Ontogenic development-associated changes in the expression of genes involved in rat bile acid homeostasis

Abstract

Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5α-reductase, 5β-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and Cyp3a11), transporters [bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter α/β (Ostα/Ostβ)], and nuclear receptors [fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4α (HNF-4α)], determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5α-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and α-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span. Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5α-reductase, 5β-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and Cyp3a11), transporters [bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter α/β (Ostα/Ostβ)], and nuclear receptors [fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4α (HNF-4α)], determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5α-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and α-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span. apical sodium-dependent bile acid transporter bile acid cytochrome P450 enzymes fetoprotein transcription factor farnesoid X receptor hepatic nuclear factor 4α sodium taurocholate-cotransporting polypeptide organic solute transporter small heterodimer partner Bile acids (BAs) are amphipathic steroids synthesized in the liver from cholesterol and are involved in important physiological functions. Among these, the best known is their role in fat digestion and absorption. This is mainly performed by their detergent activity, which is closely related to their chemical structure, which varies among many different molecular species present in the BA pool. The composition of the BA pool is not the same in all species, and in each species it probably changes during its life span. All BA variants share the presence of the steroid ring and the side chain, but they differ mainly in the degree of hydroxylation and the position of their hydroxyl groups. The total amount of BAs in the body and the composition of this pool are determined by the expression and activity of enzymes involved in their synthesis and biotransformation in concerted action with plasma membrane transporters, which account for their retention in the enterohepatic circulation. BA metabolism by intestinal bacteria and elimination by the kidney may also contribute to the final BA pool size and composition. Owing to the key role of the liver in BA homeostasis, it is easy to understand why phenotypic changes in this organ affect the BA pool. Thus, proliferative processes occurring in the liver after partial hepatectomy or during hepatocarcinogenesis are accompanied by important changes in the composition of the BA pool (1.El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Crossref PubMed Scopus (32) Google Scholar, 2.Monte M.J. Martinez-Diez M.C. El-Mir M.Y. Mendoza M.E. Bravo P. Bachs O. Marin J.J.G. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.J. Hepatol. 2002; 36: 534-542Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 3.Mendoza M.E. Monte M.J. El-Mir M.Y. Badia M.D. Marin J.J.G. Changes in the pattern of bile acids in the nuclei of rat liver cells during hapatocarcinogenesis.Clin. Sci. 2002; 102: 143-150Crossref PubMed Scopus (21) Google Scholar). Such changes are closely related to changes in the expression profiles of enzymes involved in BA metabolism (4.Monte M.J. Fernandez-Tagarro M. Marin J.J.G. Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration.Biochim. Biophys. Acta. 2005; 1734: 127-135Crossref PubMed Scopus (9) Google Scholar, 5.Monte M.J. Fernandez-Tagarro M. Macias R.I. Jimenez F. Gonzalez-San Martin F. Marin J.J.G. Changes in the expression of genes related to bile acid synthesis and transport by the rat liver during hepatocarcinogenesis.Clin. Sci. 2005; 109: 199-207Crossref PubMed Scopus (12) Google Scholar). Although ontogenic development also involves proliferation and differentiation, in addition to adaptive changes to modifications in the diet, little is known about the expression of enzymes involved in BA synthesis associated with this development (6.Massimi M. Lear S.R. Huling S.L. Jones A.L. Erickson S.K. Cholesterol 7α-hydroxylase (CYP7A): patterns of messenger RNA expression during rat liver development.Hepatology. 