High-fat-diet induced inflammation and apoptosis via activation of Ire1α in liver and hepatocytes of black seabream (Acanthopagrus schlegelii).

The present study aimed to reveal the role of inositol-requiring enzyme 1α (Ire1α) in mediating high-fat-diet (HFD) induced inflammation and apoptosis in fish and elucidate underling mechanisms of action. In experiment 1, black seabream juveniles were fed a control diet (Control, 12 % dietary lipid) or a high fat diet (HFD, 19 % dietary lipid) for eight weeks. In experiment 2, primary hepatocytes were isolated from black seabream juveniles and treated with oleic acid (OA, 200 µmol/L), OA + transfection with non-silencing control siRNA (negative control) (OA + NC), and OA + transfection with ire1α-small interfering RNA (OA + siire1α) for 48 h versus untreated (Control). Results indicated that fish fed HFD increased lipid deposition in the liver and caused hepatic steatosis. HFD group had significantly higher ire1α/Ire1α mRNA and phosphorylated protein expression and endoplasmic reticulum stress (ERS) related genes expression compared to the Control group, indicating that ERS was triggered. Meanwhile, feeding HFD induced inflammation and apoptosis by evaluated nuclear factor kappa B (nf-κb) mRNA and phosphorylated Nf-κb p65 protein expression, and c-Jun N-terminal kinase (jnk) mRNA and protein expression. However, knock down of ire1α (OA + siire1α) in primary hepatocytes alleviated OA-induced increased expression of ire1α/Ire1α mRNA and protein expression, nf-κb/Nf-κb p65 mRNA and phosphorylated protein expression, and jnk/Jnk mRNA and phosphorylated protein expression. These findings revealed the underling mechanism of action of HFD in fish, confirming that HFD increased ESR stress and Ire1α that, in turn, activated Nf-κb and Jnk pathways in hepatocytes and liver mediating HFD-induced inflammation and apoptosis.

Molecular cloning and characterization of endoplasmic reticulum stress related genes grp78 and atf6α from black seabream (Acanthopagrus schlegelii) and their expressions in response to nutritional regulation.

Glucose-regulated protein 78 (grp78) and activating transcription factor 6α (atf6α) are considered vital endoplasmic reticulum (ER) molecular chaperones and ER stress (ERS) sensors, respectively. In the present study, the full cDNA sequences of these two ERS-related genes were first cloned and characterized from black seabream (Acanthopagrus schlegelii). The grp78 cDNA sequence is 2606 base pair (bp) encoding a protein of 654 amino acids (aa). The atf6α cDNA sequence is 2168 base pair (bp) encoding a protein of 645 aa. The predicted aa sequences of A. schlegelii grp78 and atf6α indicated that the proteins contain all the structural features, which were characteristic of the two genes in other species. Tissues transcript abundance analysis revealed that the mRNAs of grp78 and atf6α were expressed in all measured tissues, but the highest expression of these two genes was all recorded in the gill followed by liver/ brain. Moreover, in vivo experiment found that fish intake of a high lipid diet (HLD) can trigger ERS by activating grp78/Grp78 and atf6α/Atf6α. However, it can be alleviated by dietary betaine supplementation, similar results were also obtained by in vitro experiment using primary hepatocytes of A. schlegelii. These findings will be beneficial for us to evaluate the regulator effects of HLD supplemented with betaine on ERS at the molecular level, and thus provide some novel insights into the functions of betaine in marine fish fed with an HLD.

Dietary cholesterol promotes growth and ecdysone signalling pathway by modulating cholesterol transport in swimming crabs (Portunus trituberculatus).

