Inhibition of mitochondrial citrate shuttle alleviates metabolic syndromes induced by high-fat diet.

The mitochondrial citrate shuttle, which relies on the solute carrier family 25 member 1 (SLC25A1), plays a pivotal role in transporting citrate from the mitochondria to the cytoplasm. This shuttle supports glycolysis, lipid biosynthesis, and protein acetylation. Previous research has primarily focused on SLC25A1 in pathological models, particularly high-fat diet (HFD)-induced obesity. However, the impact of SLC25A1 inhibition on nutrient metabolism under HFD remains unclear. To address this gap, we used zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) to evaluate the effects of inhibiting Slc25a1. In zebrafish, we administered Slc25a1-specific inhibitors (CTPI-2) for 4 wk, whereas Nile tilapia received intraperitoneal injections of dsRNA to knock down slc25a1b for 7 days. Inhibition of the mitochondrial citrate shuttle effectively protected zebrafish from HFD-induced obesity, hepatic steatosis, and insulin resistance. Of note, glucose tolerance was unaffected. Inhibition of Slc25a1 altered hepatic protein acetylation patterns, with decreased cytoplasmic acetylation and increased mitochondrial acetylation. Under HFD conditions, Slc25a1 inhibition promoted fatty acid oxidation and reduced hepatic triglyceride (TAG) accumulation by deacetylating carnitine palmitoyltransferase 1a (Cpt1a). In addition, Slc25a1 inhibition triggered acetylation-induced inactivation of Pdhe1α, leading to a reduction in glucose oxidative catabolism. This was accompanied by enhanced glucose uptake and storage in zebrafish livers. Furthermore, Slc25a1 inhibition under HFD conditions activated the SIRT1/PGC1α pathway, promoting mitochondrial proliferation and enhancing oxidative phosphorylation for energy production. Our findings provide new insights into the role of nonhistone protein acetylation via the mitochondrial citrate shuttle in the development of hepatic lipid deposition and hyperglycemia caused by HFD.NEW & NOTEWORTHY The mitochondrial citrate shuttle is a crucial physiological process for maintaining metabolic homeostasis. In the present study, we found that inhibition of mitochondrial citrate shuttle (Slc25a1) could alleviate metabolic syndromes induced by high-fat diet (HFD) through remodeling hepatic protein acetylation modification. Briefly, Slc25a1 inhibition reduces hepatic triglyceride deposition by deacetylating Cpt1a and reduces glucose oxidative catabolism by acetylating Pdhe1α. Our study provides new insights into the treatment of diet-induced metabolic syndromes.

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.

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 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.

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.

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.

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.

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.

Peroxisomal proliferator-activated receptor α-b deficiency induces the reprogramming of nutrient metabolism in zebrafish.

KEY POINTS: The pparab subtype in zebrafish is much more highly expressed in tissues with high oxidative activity than pparaa. The pparab deficiency in zebrafish reduces fatty acid ß-oxidation both in liver and muscle, illustrating its functional homology as a mammalian peroxisome proliferator-activated receptor α (PPARα). pparab deficiency promotes metabolic reprogramming by increasing glucose utilization and inhibiting amino acid breakdown. The present study brings new insights into the comprehensive regulatory roles of PPARα in the cellular fuel selection and provides a valuable animal model for PPARα studies from a viewpoint of comparative physiology. ABSTRACT: Dysfunction of lipid metabolism is involved in the pathogenesis of several chronic metabolic diseases. Peroxisome proliferator-activated receptor α (PPARα) is essential for normal metabolic homeostasis and, in particular, for the regulation of fatty acid ß-oxidation (FAO). However, little is known about its regulation roles in systemic nutrient metabolism. To explore the underlying modulation role of PPARα in metabolic homeostasis, we generated a pparab-knockout zebrafish (Danio rerio) model. The pparab mutants demonstrated lower expression of key enzymes involved in FAO, as well as lower mitochondrial and peroxisomal FAO in tissues, which was associated with lipid accumulation in liver and visceral mass. Conversely, glucose utilization was higher because they demonstrated lower blood glucose and tissue glycogen concentrations, as well as activation of the phosphoinositide 3-kinase/AKT pathway. In addition, pparab-deficient zebrafish demonstrated activation of AKT/mammalian target of rapamycin signalling and higher protein content, implying greater protein synthesis and/or lower amino acid breakdown. These data clearly revealed that pparab deletion reduces FAO but increases glucose utilization and protein deposition to maintain energy homeostasis. The present study provides new insights into the comprehensive regulatory role of PPARα in systemic energy metabolism in fish, and this pparab-deficient zebrafish also constitutes a valuable model for investigating the functions of PPARα in mammals from comparative physiology aspects.

