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.

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.

Vitamin D influences gut microbiota and acetate production in zebrafish (Danio rerio) to promote intestinal immunity against invading pathogens.

Although evidence has shown that vitamin D (VD) influences gut homeostasis, limited knowledge is available how VD regulates intestinal immunity against bacterial infection. In the present study, cyp2r1 mutant zebrafish, lacking the capacity to metabolize VD, and zebrafish fed a diet devoid of VD, were utilized as VD-deficient animal models. Our results confirmed that the expression of antimicrobial peptides (AMPs) and IL-22 was restrained and the susceptibility to bacterial infection was increased in VD-deficient zebrafish. Furthermore, VD induced AMP expression in zebrafish intestine by activating IL-22 signaling, which was dependent on the microbiota. Further analysis uncovered that the abundance of the acetate-producer Cetobacterium in VD-deficient zebrafish was reduced compared to WT fish. Unexpectedly, VD promoted the growth and acetate production of Cetobacterium somerae under culture in vitro. Importantly, acetate treatment rescued the suppressed expression of ß-defensins in VD-deficient zebrafish. Finally, neutrophils contributed to VD-induced AMP expression in zebrafish. In conclusion, our study elucidated that VD modulated gut microbiota composition and production of short-chain fatty acids (SCFAs) in zebrafish intestine, leading to enhanced immunity.

Vitamin D3 enhances the antibacterial ability in head-kidney macrophages of turbot (Scophthalmus maximus L.) through C-type lectin receptors.

It has been known that vitamin D3 (VD3) not only plays an important role in regulating calcium and phosphorus metabolism in animals, but also has extensive effects on immune functions. In this study, the mechanism how VD3 influences bactericidal ability in turbot was explored. The transcriptomic analysis identified that dietary VD3 significantly upregulated the gene expression of C-type lectin receptors (CLRs), including mannose receptors (mrc1, mrc2, pla2r1) and collectins (collectin 11 and collectin 12) in turbot intestine. Further results obtained from in vitro experiments confirmed that the gene expression of mannose receptors and collectins in head-kidney macrophages (HKMs) of turbot was induced after the cells were incubated with different concentrations of VD3 (0, 1, 10 nM) or 1,25(OH)2D3 (0, 10, 100 pM). Meanwhile, both phagocytosis and bactericidal functions of HKMs were significantly improved in VD3 or 1,25(OH)2D3-incubated HKMs. Furthermore, phagocytosis and bacterial killing of HKMs decreased after collectin 11 was knocked down. Moreover, VD3-enhanced antibacterial activities diminished in collectin 11-interfered cells. Interestingly, the evidence was provided in the present study that inactive VD3 could be metabolized into active 1,25(OH)2D3 via hydroxylases encoded by cyp27a1 and cyp27b1 in fish macrophages. In conclusion, VD3 could be metabolized to 1,25(OH)2D3 in HKMs, which promoted the expression of CLRs in macrophages, leading to enhanced bacterial clearance.

Vitamin D regulates insulin pathway and glucose metabolism in zebrafish (Danio rerio).

1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], the most active vitamin D (VD) metabolite, is a steroid hormone playing an important role in many physiological functions in addition to maintaining mineral homeostasis. In this study, we explored the mechanism that the VD regulated insulin pathway and glucose metabolism in zebrafish in vitro and in vivo. Our results show that 1,25(OH)2 D3  significantly enhances the expression of insulin receptor a (insra), insulin receptor substrate 1 (irs1) and glucose transporter 2 (glut2), and promotes glycolysis and glycogenesis, while suppressing gluconeogenesis in zebrafish liver cell line (ZFL) under the condition of high glucose (20 mM), instead of the normal glucose (10 mM). Moreover, consistent results were obtained from the zebrafish fed with VD3 -deficient diet, as well as the cyp2r1-/- zebrafish, in which endogenous VD metabolism is blocked. Furthermore, results from dual-luciferase reporting system exhibited that 1,25(OH)2 D3 directly activated the transcription of insra, rather than insrb in zebrafish by binding to vitamin D response element (VDRE) located at -181 to -167 bp in the promoter region of insra. Importantly, the 1,25(OH)2 D3 treatment significantly alleviated the symptoms of hyperglycemia in diabetic zebrafish. In conclusion, our study demonstrated that VD activates VDRE located in the promoter area of insra in zebrafish to promote insulin/insra signaling pathway, thereby contributing to the maintenance of glucose homeostasis.

Vitamin D impacts on the intestinal health, immune status and metabolism in turbot (Scophthalmus maximus L.).

