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L'acide docosahexaénoïque stimule la désintoxication des acides biliaires chez la souris.
RÉSUMÉ
La présente étude avait pour objectif d’analyser l’effet de l’acide gras polyinsaturé (AGPI), acide docosahexaenoique (ADH) sur l’homéostasie des acides biliaires (AB) chez la souris. Des mâles et femelles de 50 jours ont reçu une diète contrôle ou une diète enrichie en ADH (0.75g/kg/jour) pour 2 ou 4 semaines. Les plasmas ont été analysés par LC-MS/MS pour leur contenu en AGPI et en AB, alors que les foies ont été utilisés pour mesurer les AGPI (LC-MS/MS) et le niveau d’expression (PCR quantitatif) de gènes codant pour des protéines de synthèse (Cyp7a1, Cyp27a1), de transport (Mrp2, 3, Ostα,
Ostβ, Bsep, Ntcp), de métabolisme (Cyp3a11, Sult2a1) ou de la signalisation (Fxr, Shp, Lrh, βKlotho) des ABs. Les mêmes gènes (sauf Cyp7a1, Cyp27a1) ont aussi été mesurés
dans les intestins et les reins.
Les résultats obtenus montrent que chez les femelles traitées 14 jours, le DHA cause une augmentation de l’expression en ARNm de Cyp7a1, Cyp27a1 et Lrh, ainsi qu’une diminution de celle de Sult2a1. Dans les mâles, le même traitement active l’expression d’Ostβ. Cyp3a11 était activé chez les 2 sexes. Après 28 jours, les foies femelles montraient une accumulation d’ARNm de Ntcp, Bsep et Mrp3, alors que les transcrits de Cyp7a1 et Cyp27a1 étaient moins abondants dans les foies des 2 sexes.
Ntcp était réduit chez les mâles. Des évènements de régulation similaires concernant les
gènes Mrps, Ostβ et/ou Cyp3a11 ont été observés dans les intestins et les reins. L’analyse du profile d’AB a montré des changements pour quelques molécules spécifique, alors que la quantité total d’AB en circulation avait tendance à diminuer. Finalement, les études de corrélation entre les niveaux plasmatiques et/ou hépatique d’AGPI et l’expression génique hépatique ou les concentrations circulantes d’AB ont révélé diverses associations significatives. Notamment entre l’expression de Mrp3 et les niveaux plasmatiques et/ou hépatiques d’AGPI tels, que l’AEP, l’ADH ou leurs dérivés 18(R/S)-HEPE et PDX.
Conclusion. La supplémentation alimentaire en ADH affect le transcriptome
hépatique, intestinal et rénal lié aux AB chez la souris, ce qui modifie le profile circulant de ces acides. Les changements observés sont potentiellement en lien avec un pool d’acide biliaire plus favorable et moins toxique, ce qui supporterait une utilisation des AGPI comme médicaments anticholestatiques.
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DOCOSAHEXAENOIC ACID STIMULATES BILE ACID DETOXIFICATION IN MICE
Anna Cieślak1, Jocelyn Trottier1, Mélanie Verreault1, Frédéric Calon2, Piotr Milkiewicz3,
Marie-Claude Vohl4 and Olivier Barbier1
1 Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the Faculty
of Pharmacy, Laval University, Québec, Canada.
2 CHU de Québec Research Centre and the Faculty of Pharmacy, Laval University, Québec,
Canada.
3 Liver and Internal Medicine Unit, Department of Transplant and Liver Surgery, Medical
University of Warsaw, Warsaw, Poland.
4 Institute of Nutrition and Functional Foods (INAF) and CHU de Québec Research Centre,
Laval University, Québec, Canada.
Corresponding author:
Olivier Barbier, Ph.D
Laboratory of Molecular Pharmacology, CHU de Québec Research Centre, 2705, boulevard Laurier,
Quebec (QC) G1V 4G2, CANADA Phone: 418 654 2296
Fax: 418 654 2769
Email: olivier.barbier@crchul.ulaval.ca
Disclosure Statement: The authors have nothing to disclose.
