Abstract
Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA–VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA–CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.
Introduction
The fat soluble vitamins A (VA) and D (VD) are essential nutrients in the diet of vertebrates. VA is crucial for vision, growth, reproduction and embryological development. VD is deeply involved in calcium (Ca) and phosphorus (P) homeostasis through modulation of Ca uptake (via gills and intestine in fish, via intestine in higher vertebrates), renal reabsorption, bone deposition/resorption and modulation of the immune response. The main active metabolites of VA and VD are retinoic acid (RA) and calcitriol (CTR; 1α, 25(OH)2D3) respectively. These metabolites induce biological effects via high-affinity nuclear transcription factors. Genomic actions of CTR are mediated by the VD receptor (VDR), while RA binds to either a RA receptor (RAR) or retinoid X receptor (RXR). The RAR family includes RARα, RARβ, and RARγ, receptors that show high affinity for the all-trans or 9-cis isomers of RA. The RXR subfamily also consists of three receptor subtypes; RXRα, RXRβ and RXRγ. These differ from RARs in that they preferentially bind 9-cis RA and may act as ‘silent partners’, i.e. without ligand binding. VDRs, RXRs and RARs require dimerisation for activity (e.g. RAR–RXR or VDR–RXR). After binding ligands and undergoing dimerisation, the receptor–ligand complexes associate with specific DNA response elements (VD response elements, VDRE; RA response elements, RARE) to activate or suppress gene transcription. Indeed, interactions between CTR and retinoids are indicated as the VDR acts as a heterodimer with the RXR. The use of RXR as a DNA-binding facilitator places VDR in a receptor class that also includes the thyroid hormone receptors, the peroxisome proliferator-activated receptor (PPAR; Pathrose et al. 2002).
Bone is a key target for RA and CTR. In fish, CTR exerts effects directly on both osteoblasts and osteoclasts (Wendelaar Bonga et al. 1983). Chronic CTR exposure increases bone formation, but may also impair mineralisation, at least in rats (Wronski et al. 1986). Short-term exposure of CTR induced similar effects, with an elevated number of osteoblasts, increased bone formation, and decreased osteoclast incidence in rat cancellous bone (Erben et al. 1997). CTR is reported to both inhibit and induce expression of collagen-I mRNA, depending on factors such as the species tested, osteoblast differentiation stage, and duration of CTR exposure (Van Leeuwen et al. 2001). CTR induces osteocalcin mRNA expression in human and rat osteoblasts, Mgp mRNA in rat osteoblasts, and osteopontin and alkaline phosphatase (Alp) mRNA in human, rat and mice osteoblasts (Van Leeuwen et al. 2001). In fish, CTR exposure increases mRNA expression of an epithelial Ca channel, (ecac listed as trpv6 in ZFIN Database) in gills (Qiu et al. 2007) and intestinal Ca transport in eel (Fenwick et al. 1984). Available reports on CTR effects in fish are ambiguous with both stimulation and degradation of calcified tissues documented (Wendelaar Bonga et al. 1983, Fenwick et al. 1994). The apparent discrepancies likely result from uncontrolled factors such as Ca and P availability and hormone doses used.
Evidence suggests that RA has opposite effects on bone when compared to CTR. RA interferes with a range of osteoblast properties, e.g. inhibition of osteocalcin mRNA expression in mouse osteoblasts (Cohen-Tanugi & Forest 1998), reduction of collagen mRNA synthesis in rat and chicken osteoblasts (Dickson & Walls 1985, Kim & Chen 1989) and increased collagenase mRNA expression and collagen degradation in rat (Varghese et al. 1994). Skeletal deformities in fish have been causally associated with dietary retinol levels (Dedi et al. 1995, Ørnsrud et al. 2002).
