Vitamin D3 Regulates Energy Homeostasis under Short-Term Fasting Condition in Zebrafish (Danio Rerio).

Vitamin D3 (VD3) is a steroid hormone that plays pivotal roles in pathophysiology, and 1,25(OH)2D3 is the most active form of VD3. In the current study, the crucial role of VD3 in maintaining energy homeostasis under short-term fasting conditions was investigated. Our results confirmed that glucose-depriving pathways were inhibited while glucose-producing pathways were strengthened in zebrafish after fasting for 24 or 48 h. Moreover, VD3 anabolism in zebrafish was significantly suppressed in a time-dependent manner under short-fasting conditions. After fasting for 24 or 48 h, zebrafish fed with VD3 displayed a higher gluconeogenesis level and lower glycolysis level in the liver, and the serum glucose was maintained at higher levels, compared to those fed without VD3. Additionally, VD3 augmented the expression of fatty acids (FAs) transporter cd36 and lipogenesis in the liver, while enhancing lipolysis in the dorsal muscle. Similar results were obtained in cyp2r1-/- zebrafish, in which VD3 metabolism is obstructed. Importantly, it was observed that VD3 induced the production of gut GLP-1, which is considered to possess a potent gluconeogenic function in zebrafish. Meanwhile, the gene expression of proprotein convertase subtilisin/kexin type 1 (pcsk1), a GLP-1 processing enzyme, was also induced in the intestine of short-term fasted zebrafish. Notably, gut microbiota and its metabolite acetate were involved in VD3-regulated pcsk1 expression and GLP-1 production under short-term fasting conditions. In summary, our study demonstrated that VD3 regulated GLP-1 production in zebrafish by influencing gut microbiota and its metabolite, contributing to energy homeostasis and ameliorating hypoglycemia under short-term fasting conditions.

Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish.

Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event.

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Sterol Regulatory Element Binding Protein 1: A Mediator for High-Fat Diet-Induced Hepatic Gluconeogenesis and Glucose Intolerance in Fish.

BACKGROUND: Sterol regulatory element binding protein (SREBP) 1 is considered to be a crucial regulator for lipid synthesis in vertebrates. However, whether SREBP1 could regulate hepatic gluconeogenesis under high-fat diet (HFD) condition is still unknown, and the underlying mechanism is also unclear. OBJECTIVES: This study aimed to determine gluconeogenesis-related gene and protein expressions in response to HFD in large yellow croaker and explore the role and mechanism of SREBP1 in regulating the related transcription and signaling. METHODS: Croakers (mean weight, 15.61 ± 0.10 g) were fed with diets containing 12% crude lipid [control diet (ND)] or 18% crude lipid (HFD) for 10 weeks. The glucose tolerance, insulin tolerance, hepatic gluconeogenesis-related genes, and proteins expressions were determined. To explore the role of SREBP1 in HFD-induced gluconeogenesis, SREBP1 was inhibited by pharmacologic inhibitor (fatostatin) or genetic knockdown in croaker hepatocytes under palmitic acid (PA) condition. To explore the underlying mechanism, luciferase reporter and chromatin immunoprecipitation assays were conducted in HEK293T cells. Data were analyzed using analysis of variance or Student t test. RESULTS: Compared with ND, HFD increased the mRNA expressions of gluconeogenesis genes (2.40-fold to 2.60-fold) (P < 0.05) and reduced protein kinase B (AKT) phosphorylation levels (0.28-fold to 0.34-fold) (P < 0.05) in croakers. However, inhibition of SREBP1 by fatostatin addition or SREBP1 knockdown reduced the mRNA expressions of gluconeogenesis genes (P < 0.05) and increased AKT phosphorylation levels (P < 0.05) in hepatocytes, compared with that by PA treatment. Moreover, fatostatin addition or SREBP1 knockdown also increased the mRNA expressions of irs1 (P < 0.05) and reduced serine phosphorylation of IRS1 (P < 0.05). Furthermore, SREBP1 inhibited IRS1 transcriptions by binding to its promoter and induced IRS1 serine phosphorylation by activating diacylglycerol-protein kinase Cε signaling. CONCLUSIONS: This study reveals the role of SREBP1 in hepatic gluconeogenesis under HFD condition in croakers, which may provide a potential strategy for improving HFD-induced glucose intolerance.

