ZnO NPs induce miR-342-5p mediated ferroptosis of spermatocytes through the NF-κB pathway in mice.

BACKGROUND: Zinc oxide nanoparticle (ZnO NP) is one of the metal nanomaterials with extensive use in many fields such as feed additive and textile, which is an emerging threat to human health due to widely distributed in the environment. Thus, there is an urgent need to understand the toxic effects associated with ZnO NPs. Although previous studies have found accumulation of ZnO NPs in testis, the molecular mechanism of ZnO NPs dominated a decline in male fertility have not been elucidated. RESULTS: We reported that ZnO NPs exposure caused testicular dysfunction and identified spermatocytes as the primary damaged site induced by ZnO NPs. ZnO NPs led to the dysfunction of spermatocytes, including impaired cell proliferation and mitochondrial damage. In addition, we found that ZnO NPs induced ferroptosis of spermatocytes through the increase of intracellular chelatable iron content and lipid peroxidation level. Moreover, the transcriptome analysis of testis indicated that ZnO NPs weakened the expression of miR-342-5p, which can target Erc1 to block the NF-κB pathway. Eventually, ferroptosis of spermatocytes was ameliorated by suppressing the expression of Erc1. CONCLUSIONS: The present study reveals a novel mechanism in that miR-342-5p targeted Erc1 to activate NF-κB signaling pathway is required for ZnO NPs-induced ferroptosis, and provide potential targets for further research on the prevention and treatment of male reproductive disorders related to ZnO NPs.

Procalcitonin/Albumin Ratio Predicts the Outcome After Severe Traumatic Brain Injury: A Propensity Score-Matched Analysis.

BACKGROUND: The procalcitonin/albumin ratio (PAR), a novel inflammation-based index, has been reported to predict the prognosis following cardiopulmonary bypass surgery and bacterial infection. However, whether PAR can predict the outcome of patients with severe traumatic brain injury (STBI) has not been fully elucidated. This study aimed to investigate the relationship between serum PAR levels and prognosis at 6 months after STBI. METHODS: We retrospectively enrolled 129 patients diagnosed with STBI and collected relevant clinical and laboratory data. Logistic regression analysis was used to estimate the association of PAR with the prognosis of STBI. The receiver operating characteristics curve was performed to examine the predictive use of PAR for prognosis. Propensity score matching (PSM) analysis was also performed to improve the reliability of the results. The primary outcome measures were expressed as a score on the modified Rankin Scale at 6 months. RESULTS: The unfavorable prognosis group had advanced age, lower Glasgow Coma Scale score, higher rate of cerebral hernia and intracranial infection, higher neutrophil/lymphocyte ratio (NLR) and C-reactive protein/albumin ratio (CAR), elevated PAR, and higher rate of pneumonia. Multivariate analysis showed that PAR (before PSM: odds ratio 3.473, 95% confidence interval 2.983-4.043, P < 0.001; after PSM: odds ratio 5.358, 95% confidence interval 3.689-6.491, P < 0.001) was independently associated with unfavorable outcome. The area under the curve of the PAR for predicting an unfavorable outcome was higher than that of the CAR and NLR. CONCLUSIONS: The PAR might be a novel independent risk factor of the outcome after STBI. Moreover, PAR was a better biomarker in predicting the outcome of patients with STBI than CAR and NLR.

Sodium oxamate reduces lactate production to improve the glucose homeostasis of Micropterus salmoides fed high-carbohydrate diets.