Data from mammalian studies indicate that excess VA levels antagonise VD's roles in Ca homeostasis. High VA intake can ameliorate toxic (overdose) effects of VD in rats (Clark & Bassett 1962), turkey (Metz et al. 1985) and chick (Aburto & Britton 1998b), and increased requirement for dietary VD has been shown with high levels of dietary VA (Metz et al. 1985, Aburto & Britton 1998a). In addition to sharing common targets, CTR and RA interact at other levels. For example, RA exhibits direct effects on key genes in the CTR signalling pathway. RA was found to stimulate Vdr expression in both tumour and non-tumour bone cells of rats (Petkovich et al. 1986) and mouse (Suzuki et al. 1993). RA also increases the expression of the VD catabolising enzyme 25(OH)D3-24-hydroxylase (Cyp24a1; Allegretto et al. 1995), and synergistic effects were shown between RA and CTR in their effect on rat renal CYP24A1 activity (Reinhardt et al. 1999). Frankel et al. (1986) found that chronically VA-overdosed rats had increased osteoclast activity, reduced osteoid formation and reduced circulating levels of 25(OH)D3, and that VD-overdosed animals lowered their serum Ca and serum 25(OH)D3 after a moderate dose of VA. In a series of studies on rats with varying VD status, reduced serum Ca levels, increased P levels, reduced mineralisation of bones (Rohde et al. 1999, Rohde & DeLuca 2005) and increased bone resorption (Rohde & DeLuca 2003) were found as an effect of VA supplementation. A VA dose corresponding to a single serving of liver antagonised the ability of CTR to increase intestinal Ca absorption in human subjects 24 h post treatment (Johansson & Melhus 2001). Thus, RA and CTR exhibit complex interactions, reflected in Ca transport phenomena and aspects of bone turnover.
Intensive aquaculture of Atlantic salmon (Salmo salar) uses formulated diets with high contents of fish meal and fish oil, and thus potentially has excessive amounts of VA. Little if anything is known regarding the effect of VA on VD (and vice versa) in salmon and other fish species.
This study was undertaken to investigate interactions between RA and CTR in Atlantic salmon, to understand the nature of a possible interaction, and to determine any effect on vitamin plasma levels, gene expression of selected bone markers, and Ca and P absorption.
Materials and Methods
Fish and experimental treatment
Atlantic salmon (Salmo salar L.) ‘Sea Farm strain’, with an average weight (±s.d.) of 35±6 g was obtained from Marine Harvest, Tveitevåg, Norway. The fish were maintained in 160-l fibreglass tanks with flowing fresh water at 9 l/min, oxygen content ∼10 mg/l and water temperature 5–7 °C. Fish were fed a commercial diet (Nutra Transfer LB3, Skretting, Norway) until 4 days before experimental treatment. The experiment was approved by the National Animal Research Authority of Norway. There was no mortality or signs of distress among the fish during the trial.
Establishment of optimal RA dose
All-trans RA (Sigma–Aldrich) was thoroughly mixed with sunflower oil (Yonca Gida San AS, Manisa, Turkey) immediately before use. Fish were anaesthetised in 40 mg/l benzocaine, and, gently held in a wet towel, before a volume ranging from 80 to 95 μl oil was injected i.p. with a 23 gauge needle and disposable syringes. Doses administered were 0, 10, 100, 250 and 500 μg RA/g fish with three fish tested per dose. Each dose was administered to three fish. Fish were individually marked with a silicon dye (Visible Implant Elastomer, Northwest Marine Technology, Shaw Island, WA, USA) and kept in the same tank for 5 days. After which the fish were anaesthetised using benzocaine (40 mg/l) and blood was collected from the caudal vessels by puncture with heparinised syringes. Blood samples were kept on ice until centrifugation (1500 g, 10 min). Plasma was flash-frozen in liquid N2 and stored at −80 °C until analysis. Liver was excised and a standardised section immediately flash-frozen in liquid N2. Gene expression of the RA catabolising enzyme cyp26 was used as a marker for RA exposure.
RA and CTR treatment
CTR (1α,25(OH)2D3) and all-trans RA were thoroughly mixed with sunflower oil to obtain two stock solutions; 3 μg CTR/ml oil and 30 mg RA/ml oil. Four treatments were tested: 1) oil (Control group), 2) CTR in oil (CTR group), 3) RA in oil (RA group) and 4) RA plus CTR (RA–CTR group) in oil. A volume between 80 and 170 μl was injected i.p. Predicted vitamin doses were 10 ng CTR/g fish (Qiu et al. 2007) and 100 μg RA/g fish (based on the experiment for establishing optimal RA dose described above). Fish were individually marked with silicon dye and experimental groups were kept in the same tank throughout the experiment. Fish were sampled prior to injection (day 0) and at days 3, 5 and 7 after injection and sampled as above. At day 0, individual plasma samples from five fish were taken (n=5) but at days 3, 5 and 7 plasma from nine fish per treatment were pooled into three pools (n=3). Liver, gill and vertebral spine from nine fish per treatment were collected and immediately flash frozen on liquid N2. Gill epithelium was scraped off the arch using a microscope slide. The vertebral spine was excised and all adhering tissue removed.