Key words: Bile acids, n-3 polyunsaturated fatty acids, Gene expression, HPLC-MS/MS
quantification
Financial Support: This study was supported by grants from the Canadian Institute of Health
Research (CIHR; grant#119331), the Canadian Foundation for Innovation (CFI; grant#25712), the Canadian Liver Foundation and the Natural Sciences and Engineering Research Council of Canada (NSERC). Anna Cieślak is holder of a scholarship from the “Fondation du CHU de
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ABSTRACT
The present study aims at investigating how the n-3 polyunstaturated fatty acid (n-3 PUFA), docosahexaenoic acid (DHA) affects bile acid (BA) homeostasis in mice. Fifty-day old male and female C57BL/6 mice were fed either a control (n=5/group/sex) or a high DHA diet (0.75g/kg/day) during 2 (n=5/group/sex) or 4 weeks (n=6/group/sex). Plasma and hepatic n-3 PUFA levels (DHA, eicosapentaenoic acid), their metabolites (17S-HDHA, 18RS-HEPE, PDX) and plasma concentration of 21 BAs were analyzed by LC-MS/MS. The expression of 14 genes coding for proteins involved in BA synthesis (Cyp7a1, Cyp27a1), transport (Mrp2, 3,
Ostα, Ostβ, Bsep, Ntcp) and metabolism (Cyp3a11, Sult2a1), or controlling these processes (Fxr, Shp, Lrh, βKlotho) was analyzed by qRT-PCR in the liver, intestine and kidneys. In 14-
day treated female livers, DHA caused significant accumulation of Cyp7a1, Cyp27a1 and Lrh transcripts, while Sult2a1 mRNA expression was reduced. In males, DHA activated Ostβ, while in both sexes Cyp3a11 was increased. After 28-day of DHA-enriched diet, female livers displayed higher levels of Ntcp, Bsep and Mrp3 mRNAs, while Cyp7a1 and Cyp27a1 transcripts were reduced in male and female tissues, and Ntcp was decreased in males. Similar regulatory events regarding Mrps, Ostβ and Cyp3a11 genes were also detected in the intestines and kidneys. Plasma BA profiling revealed that DHA caused significant changes in specific species, and that these changes tended to reduce the amount of circulating BAs. Finally, correlation studies evidenced several associations between n-3 PUFA derivatives, hepatic BA-related gene expression and BA levels: Mrp3 was positively correlated to plasma levels of EPA, DHA, 18(R/S)-HEPE and PDX, and to hepatic levels of EPA, 18(R/S)-HEPE and PDX; Ostβ mRNA levels were negatively correlated to EPA. The circulating 18(R/S)- HEPE was negatively associated with CA concentration, while plasma HDCA correlated with both 18(R/S)-HEPE and PDX hepatic levels.
Conclusion. Supplementation with DHA affects the hepatic, intestinal and renal BA-
related transcriptome, and modifies the circulating BA profile. The observed changes are proposed to favor the detoxification of BAs, and support the implementation of n-3 PUFAs as an anticholestatic approach.