Vitamin D (VD) plays a vital role in various physiological processes in addition to its classic functions on maintaining the balance of Ca and P metabolism. However, there still are gaps to understand in depth the issues on the precise requirement, metabolic processes and physiological functions of VD in fish. In this study, we investigated the effects of VD on the growth, intestinal health, host immunity and metabolism in turbot (Scophthalmus maximus L.), one important commercial carnivorous fish in aquaculture, through the supplementation of different doses of dietary VD3 (0, 200, 400, 800 and 1600 µg VD3/kg diet). According to our results, the optimal VD3 level in the feed for turbot growth was estimated to be around 400 IU/kg, whereas VD3 deficiency or overdose in diets induced the intestinal inflammation, lowered the diversity of gut microbiota and impaired the host resistance to bacterial infection in turbot. Moreover, the level of 1α,25(OH)2D3, the active metabolite of VD3, reached a peak value in the turbot serum in the 400 µg group, although the concentrations of Ca and phosphate in the turbot were stable in all groups. Finally, the deficiency of dietary VD3 disturbed the nutritional metabolism in turbot, especially the metabolism of lipids and glucose. In conclusion, this study evaluated the optimal dose of dietary VD3 for turbot and provided the evidence that VD has a significant impact on intestinal health, host immunity and nutritional metabolism in fish, which deepened our understanding on the physiological functions and metabolism of VD3 in fish.

Vitamin D Enhances Neutrophil Generation and Function in Zebrafish (Danio rerio).

Vitamin D (VD) is a major regulator of calcium metabolism in many living organisms. In addition, VD plays a key role in regulating innate and adaptive immunity in vertebrates. Neutrophils constitute an important part of the first line of defense against invading microbes; however, the potential effect of VD on neutrophils remains elusive. Thus, in this study zebrafish in different developmental stages were utilized to identify the potential role of VD in the basal homeostasis and functions of neutrophils. Our results showed that addition of exogenous VD3 promoted granulopoiesis in zebrafish larvae. Reciprocally, neutrophil abundance in the intestine of adult zebrafish with a cyp2r1 mutant, lacking the capacity to 25-hydroxylate VD, was reduced. Moreover, VD-mediated granulopoiesis was still observed in gnotobiotic zebrafish larvae, indicating that VD regulates neutrophil generation independent of the microbiota during early development. In contrast, VD was incapable to influence granulopoiesis in adult zebrafish when the commensal bacteria were depleted by antibiotic treatment, suggesting that VD might modulate neutrophil activity via different mechanisms depending on the developmental stage. In addition, we found that VD3 augmented the expression of il-8 and neutrophil recruitment to the site of caudal fin amputation. Finally, VD3 treatment significantly decreased bacterial counts and mortality in zebrafish infected with Edwardsiella tarda (E. tarda) in a neutrophil-dependent manner. Combined, these findings demonstrate that VD regulates granulopoiesis and neutrophil function in zebrafish immunity.

Vitamin D3 protects turbot (Scophthalmus maximus L.) from bacterial infection.

Accumulating evidence supports that vitamin D3 (VD3) possesses immunomodulatory properties besides its classical actions in calcium and bone homeostasis. In this study, juvenile turbots were fed with the diets containing 0 IU/kg VD3 or the optimum dose of 400 IU/kg VD3 for 8 weeks. To investigate the effects of VD3 on anti-infectious immunity in fish, 107 CFU Edwardsiella tarda was injected intraperitoneally to each juvenile turbot after the feeding trial. Our results showed that the mortality of infected turbots with dietary VD3 was much lower than that in VD3 deficient group, and the supplementation of dietary VD3 significantly reduced the bacterial load in the spleen of infected turbots. Further analysis demonstrated that the production of reactive oxygen species (ROS) in haemocytes and lysozyme activity in serum was elevated, and the responses of T cells and B cells were modulated in VD3-supplemented turbots. Moreover, the inflammation was significantly exacerbated in the infected turbots fed with 0 IU/kg VD3 compared to the fish fed with 400 IU/kg VD3. In addition, the head kidney macrophages (HKMs) in turbots were isolated and incubated with VD3in vitro, the results showed that VD3 significantly promoted the bactericidal activity in HKMs. In conclusion, our study has shown clear evidence that VD3 positively regulates the innate and adaptive immunity in fish, which is beneficial to the defense in fish against pathogen infection.

Spatio-Temporal Sequential Memory Model With Mini-Column Neural Network.

Memory is an intricate process involving various faculties of the brain and is a central component in human cognition. However, the exact mechanism that brings about memory in our brain remains elusive and the performance of the existing memory models is not satisfactory. To overcome these problems, this paper puts forward a brain-inspired spatio-temporal sequential memory model based on spiking neural networks (SNNs). Inspired by the structure of the neocortex, the proposed model is structured by many mini-columns composed of biological spiking neurons. Each mini-column represents one memory item, and the firing of different spiking neurons in the mini-column depends on the context of the previous inputs. The Spike-Timing-Dependant Plasticity (STDP) is used to update the connections between excitatory neurons and formulates association between two memory items. In addition, the inhibitory neurons are employed to prevent incorrect prediction, which contributes to improving the retrieval accuracy. Experimental results demonstrate that the proposed model can effectively store a huge number of data and accurately retrieve them when sufficient context is provided. This work not only provides a new memory model but also suggests how memory could be formulated with excitatory/inhibitory neurons, spike-based encoding, and mini-column structure.

