ω-6 Polyunsaturated fatty acids (PUFAs) are essential fatty acids that participate in macroautophagy (hereafter referred to as autophagy) and the Kelch ECH-associating protein 1 (Keap1)-nuclear ...factor erythroid 2-related factor 2 (Nrf2) antioxidant system in organisms. However, the molecular mechanisms by which ω-6 PUFAs (linoleic acid) regulate autophagy and Keap1-Nrf2 antioxidant system are not completely understood. Therefore, the purposes of this study were to explore the molecular mechanisms by which ω-6 PUFAs (linoleic acid) regulate autophagy and antioxidant system and to investigate the potential relationship between autophagy and antioxidant system through transcriptomic analysis, quantitative real-time polymerase chain reaction (RT-qPCR), western blot analysis, coimmunoprecipitation (Co-IP) and electrophoretic mobility shift assays (EMSAs) in vivo and in vitro. The results of the present study indicated that ω-6 PUFAs in diets induced autophagy but decrease antioxidant ability in vivo. However, the results also provided evidence, for the first time, that ω-6 PUFAs (linoleic acid) induced autophagy and increased antioxidant ability through the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway and the AMPK-target of rapamycin (TOR) signaling pathway in hepatocytes in vitro. Interestingly, the findings revealed a ω-6 PUFA-induced synergistic feedback loop between autophagy and antioxidant system, which are connected with each other through the P62 and Keap1 complex. These results suggested that ω-6 PUFAs (linoleic acid) could be useful for activating a synergistic feedback loop between autophagy and antioxidant system and could greatly aid in the prevention and treatment of multiple pathologies.
Ectopic lipid accumulation has been observed in fish fed a high-lipid diet. However, no information is available on the mechanism by which dietary lipid levels comprehensively regulate lipid ...transport, uptake, synthesis and catabolism in fish. Therefore, the present study aimed to gain further insight into how dietary lipids affect lipid deposition in the liver of large yellow croaker(Larimichthys crocea). Fish (150.00±4.95 g) were fed a diet with a low (6%), moderate (12%, the control diet) or high (18%) crude lipid content for 10 weeks. Growth performance, plasma biochemical indexes, lipid contents and gene expression related to lipid deposition, including lipoprotein assembly and clearance, fatty acid uptake and triacylglycerol synthesis and catabolism, were assessed. Growth performance was not significantly affected. However, the hepato-somatic and viscera-somatic indexes as well as plasma triacylglycerol, non-esterified fatty acids and LDL-cholesterol levels were significantly increased in fish fed the high-lipid diet. In the livers of fish fed the high-lipid diet, the expression of genes related to lipoprotein clearance (LDLR) and fatty acid uptake (FABP11) was significantly up-regulated, whereas the expression of genes involved in lipoprotein assembly (apoB100), triacylglycerol synthesis and catabolism (DGAT2, CPT I) was significantly down-regulated compared with fish fed the control diet, and hepatic lipid deposition increased. In fish fed the low-lipid diet, the expression of genes associated with lipoprotein assembly and clearance (apoB100, LDLR, LRP-1), fatty acid uptake (CD36, FATP1, FABP3) and triacylglycerol synthesis (FAS) was significantly increased, whereas the expression of triacylglycerol catabolism related genes (ATGL, CPT I) was reduced compared with fish fed the control diet. However, hepatic lipid content in fish fed the low-lipid diet decreased mainly due to low dietary lipid intake. In summary, findings of this study provide molecular insight into the role of lipid deposition in the liver in response to different dietary lipid contents.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dietary phospholipid (PL) supplementation has been shown to reduce lipid accumulation in the tissues of farmed fish; however, the mechanisms underlying this effect are largely unknown. Thus, the ...present study was conducted to evaluate the potential impacts of PL on hepatic lipid metabolism both in vivo and in vitro. For in vivo study, four experimental diets - low lipid and low PL diet, as control diet (LL-LP diet, containing 12 % lipid and 1·5 % PL), low-lipid and high-PL diet (containing 12 % lipid and 8 % PL), high-lipid and low-PL diet (HL-LP diet, containing 20 % lipid and 1·5 % PL) and high-lipid and high-PL diet (HL-HP diet, containing 20 % lipid and 8 % PL) - were randomly allocated to four groups of large yellow croaker (Larimichthys crocea) (three cages per group) with similar initial body weight (approximately 8 g). For in vitro study, primary hepatocytes isolated from large yellow croaker were incubated either with graded levels of phosphatidylcholine (PC) (0-250 μm) or small interfering RNA (siRNA) for CTP: choline phosphate cytidylyltranferase α (CCTα) (siRNA-CCTα). Results showed that survival was independent of dietary treatments (P>0·05). Weight gain and feed efficiency in the HL-HP group were significantly higher than in the LL-LP and HL-LP groups (P<0·05). High level of dietary PL could markedly reduce abnormal hepatic lipid accumulation induced by the HL-LP diet (P<0·05). Similarly, compared with the corresponding controls, a significant decrease/increase in lipid content was observed in primary hepatocytes incubated with PC/siRNA-CCTα (P<0·05). High level of dietary PL reversed the HL-LP diet-induced increased levels of mRNA of fatty acid uptake and lipid synthesis related genes (P<0·05). In addition, High level of dietary PL markedly down-regulated the transcript levels of fatty acid oxidation-related genes and enhanced the transcript levels of VLDL assembly-related genes regardless of dietary lipid levels (P<0·05). Compared with corresponding controls, primary hepatocytes treated with PC showed significantly higher mRNA expression of lipid synthesis and VLDL assembly-related genes and lower mRNA expression of fatty acid oxidation-related genes, with hepatocytes treated with siRNA-CCTα exhibiting the opposite trend (P<0·05). In summary, these results demonstrated that high level of dietary PL might reverse the HL-LP diet-induced abnormal lipid accumulation in the liver through inhibiting fatty acid uptake and lipid synthesis, together with promoting the lipid export at the transcriptional level. Lipid export-promoting effect of PC was confirmed by in vitro studies. The present study showed for the first time that PL or PC could influence various metabolic pathways to regulate hepatic lipid deposition in fish at least at the transcriptional level.