Optimal dietary lipid levels alleviated adverse effects of high temperature on growth, lipid metabolism, antioxidant and immune responses in juvenile turbot (Scophthalmus maximus L.).

Fish physiological health is often negatively impacted by high-temperature environments and there are few studies on how dietary lipids affect fish growth and physiology when exposed to heat stress. The main objective of this research was to examine the impact of dietary lipid levels on growth and physiological status of juvenile turbot (Scophthalmus maximus L.) and determine if dietary lipid concentration could alleviate the possible adverse effects of heat stress. Five diets containing 6.81%, 9.35%, 12.03%, 14.74%, and 17.08% lipid, respectively, were formulated and fed to turbot (initial weight 5.13 ± 0.02 g) under high-temperature conditions (24.0-25.0 °C). Meanwhile, the diet with 12.03% lipid (considered by prior work to be an optimal dietary lipid level) was fed to turbot of the same size at normal temperature. Results suggested that, among the different dietary lipid levels under high-temperature conditions, fish fed the optimal lipid (12.03%) exhibited better growth compared to non-optimal lipid groups, as evidenced by higher weight gain and specific growth rate. Simultaneously, the optimal lipid diet may better maintain lipid homeostasis, as attested by lower liver and serum lipid, along with higher liver mRNA levels of lipolysis-related genes (pgc1α, lipin1, pparα, lpl and hl) and lower levels of synthesis-related genes (lxr, fas, scd1, pparγ, dgat1 and dgat2). Also, the optimal lipid diet might mitigate oxidative damage by improving antioxidant enzyme activity, decreasing malondialdehyde levels, and up-regulating oxidation-related genes (sod1, sod2, cat, gpx and ho-1). Furthermore, the optimal lipid may enhance fish immunity, as suggested by the decrease in serum glutamic-oxalacetic/pyruvic transaminase activities, down-regulation of pro-inflammatory genes and up-regulation of anti-inflammation genes. Correspondingly, the optimal lipid level suppressed MAPK signaling pathway via decreased phosphorylation levels of p38, JNK and ERK proteins in liver. In summary, the optimal dietary lipid level facilitated better growth and physiological status in turbot under thermal stress.

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

Dietary methanotroph bacteria meal alleviates soybean meal-induced enteritis by improving immune tolerance and intestinal flora profile of juvenile turbot (Scophthalmus maximus L.).

An 8-week growth trial was performed to investigate the protective effects of methanotroph bacteria meal (MBM) produced from methane against soybean meal-induced enteritis (SBMIE) in juvenile turbot (Scophthalmus maximus L.). Five isonitrogenous and isolipidic diets were formulated: fishmeal-based diet (FM, the control group); FM with approximate 50% of fishmeal substituted by 399.4 g/kg soybean meal (SBM); SBM supplemented with 63.6, 127.2 and 190.8 g/kg MBM (named MBM1, MBM2 and MBM3), each diet was randomly assigned to triplicate fibreglass tanks. Results showed that fish fed with SBM exhibited enteritis, identified by reduced relative weight of intestine (RWI), as well as expanded lamina propria width and up-regulated gene expression of pro-inflammatory cytokines (tnf-α, il-6 and il-8) in intestine. While the above symptoms were reversed when diet SBM supplemented with MBM at the levels of 63.6 and 127.2 g/kg, as well as characterized by up-regulated gene expression of anti-inflammatory cytokines (tgf-ß and il-10) and tight junction protein (claudin3, claudin4 and claudin7) in intestine. Intestinal transcriptome analysis showed that the differentially expressed genes between groups FM and SBM predominantly enriched in the JAK-STAT signaling pathway, and the enrichment of differentially expressed genes between groups SBM and SBM supplemented with 63.6 g/kg MBM was in the inflammatory bowel disease (IBD) and JAK-STAT signaling pathway. To be specific, the expression of jak1, jak2b, stat1 and stat5a was significantly up-regulated when fish fed with SBM, suggested the activation of JAK-STAT signaling pathway, while the expression of these above genes was depressed by providing MBM to diet SBM, and the gene expression of toll-like receptors tlr2 and tlr5b showed a similar pattern. Moreover, intestinal flora analysis showed that community richness and abundance of beneficial bacteria (Cetobacterium and acillus_coagulans) were improved when fish fed with SBM supplemented with 63.6 g/kg MBM. Overall, methanotroph bacteria meal may alleviate SBMIE by regulating the expression of tight junction protein, toll-like receptors and JAK-STAT signaling pathway, as well as improving intestinal flora profile, which would be beneficial for enhancing the immune tolerance and utilization efficiency of turbot to dietary soybean meal.