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.

Gemfibrozil improves lipid metabolism in Nile tilapia Oreochromis niloticus fed a high-carbohydrate diet through peroxisome proliferator activated receptor-α activation.

High carbohydrate diet (HCD) can induce lipid metabolism disorder, characterized by excessive lipid in farmed fish. Peroxisome proliferator activated receptor-α (PPARα) plays an important role in lipid homeostasis. In this study, we hypothesize that PPARα can improve lipid metabolism in fish fed HCD. Fish (3.03 ± 0.11 g) were fed with three diets: control (30% carbohydrate), HCD (45% carbohydrate) and HCG (HCD supplemented with 200 mg/kg gemfibrozil, an agonist of PPARα) for eight weeks. The fish fed HCG had higher growth rate and protein effiency than those fed the HCD diet, whereas the opposite trend was observed in feed conversion ratio, hepatosomatic index and mesenteric fat index. Additionally, fish fed HCG significantly decreased lipid accumulation in the whole body, liver and adipose tissues compared to those fed the HCD diet. Furthermore, fish in the HCG group significantly increased the mRNA and protein expression and protein dephosphorylation of PPARα. The HCG group also significantly increased the mRNA level of the downstream target genes of PPARα, whereas the opposite trend occured in the mRNA level of lipolysis-related genes compared to the HCD group. Besides, fish in the HCG group remarkably decreased the contents of alanine aminotransferase, aspartate aminotransferase and malondialdehyde, whereas the opposite occured in the activities of antioxidative enzymes and anti-inflammatory cytokine genes compared to the HCD group. This study indicates that gemfibrozil can improve lipid metabolism and maintain high antioxidant and anti-inflammatory capacity through activating PPARα in Nile tilapia fed a high carbohydrate diet.

High-carbohydrate diet promotes the adaptation to acute hypoxia in zebrafish.

Oxygen deprivation (hypoxia) is a common challenge in water environment, which causes lack of energy and oxidative damage in organisms. Many studies have indicated a number of physiological and metabolic changes under hypoxia, but the effects of dietary nutrients on hypoxia tolerance have not been well evaluated. In the present 7-week feeding trial, we fed zebrafish with low-protein diet (LP), high-protein diet (HP), low-fat diet (LF), high-fat diet (HF), low-carbohydrate diet (LC), and high-carbohydrate diet (HC), respectively. Afterward, the resistance to acute hypoxia challenge, growth, body composition, activities of metabolic enzymes, and expressions of energy homeostasis-related genes and six hifαs genes were measured. The results indicated that only the HC diet could significantly improve the resistance to hypoxia challenge. Moreover, the HC diet feeding caused higher glycogen deposition in the liver and muscle, and these glycogens were significantly reduced after 6-h acute hypoxia challenge. Meanwhile, the lactate content in the liver and blood was increased in the HC groups. At hypoxia status, the relative mRNA expressions of the genes related to glycolysis, ATP production, insulin signaling pathway, and hif-3a (hif1al) were all significantly increased in the muscle of the HC diet-fed fish. This study revealed that high-carbohydrate diet could improve the resistance to hypoxia by activating glycolysis and hif/insulin signaling pathway in zebrafish, mainly in the muscle, to efficiently supply energy. Therefore, our results highlight the importance of dietary carbohydrate in resisting hypoxia in fish.

Impaired peroxisomal fat oxidation induces hepatic lipid accumulation and oxidative damage in Nile tilapia.