Cholesterol, as an indispensable nutrient, regulates molting and growth in crustacean. As crustaceans are unable to biosynthesize cholesterol de novo, it is central to understand how dietary cholesterol affects molting in crustaceans. An 8-week feeding trial was conducted to evaluate the effects of dietary cholesterol level (0.12%, 0.43%, 0.79%, 1.00%, 1.30% and 2.50%) on growth, cholesterol metabolism and expression of genes related to lipid and ecdysone metabolism in female swimming crabs (Portunus trituberculatus). A total of 192 crabs (1.41 ± 0.05 g) were randomly distributed into 192 aquaria. Each treatment had 4 replicates with each replicate containing 8 crabs. Crabs fed the 1.00% cholesterol diet showed best growth performance, and thus based on percent weight gain, the optimal dietary cholesterol requirement was calculated at 1.01%. Tissue cholesterol concentrations were positively correlated with dietary cholesterol level. The contents of functional fatty acids in hepatopancreas significantly increased as dietary cholesterol increased from 0.12% to 2.50% (P < 0.05). The expression levels of genes related to lipogenesis pathway, lipid catabolism and fatty acid oxidation were significantly down-regulated with increased dietary cholesterol level (P < 0.05). The highest expression levels of cholesterol transport genes, low-density lipoprotein receptor (ldlr) and low-density lipoprotein receptor-related protein 2 (lrp2) occurred in crabs fed the 1.30% cholesterol diet. Moreover, hormones related to molting such as crustacean hyperglycemic hormone (CHH), methyl farnesoate (MF), molt-inhibiting hormone (MIH), and ecdysone in hemolymph were significantly influenced by dietary cholesterol level (P < 0.05). The highest expression levels of ecdysone receptor (ecr) and chitinase 1 (chi1) in eyestalk and hepatopancreas were found in crabs fed the diet containing 1.00% cholesterol (P < 0.05). In conclusion, the optimal dietary level was beneficial to functional fatty acid accumulation, regulated lipid metabolism, promoted the ecdysone signalling pathway by improving the cholesterol transport, and improved the molting rate and growth of swimming crabs.

Lipid metabolic disorders and physiological stress caused by a high-fat diet have lipid source-dependent effects in juvenile black seabream Acanthopagrus schlegelii.

This study was conducted to evaluate the effects of different dietary lipid sources on growth performance, lipid metabolism, and physiological stress responses including oxidative stress (OS) and endoplasmic reticulum stress (ERS) of juvenile Acanthopagrus schlegelii (initial weight 0.88 ± 0.01 g) fed a high-fat diet (HFD). Four isonitrogenous and isolipidic experimental diets containing different lipid sources were formulated: fish oil (FO), palm oil (PO), linseed oil (LO), and soybean oil (SO), respectively. Results indicated that fish fed HFD supplemented with FO significantly improved growth than SO treatment. The high concentrations of aspartate aminotransferase and alanine transaminase were found in HFD supplemented with SO. Fish fed dietary LO supplementation showed significantly lower serum cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein contents than those in SO group. Likewise, hepatic paraffin section analysis indicated that HFD with PO or SO supplementation increased fat drop. The expression levels of peroxisome proliferators-activated receptor alpha (pparα) and silent regulator 1 (sirt1) were significantly elevated by HFD with FO or LO supplementation. Additionally, the key marker of OS malonaldehyde was significantly increased in FO and SO groups. ERS-related genes were activated in dietary PO or SO supplementation and, hence, triggering inflammation and apoptosis by promoting the expression levels of nuclear factor kappa B (nf-κb) and c-Jun N-terminal kinase (jnk). Overall, the present study reveals that lipid metabolic disorders and physiological stress caused by a HFD have significant lipid source-dependent effects, which have important guiding significance for the use of HFD in marine fish.

A New Insight Into the Underlying Adaptive Strategies of Euryhaline Marine Fish to Low Salinity Environment: Through Cholesterol Nutrition to Regulate Physiological Responses.