The study was performed to evaluate the effects of the reduced lactate production by sodium oxamate (SO) on growth performance, lactate and glucose and lipid metabolism, and glucose tolerance of Micropterus salmoides fed high-carbohydrate (CHO) diets. In in vitro study, primary hepatocytes were incubated for 48 h in a control medium (5.5 mM glucose), a high-glucose medium (25 mM glucose, HG), or a SO-containing high-glucose medium (25 mM glucose + 50 mM SO, HG-SO). Results indicated lactate and triglyceride (TG) levels, and lactate dehydrogenase a (LDH-a) expression in the HG-SO group were remarkably lower than those of the HG group. In in vivo study, M. salmoides (5.23 ± 0.03 g) were fed four diets containing a control diet (10% CHO, C) and three SO contents [0 (HC), 100 (HC-SO1), and 200 (HC-SO2) mg·kg-1, respectively] of high-CHO diets (20% CHO) for 11 wk. High-CHO diets significantly reduced weight gain rate (WGR), specific growth rate (SGR), p-AMPK-to-t-AMPK ratio, and expression of insulin receptor substrate 1 (IRS1), insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), fructose-1,6-biphosphatase (FBPase), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl transferase 1α (CPT1α) compared with the C group, whereas the opposite was true for plasma levels of glucose, TG, lactate, tissue glycogen, and lipid contents, and expression of LDH-a, monocarboxylate transporter 1 and 4 (MCT1 and MCT4), insulin, glucokinase (GK), pyruvate dehydrogenase E1 subunit (PDH), sterol-regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS). The HC-SO2 diets remarkably increased WGR, SGR, p-AMPK-to-t-AMPK ratio, and expression of IRS1, IGF-I, IGF-IR, GK, PDHα, PDHß, FAS, acetyl-CoA carboxylase 1 (ACC1), PPARα, and CPT1α compared with the HC group. Besides, HC-SO2 diets also enhanced glucose tolerance of fish after a glucose loading. Overall, the reduced lactate production by SO benefits growth performance and glucose homeostasis of high-CHO-fed M. salmoides through the enhancement of glycolysis, lipogenesis, and fatty acid ß-oxidation coupled with the suppression of glycogenesis and gluconeogenesis.

Terrestrial Compound Protein Replacing Dietary Fishmeal Improved Digestive Enzyme Activity, Immune Response, Intestinal Microflora Composition, and Protein Metabolism of Golden Pompano (Trachinotus ovatus).

Terrestrial compound protein (Cpro) can be potentially used to replace fishmeal (FM) in the marine carnivorous teleost, golden pompano (Trachinotus ovatus). Four isonitrogenous (45%) and isolipidic (12%) diets named FM30, AP80, PP80, and CP80 were formulated. FM30 (control) contained 30% FM and 25% basic protein, while AP80, PP80, and CP80 only contained 6% FM, where 80% FM and 25% basic protein of control diet were completely replaced by animal protein, plant protein, and Cpro, respectively. After golden pompano juveniles (initial weight: 10.32 ± 0.09 g) were, respectively, fed the four diets in floating sea cages for 10 weeks, the growth performance, intestinal digestive enzyme activity, and immune responses, protein metabolism indices of the CP80 group were similar to or better than those of the FM30 group (P > 0.05), and significantly better than those of the AP80 and PP80 groups. Specifically, the weight gain (WG), feed conversion ratio (FCR), activity of alanine transaminase (ALT), growth hormone (GH), and insulin-like growth factor-1 (IGF-1) contents of serum, mRNA level of interleukin-10 (il-10), zonula occludens-2 (zo-2), claudin-3, claudin-12, and eukaryotic translation initiation factor 4G (eif4g) were significantly higher, and the activity of α-amylase (AMS), lipase (LPS) in the foregut and midgut, interleukin-8 (il-8) expression in the intestine was significantly lower than that in the CP80 group, compared with those in AP80 and PP80 groups (P < 0.05). Moreover, the intestinal microflora composition of golden pompano fed with the CP80 diet was improved. Specifically, at the phylum level, the relative abundance of harmful bacterial strains cyanobacteria and TM7 of CP80 group was similar to those of FM30 group (P > 0.05), but was significantly lower than those of AP80 and PP80 groups (P < 0.05). At the genus level, the beneficial bacterial strains Agrobacterium and Blantia of CP80 group were also similar to those of FM30 group (P < 0.05), which were significantly higher than those of AP80 and PP80 groups, but the beneficial bacterial strains Bifidobacterium and Devosia of CP80 group were significantly higher than that in the other groups (P < 0.05). Besides, in diet CP80, the contents of amino acids and anti-nutritional factor, as well as the in vitro digestion rate were comparable to those of FM30, and the anti-nutritional factor content was between AP80 and PP80; total essential amino acids (EAAs) and methionine contents were higher than those in AP80, the glycine content was higher than that in PP80. Taken together, these results indicated that the CP80 diet had better amino acid composition and relatively low content of anti-nutritional factors, as well as high-digestion rate, and thus leads to the fish fed CP80 displaying improved effects in digestive enzyme activity, immune response, protein metabolism, and intestinal microbiota composition, which may be the important reasons to explain why that 80% of FM can be replaced by Cpro in the diet of golden pompano.