Many metabolic diseases in fish are often associated with lowered peroxisomal fatty acid (FA) ß-oxidation. However, the physiological role of peroxisomal FA oxidation in lipid metabolism in fish still remains unclear. In the present study, a specific peroxisomal FA ß-oxidation inhibitor, 10,12-tricosadiynoic acid (TDYA), was used to investigate the effects of impaired peroxisomal ß-oxidation on growth performance, health status, and lipid metabolism in Nile tilapia. The results showed that the dietary TDYA treatment did not affect weight gain, but significantly decreased peroxisomal ß-oxidation in the liver, and increased body fat accumulation. The fish with impaired peroxisomal ß-oxidation exhibited higher contents of serum lipid and peroxidation products, and alanine aminotransferase activity, and significantly lowered hepatic activities of superoxide dismutase and catalase. The inhibited peroxisomal ß-oxidation did not enhance mitochondrial ß-oxidation activity, but compensatorily upregulated FA ß-oxidation-related gene expression, and downregulated the gene expressions in lipolysis and lipogenesis. Taken together, TDYA treatment markedly induced lipid accumulation and hepatic oxidative damage via systemically depressing lipid catabolism and antioxidant capacity. Our findings reveal the pivotal roles of peroxisomal ß-oxidation in maintaining health and lipid homeostasis in fish, and could be helpful in understanding metabolic diseases in fish.

Fasting enhances cold resistance in fish through stimulating lipid catabolism and autophagy.

KEY POINTS: In a cold environment, mammals increase their food intake while fish decrease or stop feeding. However, the physiological value of fasting during cold resistance in fish is currently unknown. Fasting for more than 48 h enhanced acute cold resistance in zebrafish, which correlated with lipid catabolism and cell damage attenuation. Lipid catabolism and autophagy were necessary for cold resistance in fish and the inhibition of mitochondrial fatty acid ß-oxidation or autophagy weakened the fasting-induced cold resistance. Repression of mechanistic target of rapamycin (mTOR) signalling pathway by rapamycin largely mimicked the beneficial effects of fasting in promoting cold resistance, suggesting mTOR signalling may be involved in the fasting-induced cold resistance in fish. Our study demonstrates that fasting may be a protective strategy for fish to survive under cold stress. ABSTRACT: In cold environments, most homeothermic animals increase their food intake to supply more energy to maintain body temperature, whereas most poikilothermic animals such as fishes decrease or even stop feeding under cold stress. However, the physiological value of fasting during cold resistance in poikilotherms has not been explained. Here, we show that moderate fasting largely enhanced cold resistance in fish. By using pharmacological (fenofibrate, mildronate, chloroquine and rapamycin) and nutritional approaches (fatty acids diets and amino acids diets) in wild-type or specific gene knock-out zebrafish models (carnitine palmitoyltransferase-1b-deficient strain, CPT1b-/- , or autophagy-related protein 12-deficient strain, ATG12-/- ), we verified that fasting-stimulated lipid catabolism and autophagy played essential roles in the improved cold resistance. Moreover, suppression of the mechanistic target of rapamycin (mTOR) pathway by using rapamycin mostly mimicked the beneficial effects of fasting in promoting cold resistance as either the physiological phenotype or transcriptomic pattern. However, these beneficial effects were largely reduced when the mTOR pathway was activated through high dietary leucine supplementation. We conclude that fasting helps fish to resist cold stress by modulating lipid catabolism and autophagy, which correlates with the mTOR signalling pathway. Therefore, fasting can act as a protective strategy of fish in resisting coldness.

Functional differences between l- and d-carnitine in metabolic regulation evaluated using a low-carnitine Nile tilapia model.