Salinity is an important environmental factor that can affect the metabolism of aquatic organisms, while cholesterol can influence cellular membrane fluidity which are vital in adaption to salinity changes. Hence, a 4-week feeding trial was conducted to evaluate the effects of water salinity (normal 23 psu and low 5 psu) and three dietary cholesterol levels (CH0.16, 0.16%, CH1.0, 1.0% and CH1.6, 1.6%) on osmoregulation, cholesterol metabolism, fatty acid composition, long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis, oxidative stress (OS), and endoplasmic reticulum stress (ERS) of the euryhaline fish black seabream (Acanthopagrus schlegelii). The results indicated that in low salinity, fish fed with the CH1.0 diet improved ion reabsorption and osmoregulation by increased Na+ concentration in serum as well as expression levels of osmoregulation-related gene expression levels in gills. Both dietary cholesterol level and water salinity significantly affected most cholesterol metabolic parameters in the serum and tissues, and the results showed that low salinity promoted cholesterol synthesis but inhibited cholesterol catabolism. Besides, in low salinity, hepatic expression levels of LC-PUFA biosynthesis genes were upregulated by fed dietary cholesterol supplementation with contents of LC-PUFAs, including EPA and DHA being increased. Malondialdehyde (MDA) was significantly increased in low-salinity environment, whereas MDA content was decreased in fish fed with dietary CH1.0 by activating related antioxidant enzyme activity and gene expression levels. A similar pattern was recorded for ERS, which stimulated the expression of nuclear factor kappa B (nf-κb), triggering inflammation. Nevertheless, fish reared in low salinity and fed with dietary CH1.0 had markedly alleviated ERS and downregulated gene expression levels of pro-inflammatory cytokines. Overall, these findings demonstrate that cholesterol, as an important nutrient, plays vital roles in the process of adaptation to low salinity of A. schlegelii, and provides a new insight into underlying adaptive strategies of euryhaline marine fish reared in low salinity.

The Benefit of Optimal Dietary Lipid Level for Black Seabream Acanthopagrus schlegelii Juveniles under Low-Salinity Environment.

The present study was aimed at evaluating the regulatory effects of dietary lipid levels on growth performance, osmoregulation, fatty acid composition, lipid metabolism, and physiological response in Acanthopagrus schlegelii under low salinity (5 psu). An 8-week feeding trial was conducted in juvenile A. schlegelii with an initial weight of 2.27 ± 0.05 g, and six isonitrogenous experimental diets were formulated with graded levels of lipid: 68.7 g/kg (D1), 111.7 g/kg (D2), 143.5 g/kg (D3), 188.9 g/kg (D4), 239.3 g/kg (D5), and 269.4 g/kg (D6), respectively. Results indicated that fish fed with diet containing 188.9 g/kg lipid significantly improved growth performance. Dietary D4 improved ion reabsorption and osmoregulation by increasing the concentrations of Na+, K+, and cortisol in serum and activities of Na+/K+-ATPase as well as expression levels of osmoregulation related to gene expression levels in the gill and intestine. The expression levels of long chain polyunsaturated fatty acid biosynthesis-related genes were dramatically upregulated when dietary lipid levels increased from 68.7 g/kg to 189.9 g/kg with levels of docosahexaenoic (DHA), eicosapentaenoic (EPA), and DHA/EPA ratio being highest in the D4 group. When fish fed dietary lipid levels from 68.7 g/kg to 188.9 g/kg, lipid homeostasis could be maintained by upregulating sirt1 and pparα expression levels, whereas lipid accumulation was observed in dietary lipid levels of 239.3 g/kg and over. Fish fed with high dietary lipid levels resulted in physiological stress related to oxidative stress and endoplasmic reticulum stress. In conclusion, based on weight gain, the optimal dietary lipid requirement of juvenile A. schlegelii reared at low-salinity water is 196.0 g/kg. These findings indicate that the optimal dietary lipid level can improve growth performance, n-3 LC-PUFA accumulation, and osmoregulatory ability and maintain lipid homeostasis and normal physiological functions of juvenile A. schlegelii.

Dietary Betaine Mitigates Hepatic Steatosis and Inflammation Induced by a High-Fat-Diet by Modulating the Sirt1/Srebp-1/Pparɑ Pathway in Juvenile Black Seabream (Acanthopagrus schlegelii).