The Importance of Fatty Acid Precision Nutrition: Effects of Dietary Fatty Acid Composition on Growth, Hepatic Metabolite, and Intestinal Microbiota in Marine Teleost Trachinotus ovatus.

Our recent study demonstrated that diet with blend oil (named BO1) as lipid, which is designed on the base of essential fatty acid requirement of Trachinotus ovatus, achieved good performance. Here, to confirm its effect and investigate the mechanism, three isonitrogenous (45%) and isolipidic (13%) diets (D1-D3) only differing in dietary lipids, which were, respectively, fish oil (FO), BO1, and blend oil 2 (BO2) consisting of FO and soybean oil at 2 : 3, were formulated and used to feed the T. ovatus juveniles (average initial weight: 7.65 g) for 9 weeks. The results showed that the weight gain rate of fish fed D2 was higher than that of fish fed D3 (P < 0.05) and had no significant difference from that of fish fed D1 (P > 0.05). Correspondingly, compared with the D3 group, fish of the D2 group exhibited better oxidative stress parameters such as lower serum malondialdehyde content and inflammatory indexes in the liver such as the lower expression level of genes encoding four interleukin proteins and tumor necrosis factor α, as well as higher hepatic immune-related metabolites such as valine, gamma-aminobutyric acid, pyrrole-2-carboxylic acid, tyramine, l-targinine, p-synephrine, and butyric acid (P < 0.05). Furthermore, the intestinal probiotic (Bacillus) proportion was significantly higher, while the pathogenic bacteria (Mycoplasma) proportion was significantly lower in the D2 group than that in the D3 group (P < 0.05). The main differential fatty acids of diet D2 were close to those of D1, while the levels of linoleic acid and n-6 PUFA, as well as the ratio of DHA/EPA of D3, were higher than those of D1 and D2. These results indicated that the better performance of D2 such as enhancing growth, reducing oxidative stress, and improving immune responses and intestinal microbial communities in T. ovatus may be mainly due to the good fatty acid composition of BO1, which indicated the importance of fatty acid precision nutrition.

PPARγ regulates fabp4 expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes.

N-3 long-chain (≥C20) PUFA (LC-PUFA) are vital fatty acids for fish and humans. As a main source of n-3 LC-PUFA for human consumers, the n-3 LC-PUFA content of farmed fish is important. Previously, we identified fatty acid-binding protein (fabp)-4 as a candidate gene for regulating the n-3 LC-PUFA content. Herein, we further assessed the role of fabp4 in this process. First, a 2059 bp promoter sequence of fabp4 in Trachinotus ovatus was cloned and, using progressive deletion, determined -2006 bp to -1521 bp to be the core promoter sequence. The PPAR-γ binding sites were predicted to occur in this region. A luciferase reporter assay showed that the promoter activity of fabp4 decreased following mutation of the PPARγ binding site and that PPARγ increased the fabp4 promoter activity in a dose-dependent manner, implying that T. ovatus fabp4 is a target of PPARγ. The overexpression of fabp4 or PPARγ increased the DHA content in hepatocytes, whereas suppression of their expression diminished this effect, suggesting that both fabp4 and PPARγ play an active role in regulating DHA content. Moreover, the inhibition of fabp4 attenuated the increase in PPARγ-mediated DHA content, and the overexpression of fabp4 alleviated this effect. Collectively, our findings indicated that fabp4, which is controlled by PPARγ, plays an important role in DHA content regulation. The new regulation axis can be considered a promising novel target for increasing the n-3 LC-PUFA content in T. ovatus.

Interactions between dietary carbohydrate and thiamine: implications on the growth performance and intestinal mitochondrial biogenesis and function of Megalobrama amblycephala.