Endoplasmic reticulum (ER) homeostasis plays a vital role in cell physiological functions. Various factors can destroy the homeostasis of the ER and cause ER stress. Moreover, ER stress is often ...related to inflammation. Glucose-regulated protein 78 (GRP78) is an ER chaperone, which plays a vital role in maintaining cellular homeostasis. Nevertheless, the potential effects of GRP78 on ER stress and inflammation is still not fully elucidated in fish. In the present study, ER stress and inflammation was induced by tunicamycin (TM) or palmitic acid (PA) in the macrophages of large yellow croakers. GRP78 was treated with an agonist/inhibitor before or after the TM/PA treatment. The results showed that the TM/PA treatment could significantly induce ER stress and an inflammatory response in the macrophages of large yellow croakers whereas the incubation of the GRP78 agonist could reduce TM/PA-induced ER stress and an inflammatory response. Moreover, the incubation of the GRP78 inhibitor could further induce TM/PA-induced ER stress and an inflammatory response. These results provide an innovative idea to explain the relationship between GRP78 and TM/PA-induced ER stress or inflammation in large yellow croakers.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A feeding experiment was conducted to examine the effects of dietary administration of
Bacillus subtilis and fructooligosaccharide (FOS) on growth performance, survival, immune responses and disease ...resistance of juvenile large yellow croaker,
Larimichthys crocea (mean initial body weight 7.82
g
±
0.68). Nine practical diets were formulated to contain three levels of
B. subtilis (0.0, 0.42
×
10
7
cfu
g
−1 and 1.35
×
10
7
cfu
g
−1), each with three FOS levels (0, 0.2% and 0.4% of dry weight). Each diet was randomly assigned to triplicate groups of 60 juveniles. The experiment was conducted in floating sea cages (1.0
×
1.0
×
1.5
m) for 10
weeks. At the termination of the feeding trial, alternative complement pathway (ACP), superoxide dismutase (SOD) and lysozyme activity of serum and respiratory burst activity of head kidney macrophage were determined and fishes were challenged intraperitoneally with
Vibrio harveyi. The results showed that at each dietary FOS level, dietary supplementation of 1.35
×
10
7
cfu
g
−1
B. subtilis significantly increased the specific growth rate (SGR) (
P
<
0.01) and feed efficiency ratio (FER) (
P
<
0.05) compared with the groups without
B. subtilis supplementation. The immune assay showed that at each FOS level, compared to the groups without
B. subtilis supplementation significantly enhanced serum lysozyme was observed in fish fed the
B. subtilis-supplemented diets (
P
<
0.05) and significantly enhanced serum SOD activity was observed in fish fed the diet with 1.35
×
10
7
cfu
g
−1
B. subtilis (
P
<
0.05), while the serum ACP activity and the respiratory burst activity of head kidney macrophage were independent of dietary treatments. The challenge experiment showed that compared to the groups without
B. subtilis supplementation the cumulative mortality after infection with
V. harveyi was significantly lower in fish fed the diet with 1.35
×
10
7
cfu
g
−1
B. subtilis (
P
<
0.05), at each FOS level. However, at each dietary
B. subtilis level, addition of FOS in diets did not significantly affect the growth performance, immune response and disease resistance of large yellow croaker. No significant interactions were observed between dietary
B. subtilis and FOS. These results showed that dietary supplementation of
B. subtilis at a dose of 1.35
×
10
7
cfu
g
−1 improved growth, feed efficiency ratio, non-specific immune responses and disease resistance of juvenile large yellow croaker,
L. crocea.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Soybean meal-induced enteropathy (SBMIE) is prevalent in aquaculture. The aim of this study is to evaluate the role of daidzein on SBMIE of juvenile turbot (Scophthalmus maximus L.) by feeding with ...fish meal diet (FM), soybean meal diet (SBM, 40% fish meal protein in FM replaced by soybean meal protein) and daidzein diet (DAID, 40 mg/kg daidzein supplemented to SBM) for 12 weeks. We found that daidzein supplementation elevated the gene expression of anti-inflammatory cytokine TGF-β, decreased gene expression of pro-inflammatory cytokines TNF-α and signal molecules p38, JNK and NF-κB. SBM up-regulated the genes expression related to oxidative stress and apoptosis, but dietary daidzein restored it to the similar level with that in FM group. Moreover, dietary daidzein up-regulated gene expression of tight junction protein, and modified the intestinal microbial profiles with boosted relative abundance of phylum Proteobacteria and Deinococcus-Thermus, genera Sphingomonas and Thermus, species Lactococcus lactis, and decreased abundance of some potential pathogenic bacteria. In conclusion, dietary daidzein could ameliorate SBM-induced intestinal inflammatory response, oxidative stress, mucosal barrier injury and microbiota community disorder of turbot. Moreover, p38, JNK and NF-κB signaling might be involved in the anti-inflammatory process of daidzein, and daidzein itself might act as an antioxidant to resist SBM-induced oxidative damage.