The present study aimed to elucidate the mechanism of dietary betaine, as a lipid-lowering substance, on the regulation of lipid metabolism and inflammation in juvenile black seabream (Acanthopagrus schlegelii) fed a high fat diet. An 8-week feeding trial was conducted in black seabream with an initial weight of 8.39 ± 0.01g fed four isonitrogenous diets including Control, medium-fat diet (11%); HFD, high-fat diet (17%); and HFD supplemented with two levels (10 and 20 g/kg) of betaine, HFD+B1 and HFD+B2, respectively. SGR and FE in fish fed HFD+B2 were significantly higher than in fish fed HFD. Liver histology revealed that vacuolar fat droplets were smaller and fewer in bream fed HFD supplemented with betaine compared to fish fed HFD. Betaine promoted the mRNA and protein expression levels of silent information regulator 1 (Sirt1), up-regulated mRNA expression and protein content of lipid peroxisome proliferator-activated receptor alpha (pparα), and down-regulated mRNA expression and protein content of sterol regulatory element-binding protein-1(srebp-1). Furthermore, the mRNA expression levels of anti-inflammatory cytokines in liver and intestine were up-regulated, while nuclear factor kB (nf-kb) and pro-inflammatory cytokines were down-regulated by dietary betaine supplementation. Likewise, in fish that received lipopolysaccharide (LPS) to stimulate inflammatory responses, the expression levels of mRNAs of anti-inflammatory cytokines in liver, intestine and kidney were up-regulated in fish fed HFD supplemented with betaine compared with fish fed HFD, while nf-kb and pro-inflammatory cytokines were down-regulated. This is the first report to suggest that dietary betaine could be an effective feed additive to alleviate hepatic steatosis and attenuate inflammatory responses in black seabream fed a high fat diet by modulating the Sirt1/Srebp-1/Pparɑ pathway.

Dietary fenofibrate attenuated high-fat-diet-induced lipid accumulation and inflammation response partly through regulation of pparα and sirt1 in juvenile black seabream (Acanthopagrus schlegelii).

An 8-week feeding trail was conducted in Acanthopagrus schlegelii with an initial body weight of 8.34 ± 0.01g. Three isonitrogenous diets were formulated, (1) Control: medium-fat diet (12%); (2) HFD: high-fat diet (18%); (3) HFD + FF: high-fat diet with fenofibrate (0.15%). Liver histological analysis revealed that, compared to HFD, vacuolar fat drops were smaller and fewer in fish fed fenofibrate. Expression of lipid catabolism regulator peroxisome proliferator-activated receptor alpha (pparα) was up-regulated by fenofibrate compared with HFD. In addition, fenofibrate significantly increased the expression level of silent information regulator 1 (sirt1). Meanwhile, the expression level of anti-inflammatory cytokine interleukin 10 (il-10) in intestine was up-regulated, while pro-inflammatory cytokine interleukin 1ß (il-1ß) in liver and intestine were down-regulated by dietary fenofibrate supplementation. Overall, the present study indicated that fenofibrate reduced fat deposition and attenuated inflammation response caused by HFD partly through a pathway involving regulation of pparα and sirt1.

Dietary choline supplementation attenuated high-fat diet-induced inflammation through regulation of lipid metabolism and suppression of NFκB activation in juvenile black seabream (Acanthopagrus schlegelii).

The present study aimed to investigate whether dietary choline can regulate lipid metabolism and suppress NFκB activation and, consequently, attenuate inflammation induced by a high-fat diet in black sea bream (Acanthopagrus schlegelii). An 8-week feeding trial was conducted on fish with an initial weight of 8·16 ± 0·01 g. Five diets were formulated: control, low-fat diet (11 %); HFD, high-fat diet (17 %); and HFD supplemented with graded levels of choline (3, 6 or 12 g/kg) termed HFD + C1, HFD + C2 and HFD + C3, respectively. Dietary choline decreased lipid content in whole body and tissues. Highest TAG and cholesterol concentrations in serum and liver were recorded in fish fed the HFD. Similarly, compared with fish fed the HFD, dietary choline reduced vacuolar fat drops and ameliorated HFD-induced pathological changes in liver. Expression of genes of lipolysis pathways were up-regulated, and genes of lipogenesis down-regulated, by dietary choline compared with fish fed the HFD. Expression of nfκb and pro-inflammatory cytokines in liver and intestine was suppressed by choline supplementation, whereas expression of anti-inflammatory cytokines was promoted in fish fed choline-supplemented diets. In fish that received lipopolysaccharide to stimulate inflammatory responses, the expression of nfκb and pro-inflammatory cytokines in liver, intestine and kidney were all down-regulated by dietary choline compared with the HFD. Overall, the present study indicated that dietary choline had a lipid-lowering effect, which could protect the liver by regulating intrahepatic lipid metabolism, reducing lipid droplet accumulation and suppressing NFκB activation, consequently attenuating HFD-induced inflammation in A. schlegelii.