Dietary palm oil enhances Sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis through inducing endoplasmic reticulum stress in muscle of large yellow croaker (Larimichthys crocea).

Sterol regulatory element-binding protein 2 (SREBP2) is considered to be a major regulator to control cholesterol homoeostasis in mammals. However, the role of SREBP2 in teleost remains poorly understand. Here, we explored the molecular characterisation of SREBP2 and identified SREBP2 as a key modulator for 3-hydroxy-3-methylglutaryl-coenzyme A reductase and 7-dehydrocholesterol reductase, which were rate-limiting enzymes of cholesterol biosynthesis. Moreover, dietary palm oil in vivo or palmitic acid (PA) treatment in vitro elevated cholesterol content through triggering SREBP2-mediated cholesterol biosynthesis in large yellow croaker. Furthermore, our results also found that PA-induced activation of SREBP2 was dependent on the stimulating of endoplasmic reticulum stress (ERS) in croaker myocytes and inhibition of ERS by 4-Phenylbutyric acid alleviated PA-induced SREBP2 activation and cholesterol biosynthesis. In summary, our findings reveal a novel insight for understanding the role of SREBP2 in the regulation of cholesterol metabolism in fish and may deepen the link between dietary fatty acid and cholesterol biosynthesis.

Molecular cloning, tissue expression pattern, responses to different fatty acids and potential functions of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in large yellow croaker (Larimichthys crocea).

In farmed fish, diets rich in palm oil have been observed to promote abnormal lipid build-up in the liver, subsequently leading to physiological harm and disease onset. Emerging research suggests that integrating phospholipids into the feed could serve as a potent countermeasure against hepatic impairments induced by vegetable oil consumption. Phosphatidylcholine is the most abundant type among phospholipids. In the metabolic processes of mammal, lysophosphatidylcholine acyltransferase 1 (LPCAT1), crucial for phosphatidylcholine remodeling, demonstrates a marked affinity towards palmitic acid (PA). Nonetheless, aspects concerning the cloning, tissue-specific distribution, and affinity of the LPCAT1 gene to diverse oil sources have yet to be elucidated in the large yellow croaker (Larimichthys crocea). Within the scope of this study, we successfully isolated and cloned the cDNA of the LPCAT1 gene from the large yellow croaker. Subsequent analysis revealed distinct gene expression patterns of LPCAT1 across ten different tissues of the species. The fully sequenced coding DNA sequence (CDS) of LPCAT1 spans 1503 bp and encodes a sequence of 500 amino acids. Comparative sequence alignment indicates that LPCAT1 shares a 69.75 % amino acid similarity with its counterparts in other species. Although LPCAT1 manifests across various tissues of the large yellow croaker, its predominance is markedly evident in the liver and gills. Furthermore, post exposure of the large yellow croaker's hepatocytes to varied fatty acids, PA has a strong response to LPCAT1. Upon the addition of appropriate lysolecithin to palm oil feed, the mRNA expression of LPCAT1 in the liver cells of the large yellow croaker showed significant variations compared to other subtypes. Concurrently, the mRNA expression of pro-inflammatory genes il-1ß, il-6, il-8, tnf-α and ifn-γ in the liver tissue of the large yellow croaker decreased. Interestingly, they exhibit the same trend of change. In conclusion, we have cloned the LPCAT1 gene on fish successfully and find the augmented gene response of LPCAT1 in hepatocytes under PA treatment first. The results of this study suggest that LPCAT1 may be associated with liver inflammation in fish and offer new insights into mitigating liver diseases in fish caused by palm oil feed.