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A 30-day feeding trial was conducted to investigate the effects of dietary Astragalus polysaccharides (APS) on survival, growth performance, activities of digestive enzyme, antioxidant responses and ...intestinal development of large yellow croaker (Larimichthys crocea) larvae. Five isonitrogenous (53% crude protein) and isolipidic (18% crude lipid) diets were formulated with graded levels of APS (0.00%, 0.05%, 0.10%, 0.15% and 0.20%). Results showed that larvae fed diets with 0.10% and 0.15% APS had significantly higher specific growth rate and body crude protein content. Meanwhile, larvae fed diets with 0.10% and 0.20% APS had significantly higher trypsin activity in intestinal segments (IS) at 45 days after hatch (DAH). Supplementation with 0.10% APS significantly improved the activity of alkaline phosphatase both in brush border membrane and IS of larvae at 45 DAH. Similarly, larvae fed the diet with 0.10% APS had significantly higher activity of leucine aminopeptidase in IS at 45 DAH than the control group. Also, larvae fed the diet with 0.10% APS showed significantly higher activity of superoxide dismutase in IS than the control group. Total antioxidant capacity in pancreatic segments (PS) were significantly higher in larvae fed diets with 0.15% and 0.20% than the control group. Malondialdehyde contents in PS were significantly lower in larvae fed the diet with 0.10% APS than the control group. Furthermore, supplementation of 0.10% APS significantly increased the length of intestinal microvillus and the height of enterocyte. The selected markers for intestinal epithelial proliferation and differentiation were higher expressed in larvae fed diets with APS than the control group, such as tight junction protein-2, proliferating cell nuclear antigen and sucrase-isomaltase. In conclusion, appropriate supplementation of APS (0.05% - 0.10%) could promote growth performance of large yellow croaker larvae through increasing the activities of digestive enzyme and antioxidant enzyme, improving intestinal morphology and promoting the intestinal development.
•Dietary Astragalus polysaccharides promotes growth performance of large yellow croaker larvae.•Digestive enzyme activities of larvae are enhanced by dietary Astragalus polysaccharides supplementation.•Dietary Astragalus polysaccharides promotes intestinal morphology of larvae.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A 92-day feeding experiment was conducted to investigate the effects of dietary soybean oil (SO) on growth performance, fatty acid composition, lipid deposition and hepatic lipid metabolism related ...gene expression in juvenile turbot (Scophthalmus maximus L.) (mean initial body weight, 5.88±0.02g). Three experimental diets were formulated with substitution of 33.3%, 66.7% and 100% fish oil (FO) by SO. Each diet was randomly fed to triplicate tanks, and each tank was stocked with 35 fish. The results showed that specific growth rate of turbot fed diets with 100% SO was significantly lower than that in the 33.3% and 66.7% SO groups. Fatty acid (FA) composition of total lipid in the liver and muscle was closely correlated with those in diets. The lipid content in the liver and muscle significantly increased with increasing dietary SO level. The activity of LPL in the liver of turbot was not significantly affected by dietary SO level. Relative gene expression of LPL, PPARα, FAS and MTP significantly increased, while relative expression of LXR and CPT I significantly decreased with increasing dietary SO level. There was no significant difference in the expression of SREBP-1 among dietary treatments. These results suggested that the growth of turbot fed diets with 100% SO was significantly lower than the low SO group. The increase of lipid deposition in the liver of turbot fed diets with higher dietary SO level would be related to the up-regulation of fatty acid synthesis-related gene (FAS) and the down-regulation of fatty acids oxidation gene (CPT I) expression.
