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Urotensin I (UI), a member of the corticotropin-releasing hormone family of peptides, regulates a diverse array of physiological functions, including appetite regulation, defensive behavior and stress response. In this study, firstly, the tissue-specific distribution of UI mRNA in olive flounder (Paralichthys olivaceus) was characterized and we found that UI mRNA was highly expressed in caudal neurosecretory system (CNSS) tissue. Secondly, alignment analysis found that a conserved cAMP response binding (CREB) site and a TATA element were located in the proximal promoter of UI gene. In addition, treatment of forskolin activatated cAMP-CREB pathway and induced the up-regulation of UI mRNA in cultured CNSS, suggesting the role of CREB in regulating the UI mRNA expression. Furthermore, plasma UI concentration and UI mRNA in CNSS showed obvious daily rhythm, with higher values in the daytime while lower values in the nighttime. Thirdly, using bold personality (BP) and shy personality (SP) flounder as an animal model, we found that flounder exhibited significantly higher locomotor activity in the nighttime than in the daytime (P

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc × Landrace × Large White crossbred piglets (12 ± 0.5 kg) were randomly allocated into four groups in a 2 × 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1β, IL-6, TNF-α, IL-10, COX-2, TLR2, TLR4, and GR (P < 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P < 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P < 0.01). Interaction of ACTH × LPS was significant for cNOS level (P = 0.011) and ssc-miR-338 expression (P = 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P < 0.01), which was significantly attenuated by ACTH pretreatment (P < 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P < 0.05). TLRs 1–10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P < 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

A neuromodulatory role for dopamine has been reported for magnocellular neuroendocrine cells in the mammalian hypothalamus. We examined its potential role as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder Paralichthys olivaceus . In vitro application of dopamine (DA) caused an increase in electrical activity (firing frequency, recorded extracellularly) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The dopamine precursor, levodopa (L-DOPA), also increased firing frequency, cell recruitment and enhanced bursting and tonic activity. The effect of dopamine was blocked by the D1, D5 receptor antagonist SCH23390, but not by the D2, D3, D4 receptor antagonist amisulpride. Transcriptome sequencing revealed that all DA receptors (D1, D2, D3, D4, and D5) were present in the flounder CNSS. However, quantitative RT-PCR revealed that D5 receptor mRNA expression was significantly increased in the CNSS following dopamine superfusion. These findings suggest that dopamine may modulate CNSS activity in vivo , and therefore neurosecretory output, through D5 receptors.

Glucocorticoid receptor (GR) is transcribed in a tissue- and cell-specific manner with multiple exon 1 mRNA variants driven by selective promoters. We recently cloned and characterized the 5.3kb proximal promoter sequence of porcine GR gene containing 7 untranslated alternative first exons each processed by a distinct promoter. In this study, we showed tissue-specific expression of total GR and its exon 1 mRNA variants in hippocampus, muscle and liver of pigs. Total GR mRNA was most abundant in liver, followed by muscle and hippocampus in descending order. Among all the GR exon 1 mRNA variants detected, GR exon 1-9/10 and 1-4 were the most predominant variants in all the three tissues. The abundance of GR exon 1-4 mRNA was similar to that of 1-10 in muscle, but was significantly lower than 1-10 in liver and hippocampus. The activities of truncated short (S) and long (L) promoters of respective GR exon 1 mRNA variants were analyzed by luciferase reporter assay in 3 representative cell lines, SY5Y, C2C12 and HepG2. S1-10 and S1-4 demonstrated significantly higher activities than other short promoters in all the cell lines examined. Nevertheless, the strongest activity and cell specificity were detected for L1-10 promoter, which was consistent with the predominant exon 1-9/10 expression in porcine tissues. Moreover, with 3 potential nGRE binding sites, L1-10 promoter was more sensitive to dexamethasone (DEX) in HepG2. Our data provide basic knowledge of the transcriptional mechanism underlying the tissue- and cell-specific expression of porcine GR under basal or ligand-stimulated conditions.

Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (P

In the present study the influence of long photoperiod (LP, 16L:8D) and short photoperiod (SP, 8L:16D) on hepatic energy metabolism in the olive flounder (Paralichthys olivaceus) was investigated. Flounders were maintained under LP or SP conditions for 2 weeks then plasmatic and hepatic parameters were assessed. At the plasmatic level, the concentration of cortisol was enhanced in flounder maintained under LP compared to SP. Alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities in plasma also increased in LP flounder. There was no significant difference in plasma glucose levels between the two experimental groups. Plasma osmotic pressure, Na and Cl levels were significantly higher in LP compared to the SP group. In liver, a significant decrease of triglycerides together with an increase in glycogen was observed in the LP group. Hepatic hsl and pepck and muscle hsl mRNA expression in LP was significantly higher in the SP group. Overall the results indicate that the LP treatment caused a mild stress response and increased hepatic energy metabolism in the flounder, which in turn could affect osmoregulation. In conclusion, it would appear that LP treatment can adversely influence hepatic energy metabolism in adult olive flounder under fasting condition.

MicroRNA (miRNA) biogenesis is determined mainly by Drosha, Dicer and Argonaute2 (Ago2). Different breeds of pigs with vast differences in serum cortisol level demonstrate distinct profiles of hepatic miRNA expression. As yet, little is known about whether glucocorticoid contributes to the breed differences in miRNA biogenesis. Here, we used newborn Large White (LW) and Erhualian (EHL) piglets to investigate the role of glucocorticoid in breed-specific hepatic miRNA biogenesis. Erhualian piglets showing significantly higher serum cortisol level, as compared to LW, demonstrated higher hepatic expression of Drosha, Dicer and Ago2 at the protein level, but not at the mRNA level. At the post-transcriptional level, miRNAs that are predicted to target these proteins may be involved in the regulation. Hepatic expression of miR-15b and miR-222 was significantly lower in EHL piglets and was associated with higher glucocorticoid receptor binding to the respective promoter regions of miR-15b and miR-222 genes. The inhibitory effect of glucocorticoid on miR-15b and miR-222 expression was further verified in HepG2 cells, in which dexamethasone significantly downregulated the expression of primary transcripts of miR-15b and miR-222 genes. In conclusion, the higher protein content of Drosha, Dicer and Ago2 in the liver of EHL piglets is post-transcriptionally regulated, at least in part, by glucocorticoid-mediated repression of miR-15b and miR-222.

Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P