A12-week experiment was conducted to evaluate the influences of thiamine ongrowth performance, and intestinal mitochondrial biogenesis and function of Megalobramaamblycephala fed a high-carbohydrate (HC) diet. Fish (24·73 (sem 0·45) g) were randomly assigned to one of four diets: two carbohydrate (CHO) levels (30 and 45 %) and two thiamine levels (0 and 1·5 mg/kg). HC diets significantly decreased DGC, GRMBW, FIMBW, intestinal activities of amylase, lipase, Na+, K+-ATPase, CK, complexes I, III and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK: T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, mitochondrial transcription factor A, Opa-1, ND-1 and COX-1 and 2, while the opposite was true for ATP, AMP and reactive oxygen species, and the transcriptions of dynamin-related protein-1, fission-1 and mitochondrial fission factor. Dietarythiamine concentrations significantly increased DGC, GRMBW, intestinal activities of amylase, Na+, K+-ATPase, CK, complexes I and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK:T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, Opa-1, ND-1, COX-1 and 2, SGLT-1 and GLUT-2. Furthermore, a significant interaction between dietary CHO and thiamine was observed in DGC, GRMBW, intestinal activities of amylase, CK, complexes I and IV, ΔΨm, the AMP:ATP ratio, the P-AMPK:T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, Opa-1, COX-1 and 2, SGLT-1 and GLUT-2. Overall, thiamine supplementation improved growth performance, and intestinal mitochondrial biogenesis and function of M. amblycephala fed HC diets.

Beneficial effects of re-feeding high α-linolenic acid diets on the muscle quality, cold temperature and disease resistance of tilapia.

To investigate the effects of re-feeding high α-linolenic acid (ALA) diets on the muscle quality, cold temperature and disease resistance of the tilapia with nutritional history of soybean oil diets, three experimental diets with linoleic aicd (LA)/ALA ratios at 9 (D1, taking soybean oil as lipid sources), 3 and 1 (D2 and D3, taking soybean and linseed oils as lipid sources) were prepared to re-feed juveniles for 10 weeks, and the growth performance, muscle quality were analyzed. After the re-feeding trial, the fish were fasted for 8 weeks at cold temperature (15°C-20 °C) and then subjected to the Aeromonas hydrophila challenge, and the cold temperature and disease resistance of the fish were evaluated. It was shown that a comparable growth performance was detected among the three dietary groups, while, the high feed efficiency and low viscerosomatic and hepatosomatic index were detected in the D2 and D3 groups compared with the D1 group. In addition, the docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (PUFA) levels of the muscle increased in a parallel pattern with the dietary ALA levels, and the muscular tenderness, adhesiveness, and chewiness were modified substantially in fish fed the diets D2 and D3. After 8-week fasting at cold temperature, the low serum total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels, and high serum lysozyme (LZM) and C3 levels were observed in the D2 and D3 groups compared with the D1 group. It was also shown that high eicosapentaenoic acid (EPA), DHA, and n-3 PUFA levels were observed in the intestine, liver, and spleen of fish from D2 and D3 groups. Correspondingly, in the fish of the D2 and D3 groups, the mRNA levels of lzm in the liver, intestine, and spleen, and c3 in the intestine and spleen were increased, while the mRNA levels of il-1ß, ifn-γ, and tnf-α in the intestine, and ifn-γ, tnf-α in the liver, as well as spleen il-1ß, were decreased. Furthermore, the survival at day 15 post-challenge of A. hydrophila in the D2 and D3 groups were higher than those of the D1 group. The results demonstrated that re-feeding high ALA diets were beneficial to the muscle quality, cold temperature and disease resistance in the tilapia, and provide a basis for selecting the dietary lipid sources of tilapia pre-winter feed.

Influence of dietary fermented Folium mori on growing performance, lipometabolism and disease resistance of golden pompano Trachinotus ovatus.

