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.

Pparα activation stimulates autophagic flux through lipid catabolism-independent route.

Autophagy is a cellular process that involves the fusion of autophagosomes and lysosomes to degrade damaged proteins or organelles. Triglycerides are hydrolyzed by autophagy, releasing fatty acids for energy through mitochondrial fatty acid oxidation (FAO). Inhibited mitochondrial FAO induces autophagy, establishing a crosstalk between lipid catabolism and autophagy. Peroxisome proliferator-activated receptor α (PPARα), a transcription factor, stimulates lipid catabolism genes, including fatty acid transport and mitochondrial FAO, while also inducing autophagy through transcriptional regulation of transcription factor EB (TFEB). Therefore, the study explores whether PPARα regulates autophagy through TFEB transcriptional control or mitochondrial FAO. In aquaculture, addressing liver lipid accumulation in fish is crucial. Investigating the link between lipid catabolism and autophagy is significant for devising lipid-lowering strategies and maintaining fish health. The present study investigated the impact of dietary fenofibrate and L-carnitine on autophagy by activating Pparα and enhancing FAO in Nile tilapia (Oreochromis niloticus), respectively. The dietary fenofibrate and L-carnitine reduced liver lipid content and enhanced ATP production, particularly fenofibrate. FAO enhancement by L-carnitine showed no changes in autophagic protein levels and autophagic flux. Moreover, fenofibrate-activated Pparα promoted the expression and nuclear translocation of Tfeb, upregulating autophagic initiation and lysosomal biogenesis genes. Pparα activation exhibited an increasing trend of LC3II protein at the basal autophagy and cumulative p62 protein trends after autophagy inhibition in zebrafish liver cells. These data show that Pparα activation-induced autophagic flux should be independent of lipid catabolism.

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.

The dysfunction of hormone-sensitive lipase induces lipid deposition and reprogramming of nutrient metabolism in fish.

Hormone-sensitive lipase (HSL) is one of the rate-determining enzymes in the hydrolysis of TAG, playing a crucial role in lipid metabolism. However, the role of HSL-mediated lipolysis in systemic nutrient homoeostasis has not been intensively understood. Therefore, we used CRISPR/Cas9 technique and Hsl inhibitor (HSL-IN-1) to establish hsla-deficient (hsla-/-) and Hsl-inhibited zebrafish models, respectively. As a result, the hsla-/- zebrafish showed retarded growth and reduced oxygen consumption rate, accompanied with higher mRNA expression of the genes related to inflammation and apoptosis in liver and muscle. Furthermore, hsla-/- and HSL-IN-1-treated zebrafish both exhibited severe fat deposition, whereas their expressions of the genes related to lipolysis and fatty acid oxidation were markedly reduced. The TLC results also showed that the dysfunction of Hsl changed the whole-body lipid profile, including increasing the content of TG and decreasing the proportion of phospholipids. In addition, the systemic metabolic pattern was remodelled in hsla-/- and HSL-IN-1-treated zebrafish. The dysfunction of Hsl lowered the glycogen content in liver and muscle and enhanced the utilisation of glucose plus the expressions of glucose transporter and glycolysis genes. Besides, the whole-body protein content had significantly decreased in the hsla-/- and HSL-IN-1-treated zebrafish, accompanied with the lower activation of the mTOR pathway and enhanced protein and amino acid catabolism. Taken together, Hsl plays an essential role in energy homoeostasis, and its dysfunction would cause the disturbance of lipid catabolism but enhanced breakdown of glycogen and protein for energy compensation.

PDK inhibition promotes glucose utilization, reduces hepatic lipid deposition, and improves oxidative stress in largemouth bass (Micropterus salmoides) by increasing pyruvate oxidative phosphorylation.

In omnivorous fish, the pyruvate dehydrogenase kinases (PDKs)-pyruvate dehydrogenase E1α subunit (PDHE1α) axis is essential in the regulation of carbohydrate oxidative catabolism. Among the existing research, the role of the PDKs-PDHE1α axis in carnivorous fish with poor glucose utilization is unclear. In the present study, we determined the effects of PDK inhibition on the liver glycolipid metabolism of largemouth bass (Micropterus salmoides). DCA is a PDK-specific inhibitor that inhibits PDK by binding the allosteric sites. A total of 160 juvenile largemouth bass were randomly divided into two groups, with four replicates of 20 fish each, fed a control diet and a control diet supplemented with dichloroacetate (DCA) for 8 weeks. The present results showed that DCA supplementation significantly decreased the hepatosomatic index, triglycerides in liver and serum, and total liver lipids of largemouth bass compared with the control group. In addition, compared with the control group, DCA treatment significantly down-regulated gene expression associated with lipogenesis. Furthermore, DCA supplementation significantly decreased the mRNA expression of pdk3a and increased PDHE1α activity. In addition, DCA supplementation improved glucose oxidative catabolism and pyruvate oxidative phosphorylation (OXPHOS) in the liver, as evidenced by low pyruvate content in the liver and up-regulated expressions of glycolysis-related and TCA cycle/OXPHOS-related genes. Moreover, DCA consumption decreased hepatic malondialdehyde (MDA) content, enhanced the activities of superoxide dismutase (SOD), and increased transforming growth factor beta (tgf-ß), glutathione S-transferase (gst), and superoxide dismutase 1 (sod1) gene expression compared with the control diet. This study demonstrated that inhibition of PDKs by DCA promoted glucose utilization, reduced hepatic lipid deposition, and improved oxidative stress in largemouth bass by increasing pyruvate OXPHOS. Our findings contribute to the understanding of the underlying mechanism of the PDKs-PDHE1α axis in glucose metabolism and improve the utilization of dietary carbohydrates in farmed carnivorous fish.