l-Carnitine is essential for mitochondrial ß-oxidation and has been used as a lipid-lowering feed additive in humans and farmed animals. d-Carnitine is an optical isomer of l-carnitine and dl-carnitine has been widely used in animal feeds. However, the functional differences between l- and d-carnitine are difficult to study because of the endogenous l-carnitine background. In the present study, we developed a low-carnitine Nile tilapia model by treating fish with a carnitine synthesis inhibitor, and used this model to investigate the functional differences between l- and d-carnitine in nutrient metabolism in fish. l- or d-carnitine (0·4 g/kg diet) was fed to the low-carnitine tilapia for 6 weeks. l-Carnitine feeding increased the acyl-carnitine concentration from 3522 to 10 822 ng/g and alleviated the lipid deposition from 15·89 to 11·97 % in the liver of low-carnitine tilapia. However, as compared with l-carnitine group, d-carnitine feeding reduced the acyl-carnitine concentration from 10 822 to 5482 ng/g, and increased lipid deposition from 11·97 to 20·21 % and the mRNA expression of the genes involved in ß-oxidation and detoxification in the liver. d-Carnitine feeding also induced hepatic inflammation, oxidative stress and apoptosis. A metabolomic investigation further showed that d-carnitine feeding increased glycolysis, protein metabolism and activity of the tricarboxylic acid cycle and oxidative phosphorylation. Thus, l-carnitine can be physiologically utilised in fish, whereas d-carnitine is metabolised as a xenobiotic and induces lipotoxicity. d-Carnitine-fed fish demonstrates increases in peroxisomal ß-oxidation, glycolysis and amino acid degradation to maintain energy homeostasis. Therefore, d-carnitine is not recommended for use in farmed animals.

The comparisons in protective mechanisms and efficiencies among dietary α-lipoic acid, ß-glucan and l-carnitine on Nile tilapia infected by Aeromonas hydrophila.

Dietary α-lipoic acid (LA), ß-glucan (Gluc) and l-carnitine (L-Ca) are commonly used additives to promote fish growth and stress resistance in aquaculture production. However their mechanisms and efficiencies in helping fish to resist diseases have not been compared before. In this study, we fed Nile tilapia (Oreochromis niloticus) with diets containing appropriate doses of LA, Gluc and L-Ca for five weeks and further intraperitoneally injected the fish with Aeromonas hydrophila. After dietary treatment, none of the additives affected the fish growth, but dietary Gluc and L-Ca reduced protein and lipid body contents in fish, respectively. After A. hydrophila challenge, all fish treated with the three dietary additives showed higher survival rate, but those fed on dietary L-Ca had lower survival than those fed on LA and Gluc diets, indicating high protection efficiency of LA and Gluc. The protective mechanisms of the three feed additives were quite different under A. hydrophila infection. Dietary LA induced higher total antioxidant capacity and higher mRNA expression of anti-oxidative genes than other additives in liver and also activated partly the immune function in serum and spleen. Gluc largely increased the immune function by activating the immunity enzymes in serum, inducing inflammation in liver and increasing the expression of immune genes in spleen and head kidney. Gluc also increased partly the antioxidant capacity in serum and liver and lipid catabolism in liver. L-Ca largely increased lipid catabolism in liver while it increased partly the antioxidant capacities in serum and liver. Taken together, these results indicate that, dietary LA, Gluc and L-Ca have various protective mechanisms and differ in their efficiencies on resisting A. hydrophila infection in Nile tilapia.

PPARα activation enhances the ability of nile tilapia (Oreochromis niloticus) to resist Aeromonas hydrophila infection.

Peroxisome proliferator-activated receptor α (PPARα) plays critical physiological roles in energy metabolism, antioxidation and immunity of mammals, however, these functions have not been fully understood in fish. In the present study, Nile tilapia (Oreochromis niloticus) were fed with fenofibrate, an agonist of PPARα, for six weeks, and subsequently challenged with Aeromonas hydrophila. The results showed that PPARα was efficiently activated by fenofibrate through increasing mRNA and protein expressions and protein dephosphorylation. PPARα activation increased significantly mitochondrial fatty acid ß-oxidation efficiency, the copy number of mitochondrial DNA and expression of monoamine oxidase (MAO), a marker gene of mitochondria. Meanwhile, PPARα activation also increased significantly the expression of NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 9 (NDUFA9, complex I) and mitochondrial cytochrome c oxidase 1 (MTCO1, complex IV). The fenofibrate-fed fish had higher survival rate when exposed to A. hydrophila. Moreover, the fenofibrate-fed fish also had higher activities of immune and antioxidative enzymes, and gene expressions of anti-inflammatory cytokines, while had lower expressions of pro-inflammatory cytokine genes. Taken together, PPARα activation improved the ability of Nile tilapia to resist A. hydrophila, mainly through enhancing mitochondrial fatty acids ß-oxidation, immune and antioxidant capacities, as well as inhibiting inflammation. This is the first study showing the regulatory effects of PPARα activation on immune functions through increasing mitochondria-mediated energy supply in fish.