1998; 28: 1064-1072Crossref PubMed Scopus (45) Google Scholar); more is known about the expression of transporters under these circumstances (7.Hardikar W. Ananthanarayanan M. Suchy F.J. Differential ontogenic regulation of basolateral and canalicular bile acid transport proteins in rat liver.J. Biol. Chem. 1995; 270: 20841-20846Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8.Tomer G. Ananthanarayanan M. Weymann A. Balasubramanian N. Suchy F.J. Differential developmental regulation of rat liver canalicular membrane transporters Bsep and Mrp2.Pediatr. Res. 2003; 53: 288-294Crossref PubMed Scopus (57) Google Scholar). In light of the foregoing, the aim of this study was to investigate the relationship between changes in the BA pool size and composition during fetal life, postnatal development, youth, maturity, and senescence and modifications in the expression profiles of the enzymes, transporters, and nuclear receptors involved in the control of BA pool size and composition. Particular attention is paid to the weaning period, when important nutritional, nervous, and endocrine factors that may affect BA homeostasis occur. BAs and 5β-cholestane used as standards in GC-MS (>95% pure by thin-layer chromatography) as well as cholylglycine hydrolase, 3α-hydroxysteroid dehydrogenase, diaphorase, and resazurin were purchased from Sigma-Aldrich (Madrid, Spain). [3H]taurocholic acid (specific radioactivity, 3.0 Ci/mmol) was obtained from New England Nuclear (Pacisa, Madrid, Spain). All other chemicals were from Sigma-Aldrich or Merck Eurolab (Barcelona, Spain). They were of high purity and were used as purchased without any further purification. Pregnant Wistar CF rats and their offspring were obtained from the University of Salamanca Animal House, except for aged animals, which were from Harlan Iberica (Barcelona, Spain). Dams and weaned animals were fed on commercial pelleted rat food (Harlan Iberica) and water ad libitum. Lighting was controlled by a timer that permitted light between 8:00 AM and 8:00 PM. All animals received humane care as described in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication). Experimental protocols were approved by the Ethical Committee for the Use of Laboratory Animals of the University of Salamanca. All litters were weaned on day 21 unless stated otherwise. Samples were obtained from fetuses (gestation days 14 and 20), neonates (on the first day after delivery), young animals of 1, 2, 3, or 4 weeks of age, adult rats of 8 weeks of age, and aged/senescent rats 9 and 24 months old. Fetuses and neonates were used without separation between males and females. Only males were used in groups of older animals. Eight weeks of age was defined as adult stage based on previous studies by us and others (9.Monte M.J. Morales A.I. Arevalo M. Alvaro I. Macias R.I.R. Marin J.J.G. Reversible impairment of neonatal hepatobiliary function by maternal cholestasis.Hepatology. 1996; 23: 1208-1217Crossref PubMed Google Scholar, 10.Balasubramaniyan N. Shahid M. Suchy F.J. Ananthanarayanan M. Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development.Am. J. Physiol. 2005; 288: G251-G260Crossref PubMed Scopus (41) Google Scholar). Sample collection was always performed at 3:00 PM on nonfasted animals anesthetized with sodium pentobarbital (5 mg/100 g body weight, ip). After laparotomy, a small sample (<30 mg) of liver tissue was obtained to measure gene expression. To extract the total BA pool, the liver, small intestine, and mesenterium were removed and processed together as reported previously (11.Vicens M. Macias R.I.R. Briz O. Rodriguez A. El-Mir M.Y. Medarde M. Marin J.J.G. Inhibition of the intestinal absorption of bile acids using cationic derivatives. Mechanism and repercussions.Biochem. Pharmacol. 2007; 73: 394-404Crossref PubMed Scopus (19) Google Scholar). Briefly, these organs were washed in saline, weighed, and homogenized in saline (1:2, w/v). A trace amount of radiolabeled [3H]taurocholic acid was added to the homogenate and used as an internal standard to evaluate the yield of the extraction procedure. BAs were extracted from the homogenate by the addition of 2 volumes of ethanol and at for 2 by for This was and were and used to measure and to BA To fetal on day 14 or of a was under pentobarbital Fetuses were removed and weighed, and their and small were processed as described The BA pool was not measured in 14 day fetuses, to the amount of fetuses to the amount of this only hepatic gene expression was measured by the of to fetuses In to investigate the of the time of weaning, the litters were weaned on day 21 or day In were obtained at and days of In these animals, a sample from the from the was also obtained to measure expression of BA obtained rat liver or small were in the Madrid, and at RNA extraction RNA was from these using from Spain). After with RNA was with the The was synthesized using and obtained from were with the of Madrid, for in and their was using The reported (4.Monte M.J. Fernandez-Tagarro M. Marin J.J.G. Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration.Biochim. Biophys. Acta. 2005; 1734: 127-135Crossref PubMed Scopus (9) Google Scholar, R.I.R. Monte M.J. Jimenez Marin J.J.G. effect of rats with acid on the impairment of bile in the by maternal Pharmacol. 2005; PubMed Scopus Google except for the apical sodium-dependent bile acid transporter hepatic nuclear factor 4α organic solute transporter and quantitative was performed using in an were as 1 at for by at for and for of was using The of or of PCR, with and permitted the of in all The of for each gene in each sample were using and as genes (4.Monte M.J. Fernandez-Tagarro M. Marin J.J.G. Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration.Biochim. Biophys. Acta. 2005; 1734: 127-135Crossref PubMed Scopus (9) Google Scholar, 5.Monte M.J. Fernandez-Tagarro M. Macias R.I. Jimenez F. Gonzalez-San Martin F. Marin J.J.G. Changes in the expression of genes related to bile acid synthesis and transport by the rat liver during hepatocarcinogenesis.Clin. Sci. 2005; 109: 199-207Crossref PubMed Scopus (12) Google Scholar). The was measured with the The BA pool size was determined by of total BAs in from liver and small BA were measured enzymatically and of total bile Acta. PubMed Scopus Google as described previously (9.Monte M.J. Morales A.I. Arevalo M. Alvaro I. Macias R.I.R. Marin J.J.G. Reversible impairment of neonatal hepatobiliary function by maternal cholestasis.Hepatology. 1996; 23: 1208-1217Crossref PubMed Google and the BA pool size was corrected by account the in the extraction of the measured in the homogenate and in the final The yield was quantitative BA species in the were and measured by as described previously (1.El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Crossref PubMed Scopus (32) Google Scholar). Briefly, after acid as a first internal BAs were enzymatically M. and biotransformation of acid in J. Physiol. Google Scholar, J. M. N. of acid associated with acid and their in liver Res. Full Text PDF PubMed Google Scholar). BAs were extracted from the by extraction in Madrid, Spain). were by with were in for at A. J. P. of profiles of bile acids in using a and Res. Full Text PDF PubMed Google Scholar). the 5β-cholestane was added to the as a internal GC-MS were on a Madrid, to a using a described previously M. and biotransformation of acid in J. Physiol. Google Scholar). are as To the of the among or the of was as To the of changes in the BA pool by body and liver weight, these were measured in all groups not The of increased in up to After liver increased body Thus, the decreased from in adult animals weeks to in aged animals months The absolute size of the BA pool increased progressively from in fetuses days of to in adult rats weeks of In older the of in the BA pool size was the BA pool size was corrected by liver a was at 1 week of age that the life of the animals, except after weaning, when a transient and peak of the in was The of the BA pool size in adult rats in this study by the liver and and BAs were to obtained previously in by bile for Monte M.J. M.C. Marin J.J.G. Bile by the rat liver during J. PubMed Scopus (12) Google Scholar). Owing to the of the of the of the genes used to the of of expression levels between groups of rats with different first the of in all groups F. N. A. and F. of quantitative by of internal control Biol. with to no among groups for genes were not was used as a this The in the expression of is with previous studies during liver as that occurring during liver and (4.Monte M.J. Fernandez-Tagarro M. Marin J.J.G. Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration.Biochim. Biophys. Acta. 2005; 1734: 127-135Crossref PubMed Scopus (9) Google Scholar, 5.Monte M.J. Fernandez-Tagarro M. Macias R.I. Jimenez F. Gonzalez-San Martin F. Marin J.J.G. Changes in the expression of genes related to bile acid synthesis and transport by the rat liver during hepatocarcinogenesis.Clin. Sci. 2005; 109: 199-207Crossref PubMed Scopus (12) Google Scholar). the expression of the key enzymes involved in BA synthesis was that the of Cyp7a1 was by that of Cyp27 that the may play a more important role in young animals in Cyp27 not to for the in the BA pool after weaning, no in the expression of Cyp27 was whereas that of Cyp7a1 was The early of the degree of Cyp7a1 expression was to that reported previously using (6.Massimi M. Lear S.R. Huling S.L. Jones A.L. Erickson S.K. Cholesterol 7α-hydroxylase (CYP7A): patterns of messenger RNA expression during rat liver development.Hepatology. 1998; 28: 1064-1072Crossref PubMed Scopus (45) Google with this the were not to Cyp7a1 in day of the of molecular species in the BA pool that complexity was high during life This was probably by the the of maternal of which by the This is for which in the fetal but that in the fetal BA pool in rats and also in M.J. Macias R.I.R. Bravo P. El-Mir M.Y. A. 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Changes in the expression of genes related to bile acid synthesis and transport by the rat liver during hepatocarcinogenesis.Clin. Sci. 2005; 109: 199-207Crossref PubMed Scopus (12) Google this relationship during rat ontogenic development The that acid was in but was present in the period, when the expression was in adult rat liver The for these is but it that the for the of this BA in liver during ontogenic development not only on the expression previously that the nuclear receptors involved in bile are and are during the fetal whereas their expression during the postnatal N. Shahid M. Suchy F.J. Ananthanarayanan M. Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development.Am. J. Physiol. 2005; 288: G251-G260Crossref PubMed Scopus (41) Google Scholar). this Thus, the expression of farnesoid X receptor small heterodimer partner and was low at 1 week and increased progressively the first postnatal The in was accompanied by a in Shp up to the when downregulation of this nuclear receptor the upregulation of Cyp7a1 The expression levels of these nuclear receptors at from to but increased during except for which The BA pool of the is mainly to the high proportion of acids, which are to for a in the amount of acids in the BA pool during senescence Thus, the in expression in aged rats may related to the of their BA pool. the expression profiles of the nuclear receptors were with of enzymes it appeared that during senescence the effect of on the transcription of Cyp7a1 and Cyp8b1 the by upregulation to the high of and the that the amount of its in of these enzymes in the liver of animals the of other mechanisms the in time of the in the expression of and in senescence the of a relationship or is an further In this it that during fetal liver development in the expression of A. F.J. of the hepatocyte nuclear factor in control of the X receptor during fetal liver development.Hepatology. 2003; PubMed Scopus Google Scholar). changes during the week of age, by of and of and J. Physiol. Google Scholar). the control of BA pool size and the changes occurring during this of a peak in Shp expression weaning, by a to to of the week This also by others N. Shahid M. Suchy F.J. Ananthanarayanan M. Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development.Am. J. Physiol. 2005; 288: G251-G260Crossref PubMed Scopus (41) Google together with the of a peak in activity at weaning in the which on day 14 by on days F.J. of hepatic bile acid in the Res. PubMed Scopus Google Scholar). the of the expression of sodium-dependent BA by the rat liver sodium taurocholate-cotransporting polypeptide early after (7.Hardikar W. Ananthanarayanan M. Suchy F.J. Differential ontogenic regulation of basolateral and canalicular bile acid transport proteins in rat liver.J. Biol. Chem. 1995; 270: 20841-20846Abstract Full Text Full Text PDF PubMed Scopus (72) Google BA transport in rat at weaning W. Suchy F.J. and molecular of the of a rat sodium-dependent bile acid 1995; PubMed Google Scholar). BA is a involved in the absorption of and that the of BA expression at weaning and is by but not by regulation of expression of bile acid in J. Physiol. Scholar). to further BA homeostasis during this of life in the To the changes were to or to changes by nutritional, nervous, and endocrine factors the after separation from their different groups were investigated. In weaning was imposed on day 21 after In the weaning was day This had no effect on body (on day of age, 21 day weaning g and day weaning on day of age, 21 day weaning g and day weaning the time course of the absolute or relative BA pool size was not it a in the BA composition. The delay in weaning was accompanied by increased and of cholic acid in the BA pool and a decreased of chenodeoxycholic acid and α-muricholic acid is that this to changes in the transporters involved in enterohepatic that are at adult levels at this stage F.J. of bile during of hepatic bile acid metabolism and PubMed Scopus Google Scholar). In this no changes were in the of of liver and bile salt export or intestinal and the at that time of life in Shp was in the weaned at days the expression of Cyp7a1 was not by weaning together with the of changes in the expression of transporters the enterohepatic is with the of changes in the BA pool the delay in weaning a upregulation of Cyp8b1 which may account for the increased proportion of cholic acid in the BA pool of these animals of time of weaning on the of BA species in the rat BA pool in 4 week animals. are from animals from different groups of rats weaned at 21 and days with the cholic chenodeoxycholic of time of weaning on the relative of the of Shp sodium taurocholate-cotransporting polypeptide and bile salt export in rat liver and apical sodium-dependent bile acid transporter organic solute transporter and in rat are from animals from different groups of rats weaned at 21 and days with the for on tissue week rat liver or as were as Shp Bsep and of time of weaning on the relative of by quantitative of Cyp7a1 and Cyp8b1 in rat are from animals from different groups of rats weaned at 21 and days with the for of Cyp7a1 and Cyp8b1 on tissue week rat were as and In during postnatal rat liver development, and more weaning, important quantitative and changes are in the BA pool. Although the quantitative changes to and not on the time of weaning, modifications in BA pool composition are probably related to this and by The J. F. J. and N. for care of the animals. by M. I. by and the of the and of the by N. are also This study was in by the and and the The is a of the for on by the and the and to the for and