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.

Effects of dietary mannan oligosaccharides (MOS) supplementation on metabolism, inflammatory response and gut microbiota of juvenile Nile tilapia (Oreochromis niloticus) fed with high carbohydrate diet.

High-carbohydrate diet could achieve cost-sparing effect in aquafeed, but it may cause adverse effects on the growth condition or health status of fish. In order to reduce the adverse effects caused by high carbohydrate diet, mannan oligosaccharides (MOS), a commonly used prebiotics, was used as the feed additive to feed juvenile Nile tilapia (Oreochromis niloticus) (1.19 ± 0.01g) for ten weeks. Three treatments including CON (35% carbohydrate diet), HC (45% carbohydrate diet) and HM (45% carbohydrate supplemented diet with 5 g/kg MOS) were involved. The results showed that MOS supplementation increased the weight gain and body length of juvenile Nile tilapia compared with the HC group. Addition of MOS decreased serum glucose and liver glycogen by increasing enzymes activity related to glycolysis. Furthermore, supplementation of MOS decreased the high carbohydrate diet induced triglycerides accumulation in liver by reducing the expression level of genes related to TG synthesis. Dietary MOS also down-regulated the gene expression level of inflammation factors in liver. Intestinal bacterial composition analyses showed that supplementation of MOS in high carbohydrate diet altered the gut microbial composition and enriched pathways related to the glucose metabolism based on KEGG analyses. In general, our results demonstrated that MOS supplementation in high carbohydrate diet could regulate glucose and lipid homeostasis which may be related to the alteration of gut microbiota. These findings shed light on the application of prebiotics to increase the growth performance, alleviate the metabolic disorders and regulate inflammatory response in aquaculture.

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.

Primary nature of brown carbon absorption in a frigid atmosphere with strong haze chemistry.

Severe haze hovered over Harbin during the heating season of 2019-2020, making it one of the ten most polluted Chinese cities in January of 2020. Here we focused on the optical properties and sources of brown carbon (BrC) during the extreme atmospheric pollution periods. Enhanced formation of secondary BrC (BrCsec) was evident as relative humidity (RH) became higher, accompanied with a decrease of ozone but concurrent increases of aerosol water content and secondary inorganic aerosols. These features were generally similar to the characteristics of haze chemistry observed during winter haze events in the North China Plain, and indicated that heterogeneous reactions involving aerosol water might be at play in the formation of BrCsec, despite the low temperatures in Harbin. Although BrCsec accounted for a substantial fraction of brown carbon mass, its contribution to BrC absorption was much smaller (6 vs. 28%), pointing to a lower mass absorption efficiency (MAE) of BrCsec compared to primary BrC. In addition, emissions of biomass burning BrC (BrCBB) were inferred to increase with increasing RH, coinciding with a large drop of temperature. Since both the less absorbing BrCsec and the more absorbing BrCBB increased as RH became higher, the MAE of total BrC were largely unchanged throughout the measurement period. This study unfolded the contrast in the source apportionment results of BrC mass and absorption, and could have implications for the simulation of radiative forcing by brown carbon.

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.

Mildronate triggers growth suppression and lipid accumulation in largemouth bass (Micropterus salmoides) through disturbing lipid metabolism.

Many metabolic diseases in fish are often associated with lowered mitochondrial fatty acid ß-oxidation (FAO). However, the physiological role of mitochondrial FAO in lipid metabolism has not been verified in many carnivorous fish species, for example in largemouth bass (Micropterus salmonids). In the present study, a specific mitochondrial FAO inhibitor, mildronate (MD), was used to investigate the effects of impaired mitochondrial FAO on growth performance, health status, and lipid metabolism of largemouth bass. The results showed that the dietary MD treatment significantly suppressed growth performance and caused heavy lipid accumulation, especially neutral lipid, in the liver. The MD-treated fish exhibited lower monounsaturated fatty acid and higher long-chain polyunsaturated fatty acids in the muscle. The MD treatment downregulated the gene expressions in lipolysis and lipogenesis, as well as the expressions of the genes and some key proteins in FAO without enhancing peroxisomal FAO. Additionally, the MD-treated fish had lower serum aspartate aminotransferase activity and lower pro-inflammation- and apoptosis-related genes in the liver. Taken together, MD treatment markedly induced lipid accumulation via depressing lipid catabolism. Our findings reveal the pivotal roles of mitochondrial FAO in maintaining health and lipid homeostasis in largemouth bass and could be hopeful in understanding metabolic diseases in farmed carnivorous fish.