Ca and P influx in gut sacs
A functional assay of Ca and P absorption through an in vitro experiment exposing gut sacs to labelled Ca or P served as a marker of protein expression, e.g. Ca channels in intestine. Since cold-water fish often show a considerable delay between gene transcription and protein expression, timing of sampling is of essence. In a study by Qiu et al (2007), after a single i.p. injection of CTR, expression of an epithelial Ca channel, VDR and a zinc transporter in rainbow trout gill was at a maximum 5 days after injection, suggesting that the protein expression would be at maximum subsequent to day 5. Accordingly, at day 7 after injection, the digestive tract was excised from the section immediately distal to the pyloric caecae and transferred to ice-cold aerated Cortland saline (composition in mM: NaCl, 124; KCl, 5.1; CaCl2.2H2O, 1; MgSO4, 1.9; NaHCO3, 11.9; NaH2PO4.H2O, 2.9; glucose, 5.5; (Mommsen & Hochachka 1994). The method for intestinal sac uptake studies followed that described by Nadella et al. (2006). The excised intestine was cut in two with the anterior (for Ca uptake determination) and posterior (for phosphorous) sections being defined by the obvious morphological distinctions between these gut regions. Measurement of unidirectional ion movement started within 2 h of gut dissection.
A flanged catheter was tied in place at one end of the intestinal sacs, and ice-cold Cortland saline used to flush the sacs of food remains. The open end was then tied with suture thread. Radionuclide containing solutions were introduced into the sacs through the catheter. These solutions consisted of either 33P or 45Ca (ARC, St Louis, MO, USA) at a radioisotope concentration of 370 kBq/ml Cortland saline. The anterior sac always contained 45Ca, while the posterior contained 33P. Solutions were added via the catheter until the gut sacs appeared firm, but not taut (∼20–70 μl per sac, varying with sac size). Catheters were heat sealed, and the sac placed in 4.5 ml of bathing solution (Cortland saline) in a 5 ml plastic tube, itself situated in a water bath with temperature controlled at 12 °C (±1 °C). Each tube was bubbled with a 95% O2, 5% CO2 gas mixture. After 2 h, the sacs were removed, the suture undone and the sacs flushed with ∼10 volumes of cold rinse solution, containing either ∼1 M Ca (for 45Ca) or ∼1 M phosphate (for 33P), to displace adsorbed radionuclide. Sacs were then split longitudinally, blotted gently on wet tissue and the mucosal surface scraped with a microscope slide. The intestinal surface area was estimated as described by Grosell & Jensen (1999).
Intestinal scrapings and the remainder of the intestinal tissue were then digested (1 M HNO3) for 2 days at room temperature, neutralised (1 M NaOH), before addition of scintillation fluor (10 ml; Ecoscint A; National Diagnostics, Atlanta, GA, USA). Duplicate 2 ml samples of the bathing medium were taken to assess 45Ca or 33P specific activities. Radionuclide activity was estimated by liquid scintillation counting (1900 TR Tri-Carb, Packard, Ramsey, MN, USA), with quenching corrected via an external standards ratio method. Unidirectional fluxes (nmol/cm2 per h) were calculated on a surface area-specific basis, over an hour using initial specific activity (c.p.m./nmol). Absorbed minerals were defined as those accumulated in the intestinal tissue and in the serosal medium (Nadella et al. 2006).
Analysis of plasma retinoids, CTR, magnesium, Ca and P
Plasma was analysed for retinoids, CTR, magnesium (Mg), Ca and P. RA, 4-oxo RA and retinol were analysed by AS Vitas (Oslo, Norway) with a LC/MS/MS (Gundersen et al. 2007). CTR was measured by the Radboud University Nijmegen Medical Centre according to van Hoof et al. (1993). Plasma was diluted 250-fold with demineralised water, and total Ca and P were measured by Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES, Plasma IL200; Thermo Electron, Waltham, MA, USA).
RNA extraction
Frozen samples of liver and gill were homogenised in Trizol using MagNA Lyser Green Beads (Roche). Vertebra samples were homogenised in liquid N2 using a mortar and pestle prior to Trizol extraction. Total RNA was purified using Trizol extraction and subjected to DNAse treatment (DNA-freeTM, Ambion, Austin, TX, USA). RNA quantity and quality (A260/280) was assessed using a NanoDrop ND100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and by gel electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
Reverse transcription-PCR
cDNA was prepared from 125 ng RNA using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA, USA) with Oligo d(T)16 primers in a total reaction volume of 30 μl. The reaction mixtures were incubated at 25 °C for 10 min, 48 °C for 60 min and 95 °C for 5 min followed by a decrease to 4 °C. Reverse transcription (RT)-PCR efficiency was validated using a six-point calibration curve from serial dilutions of one RNA sample from 1000 to 15 ng per reaction. ‘No template’ and ‘no activity’ controls were made for each RT master mix.