Palmitic acid activates NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1ß production in large yellow croaker (Larimichthys crocea).

Studies on marine fish showed that vegetable oils substituted for excessive fish oil increased interleukin-1ß (IL-1ß) production. However, whether the nucleotide-binding oligomerization domain, leucine-rich repeat-containing family, pyrin domain-containing-3 (NLRP3) inflammasome has a substantial role in fatty acid-induced IL-1ß production in fish remains unclear. The associated specific mechanism is also unknown. In this study, nlrp3, caspase-1 and apoptosis-associated speck-like protein containing a CARD (asc) were successfully cloned, and NLRP3 inflammasome consisted of NLRP3, caspase-1 and ASC in large yellow croaker. Primary hepatocytes of fish incubated with palmitic acid (PA) exhibited the highest expression of pro-inflammatory genes (il-1ß and tnfα) and NLRP3 inflammasome related genes (nlrp3, caspase-1 and asc), caspase-1 activity and IL-1ß production among different treatments. Furthermore, PA-induced NLRP3 inflammasome activation was confirmed to require two signals: the first signal was that PA promoted the NF-κB (P65) protein into the nucleus, and NF-κB increased NLRP3 promoter activity and nlrp3 transcription. The second signal was that PA inhibited AMPK phosphorylation and decreased mitophagy by inhibiting the expression of PINK and parkin proteins, thereby damaging the mitochondria that could not be effectively cleared. Mitochondrial damage generated excessive amounts of reactive oxygen species, which activated the NLRP3 inflammasome and then induced caspase-1 activity and IL-1ß production. Therefore, excessive dietary PA activated NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1ß production, thereby leading to inflammation in fish.

Vitamin D regulates glucose metabolism in zebrafish (Danio rerio) by maintaining intestinal homeostasis.

Vitamin D (VD) is a steroid hormone that is widely known to play an important role in maintaining mineral homeostasis, and regulating various physiological functions. Our previous results demonstrated that the interruption of VD metabolism caused hyperglycemia in zebrafish. In the present study we further explored the mechanism that VD regulates glucose metabolism by maintaining intestinal homeostasis in zebrafish. Our results showed that the expression of several peptide hormones including gastric inhibitory peptide, peptide YY, and fibroblast growth factor 19 in the intestine decreased, while the expression of sodium glucose cotransporter-1 and gcg was increased in the intestine of the zebrafish fed with the VD3-deficient diet. Consistently, similar results were obtained in cyp2r1-/- zebrafish, in which endogenous VD metabolism is blocked. Furthermore, the results obtained from germ-free zebrafish exhibited that VD-regulated glucose metabolism was partly dependent on the microbiota in zebrafish. Importantly, the transplantation of gut microbiota collected from cyp2r1-/- zebrafish to germ-free zebrafish led to hyperglycemic symptoms in the fish, which were associated with the altered structure and functions of the microbiota in cyp2r1-/- zebrafish. Interestingly, the treatments with acetate or Cetobacterium somerae, a potent acetate producer, lowered the glucose contents whereas augmented insulin expression in zebrafish larvae. Notably, acetate supplementation alleviated hyperglycemia in cyp2r1-/- zebrafish and other diabetic zebrafish. In conclusion, our study has demonstrated that VD modulates the gut microbiota-SCFAs-gastrointestinal hormone axis, contributing to the maintenance of glucose homeostasis.

Exploring the role of SWI/SNF complex subunit BAF60c in lipid metabolism and inflammation in fish.