Influence of dietary sodium taurocholate on the growth performance and liver health of Nile tilapia (Oreochromis niloticus).

Bile acids (BAs) are a class of cholesterol-derived amphipathic molecules approved as new animal feed additives. However, the functional researches mainly focused on BAs mixture, and the influence of the individual BA on fishes was still limited. In the present study, Nile tilapia were fed basal diet with three levels of sodium taurocholate at 0 mg/kg (CON), 300 mg/kg (TCAL), and 600 mg/kg (TCAH) for 8 weeks. The results indicated that addition of sodium taurocholate did not significantly influence the growth performance. Instead, TCAH group had higher cholesterol accumulation with liver fibrosis. In TCAH group, the level of nuclear factor E2-related factor 2 (nrf2) signaling-associated oxidative stress factors significantly increased in the liver. Additionally, fish in TCAH group had the highest expression level of genes encoding endoplasmic reticulum (ER) stress and inflammatory cytokines in the liver. In conclusion, 300 mg/kg of sodium taurocholate did not significantly influence the growth performance of fish, while 600 mg/kg of sodium taurocholate markedly induced cholesterol accumulation and liver injury, suggesting that the application of taurocholic acid in aquafeed should be re-evaluated.

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.

Sodium acetate alleviated high-carbohydrate induced intestinal inflammation by suppressing MAPK and NF-κB signaling pathways in Nile tilapia (Oreochromis niloticus).

With the development of aquaculture industry, high-carbohydrate diet is used to stimulate protein-sparing effect and reduce feed cost. However, fish utilize carbohydrates poorly in general, and instead, high level of carbohydrates in the diet influence the growth condition of fish. How to alleviate the side effects of high carbohydrate diet on fish health has attracted more and more attentions. In the present study, Nile tilapia (Oreochromis niloticus) were fed with 25% and 45% of carbohydrate diet for eight weeks. Higher body weight but lower resistance to pathogen was found in 45% carbohydrate diet group. Higher expression level of inflammation cytokines, increased expression of total NF-κB protein and phosphorylated NF-κB protein (p-NF-κB) were detected in higher carbohydrate group. Concentration of short-chain fatty acids (SCFAs) was measured and the results indicated that high-carbohydrate diet decreased acetate content in the intestine. In order to detect the relationship between the decreased concentration of acetate and lower resistance to pathogen in high-carbohydrate group, 45% of carbohydrate diets (HC) supplemented with different concentrations of sodium acetate (HC + LA, 100 mmol/L; HC + MA, 200 mmol/L; HC + HA, 400 mmol/L) were used to raise Nile Tilapia for eight weeks. The results indicated that addition of 200 mmol/L sodium acetate (HC + MA) reduced the mortality when fish were challenged with Aeromonas hydrophila. Furthermore, we also found that addition of 200 mmol/L sodium acetate mainly inhibited p38 mitogen-activated protein kinase (p38MAPK) and NF-κB phosphorylation to decrease the expression level of inflammation cytokines (IL-8, IL-12, TNF-α and IL-1ß) in the intestine. The present study indicated that certain concentration of sodium acetate could alleviate high-carbohydrate induced intestinal inflammation mainly by suppressing MAPK activation and NF-κB phosphorylation.

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.

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.

Inhibition of intestinal lipases alleviates the adverse effects caused by high-fat diet in Nile tilapia.

Intestinal lipases are fat-digesting enzymes, which play vital roles in lipid absorption in the intestine. To study the regulation of intestinal lipase activity in systemic lipid metabolism in fish, especially in the metabolic diseases caused by high-fat diet (HFD) feeding, we inhibited intestinal lipases in Nile tilapia to investigate the physiological consequences. In the present study, Nile tilapia were firstly fed with HFD (12% fat) for 6 weeks to establish a fatty fish model. Afterwards, Orlistat as a potent intestinal lipase inhibitor was added into the HFD for the following 5-week feeding trial, with two dietary doses (Orlistat16 group, 16 mg/kg body weight; Orlistat32 group, 32 mg/kg body weight). After the trial, both doses of Orlistat treatment significantly reduced intestinal lipase activity, fat absorption, hepatic lipid accumulation, and gene expression of lipogenesis, whereas increased gene expression of lipid catabolism. Moreover, intestinal lipase inhibition increased immune enzyme activities, antioxidant capacity, and gene expression of anti-inflammatory cytokines, whereas lowered gene expression of pro-inflammatory cytokines. Besides, Orlistat could also improve the structure of the intestine and increase expression of intestinal tight-coupling protein. Taken together, intestinal lipase inhibition alleviated the adverse effects caused by HFD in Nile tilapia. Thus, intestinal lipases played key roles in absorbing dietary lipid and could be a promising target in regulating systemic lipid metabolism in fish.