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INTRODUCTION
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are prominent nutrients of marine origin with an array of bioactive properties [407]. Of particular interest is docosahexaenoic acid (DHA) supplemented through an adequate diet, as a widely abundant n-3 PUFA distributed in most human tissues [399]. DHA exerts extensive metabolic, structural and functional effects [406], which determine their health benefits in the onset of chronic human disorders, including cancer, inflammatory and cardiovascular diseases [477-479]. Recently, dietary DHA has also been revealed effective at preventing liver injury and fibrosis [480]. Indeed, DHA supplementation improved biochemical parameters of liver damage in total parenteral nutrition, as well as in cholestatic patients with impaired bile flow suffering from Primary Sclerosing Cholangitis (PSC) [431, 432, 481]. Since cholestasis results in a dramatic increase in hepatic and serum bile acids (BAs), which are toxic at high concentrations and participate to the development of liver failure [482], n-3 PUFAs have been therefore investigated for their potential to attenuate BA toxicity. The in vitro [433, 434] and in vivo [483] studies have demonstrated a clear benefit in the use of DHA in decreasing deleterious effects induced by chronic exposure of BAs in the liver. Finally, our recent data indicated n-3 PUFAs as the transcriptomic regulators of BA detoxification in human cells ex vivo [Cieślak A. et al., in preparation]. However, the role of n-3 PUFAs in regulating BA homeostasis in vivo remains unknown.
Besides their well-established role in cholesterol elimination and intestinal absorption of dietary nutrients, BAs are signaling molecules that regulate lipid, energy and glucose metabolism [24, 484]. Primary BAs (cholic [CA] and chenodeoxycholic acid [CDCA] in humans and CA, alpha- [αMCA] and beta-muricholic [βMCA] in rodents) are synthesized in the liver from cholesterol, and conjugated with the amino acids glycine or taurine before being secreted into bile for storage in the gallbladder [22]. Once released into the intestine during meals, discharged BAs are converted by gut flora into chemically diverse secondary species such as deoxycholic (DCA), lithocholic (LCA) and ursodeoxycholic (UDCA) acids in humans, as well as ω-muricholic (ωMCA), hyodeoxycholic (HDCA) and hyocholic (HCA) acids in rodents [24]. Most of the BAs (95%) are then secreted to the portal circulation and reabsorbed to the liver completing a cycle of enterohepatic circulation [22].
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Due to their detergent properties, BAs are toxic when accumulated at high concentrations [22]. The degree of BA hydrophobicity defined by the number of hydroxyl groups present in BA structure has been identified as the major factor which correlates with BA toxicity [243]. The reduced BA toxicity may be enhanced by increasing the systemic amount of hydrophilic BAs either by direct application, or by promoting metabolic conjugation reactions to form the hydrophilic derivatives [244]. Moreover, the regulation of BA hepatic synthesis and transport within the liver, intestine and kidneys plays an essential role in maintaining BA homeostasis [274] Consequently, compensatory mechanisms that aimed at lowering intracellular BAs concentrations and favoring the formation of less hydrophobic BA profile become active in cholestasis [240]. The expression of key BA- synthesizing enzymes (cytochrome P450 (CYP)7A1 and 27A1) is transcriptionally repressed leading to a decreased formation of primary BAs in the liver [439]. An adaptive response of BA transporters includes a reduction of the sodium-dependent (NTCP) uptake system, preventing the influx of BA from bloodstream [103, 443]. Moreover, cholestasis also enhances biliary excretion of BAs by the bile salt export pump (BSEP), organic solute transporter alpha/beta (OSTα/β) and multi-drug resistance protein (MRP) 3 and 4 proteins which promote BAs elimination from the liver [103, 485]. Furthermore, the expression of phase I (i.e CYP3A4/CYP3A11 in rodents) and II ((i.e sulfotransferase SULT2A1) BA detoxification enzymes is induced, which favors generation of less toxic BA species easily secreted into the urine [444, 445]. These mechanisms are regulated by a series of ligand- activated transcription factors, called nuclear receptors (NRs) such as farnesoid X receptor (FXR, NR1H4), small heterodimer partner (SHP, NR0B2), liver receptor homolog-1 (LRH- 1, NR5A2), Klotho beta-like protein (βKlotho, KLB) [439, 447].