Forskolin reduces fat accumulation in Nile tilapia (Oreochromis niloticus) through stimulating lipolysis and beta-oxidation.

High fat diets are commonly used in aquaculture to reduce feed cost in Nile tilapia, but impair its lipid homeostasis. This study evaluated the role of forskolin on reducing fat accumulation in Nile tilapia (Oreochromis niloticus) by using in vitro and in vivo experiments. The use of 50 µM forskolin in vitro increased free fatty acid and glycerol release, but decreased triglyceride in adipocytes and hepatocytes. The adipose triglyceride lipase (ATGL), protein kinase cAMP-dependent type I regulatory subunit alpha (PKAR I) and other genes related to ß-oxidation (peroxisome proliferator activated receptor alpha, PPARα and carnitine O-palmitoyltransferase 1, CPT1) were significantly up-regulated. After feeding a high-fat diet for six weeks, O. niloticus were fed with 0 (control), 0.5 and 1.5 mg/kg forskolin for two weeks to determine whether forskolin could reduce fat accumulation in vivo. Fish fed the two levels of forskolin decreased significantly the hepatosomatic and mesenteric fat indices. The total lipid in the whole fish and liver together with the serum glycerol content were lower in fish fed on forskolin than in the control. The fish fed on forskolin diets exhibited smaller areas of lipid droplets in adipose and liver tissues. Lipolysis related genes (ATGL, hormone-sensitive lipase, HSL; monoacylglycerol lipase, MGL; and protein kinase cAMP-activated catalytic subunit, PKAC) and ß-oxidation genes (PPARα; fatty acid binding protein 1, FABP1; and CPT1) in the adipose were up-regulated. Similarly, in the liver lipolysis genes such as ATGL and PKAR I and ß-oxidation genes (PPARα, FABP1, CPT1 and acyl-CoA oxidase, ACO) showed an increasing trend with the increase of forskolin doses. This study indicates that forskolin can reduce fat accumulation in the adipose and liver by stimulating lipolysis and ß-oxidation in O. niloticus.

Inhibited autophagy impairs systemic nutrient metabolism in Nile tilapia.

Autophagy is a conserved cellular degradation process through which intracellular components are degraded by the lysosome, but its roles in fish metabolism have not been studied in depth. Therefore, the present study aimed to investigate whether autophagy plays a key role in maintaining metabolic homeostasis in fish. In an 8-week feeding trial, Nile tilapia were fed either a control diet with medium fat and medium carbohydrate (Control), or a control diet supplemented with a classic autophagy inhibitor (chloroquine, CQ). CQ supplementation significantly inhibited autophagy and impaired fish growth and protein synthesis, and the glycolysis was stimulated, accompanied by fat accumulation, high oxidative stress and inflammation. Physiological status and gene expressions suggested that impaired autophagy might be at least one cause of the metabolic diseases which has been commonly seen in aquaculture. These results indicate that inhibition of autophagy could significantly affect the metabolism of lipid, carbohydrate and protein in fish; hence, autophagy could play important roles in maintaining homeostasis of nutrient metabolism in cultured fish.

Reduced oxidative stress increases acute cold stress tolerance in zebrafish.

Cold stress is a major threat to fish in both nature and aquaculture, and can induce oxidative stress in various fish. While the exact role of oxidative stress in cold-caused mortality is still unknown. The purpose of the present study was to evaluate the effects of oxidative stress on cold tolerance in fish and verify whether changing oxidative status could affect cold tolerance. We firstly demonstrated that acute cold exposure induced high oxidative stress in zebrafish liver, which may lead to mortality. Then we performed in vivo and in vitro experiments to determine the effects of the altered oxidative status on cold tolerance in zebrafish and zebrafish liver cell line (ZFL), respectively. In the in vivo study, the zebrafish which were fed with α-lipoic acid or reduced glutathione had lower cold-caused oxidative stress and tissues damage, and showed higher cold tolerance. In the experiment using zebrafish cells, increasing oxidative stress by H2O2 decreased the cellular cold tolerance, and the cold tolerance was partly recovered when oxidative stress was reduced by the addition of Vitamin C (VC). Taken together, we conclude that the reduction of oxidative stress increases cold tolerance in fish.