Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

Molecular and functional characterization of sirt4 and sirt6 in Megalobrama amblycephala under high glucose metabolism.

We analyzed the sequences of sirt4 and sirt6 and their changes in expression after oral glucose administration in blunt snout bream (Megalobrama amblycephala). We cloned sirt4 and sirt6 and found that their full-length cDNA sequences were 1530 bp and 1723 bp, respectively; their amino acid sequences were 93% and 92% identical to Danio rerio. Megalobrama amblycephala were fed a high glucose solution (3 g/kg). Normally, sirt4 expression is higher in spleen, intestine, and gill (P < .05), and sirt6 expression is higher in intestine and gill (P < .05). After oral glucose administration, sirt4 and sirt6 expression increased in liver and gill, and sirt4 expression increased in intestine at 0.5 h (P < .05). In contrast, sirt4 in kidney and sirt6 in head kidney were downregulated at 1 h (P < .05). Expression of sirt4 was upregulated in brain, head kidney, spleen, muscle, and liver 2 h, 4 h, 4 h/24 h, 8 h, and 24 h, respectively (P < .05). Expression of sirt4 was downregulated in kidney at 8 h-48 h (P < .05). Expression of sirt6 was upregulated in intestine, liver, muscle, kidney, and spleen at 4 h/24 h, 8 h/24 h, 12 h, 12 h, and 24 h, respectively (P < .05). We report that sirt4 and sirt6 are highly conserved in evolution and exhibit tissue-specific expression profiles. We demonstrate that the expression of sirt4 and sirt6 are tissue-specific, and depend upon tissue-specific responses to glucose metabolism.

High glucose affected respiratory burst activity of peripheral leukocyte via G6PD and NOX inhibition in Megalobrama amblycephala.

High glucose levels are known to impair growth and immune function in fish. Here we investigated the role of glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidase (NOX) in high glucose-associated impairment of leukocyte respiratory burst activity in Megalobrama amblycephala. We cultured peripheral leukocytes isolated from M. amblycephala with media containing no glucose (non-glucose group), 11.1 mmol/L d-glucose (physiologic glucose group), 22.2 mmol/L d-glucose (high-glucose group), or 11.1 mmol/L d-glucose + 100 µmol/L dehydroepiandrosterone (DHEA) (DHEA-treated group). After 24 h, we assayed production of reactive oxygen species (ROS) as a measure of respiratory burst function as well as activity of G6PD and NOX. The high-glucose group and DHEA-treated group showed significantly reduced respiratory burst function, reduced production of ROS, and reduced G6PD and NOX activity at 24 h, compared to the non-glucose and physiologic glucose groups (P < 0.05). The degree of impairment was similar between high-glucose and DHEA-treated groups (P > 0.05). These findings suggest that reduced NADPH availability likely underlies the suppression of respiratory burst function in M. amblycephala leukocytes exposed to high glucose levels.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

Comparative transcriptome analysis reveals the gene expression profiling in bighead carp (Aristichthys nobilis) in response to acute nitrite toxicity.

OBJECTIVE: Nitrite exposure induces growth inhibition, metabolic disturbance, oxidative stress, organic damage, and infection-mediated mortality of aquatic organism. This study aimed to investigate the mechanism in responses to acute nitrite toxicity in bighead carp (Aristichthys nobilis, A. nobilis) by RNA-seq analysis. METHODS: Bighead carps were exposed to water with high nitrite content (48.63 mg/L) for 72 h, and fish livers and gills were separated for RNA-seq analysis. De novo assembly was performed, and differentially expressed genes (DEGs) between control and nitrite-exposed fishes were identified. Furthermore, enrichment analysis was performed for DEGs to annotate the molecular functions. RESULTS: A total of 406,135 transcripts and 352,730 unigenes were tagged after de novo assembly. Accordingly, 4108 and 928 DEGs were respectively identified in gill and liver in responses to nitrite exposure. Most of these DEGs were up-regulated DEGs. Enrichment analysis showed these DEGs were mainly associated with immune responses and nitrogen metabolism. CONCLUSIONS: We suggested that the nitrite toxicity-induced DEGs were probably related to dysregulation of nitrogen metabolism and immune responses in A. nobilis, particularly in gill.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Identification of Differentially Expressed Micrornas Associate with Glucose Metabolism in Different Organs of Blunt Snout Bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is a widely favored herbivorous fish species and is a frequentlyused fish model for studying the metabolism physiology. This study aimed to provide a comprehensive illustration of the mechanisms of a high-starch diet (HSD) induced lipid metabolic disorder by identifying microRNAs (miRNAs) controlled pathways in glucose and lipid metabolism in fish using high-throughput sequencing technologies. Small RNA libraries derived from intestines, livers, and brains of HSD and normal-starch diet (NSD) treated M. amblycephala were sequenced and 79, 124 and 77 differentially expressed miRNAs (DEMs) in intestines, livers, and brains of HSD treated fish were identified, respectively. Bioinformatics analyses showed that these DEMs targeted hundreds of predicted genes were enriched into metabolic pathways and biosynthetic processes, including peroxisome proliferator-activated receptor (PPAR), glycolysis/gluconeogenesis, and insulin signaling pathway. These analyses confirmed that miRNAs play crucial roles in glucose and lipid metabolism related to high wheat starch treatment. These results provide information on further investigation of a DEM-related mechanism dysregulated by a high carbohydrate diet.

Chronic stress effects of high doses of vitamin D3 on Megalobrama amblycephala.

Dietary vitamin D3 plays an important role in the growth of aquatic animals, but long-term excessive feeding has potential hazards. In this study, Megalobrama amblycephala specimens were fed different experimental diets with 2000 IU/kg or 200,000 IU/kg of vitamin D3 for 90 days, in order to evaluate chronic stress effects of high doses of vitamin D3 on growth, immunity, and structural damage to enterohepatic tissues. The results showed that high doses of vitamin D3 did not have a significant influence on the growth performance of M. amblycephala (P > 0.05), but it significantly reduced the survival rate after infection by Aeromonas hydrophila (P < 0.05). Serum albumin, alkaline phosphatase, and insulin levels, as well as hepatic total antioxidant capacity, were also significantly reduced (P < 0.05). Serum cortisol levels and hepatic heat stress protein 70 expression in M. amblycephala showed that high doses of vitamin D3 significantly inhibit the anti-stress ability of M. amblycephala (P < 0.05). Paraffin tissue sections and electron microscopy showed that high doses of vitamin D3 could cause different degrees of structural damage to enterohepatic tissues of M. amblycephala. Our results indicate that, although M. amblycephala can tolerate high doses of dietary vitamin D3 over a long period, its glycolipid metabolism, immune function, anti-stress function, and resistance to pathogenic infections are adversely affected.