Folium mori, as a plant unconventional feedstuff, are comparatively available due to cost-effectiveness, whereas their usage as aquafeed in pure form is restricted owing to the great fibre and antinutritional factors (ANFs) levels. Thereof, several methods of processing are introduced to remove antinutrient factors from the plant products, leading to improvement of bioactivity and digestibility. The assay was completed to evaluate the method of fermentation and the role of dietary fermented Folium mori (FFM) in golden pompano. Each of 5 diets with FFM at contents of 0.0%, 2.0%, 4.0%, 6.0% and 8.0% (D0.0, D2.0, D4.0, D6.0 and D8.0) was fed to the fishes with original body weight of 9.02g in triplicate sea cages for 56 days. The outcomes revealed that FFM in D4.0 and D6.0 elevated the growing performance of the fishes and the growing performance of D4.0 was remarkably improved in contrast to D0.0 and D2.0(P < 0.05). Whole body lipidic levels were obviously elevated when the diet FFM contents were below 8.0% (P < 0.05), whereas the contents of muscular moisture were generally reduced. In addition, FFM significantly increased serum high density lipoprotein (HDL) and remarkably reduced overall triglyceride (TG) in D2.0 to D6.0(P < 0.05). Moreover, FFM remarkably elevated the activities of lipase of stomach and hepatopancreas in contrast to D0.0 (P < 0.05) as well as intestinal tryptic enzyme in the entire FFM groups (P < 0.05). Eventually, FFM remarkably ameliorated disease-resistant characters of golden pompano to Vibrio harveyi in D4.0 and D6.0 (P < 0.05) and the RPS in D4.0 was optimal. To sum up, the present research displayed favorable role of FFM in growing performance, digestion, lipometabolism and disease-resistant characters, and the recommendation as to the supplementation content of diet FFM in compound feed of juvenile golden pompano is 4.0% as per the experiment status herein.

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

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

Controlling nutritional status score and prognostic nutrition index predict the outcome after severe traumatic brain injury.

OBJECTIVES: Immune-nutritional status is correlated with a clinical outcome in critical illness. Recently, controlling nutritional status (CONUT) score and prognostic nutrition index (PNI) has been reported to predict prognosis following cancer and other diseases. The aim of this study was to explore the relationship between the CONUT score and PNI and 6-month outcome in patients with severe traumatic brain injury (STBI). METHODS: We retrospectively analyzed the clinical data of 78 patients with STBI, including the CONUT score and PNI. Patients were divided into high CONUT group and low CONUT group. Patients were also divided into high PNI and low PNI group respectively. The 6-month outcome was evaluated by the modified Rankin scale (mRS). The unfavorable outcome was defined as mRS score ≥3. RESULTS: The unfavorable outcome group had lower Glasgow coma scale (GCS) scores, serum albumin, total cholesterol, PNI, and higher CONUT scores (P < 0.05). Both CONUT scores and PNI were strongly correlated with mRS (r = 0.429, P < 0.05; r = -0.590, P < 0.05, respectively). After adjustment for confounding factors, the odds ratios of CONUT scores and PNI for predicting unfavorable outcome were 10.478 (95% CI: 2.793-39.301) and -0.039 (95% CI: 0.008-0.204), respectively. The area under the curve (AUC) of CONUT scores for predicting unfavorable outcome was 0.777 (95% CI: 0.674-0.880, P < 0.01), which was similar to PNI (0.764, 95% CI: 0.657-0.87, P < 0.01). CONCLUSION: Both CONUT scores and PNI might be novel independent predictors of the poor outcome in STBI.

Systemic immune-inflammation index predicts the outcome after aneurysmal subarachnoid hemorrhage.

Systemic inflammatory response is closely related to the pathogenesis and prognosis in critical patients. Recently, systemic immune-inflammation index (SII), an indicator of systemic inflammatory response, was proved to predict the outcome in cancerous and non-cancerous diseases. The aim of this study is to investigate the association between SII on admission and 6-month outcome in patients with aneurysmal subarachnoid hemorrhage (aSAH). The clinical data and prognosis of 76 patients with aSAH were analyzed. The 6-month outcome was assessed by the modified Rankin scale(mRS). The unfavorable outcome was defined as mRS score ≥ 3. In addition, multivariate analysis was conducted to investigate factors independently associated with the favorable outcome. Receiver operating characteristic (ROC) curve analysis was undertaken to identify the best cut-off value of SII for the discriminate between favorable and unfavorable outcome in these patients. Thirty-six patients (47.4%) in our study had an unfavorable outcome (mRS ≥ 3) at 6 months, and twenty-four (66.7%) of them were in the high-SII group. A significantly higher SII on admission was observed in patients with unfavorable functional outcome at 6 months. Binary logistic regression analysis showed that there was an independent association between SII on admission and 6-month clinical outcome (adjusted OR = 4.499, 95%CI: 1.242-16.295, P < 0.05). The AUC of the SII for predicting unfavorable outcome was 0.692 (95% CI: 0.571-0.814, P < 0.05). Systemic immune-inflammation index (SII) could be a novel independent prognostic factor for aSAH patients at the early stage of the disease.