The present study aimed at investigating the effects of a DHA-enriched diet on BA homeostasis in vivo. We first evaluated how DHA supplementation impacts the expression of key genes related to BA synthesis, metabolism and transport in mouse liver, intestine and kidneys in healthy male and female C57BL/6 mice after 2- and 4-week DHA-enriched diet. Next, we evaluated the impact of the transcriptomic changes on the circulating BA profile. The present observations provide the first in vivo evidence that DHA supplementation impacts both BA-related genes expression and the circulating BA profile and therefore could be considered as a therapeutic option for cholestatic liver diseases such as PSC.
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MATERIALS AND METHODS
Materials
EPA and DHA were obtained from Sigma (St. Louis, MO, USA). The source of DHA in an animal diet was from Ocean Nutrition Inc (Halifax, NS, Canada). Normal and deuterated BAs were purchased from Steraloids Inc. (Newport, RI, USA) and C/D/N Isotopes Inc. (Pointe-Claire, QC, Canada), respectively. Strata X and Synergie RP Hydro columns were from Phenomenex (Torrance, CA, USA). The Taqman reagents (PCR Master Mix and probes) were purchased from Thermo (Life Technologies Division, Foster City, CA, USA).
Ethics statement
The use of animals was approved by the Laval University Animal Ethics Committee (approval ID 2012027-1) and all procedures were performed according to the guidelines of the Canadian Council on Animal Care.
Animals and diets
Fifty days old male and female C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed in a room with controlled temperature (20–23°C) and lighting (12H light/dark cycles). Mice were randomly assigned to 2 isocaloric diets of previously determined formula [486]: (i) a control diet (n=5/group/sex) containing 7 and 67 µmole/g of α-linolenic (LNA, 18∶3 n-3) and linoleic (LA, 18∶2 n-6) acid, respectively, instead of n-3 PUFAs (DHA and eicosapentaenoic acid, EPA); or (ii) a high-DHA diet (0.6g/kg/day, DHA:EPA ratio of 4∶1) composed of 9.5, 0.5 and 34 µmole/g of DHA, LNA and LA, respectively, during 2 (n=5/group/sex) or 4 weeks (n=6/group/sex). The n-6∶n-3 PUFA ratios were of 10.4 and 2.8 in the control and high-DHA diets, respectively. The source of DHA was a microencapsulated formulation (MEG-3) of fish oils from Ocean Nutrition Inc (Halifax, NS, Canada). Moreover, both diets contained equal quantity of total fat (5% w/w), total carbohydrates (66% w/w), total protein (20% w/w), total calorie (4 kcal/g), fibers, vitamins, minerals, antioxidants (except for vitamin C) and were deprived of phytoestrogens, which are known to influence the hormonal status in animals [487]. The mice were allowed free access to food and water, and their body weight was measured daily. At the end of the experimental period, mice were sacrificed to take blood by cardiac puncture and organs (liver, kidneys, intestine). All samples were stored at – 80°C until being analyzed.
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RNA isolation, reverse transcription and quantitative real time PCR (qRT-PCR)
Total RNA was isolated from treated or control mice tissues (50-80 mg) according to the TriReagent acid:phenol protocol as recommended by the supplier (Molecular Research Center Inc., Cincinnati, OH, USA). The reverse transcription (RT) reaction was performed using 200 units of Superscript II (Invitrogen) and random hexamer primers (150 ng) to 1 µg of total RNA (Invitrogen, Burlington, ON, Canada) at 42°C for 50 min, as described [60]. Real-time PCR quantifications were performed using an ABI ViiA 7 qRT- PCR system from Thermo-Life Technologies (Carlsbad, ON, CA). For each reaction, the final volume of 10 µL was composed of 5 µL of PCR Mix, 0.5 µL of probe (Suplemental Table 1), 1.5 H2O and 3 µL of a RT product diluted 50 times. Conditions for qRT-PCR were
95°C for 10 min, 95°C for 15 sec, and annealing temperature for 1 min for 40 cycles. Threshold cycle (Ct) values were analyzed using the comparative Ct (ΔΔCt) method as recommended by the manufacturer (Thermo-Life Technologies). The amount of target gene was obtained by normalizing to the endogenous reference 18S and was expressed relatively to vehicle-treated cells set at 1.