Q-PCR
Real-time qPCR primers for ecac, cyp24 and cyp26 were designed using Primer Express 2.0 software (Applied Biosystems). Primers for col1a2, mgp and alp were from Wargelius et al. (2009), and primers for ef1aA and arp were from Olsvik et al. (2005, 2007) respectively. Primers were based on salmonid sequences when available or on sequences from related species (Table 1, primer sequences). Real-time qPCR was performed using SYBRGreen PCR Master mix (Applied Biosystems), 2.5 μl RT template and gene specific primers (0.9 μM) and run on the ABI Prism 7000 Sequence Detection system (Applied Biosystems). All samples were run in triplicate with accompanying ‘no template’ and ‘no activity’ controls. Reaction conditions were 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min and a final dissociation step with 95 °C for 15 s, 60 °C for 20 s and 95 °C for 15 s. All Q-PCR products yielded a single-peak melting curve indicating that no primer–dimer formation occurred.
Primer sequences for matrix Gla protein (mgp), collagen 1 α2 (col1a2), alkaline phosphatase (alp), epithelial calcium channel (ecac), 25-hydroxyvitamin D3-24-hydroxylase (cyp24), retinoic acid-metabolising enzyme (cyp26), elongation factor 1A (ef1aA) and acidic ribosomal protein (arp) in Atlantic salmon (S. salar L.)
| Primer sequence (5′→3′) | Accession number | |
|---|---|---|
| Primer | ||
| col1a 2 | Fw: GAG GGT GGA TGC AGG TGT GT | CA064459 |
| Rv: TAC TGG ATC GAC CCC AAC CA | ||
| mgp | Fw: GAA AGC ACA GAA TCC TTT GAA GAT GT | AY182239 |
| Rv: GTG GAC TCT GTG GGT TGA TGA A | ||
| ecac | Fw: TGG GTG CCC TGG TTA TTC TG | AY256348 |
| Rv: ATC GCA TAG GCA ATA AGA ATG ACA | ||
| alp | Fw: CTA CAC GCC AAG AGG GAA CAC | CO472235 |
| Rv: GGT AAA GGG TTT CTG GTC CAC AT | ||
| cyp26 | Fw: GAG GAC TCG TCG CGT TTT AAC T | CK890206 |
| Rv: TTG GCG AAC TCT TTC CCT ACA | ||
| cyp24 | Fw: GGA GAC CAT TTG CTT AGT GC | AY526907 |
| Rv: CCA AAT GTG CTC ATC ATC G | ||
| arp | Fw: TCA TCC AAT TGC TGG ATG ACT ATC | AY255630 |
| Rv: CTT CCC ACG CAA GGA CAG A | ||
| ef1aA | Fw: CCC CTC CAG GAC GTT TAC AAA | AF321836 |
| Rv: CAC ACG GCC CAC AGG TAC A |
Normalisation of gene expression and statistical analyses
Elongation factor 1A (ef1aA) and acidic ribosomal protein (arp) were used as reference genes. The stability of these genes was estimated using the geNorm VBA applet for Microsoft Excel (Vandesompele et al. 2002). ef1aA proved to be the more stable and all investigated genes are presented relative to ef1aA. Assessment of statistically significant differences between treatments for gene expression data was performed using non-parametric Kruskal–Wallis ANOVA followed by the Mann–Whitney U-test. Regression analysis was performed for correlation between P and Ca uptake in gut sacs using GraphPad Prism 5.02 (GraphPad software, La Jolla, CA, USA). Assessment of statistically significant differences between treatments for plasma values and the gut sac experiment were determined by one-way ANOVA, followed by a least significant difference post-hoc test at the α=0.05 level.