Activin A attenuates apoptosis of granulosa cells in atretic follicles through ERß-induced autophagy.

It is well known that approximately 99% of ovarian follicles in mammals suffer from a degenerative process known as atresia, which is a huge waste of genetic resource in female animals. Studies have shown that activin A (ACT-A) is located in ovarian granulosa cells and has different effects in granulosa cell depending on species. Although granulosa cells play a critical role during follicular atresia, the mechanism of action of ACT-A in bovine ovarian granulosa cells (BGC) is poorly understood. In this study, we first determined the apoptosis of BGCs isolated from growth follicles and atretic follicles respectively. Then, BGC isolated from atretic follicles were used as a model to elucidate the role of ACT-A in cattle ovary. The results showed that apoptosis occurred in both growing follicles and atretic follicles, and the percentage of apoptotic cells in atretic follicles was higher than that in growing follicles. The current results indicated that ACT-A can attenuate apoptosis of BGC by maintaining the function of BGC in atretic follicles. Increased ERß induced by ACT-A promoted BGC autophagy but had no effect on apoptosis. In summary, this study suggests that ACT-A attenuates BGC apoptosis in atretic follicles by ERß-mediated autophagy signalling.

Dietary calcium pyruvate could improve growth performance and reduce excessive lipid deposition in juvenile golden pompano (Trachinotus ovatus) fed a high fat diet.

Excessive lipid deposition in farmed fish is a challenge in the aquaculture industry. To study the effect of dietary calcium pyruvate (CaP) on lipid accumulation in fish, we used a high fat diet (HFD) to establish a lipid accumulation model in juvenile golden pompano (Trachinotus ovatus) and supplemented with 0%, 0.25%, 0.50%, 0.75% and 1.0% CaP (diets D0-D4, respectively). After 8-week feeding in floating cages, dietary CaP significantly improved growth performance, which peaked in fish fed diet D3. Supplementation of CaP significantly decreased whole body lipid content in fish fed D2-D4 and hepatosomatic index and liver lipid content in fish fed D3 and D4. Serum and hepatic antioxidant indices, including glutathione, catalase and superoxide dismutase, showed generally increasing trends in fish fed diets with CaP. In addition, increasing dietary CaP increasingly reduced hepatic activities of hexokinase, phosphofructokinase and pyruvate kinase involved in glycolysis, and increased glycogen contents of the liver and muscle. Dietary CaP up-regulated the liver mRNA expression of pparα, cpt1, hsl and fabp1, but down-regulated expression of srebp-1, fas and acc. In conclusion, 0.75% CaP improved growth performance and reduced excessive lipid deposition by affecting fatty acid synthesis and lipolysis in juvenile T. ovatus fed HFD.

miR-26a mediates LC-PUFA biosynthesis by targeting the Lxrα-Srebp1 pathway in the marine teleost Siganus canaliculatus.

MicroRNAs have been recently shown to be important regulators of lipid metabolism. However, the mechanisms of microRNA-mediated regulation of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis in vertebrates remain largely unknown. Herein, we for the first time addressed the role of miR-26a in LC-PUFA biosynthesis in the marine rabbitfish Siganus canaliculatus The results showed that miR-26a was significantly down-regulated in liver of rabbitfish reared in brackish water and in S. canaliculatus hepatocyte line (SCHL) incubated with the LC-PUFA precursor α-linolenic acid, suggesting that miR-26a may be involved in LC-PUFA biosynthesis because of its abundance being regulated by factors affecting LC-PUFA biosynthesis. Opposite patterns were observed in the expression of liver X receptor α (lxrα) and sterol regulatory element-binding protein-1 (srebp1), as well as the LC-PUFA biosynthesis-related genes (Δ4 fads2, Δ6Δ5 fads2, and elovl5) in SCHL cells incubated with α-linolenic acid. Luciferase reporter assays revealed rabbitfish lxrα as a target of miR-26a, and overexpression of miR-26a in SCHL cells markedly reduced protein levels of Lxrα, Srebp1, and Δ6Δ5 Fads2 induced by the agonist T0901317. Moreover, increasing endogenous Lxrα by knockdown of miR-26a facilitated Srebp1 activation and concomitant increased expression of genes involved in LC-PUFA biosynthesis and consequently promoted LC-PUFA biosynthesis both in vitro and in vivo These results indicate a critical role of miR-26a in regulating LC-PUFA biosynthesis through targeting the Lxrα-Srebp1 pathway and provide new insights into the regulatory network controlling LC-PUFA biosynthesis and accumulation in vertebrates.