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)
For BAs measurements in plasma, 25 BA species (free, taurine- or glycine- conjugated) were profiled in 100 µL of mice plasma using LC-MS/MS as extensively described elsewhere [312]. The chromatographic system consisted of an Alliance 2690 Separations Module (Waters, Milford, MA) coupled to an API4000 instrument equipped with an electrospray ionization source (Applied Biosystems, Concord, Canada). Lipidomic analysis of n-3 PUFAs and their metabolites such as 18-hydroxyeicosapentaenoic acid (18(R/S)-HEPE), 7(S)-hydroxydocosahexaenoic acid (17(S)-HDHA) and protectin DX (PDX) were performed as described [488] using API16500 instrument (Applied Biosystems, Concord, Canada).
Statistics
All data are presented as mean ± SEM. Statistical differences between 2 groups were analyzed using unpaired two-side t-test. Correlation between n-3 PUFAs, their metabolites in mice serum and liver and expression of BA genes and BAs concentrations in serum mice was analyzed using the Spearman's rank correlation coefficient adjusted for
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the effects of sex, weight and treatment period. Statistical analysis was carried out using SAS statistical software, v9.4 (SAS Institute, Cary, NC, USA).
For BA analyses, the total BA concentration corresponds to the sum of the 21 BA levels. The sum of glyco- and tauroconjugates was calculated by adding the concentrations of conjugated CDCA, CA, DCA, LCA, UDCA, αMCA, βMCA and ωMCA. The sum of unconjugated BAs also included HDCA and HCA levels.
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RESULTS
DHA modulates the hepatic expression of key genes for bile acid homeostasis
Our group recently reported in vitro evidences that, as the other n-3 PUFAs EPA, DHA activates a BA detoxification system in human cell models for the liver, intestine and kidney [Cieślak A. et al., in preparation]. This activation involves the transcriptional modulation of genes involved in BA synthesis, metabolism and transport [Cieślak A. et al., in preparation]. To evaluate whether such processes are still present when DHA treatment is applied to living animals, we initiated an in vivo protocol in which 50-day old male and female mice were fed with a control or an isocaloric DHA-enriched diet for 14 or 28 days. The hepatic expression of key genes controlling BA synthesis, transport, metabolism and signalling was analysed through quantitative qRT-PCR (Figures 1-3).
After 14-day exposure, DHA caused significant (p values <0.05 or >0.01) accumulation of Cyp7a1, Cyp27a1 and Lrh transcripts only in female livers, while Ostβ mRNA levels were increased in male samples (Figure 1A). Interestingly, Sult2a1 transcripts, undetected in female samples, were less abundant in treated male livers than in controls. Finally, DHA also caused a significant induction of Cyp3a11 mRNA levels in livers from both male and female mice (Figure 1A).
Interestingly, tissues from animals fed with DHA for a longer duration (i.e 28 instead of 14 days) exhibited a relatively different response profile to DHA (Figure 1B). Indeed,
Cyp7a1 and Cyp27a1 mRNA levels were found to be reduced in DHA-treated samples
from both males and females (but significantly reduced [i.e p<0.05] only in males). In the same vein, several changes in BA transporter expression which did not reached statistical significance in 14-day treated tissues were significantly modulated after 28 days (Figure 1B). Indeed, the up-regulation of Ntcp, Mrp3 and Bsep mRNA expression in the liver from DHA-treated female animals was statistically significant only after 28 days of DHA diet. Interestingly, Ntcp mRNA level which accumulated in 28-day DHA-treated female samples was significantly reduced in male livers. Such a sexual dimorphism is also observed when considering the modulation of Ostβ which was significantly up-regulated in male livers after 14 days of treatment (Figure 1A), while the response in female livers was statistically significant only after 28 days of DHA exposure (Figure 1B).