Results
Optimal RA dose
A hyperbolic dose–response relation (correlation r2=0.90) for injected RA dose and the resulting cyp26 mRNA expression was observed (Fig. 1). Plasma RA concentrations ranged from 30 pM in untreated control fish to 50 nM in fish injected with 500 μg RA/g body weight (BW). cyp26 mRNA expression increased with increasing RA concentrations with a ∼430-fold higher expression in groups injected with 500 μg RA/g BW compared to those fish injected with pure sunflower oil. Furthermore, no all-trans or 13-cis 4-oxo RA, products from Cyp26 RA catabolism, were seen in the groups given 0 or 10 μg RA/g BW, while groups exposed to 100, 250 and 500 μg RA/g BW showed increasing 4-oxo RA concentrations in plasma with increasing RA exposure (data not shown). cyp26 expression plateaued at doses above 100 μg/kg BW and therefore 100 μg RA/g BW was chosen as the RA dose for combination treatment.
Gene expression (fold difference compared with non-treated fish) of cyp26 relative to elongation factor 1A (ef1aA) and plasma retinoic acid (nM) in Atlantic salmon (Salmo salar L.) 5 days after i.p. injection of 0 (•), 10 (□), 100 (▿), 250 (▵) and 500 (♦) μg/g fish of all-trans retinoic acid in sunflower oil.
Citation: Journal of Endocrinology 202, 3; 10.1677/JOE-09-0199
RA and CTR treatment
I.p. injection of CTR reduced plasma CTR levels approximately fourfold (P<0.05) after 3 days compared to controls (Fig. 2). Although still approximately twofold lower than controls (P<0.05), CTR plasma levels increased again after 5 days and were not significantly different from controls at day 7. Plasma VA metabolites were not affected by CTR injection. RA injection decreased plasma CTR approximately fivefold after 5 and 7 days (P<0.05) but not after 3 days. In the RA–CTR group, plasma CTR levels decreased approximately four- to sixfold at day 3, 5 and 7. All-trans retinol plasma concentration (the plasma transport form of retinoids), was significantly (P<0.05) lower, while plasma 9-cis, 13-cis and all-trans RA levels and the RA degradation products 13-cis and all-trans 4-oxo RA were significantly (P<0.05) higher at days 3, 5 and 7 in the RA and RA–CTR groups (Table 2). There were no differences among groups in plasma total Ca, Mg or Pi at any time point (Table 2).
Plasma values of calcitriol (pM) in Atlantic salmon (Salmo salar L.) prior to and at 3, 5 and 7 days after i.p. injection of pure sunflower oil (Control), calcitriol in sunflower oil (CTR), retinoic acid in sunflower oil (RA) or CTR and RA in sunflower oil (RA–CTR). Data reported as means±s.e.m., n=3, where each replicate represents a pool of plasma from three fish. Different superscript letters denote significant differences within each time point, P<0.05 (ANOVA).
Citation: Journal of Endocrinology 202, 3; 10.1677/JOE-09-0199
Plasma values of retinol, retinoic acid, Mg, total Ca and Pi in Atlantic salmon (Salmo salar L.) prior to and 3, 5 and 7 days after after i.p. injection of pure sunflower oil (Control), calcitriol in sunflower oil (CTR), retinoic acid in sunflower oil (RA) or CTR and RA in sunflower oil (RA–CTR). Data reported as means±s.e.m., n=3, where each replicate represents a pool of plasma from three fish
| Days post-treatment | |||||
|---|---|---|---|---|---|
| Treatment | 0 | 3 | 5 | 7 | |
| Analyte | |||||
| All-trans retinol (nmol/l) | Control | 142±39 | 148±16a | 181±46a | 154±15a |
| CTR | 151±1a | 166±16a | 115±16a | ||
| RA | 12±1b | 7±1b | 11±2b | ||
| RA–CTR | 12±3b | 10±2b | 10±1b | ||
| 13-cis retinoic acid (nmol /l) | Control | 2.8±0.4 | 9±3b | 4±1b | 3±1b |
| CTR | 14±4b | 5±1b | 4±2b | ||
| RA | 1616±396a | 2455±696a | 454±270a | ||
| RA–CTR | 1812±656a | 2982±302a | 613±299a | ||
| 9-cis retinoic acid (nmol /l) | Control | 1.4±0.1 | ND | ND | 1.2‡ |