Chromatin remodeling plays an important role in regulating gene transcription, in which chromatin remodeling complex is a crucial aspect. Brg1/Brm-associated factor 60c (BAF60c) subunit forms a bridge between chromatin remodeling complexes and transcription factors in mammals; hence, it has received extensive attention. However, the roles of BAF60c in fish remain largely unexplored. In this study, we identified BAF60c-interacting proteins by using HIS-pull-down and LC-MS/MS analysis in fish. Subsequently, the RNA-seq analysis was performed to identify the overall effects of BAF60c. Then, the function of BAF60c was verified through BAF60c knockdown and overexpression experiments. We demonstrated for the first time that BAF60c interacts with glucose-regulated protein 78 (GRP78) and regulates lipid metabolism, endoplasmic reticulum (ER) stress, and inflammation. Knockdown of BAF60c reduces fatty acid biosynthesis, ER stress, and inflammation. In conclusion, the results enriched BAF60c-interacting protein network and explored the function of BAF60c in lipid metabolism and inflammation in fish.

Butyrate induces STAT3/HIF-1α/IL-22 signaling via GPCR and HDAC3 inhibition to activate autophagy in head kidney macrophages from turbot (Scophthalmus maximus L.).

As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages.

Effects of dietary bile acids levels on growth performance, anti-oxidative capacity, immunity and intestinal microbiota of abalone Haliotis discus hannai.

Abalone Haliotis discus hannai (initial weight: 38.79 ± 0.70 g) was used as the experimental animal in a 105-day feeding trial to investigate the influence of dietary bile acids levels on the growth, anti-oxidation, immune response and intestinal microbiota. Six isonitrogenous and isolipidic diets were prepared by adding 0 (control group), 15, 30, 60, 120 and 240 mg/kg of bile acids, respectively (named BA0, BA15, BA30, BA60, BA120 and BA240, respectively). It was found that survival of abalone between groups had no significant difference (P > 0.05). Compared to the control, significant improvements in weight gain rate (WGR) were observed in the groups of BA30 and BA60 (P < 0.05). Based on WGR, the broken line regression model analysis showed that the optimum demand for dietary bile acids for abalone was 35.47 mg/kg. Dietary bile acids increased the total anti-oxidative capacity and activities of catalase, superoxide dismutase, lysozyme and alkaline phosphatase, meanwhile decreased the content of malondialdehyde, alanine aminotransferase and aspartate aminotransferase activities in the cell-free hemolymph (P < 0.05). When bile acids were added to the diets, mRNA levels of genes related to pro-inflammatory factors and apoptosis in the digestive gland were down-regulated (P < 0.05). In contrast, the expression of genes related to anti-oxidation was significantly up-regulated (P < 0.05). The Firmicutes, Actinobacteriota and Proteobacteria were the most abundant phyla in intestine. And dietary bile acids significantly decreased the abundance of Actinobacteria and increased the abundance of Firmicutes (P < 0.05). In conclusion, supplementation of dietary bile acids within 120 mg/kg significantly increased the growth of abalone. The 34.62 mg/kg of dietary bile acids significantly increased the anti-oxidative capacity of abalone. Appropriate levels of dietary bile acids (34.62-61.75 mg/kg) promote the immunity of abalone. Application of appropriate levels of bile acids in diets (34.62 mg/kg) changed the intestinal microbiota and promoted the intestinal health of abalone.

Regulation of low-density lipoprotein on lipid metabolism in macrophages of large yellow croaker (Larimichthys crocea).