A single intronic single nucleotide polymorphism in splicing site of steroidogenic enzyme hsd17b1 is associated with phenotypic sex in oyster pompano, Trachinotus anak.

Teleosts show varied master sex determining (MSD) genes and sex determination (SD) mechanisms, with frequent turnovers of sex chromosomes. Tracing the origins of MSD genes and turnovers of sex chromosomes in a taxonomic group is of particular interest in evolutionary biology. Oyster pompano (Trachinotus anak), a marine fish, belongs to the family Carangidae, in which 17b-hydroxysteroid dehydrogenase 1 (hsd17b1) has repeatedly evolved to an MSD gene. Whole-genome resequencing identified a single nucleotide polymorphism (SNP) at chromosome 24 to be strictly associated with phenotypic sex, with females being the heterozygous sex. This SNP is located in a splicing site at the first exon/intron boundary of hsd17b1. The Z-linked SNP results in malfunction of all spliced isoforms, whereas the W-linked isoforms were predicted to have open reading frames that are conserved among vertebrates, suggesting that hsd17b1 is a female-determining gene. The differential alternative splicing patterns of ZZ and ZW genotypes were consistently observed both in undifferentiated stages and differentiated gonads. We observed elevated recombination around the SD locus and no differentiation between Z and W chromosomes. The extreme diversity of mutational mechanisms that hsd17b1 evolves to an MSD gene highlights frequent in situ turnovers between sex chromosomes in the Carangidae.

Characterization of two kcnk3 genes in Nile tilapia (Oreochromis niloticus): Molecular cloning, tissue distribution, and transcriptional changes in various salinity of seawater.

As one important member of the two-pore-domain potassium channel (K2P) family, potassium channel subfamily K member 3 (KCNK3) has been reported for thermogenesis regulation, energy homeostasis, membrane potential conduction, and pulmonary hypertension in mammals. However, its roles in fishes are far less examined and published. In the present study, we identified two kcnk3 genes (kcnk3a and kcnk3b) in an euryhaline fish, Nile tilapia (Oreochromis niloticus), by molecular cloning, genomic survey and laboratory experiments to investigate their potential roles for osmoregulation. We obtained full-length coding sequences of the kcnk3a and kcnk3b genes (1209 and 1173 bp), which encode 402 and 390 amino acids, respectively. Subsequent multiple sequence alignments, putative 3D-structure model prediction, genomic survey and phylogenetic analysis confirmed that two kcnk3 paralogs are widely presented in fish genomes. Interestingly, a DNA fragment inversion of a kcnk3a cluster was found in Cypriniforme in comparison with other fishes. Quantitative real-time PCRs demonstrated that both the tilapia kcnk3 genes were detected in all the examined tissues with a similar distribution pattern, and the highest transcriptions were observed in the heart. Meanwhile, both kcnk3 genes in the gill were proved to have a similar transcriptional change pattern in response to various salinity of seawater, implying that they might be involved in osmoregulation. Furthermore, three predicted transcription factors (arid3a, arid3b, and arid5a) of both kcnk3 genes also showed a similar pattern as their target genes in response to the various salinity, suggesting their potential positive regulatory roles. In summary, we for the first time characterized the two kcnk3 genes in Nile tilapia, and demonstrated their potential involvement in osmoregulation for this economically important fish.

Effects of dietary n-3 highly unsaturated fatty acids levels on growth, lipid metabolism and innate immunity in juvenile golden pompano (Trachinotus ovatus).