| CTR | 1.4±0.1† | 2‡ | 1.2‡ | ||
| RA | 413±87 | 478±135 | 150±91 | ||
| RA–CTR | 442±110 | 579±54 | 161±75 | ||
| All-trans retinoic acid (nmol /l) | Control | 0.6±0.1 | 19±3b | 11±4b | 5±1b |
| CTR | 12±2b | 20±9b | 8±6b | ||
| RA | 51480±5118a | 44157±6549a | 14825±7119a | ||
| RA–CTR | 54031±5591a | 46043±5399a | 15544±4620a | ||
| 13-cis 4-oxo retinoic acid (nmol/l) | Control | ND | ND | ND | 1‡ |
| CTR | ND | ND | ND | ||
| RA | 9±1 | 21±4 | 9±7† | ||
| RA–CTR | 9±2 | 26±4 | 6±2† | ||
| All-trans 4-oxo retinoic acid (nmol/l) | Control | ND | ND | ND | 1‡ |
| CTR | ND | ND | ND | ||
| RA | 120±27 | 175±31 | 56±32 | ||
| RA–CTR | 124±29 | 234±64 | 61±24 | ||
| Total Ca2+ (mol/l) | Control | 2149±18* | 2261±34 | 2229±48 | |
| CTR | NA | 2134±7* | 2137±56 | 2249±36 | |
| RA | 2159±51* | 2161±81 | 2198±27 | ||
| RA–CTR | 2245±82* | 2136±75 | 2199±39 | ||
| Mg (mol/l) | Control | 272±1* | 294±2 | 286±12 | |
| CTR | NA | 270±3* | 263±8 | 285±6 | |
| RA | 296±22* | 274±12 | 272±7 | ||
| RA–CTR | 298±27* | 277±17 | 277±3 | ||
| Pi (mol/l) | Control | 1797±55* | 2026±164 | 1760±257 | |
| CTR | NA | 1599±171* | 1656±145 | 1491±338 | |
| RA | 1490±453* | 1851±52 | 1986±104 | ||
| RA–CTR | 1696±54* | 1951±194 | 1038±431 | ||
NA, not available; ND, not detected. Different superscript letters denote significant differences in each time point, P<0.05 (ANOVA).
n=1, not detected.
n=2, not detected.
n=2, loss of sample.
Gene expression
Figure 3 shows the mRNA expression of mgp, col1a2 and alp in bone, ecac in gill and cyp24 in liver. CTR injection reduced mgp mRNA expression 1.4-fold (P<0.05) relative to controls at day 5 after injection. RA- and RA–CTR-injection resulted in a 3.8- and 3.2-fold reduction (P<0.05) at day 5 and 3.0- and 3.9-fold reduction (P<0.05) at day 7 compared to controls. CTR injection reduced col1a2 mRNA expression 1.4-fold compared to controls (P<0.05) at day 7 after injection. RA and RA–CTR injection reduced col1a2 mRNA expression 2.4- and 1.8-fold (P<0..05) relative to control expression at day 5, and by 3.4- and 5.1-fold (P<0.05) at day 7. CTR injection reduced alp expression 1.3-fold (P<0.05) at day 5. Conversely, injection of RA increased alp expression 1.3-fold relative to controls at day 5 (P<0.05). CTR and RA–CTR injection did not induce expression of ecac in gills. RA injection increased expression of ecac 1.3-fold compared to control expression at day 7 (P<0.05). Expression of cyp24 was not affected by any of the treatments at any tested day.
Gene expression (fold difference relative to Control) in Atlantic salmon (Salmo salar L.) at 3, 5 and 7 days after i.p. injection of pure sunflower oil (Control), calcitriol in sunflower oil (CTR), retinoic acid in sunflower oil (RA) or calcitriol and retinoic acid in sunflower oil (RA–CTR). (A) Matrix Gla protein (mgp) in bone; (B) collagen 1a chain 2 (col1a2) in bone; (C) alkaline phosphatase (alp) in bone; (D) epithelial calcium channel (ecac) in gills and ecyp24 in liver. Data presented as median (horizontal line), 95% confidence interval (box) and min–max values (whiskers), n=9. Different superscript letters denote significant differences within each time point, P<0.05 (Kruskal–Wallis ANOVA).
Citation: Journal of Endocrinology 202, 3; 10.1677/JOE-09-0199
Ca and P uptake
P uptake across gut sacs 7 days after injection (Fig. 4) ranged between 2 and 50 nmol/cm2 per hour and was not affected by any of the treatments. Ca uptake ranged between 1 and 20 nmol/h per cm2 and was enhanced significantly in RA–CTR treated fish compared to both the controls (P<0.05) and CTR group (P<0.05). In RA treated fish, P uptake and Ca uptake was significantly and positively correlated (P<0.05, r2=0.49), while no such correlation was seen for the other experimental treatments (data not shown).