•Higher dietary SO level (>66.7%) reduces growth and muscle quality of turbot.•Increasing hepatic lipid deposition of turbot fed with high SO relates to FAS.•Increasing hepatic lipid deposition of turbot fed with high SO relates to CPT I.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The objective of this study was to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related with phosphatidylcholine (PC) ...metabolism in larval and juvenile large yellow croaker, Larimichthys crocea. Three isonitrogenous and isolipidic diets with graded levels of PLs (2.53%, 6.32% and 12.7%) were randomly allocated to triplicate groups of larvae (initial body weight: 3.86±0.24mg) eight times daily for 30days. Five isonitrogenous and isolipidic diets with graded levels of PLs (1.32%, 3.05%, 5.86%, 7.4% and 9.63%) were randomly allocated to triplicate groups of juveniles (initial body weight: 7.36±0.33g) twice daily to apparent satiation for 60days. Results showed that survival rate and specific growth rate of larvae fed the diet with 2.53% PLs were significantly lower compared with those of larvae in other groups (P<0.05). However, specific growth rate and survival rate were not significantly different among dietary treatments in juveniles. No significant difference was observed in the transcript levels of key genes involved in PC de novo synthesis, including CTP: choline phosphate cytidylyltransferase and phosphatidylethanolamine N-methyltransferase, in both the whole larvae body and the livers of juveniles fed diets with different concentrations of PLs. Although no significant difference was found, the mRNA expression levels of 1, 2-diacylglycerol choline phosphotransferase increased with the increasing levels of dietary PLs in the livers of juveniles, with acyl-CoA: diacylglycerol acyltransferase 2 showing the opposite trend. These results confirmed that the beneficial effects of dietary PLs on survival and growth performance of large yellow croaker were restricted to fish larvae. Dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis.
The present study was conducted to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related to phosphatidylcholine (PC) biosynthesis in larval and juvenile large yellow croaker, (Larimichthys crocea). The results are reliable and of both theoretical and practical importance. For “theoretical importance”, our study provided the first evidence that dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis, which could contribute to better understanding the mechanisms underlying the discrepancy in requirements for phospholipids between larval and juvenile large yellow croaker. For “practical importance”, our study confirmed that the beneficial effects of dietary PLs on growth performance of large yellow croaker were restricted to larvae rather than juveniles (at least 7.36g) and these findings could be applied to reduce the supplementation of PLs in commercial feeds for large yellow croaker, which in turn might reduce the cost of the commercial feeds for large yellow croaker.
The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed. I have read and have abided by the statement of ethical standards for manuscripts submitted to Aquaculture.
•The requirement for phospholipids is restricted to large yellow croaker larvae.•Dietary phospholipids could reduce lipid accumulation in liver of juvenile large yellow croaker.•No major effects of dietary phospholipids on mRNA expression of key genes related to phosphatidylcholine synthesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The effects of conjugated linoleic acid (CLA) on growth performance, non-specific immunity, antioxidant capacity, lipid deposition and related gene expression were investigated in the large yellow ...croaker (Larmichthys crocea). Fish (7·56 (sem 0·60) g) were fed soyabean oil-based diets with graded levels of CLA (0, 0·42, 0·83, 1·70 %) for 70 d. Quantitative PCR was used to assess the effects of CLA on the transcription of inflammation- and fatty acid oxidation-related genes. Growth in fish fed the diet with 0·42 % CLA was significantly higher. Also, phagocytic index and respiratory burst activity were significantly higher in fish fed the diets containing 0·42 and 0·83 % CLA, respectively. Hepatic total antioxidative capacity and catalase activities increased significantly when CLA increased from 0 to 0·83 %, and then decreased with further increase of CLA. However, hepatic malondialdehyde content decreased significantly as dietary CLA increased. Lipid concentration in the whole body and muscle increased significantly with increasing dietary CLA. Transcription of genes related to inflammation (cyclo-oxygenase-2 and IL-β) in the liver and kidney and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in the kidney decreased significantly as dietary CLA increased. PPARα and acyl CoA oxidase expression in the liver decreased significantly as CLA increased from 0·42 to 1·70 %. These results strongly suggest that dietary CLA could significantly affect growth performance, non-specific immunity, antioxidant capacity, lipid deposition and transcription of inflammation- and fatty acid oxidation-related genes of the large yellow croaker. This may contribute to our understanding of the mechanisms related to the physiological effects of dietary CLA in fish.
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