Short-Chain Fatty Acids Promote Intracellular Bactericidal Activity in Head Kidney Macrophages From Turbot (Scophthalmus maximus L.) via Hypoxia Inducible Factor-1α.

Short-chain fatty acids (SCFAs) are mainly produced by microbiota through the fermentation of carbohydrates in the intestine. Acetate, propionate, and butyrate are the most abundant SCFA metabolites and have been shown to be important in the maintenance of host health. In this study, head kidney macrophages (HKMs) were isolated and cultured from turbots. We found that the antibacterial activity of HKMs was increased after these cells were incubated with sodium butyrate, sodium propionate or sodium acetate. Interestingly, our results showed that all three SCFAs enhanced the expression of hypoxia inducible factor-1 α (HIF-1α) in HKMs, and further study confirmed that butyrate augmented the oxygen consumption of these cells. Moreover, HIF-1α inhibition diminished the butyrate-promoted intracellular bacterial killing activity of macrophages, and SCFAs also raised the gene expression and activity of lysozymes in HKMs via HIF-1α signaling. In addition, our results suggested that butyrate induced HIF-1α expression and the bactericidal activity of HKMs through histone deacetylase inhibition, while G protein-coupled receptors did not contribute to this effect. Finally, we demonstrated that butyrate induced a similar response in the murine macrophage cell line RAW264.7. In conclusion, our results demonstrated that SCFAs promoted HIF-1α expression via histone deacetylase inhibition, leading to the enhanced production of antibacterial effectors and increased bacterial killing of macrophages.

Synergistic Effects of Nano-ZnO and Low pH of Sea Water on the Physiological Energetics of the Thick Shell Mussel Mytilus coruscus.

In order to investigate the ecotoxicological effects of nano-ZnO particles and seawater acidification on marine bivalves, the thick shell mussels, Mytilus coruscus were subjected to joint treatments with different nano-ZnO concentrations (0 [control], 2.5 [medium] and 10 mg L-1 [high]) under two pH levels (7.7 [low]and 8.1 [control]) for 14 days. The results showed that respiration rate (RR), absorption efficiency (AE), clearance rate (CR), O:N ratio and scope for growth (SFG) were significantly reduced with nano-ZnO concentration increase, but ammonium excretion rate (ER) was increased. Low pH significantly reduced CR, RR, SFG, and O:N ratio of the mussels especially under high nano-ZnO conditions, and significantly increased ER. Principal component analysis (PCA) showed consistent relationships among most tested parameters, especially among SFG, RR, O:N ratio and CR under the normal pH and 0 nano-ZnO conditions. Therefore, seawater acidification and nano-ZnO interactively impact the ecophysiological responses of mussels and cause more severe effects when they appear concurrently.

Salinity mediates the toxic effect of nano-TiO2 on the juvenile olive flounder Paralichthys olivaceus.

Increased production of engineered nanoparticles has raised extensive concern about the potential toxic effects on marine organisms living in estuarine and coastal environments. Meanwhile, salinity is one of the key environmental factors that may influence the physiological activities in flatfish species inhabiting in those waters due to fluctuations caused by freshwater input or rainfall. In this study, we investigated the oxidative stress and histopathological alteration of the juvenile Paralichthys olivaceus exposed to nano-TiO2 (1 and 10 mg L-1) under salinities of 10 and 30 psu for 4 days. In the gills, Na+-K+-ATPase activity significantly deceased after 4 days 10 psu exposure without nano-TiO2 compared with 1 day of acclimating the salinity from the normal salinity (30 psu) to 10 psu. Under this coastal salinity, low concentration (1 mg L-1) of nano-TiO2 exerted significant impacts. In the liver, the activities of superoxide dismutase, catalase, the levels of lipid peroxide and malondialdehyde increased with nano-TiO2 exposed under 30 psu. Such increase indicated an oxidative stress response. The result of the integrated biomarker responses showed that P. olivaceus can be adversely affected by high salinity and high concentration of nano-TiO2 for a short-term (4 days) exposure. The histological analysis revealed the accompanying severe damages for the gill filaments. Principal component analysis further showed that the oxidative stress was associated with the nano-TiO2 effect at normal salinity. These findings indicated that nano-TiO2 and normal salinity exert synergistic effects on juvenile P. olivaceus, and low salinity plays a protective role in its physiological state upon short-term exposure to nano-TiO2. The mechanism of salinity mediating the toxic effects of NPs on estuarine fish should be further considered.

Short-Term Exposure of Mytilus coruscus to Decreased pH and Salinity Change Impacts Immune Parameters of Their Haemocytes.

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.