Unidirectional influx (nmol/cm2 per h) of phosphorus and calcium across gut sacs isolated from Atlantic salmon (Salmo salar L.) 7 days after i.p. injection of pure sunflower oil (Control), calcitriol in sunflower oil (CTR), retinoic acid in sunflower oil (RA) or calcitriol and retinoic acid in sunflower oil (RA–CTR). Bars reported as means (±s.e.m.), n=9. Different superscript letters denote significant differences-within each treatment, P<0.05 (ANOVA).
Citation: Journal of Endocrinology 202, 3; 10.1677/JOE-09-0199
Discussion
The effect of RA on plasma CTR and mRNA expression of selected genes provided strong evidence for an interaction between VA and VD in Atlantic salmon. We showed that artificially induced (by i.p. injection) elevations in plasma RA interfered with plasma CTR and bone physiology. We base this conclusion on three key observations. First, increased RA plasma levels reduced plasma CTR levels. Second, the altered plasma RA and CTR levels resulted in predictable alterations in mRNA expression of several genes involved in bone formation. Third, intestinal Ca absorption increased in RA treated fish, possibly indicating increased demand of this mineral. We will elaborate on these findings below.
Injection of CTR resulted in unexpected dynamics of this VD metabolite in vivo, with CTR plasma levels dropping below control values 3 days after CTR injection. Circulating CTR plasma levels are regulated by both synthesis and degradation. The enzyme primarily responsible for CTR synthesis (from 25(OH)D3) in liver and kidney) is 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). CTR degradation to biologically inactive products, e.g. 1,24,25(OH)3D3, is mediated primarily through 25-hydroxyvitamin D-24-hydroxylase (CYP24; Sakaki et al. 2005). CTR degradation through CYP24 has a multifactorial regulation, being modulated by RA, CTR (Zou et al. 1997, Reinhardt et al. 1999) and parathyroid hormone (PTH; Zierold et al. 2001) for example. (Lechner et al. 2007) demonstrated that CTR treatment of cells derived from human colon, prostate and mammary tissue diminished cyp27b1 transcription and elevated cyp24 mRNA levels. We were not able to demonstrate any increase in CYP24 at mRNA level at 3, 5 or 7 days. It is possible that changes in cyp24 expression occurred prior to day 3, and would not have been detected under our sampling regime.
Injection of RA resulted in a predictable VA profile. After injection, plasma RA levels increased quickly initially, and decreased slowly during the following days. Plasma RA is broken down by the RA-specific CYP26 (Luu et al. 2001). Indeed, transcription of cyp26 was increased in RA-injected fish when establishing optimal RA dose (Fig. 1). An important oxidation product of the Cyp26 mediated degradation of RA is 4-oxo RA (Chithalen et al. 2002). This metabolite was elevated in RA- and RA–CTR-injected fish (Table 2), and indicates that pathways for regulating excess RA concentrations had been activated. Furthermore, the availability of the RA precursor retinol was reduced 14-fold in plasma of the RA- and RA–CTR-injected fish (Table 2) suggesting that mechanisms for reducing production of RA through retinol oxidation were down-regulated.
A negative association between high plasma retinol and plasma 25(OH)D3 concentrations has been found in human subjects (Mata-Granados et al. 2008). In our study, a persistently elevated plasma RA level in Atlantic salmon decreased plasma CTR levels by at least 80%. All-trans RA and 9-cis RA are potent stimulators of Cyp24 expression in vivo in mice (Allegretto et al. 1995). A similar mechanism in salmon could explain the decrease in plasma CTR levels at day 5 and 7 in RA-treated fish. The fastest clearance of plasma CTR levels was observed in the RA–CTR group, a possible additive effect of both RA and CTR on cyp24. Accordingly, a twofold increase in rat renal CYP24 activity when co-dosing CTR and RA compared to CTR alone has previously been reported (Reinhardt et al. 1999).
