and ammonia nitrogen stresses. Activating pparg in the wild-type female fish via dietary supplementation with rosiglitazone (a pparg agonist), or reducing oxidative stress in the female pparg mutants via dietary supplementation with N-acetylcysteine (an antioxidant), or promoting mitochondrial fatty acid ß-oxidation in the female pparg mutants via dietary supplementation with l-carnitine, resulted in significantly reduced cellular injury, and improved environmental stress resistance. Collectively, our findings reveal that the regulative function of pparg in FFA esterification is important in stress resistance in female fish, and highlight the tight correlation existing between lipotoxicity and environmental adaptation.

Dietary l-carnitine supplementation recovers the increased pH and hardness in fillets caused by high-fat diet in Nile tilapia (Oreochromis niloticus).

The wide use of high-fat diet (HFD) causes negative effects on flesh quality in farmed fish. l-carnitine, a lipid-lowering additive, enhances mitochondrial fatty acid ß-oxidation. However its roles in alleviating the effects of HFD on flesh quality in fish are unknown. We fed Nile tilapia with medium-fat diet (MFD, 6% dietary lipid), high-fat diet (HFD, 12% dietary lipid) and HFCD supplemented with l-carnitine (HFCD + 400 mg/kg l-carnitine) for 10 weeks. The HFD-fed fish had higher fat deposition, pH value, myofiber density and flesh hardness than those fed on MFD. However, feeding the fish with the HFCD improved lipid catabolism, which increased significantly lactic acid content and myofiber diameter in muscle, thus reduced pH and hardness values. HFCD also reduced endoplasmic reticulum stress and myofiber apoptosis caused by HFD in the fish. Our study suggests that dietary l-carnitine supplementation alleviates the negative effects of HFD on flesh quality of farmed fish.

The Adaptive Characteristics of Cholesterol and Bile Acid Metabolism in Nile Tilapia Fed a High-Fat Diet.

Since high-fat diet (HFD) intake elevates liver cholesterol and enhanced cholesterol-bile acid flux alleviates its lipid deposition, we assumed that the promoted cholesterol-bile acid flux is an adaptive metabolism in fish when fed an HFD. The present study investigated the characteristic of cholesterol and fatty acid metabolism in Nile tilapia (Oreochromis niloticus) after feeding an HFD (13% lipid level) for four and eight weeks. Visually healthy Nile tilapia fingerlings (average weight 3.50 ± 0.05 g) were randomly distributed into four treatments (4-week control diet or HFD and 8-week control diet or HFD). The liver lipid deposition and health statue, cholesterol/bile acid, and fatty acid metabolism were analyzed in fish after short-term and long-term HFD intake. The results showed that 4-week HFD feeding did not change serum alanine transaminase (ALT) and aspartate transferase (AST) enzyme activities, along with comparable liver malondialdehyde (MDA) content. But higher serum ALT and AST enzyme activities and liver MDA content were observed in fish fed 8-week HFD. Intriguingly, remarkably accumulated total cholesterol (mainly cholesterol ester, CE) was observed in the liver of fish fed 4-week HFD, along with slightly elevated free fatty acids (FFAs) and comparable TG contents. Further molecular analysis in the liver showed that obvious accumulation of CE and total bile acids (TBAs) in fish fed 4-week HFD was mainly attributed to the enhancement of cholesterol synthesis, esterification, and bile acid synthesis. Furthermore, the increased protein expressions of acyl-CoA oxidase 1/2 (Acox1 and Acox2), which serve as peroxisomal fatty acid ß-oxidation (FAO) rate-limiting enzymes and play key roles in the transformation of cholesterol into bile acids, were found in fish after 4-week HFD intake. Notably, 8-week HFD intake remarkably elevated FFA content (about 1.7-fold increase), and unaltered TBAs were found in fish liver, accompanied by suppressed Acox2 protein level and cholesterol/bile acid synthesis. Therefore, the robust cholesterol-bile acid flux serves as an adaptive metabolism in Nile tilapia when fed a short-term HFD and is possibly via stimulating peroxisomal FAO. This finding enlightens our understanding on the adaptive characteristics of cholesterol metabolism in fish fed an HFD and provides a new possible treatment strategy against metabolic disease induced by HFD in aquatic animals.

Bacillus amyloliquefaciens ameliorates high-carbohydrate diet-induced metabolic phenotypes by restoration of intestinal acetate-producing bacteria in Nile Tilapia.

Poor utilisation efficiency of carbohydrate always leads to metabolic phenotypes in fish. The intestinal microbiota plays an important role in carbohydrate degradation. Whether the intestinal bacteria could alleviate high-carbohydrate diet (HCD)-induced metabolic phenotypes in fish remains unknown. Here, a strain affiliated to Bacillus amyloliquefaciens was isolated from the intestine of Nile tilapia. A basal diet (CON), HCD or HCD supplemented with B. amy SS1 (HCB) was used to feed fish for 10 weeks. The beneficial effects of B. amy SS1 on weight gain and protein accumulation were observed. Fasting glucose and lipid deposition were decreased in the HCB group compared with the HCD group. High-throughput sequencing showed that the abundance of acetate-producing bacteria was increased in the HCB group relative to the HCD group. Gas chromatographic analysis indicated that the concentration of intestinal acetate was increased dramatically in the HCB group compared with that in the HCD group. Glucagon-like peptide-1 was also increased in the intestine and serum of the HCB group. Thus, fish were fed with HCD, HCD supplemented with sodium acetate at 900 mg/kg (HLA), 1800 mg/kg (HMA) or 3600 mg/kg (HHA) diet for 8 weeks, and the HMA and HHA groups mirrored the effects of B. amy SS1. This study revealed that B. amy SS1 could alleviate the metabolic phenotypes caused by HCD by enriching acetate-producing bacteria in fish intestines. Regulating the intestinal microbiota and their metabolites might represent a powerful strategy for fish nutrition modulation and health maintenance in future.