Even when taking into account the sex-related differences described above, overall those results revealed that DHA regulates the hepatic expression of key genes involved in the control of BA homeostasis in the mice liver, particularly through the down-regulation of
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genes coding for BA synthesis, and the up-regulation of those controlling BA metabolism and transport.
DHA also modulates genes controlling bile acid transport and metabolism in mice intestine and kidney
To further evaluate the effects of DHA diet on genes implicated in BA homeostasis, we investigated the change in transcriptomic profiles of targeted genes in several parts of the intestine and kidney (Figures 2&3).
No significant changes were found in females after 14 days of DHA supplementation apart from the increased Mrp2 expression in small intestine (Figure 2A). In male mice, Mrp2, 3 and Ostα/β transporters were highly up-regulated throughout all parts of the intestine, as were Ostα and β in the ileum and caecum (Figure 2A). Moreover,
Fxr mRNA levels appeared to be increased in the large intestine, while Shp was activated
only in colon. A part of signaling changes related to Fxr was also maintained after 28 days of DHA supplementation. In contrast, DHA feeding resulted in reduced Ostβ and increased
Mrp2, 3 expression (Figure 2B).
Finally in kidneys, Mrp3 was up-regulated in male after 14 days of DHA supplementation, while Cyp3a11 expression increased after both 2 and 4-week treatment (Figure 3A&B). Interestingly, in females mice the mRNA level of phase I (Cy3a11) and phase II (Sult2a1) BA-metabolizing enzymes were up-regulated only after the longer DHA exposure (Figure 3B). Moreover, DHA feeding leaded to reduction of Mrp3, but increased
Ostα transporter in 2-week treatments.
Overall those results indicate that DHA also modulates the extrahepatic expression of genes controlling BA homeostasis and signaling.
DHA affects the circulating bile acid profile in mice
To further evaluate whether the changes observed in BA-related genes caused significant alterations in their homeostasis, we next profiled 25 BA species in plasma from control and DHA-treated mice (Tables 1&2).
After 2 weeks of treatment significant changes were detected in the circulating BA profile of animals fed the DHA-enriched diet (Table 1). In males, DHA caused significant changes in individual BAs when compared to the isocaloric diet: CDCA, UDCA, ωMCA and DCA concentrations were reduced by 3.84, 2.96, 2.52 and 1.65%, respectively. Other changes failed to reach the statistical significance. In female, the significant reduction was
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observed only in ωMCA (-4.94%) and βMCA (-6.54%), while other species such as CDCA, UDCA, LCA and DCA were drastically reduced but without reaching statistical significance. Consequently to these modifications, the total of unconjugated BAs was significantly (p<0.05) decreased from 9,287±2,150 in controls to 1,504±364 nM in DHA-treated female mice (Table 1). The specific changes described above also resulted in a significant 3.4-fold reduction in the total plasma BA contents in the same animals, thus demonstrating that a 14-day DHA treatment reduces the amounts of circulating BAs in female mice. However, due to gender-related differences observed in the response to this 2-week treatment, none of the changes in total, taurine- and glycine-conjugated and unconjugated BAs reached the statistical significance (Table 1).
Similar analyses performed with plasma samples were undertaken in animals treated for 4 instead of 2 weeks, and revealed a similar response to DHA both in terms of individual specific molecules or of BA groups (i.e taurine-, glycine-conjugated, or unconjugated) (Table 2). Indeed, with the notable exception of glycine species which were increased in male samples after DHA exposure, all families displayed strong reduction in both male and female samples. However, due to a large inter-individual variability in either baseline or post-treatment none of the alterations observed in these samples reached the statistical significance (Table 2).
On the other hand, it should be noted that in both series of experiments, control female animals displayed much higher BA levels than their male counterparts (Tables 1&2). Similarly, the response to DHA was also stronger in female samples than in male ones (Tables 1&2), suggesting that DHA is more efficient in reducing plasma BA levels in