The extracellular matrix proteins MGP and collagen perform a regulatory and a structural role respectively. In fish species, data on mgp transcription are scant. In the teleost, Sparus auratus, a negative RARE in the promoter region of the mgp gene has been identified and inhibition of mgp transcription by RA has been demonstrated (Conceicao et al. 2008). In the current study, transcription of mgp was significantly inhibited in the CTR, RA- and RA–CTR groups. The results obtained in the current study are, to our knowledge, the first to describe an effect of CTR on mgp in a teleost fish. Numerous studies have investigated the effect of CTR on collagen type- I formation, and both up- and down-regulation of col1 has been found, depending on species and model system (van Driel et al. 2004). The decrease in col1a2 transcription in the CTR-group at day 7 can be explained by lower plasma CTR values at days 3 and 5. RA is an inhibitor of collagen synthesis and the reduction in col1a2 transcription is in line with other studies (Wang et al. 2002).
alp is a known marker of preosteoblasts and osteoblasts in both mammals (Whyte 1994) and fish (Witten 1997) and is commonly used as a marker of mineralisation processes. Transcription of alp is stimulated by both RA (Heath et al. 1992) and CTR (Whyte 1994). Conversely, the reduction of CTR in plasma transiently reduced the expression of alp in the CTR-group in our study. The low plasma concentration of CTR in the RA- and RA–CTR groups possibly counteracted the effect of RA, and only a small increase in expression of alp (∼1.3-fold) was seen in these groups. For the RA-groups a picture emerged whereby matrix protein transcription (mgp and col1a2) was inhibited and transcription of bone alp (important for mineralisation of the matrix) was stimulated. However, a caveat applies. Gene expression data do not necessarily reflect the actual protein expression or activity. If mineralisation does increase under RA treatment, then the recruitment of Ca, P and Mg from plasma might be expected. These are the primary minerals required to deposit apatites. However, no changes in plasma levels of these minerals were seen, which we do not find surprising given the strict homeostatic control of plasma levels of these elements, especially Ca, in vertebrates. Also in fish, plasma Ca levels are tightly regulated by a variety of hormones including stanniocalcin (Pierson et al. 2004), PTH-related protein (Abbink et al. 2007), calcitonin (Najib & Martine 1996), prolactin (Flik et al. 1984) and VD. Increased deposition of Ca (and also to a lesser extent P and Mg) would quickly be compensated for by increased uptake from the environment. The gills are an important organ for Ca uptake from the water (Flik & Verbost 1993), and in many fish are more important than the intestinal uptake route. A gene likely to be involved in transcellular Ca transport is the ecac (Hoenderop et al. 1999); a member of the Transient Receptor Potential Vanilloid, TRPV, class of ion channels (Hoenderop et al. 2002). In rainbow trout, the branchial ecac responds to variations in external Ca concentrations, and is pivotal in regulating Ca influx via the gills (Shahsavarani & Perry 2006). Qiu & Hogstrand (2004) identified two VDREs in the promoter region of branchial ecac of pufferfish (Fugu rubripes) and later demonstrated an increased expression of ecac in rainbow trout (Oncorhynchus mykiss) gills after i.p. injection of CTR (Qiu et al. 2007). The increase in ecac mRNA expression in the gill in the RA-group in the current study could relate to an increased Ca demand. Lending support to changes in mineral metabolism, the absorption of Ca across the intestine was increased after 7 days in the RA–CTR groups (Fig. 4). A positive correlation between intestinal P uptake and Ca uptake in RA-treated fish was also observed, supporting the hypothesis of an increased need for Ca and/or P. Since no major changes in plasma levels of Ca and P occurred, either the excretion of the minerals had increased or more minerals were deposited. The increase in alp transcription in these same groups would be in line with the latter process. Taking into account the apparent decrease in matrix formation, constant high plasma levels of RA in Atlantic salmon may result in abnormal bone growth and could explain bone malformations seen in fish overfed VA (Ørnsrud et al. 2002).
Although i.p. injection of high RA doses can be considered a non-physiological situation, this study does provide mechanistic evidence for an interaction between RA and CTR that may form a basis for an understanding of some types of bone deformities seen in aquaculture. In support of this, we recently found evidence that feeding graded doses of VA as retinol for a prolonged period of time decreases plasma CTR, increases Alp activity in bone, increases mineral content of bone and causes bone deformities in Atlantic salmon (Ørnsrud et al. 2008).
The data from this study indicate a dual effect of RA on bone. While RA inhibits matrix formation, it also appears to activate genes involved in matrix mineralisation. Suppression of plasma CTR levels could partly explain the effects of VA on bone. The results suggest a more integrated approach when studying the effect of fat-soluble vitamins on bone could be a key to a more complete understanding of bone physiology.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Norwegian Research Council (project no. 153472).
Acknowledgements
We gratefully acknowledge the technical assistance of Tom Spanings, Hari Rudra and Rolf Hetlelid Olsen. Many thanks to Dr Anna Wargelius for constructive criticism and for helping out with the primer design and validation.
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(R Ørnsrud and E J Lock contributed equally to this work)