Lipolysis and lipophagy play individual and interactive roles in regulating triacylglycerol and cholesterol homeostasis and mitochondrial form in zebrafish.

Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl-/-; AKO fish) or lysosomal acid lipase (lal-/-; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.

The reduction of lipid-sourced energy production caused by ATGL inhibition cannot be compensated by activation of HSL, autophagy, and utilization of other nutrients in fish.

The adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL)-mediated lipolysis play important roles in lipid catabolism. ATGL is considered the central rate-limiting enzyme in the mobilization of fatty acids in mammals. Currently, severe fat accumulation has been commonly detected in farmed fish globally. However, the ATGL-mediated lipolysis and the potential synergy among ATGL, HSL, and autophagy, which is another way for lipid breakdown, have not been intensively understood in fish. In the present study, we added Atglistatin as an ATGL-specific inhibitor into the zebrafish diet and fed to the fish for 5 weeks. The results showed that the Atglistatin-treated fish exhibited severe fat deposition, reduced oxygen consumption, and fatty acid ß-oxidation, accompanied with increased oxidative stress and inflammation. Furthermore, the Atglistatin-treated fish elevated total and phosphorylation protein expressions of HSL. However, the free fatty acids and lipase activities in organs were still systemically reduced in the Atglistatin-treated fish, and the autophagy marker LC3 was also decreased in the liver. On the other hand, glycogenolysis was stimulated but blood glucose was higher in the Atglistatin-treated fish. The transcriptomic analysis also provided the hint that the protein turnover efficiency in Atglistatin-treated fish was likely to be accelerated, but the protein content in whole fish was not affected. Taken together, ATGL plays crucial roles in energy homeostasis such that its inhibition causes loss of lipid-sourced energy production, which cannot be compensated by activation of HSL, autophagy, and utilization of other nutrients.

The individual and combined effects of hypoxia and high-fat diet feeding on nutrient composition and flesh quality in Nile tilapia (Oreochromis niloticus).

Hypoxia and high-fat diet (HFD) feeding are two factors commonly existing in aquaculture. However, their individual and combined effects on nutrient composition and flesh quality in fish have not been investigated. The present study evaluated the alterations of growth, nutrient composition and flesh quality in Nile tilapia (initially 7.0 ± 0.1 g and 5.6 ± 0.2 cm) fed with normal fat diet (5.95% fat) or HFD (11.8% fat) at two dissolved oxygen levels (1.1 ± 0.1 and 7.2 ± 0.1 mg/L) for 8 weeks. The results showed that hypoxia and HFD had similar effects in inducing lipid deposition, reducing flesh protein and amino acids content, pH values and water holding ability. Hypoxia had additional adverse effects in decreasing meat yield, flesh contents of n-3 PUFA and glycogen, increasing flesh fragmentation and causing liver damages. The combination of hypoxia and HFD significantly decreased feed intake, survival rate and muscle protein content, but didn't affect flesh quality-related parameters.

Inulin alleviates adverse metabolic syndrome and regulates intestinal microbiota composition in Nile tilapia (Oreochromis niloticus) fed with high-carbohydrate diet.

A high-carbohydrate diet could achieve a protein-sparing effect, but it may cause negative impacts on the growth condition of fish due to their poor utilisation ability of carbohydrate. How to reduce the adverse effects caused by a high-carbohydrate diet is important for the development of aquaculture. In the present study, we aimed to identify whether inulin could attenuate the metabolic syndrome caused by a high-carbohydrate diet in fish. Nile tilapia (Oreochromis niloticus) (1·19 (sd 0·01) g) were supplied with 35 % carbohydrate (CON), 45 % carbohydrate (HC) and 45 % carbohydrate + 5 g/kg inulin (HCI) diets for 10 weeks. The results showed that addition of inulin improved the survival rate when fish were challenged with Aeromonas hydrophila, indicating that inulin had an immunostimulatory effect. Compared with the HC group, the HCI group had lower lipid accumulation in liver and the gene expression analyses indicated that addition of inulin down-regulated genes related to lipogenesis and up-regulated genes relevant to ß-oxidation significantly (P < 0·05). Higher liver glycogen and glucose tolerance were found in the HCI group compared with the HC group (P < 0·05). These results indicated that inulin could alleviate the metabolic syndrome induced by a high-carbohydrate diet. Furthermore, addition of inulin to a high-carbohydrate diet changed the intestinal bacterial composition and significantly increased the concentration of acetic acid and propionic acid in fish gut which have the potential to increase pathogen resistance and regulate metabolic characteristics in fish. Collectively, our results demonstrated a possible causal role for the gut microbiome in metabolic improvements induced by inulin in fish.