Low-density lipoprotein (LDL) is the main carrier of cholesterol transport in plasma, which participates in regulating lipid homeostasis. Studies in mammals have shown that high levels of LDL in plasma absorbed by macrophages trigger the formation of lipid-rich foam cells, leading to the development of atherosclerotic plaques. Although lipid-rich atherosclerosis-like lesions have been discovered in the aorta of several fish species, the physiological function of LDL in fish macrophages remains poorly understood. In the present study, LDL was isolated from the plasma of large yellow croaker (Larimichthys crocea), and mass spectrometry analysis identified two truncated forms of apolipoprotein B100 in the LDL protein profile. Transcriptomic analysis of LDL-stimulated macrophages revealed that differentially expressed genes (DEGs) were enriched in various pathways related to lipid metabolism, as confirmed by the fact that LDL increased total cholesterol and cholesteryl esters content. Meanwhile, the gene and protein expression levels of perilipin2 (PLIN2), a DEG enriched in the PPAR signaling pathway, were upregulated in response to LDL stimulation. Importantly, knocking down plin2 significantly attenuates LDL-induced cholesterol accumulation and promotes cholesterol efflux. Furthermore, the transcription factor PPARγ, which is upregulated in response to LDL stimulation, can enhance the promoter activity of plin2. In conclusion, this study suggests that LDL may upregulate plin2 expression through PPARγ, resulting in cholesterol accumulation in fish macrophages. This study will facilitate the investigation of the function of LDL in regulating lipid homeostasis in macrophages and shed light on the evolutionary origin of LDL metabolism in vertebrates.

Effects of dietary chloroquine on fish growth, hepatic intermediary metabolism, antioxidant and inflammatory responses in turbot.

Autophagy is a conserved cellular self-digestion process and is essential for individual growth, cellular metabolism and inflammatory responses. It was responsive to starvation, pathogens infection and environmental stress. However, the information on the regulation of autophagy in fish hepatic intermediary metabolism, antioxidant system, and immune responses were limited. In the present study, turbot with inhibited autophagy flux was built by dietary chloroquine. The hepatic metabolic response, antioxidant enzymes and immune responses were explored. Results showed that dietary chloroquine induced the expression of Beclin 1, SQSTM and LC-3II, and effectively inhibited autophagy flux. Autophagy dysfunction depressed fish growth and feed utilization, while it induced clusters of liver lipid droplets. The genes involved in lipolysis and fatty acid ß-oxidation, as well as the lipogenesis-related genes in chloroquine group were depressed. The phosphorylation of AMPK was activated in chloroquine group, and the genes involved in glycolysis were induced. The hepatic content of malonyldialdehyde and the activities of SOD and CAT were induced when autophagy was inhibited. The content of Complement 3, Complement 4 and Immunoglobulin M, as well as the activity of lysozyme in plasma were depressed in chloroquine group. Dietary chloroquine induced the expression of toll-like receptors and stimulated the expression of myd88 and nf-κb p65, as well as the pro-inflammatory cytokines, such as tnf-α and il-1ß. The expression of anti-inflammatory cytokine tgf-ß was depressed in the chloroquine group. Our results would extend the knowledge on the role of autophagy in teleost and assist in improving fishery production.

Comprehensive study on the effect of dietary leucine supplementation on intestinal physiology, TOR signaling and microbiota in juvenile turbot (Scophthalmus maximus L.).

Intestinal damage and inflammation are major health and welfare issues in aquaculture. Considerable efforts have been devoted to enhancing intestinal health, with a specific emphasis on dietary additives. Branch chain amino acids, particularly leucine, have been reported to enhance growth performance in various studies. However, few studies have focused on the effect of leucine on the intestinal function and its underlying molecular mechanism is far from fully illuminated. In the present study, we comprehensively evaluated the effect of dietary leucine supplementation on intestinal physiology, signaling transduction and microbiota in fish. Juvenile turbot (Scophthalmus maximus L.) (10.13 ± 0.01g) were fed with control diet (Con diet) and leucine supplementation diet (Leu diet) for 10 weeks. The findings revealed significant improvements in intestinal morphology and function in the turbot fed with Leu diet. Leucine supplementation also resulted in a significant increase in mRNA expression levels of mucosal barrier genes, indicating enhanced intestinal integrity. The transcriptional levels of pro-inflammatory factors il-1ß, tnf-α and irf-1 was decreased in response to leucine supplementation. Conversely, the level of anti-inflammatory factors tgf-ß, il-10 and nf-κb were up-regulated by leucine supplementation. Dietary leucine supplementation led to an increase in intestinal complement (C3 and C4) and immunoglobulin M (IgM) levels, along with elevated antioxidant activity. Moreover, dietary leucine supplementation significantly enhanced the postprandial phosphorylation level of the target of rapamycin (TOR) signaling pathway in the intestine. Finally, intestinal bacterial richness and diversity were modified and intestinal bacterial composition was re-shaped by leucine supplementation. Overall, these results provide new insights into the beneficial role of leucine supplementation in promoting intestinal health in turbot, offering potential implications for the use of leucine as a nutritional supplement in aquaculture practices.