To investigate the effects of dietary n-3 highly unsaturated fatty acids (HUFA) levels on growth, lipid metabolism and innate immunity in juvenile golden pompano Trachinotus ovatus, a marine carnivorous teleost, a total of 450 fish (average body weight: 14.84 g) were randomly distributed into 18 cages at sea, each dietary group with three cages and respectively fed six diets (D1-D6) with 2.30% (D1), 0.64% (D2), 1.00% (D3), 1.24% (D4), 1.73% (D5), or 2.10% (D6) n-3 HUFA. Here, D1 with fish oil as lipid source was set as control, while D2-D6 used a mixed vegetable oil as lipid source and supplemented with docosahexaenoic acid- (DHA) and eicosapentaenoic acid- (EPA) enriched oils to adjust the n-3 HUFA levels. After 8 weeks feeding, the daily growth coefficient (DGC), specific growth rate (SGR) and feed efficiency ratio (FER) showed no significant difference among the six dietary groups (P > 0.05). The levels of EPA and DHA in serum and liver increased with the dietary n-3 HUFA levels. The activity of total superoxide disumutase (T-SOD) in serum of fish fed D4 and D5 were significantly higher than that of the other groups, whereas the opposite was true for serum IL-1ß and IL-6 levels as well as liver malondialdehyde (MDA) content. The mRNA levels of genes related to hepatic lipid metabolism including sterol regulatory element-binding protein-1 (srebp-1), fatty acid binding protein 1 (fabp1), peroxisome proliferators-activated receptor alpha (pparα), elongase of very long-chain fatty acids 5 (elovl5) and fatty acyl desaturase 2 (fads2) were down-regulated in fish fed the diets with high n-3 HUFA levels, while those of apolipoprotein b 100 (aprob 100) and carnitine palmitoyl transferase 1 (cpt1) increased significantly as increasing n-3 HUFA levels up to 1.73% (D2-D5), but decreased in the 2.10% n-3 HUFA group (D6). In addition, the expression levels of genes related to innate immunity including interleukin-10 (il-10) and transforming growth factor ß1 (tgf-ß1) increased significantly when dietary n-3 HUFA increased from 0.64% to 1.73%, whereas the opposite was true for the expression levels of nuclear factor kappa-B (nf-κb), interleukin-1ß (il-1ß), interleukin-6 (il-6) and interleukin-8 (il-8). Overall, the results indicated that dietary n-3 HUFA at 1.24-1.73% (D4-D5) can effectively improve fatty acid profiles, lipid metabolism, antioxidant capacity and immune response of golden pompano.

Characterization of two kcnk3 genes in rabbitfish (Siganus canaliculatus): Molecular cloning, distribution patterns and their potential roles in fatty acids metabolism and osmoregulation.

KCNK3 is a two-pore-domain (K2P) potassium channel involved in maintaining ion homeostasis, mediating thermogenesis, controlling breath and modulating electrical membrane potential. Although the functions of this channel have been widely described in mammals, its roles in fishes are still rarely known. Here, we identified two kcnk3 genes from the euryhaline rabbitfish (Siganus canaliculatus), and their roles related to fatty acids metabolism and osmoregulation were investigated. The open reading frames of kcnk3a and kcnk3b were 1203 and 1176 bp in length, encoding 400 and 391 amino acids respectively. Multiple sequences alignment and phylogenetic analysis revealed that the two isotypes of kcnk3 were extensively presented in fishes. Quantitative real-time PCRs indicated that both genes were widely distributed in examined tissues but showed different patterns. kcnk3a primary distributed in adipose, eye, heart, and spleen tissues, while kcnk3b was mainly detectable in heart, kidney, muscle and spleen tissues. In vivo experiments showed that fish fed diets with fish oil as dietary lipid (rich in long chain polyunsaturated fatty acids, LC-PUFA) induced higher mRNA expression levels of kcnk3 genes in comparison with fish fed with plant oil diet at two different salinity environments (32 and 15‰). Meanwhile, the expression levels of kcnk3 genes were higher in seawater (32‰) than that in brackish water (15‰) when fishes were fed with both types of feeds. In vitro experiments with rabbitfish hepatocytes showed that LC-PUFA significantly improved hepatic kcnk3a expression level compared with treatment of linolenic acid. These results suggest that two kcnk3 genes are widely existed and they might be functionally related to fatty acids metabolism and osmoregulation in the rabbitfish.

Molecular Cloning, Characterization, and Expression Regulation of Acyl-CoA Synthetase 6 Gene and Promoter in Common Carp Cyprinus carpio.

Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBPα), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor α (PPARα), and PPARγ were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPARγ agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPARα agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPARγ and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.