Phosphatidylethanolamine alleviates OX-LDL-induced macrophage inflammation by upregulating autophagy and inhibiting NLRP1 inflammasome activation.

Oxidized low-density lipoprotein (OX-LDL)-induced inflammation and autophagy dysregulation are important events in the progression of atherosclerosis. Phosphatidylethanolamine (PE), a multifunctional phospholipid that is enriched in cells, has been proven to be directly involved in autophagy which is closely associated with inflammation. However, whether PE can influence OX-LDL-induced autophagy dysregulation and inflammation has not been reported. In the present study, we revealed that OX-LDL significantly induced macrophage inflammation through the CD36-NLRP1-caspase-1 signaling pathway in fish. Meanwhile, cellular PE levels were significantly decreased in response to OX-LDL induction. Based on the relationship between PE and autophagy, we then examined the effect of PE supplementation on OX-LDL-mediated autophagy impairment and inflammation induction in macrophages. As expected, exogenous PE restored impaired autophagy and alleviated inflammation in OX-LDL-stimulated cells. Notably, autophagy inhibitors reversed the inhibitory effect of PE on OX-LDL-induced maturation of IL-1ß, indicating that the regulation of PE on OX-LDL-induced inflammation is dependent on autophagy. Furthermore, the positive effect of PE on OX-LDL-induced inflammation was relatively conserved in mouse and fish macrophages. In conclusion, we elucidated the role of the CD36-NLRP1-caspase-1 signaling pathway in OX-LDL-induced inflammation in fish and revealed for the first time that altering PE abundance in OX-LDL-treated cells could alleviate inflammasome-mediated inflammation by inducing autophagy. Given the relationship between OX-LDL-induced inflammation and atherosclerosis, this study prompts that the use of PE-rich foods promises to be a new strategy for atherosclerosis treatment in vertebrates.

Transcriptome-based nutrigenomics analysis reveals the roles of dietary taurine in the muscle growth of juvenile turbot (Scophthalmus maximus).

The present study explored transcriptomics and gene regulation variations in the muscle of turbot fed with dietary taurine. A 70-day feeding trial was conducted using turbot (initial body weight: 3.66 ± 0.02 g) fed with different levels of dietary taurine: 0 % (C), 0.4 % (T2), 1.2 % (T4) and 2.0 % (T6). Two methods were used to analyze and verify the taurine effects on muscle growth: (1) real-time quantitative PCR (qRT-PCR) for the key muscle growth-related genes and (2) transcriptomic analysis by next-generation sequencing (NGS). The results showed that 1.2 % of dietary taurine supplementation significantly increased the expression of muscle growth stimulatory genes, including TauT, myoD, Myf5, myogenin and follistatin. And also, the 1.2 % level significantly decreased the expression of the muscle growth-restricting gene (myostatin). Meanwhile, transcriptomics analysis found that 1.2 % dietary taurine supplementation significantly increased the number of up-regulated genes linked to metabolic pathways. In contrast, taurine significantly enriched the actin cytoskeleton and metabolic pathways in the T4 and T2 groups, respectively. These findings align with the gene ontology (GO) analysis, which indicated a higher number of cellular component (CC) gene expressions at a 1.2 % of dietary taurine compared to a 0.4 % of dietary taurine supplementation. In conclusion, dietary taurine had positive impacts on the growth-stimulatory genes. Moreover, 1.2 % of dietary taurine supplementation is important to the metabolic pathway enrichment.