University of Groningen The enterohepatic circulation of bile salts in health and disease Hulzebos, Christian Victor IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2004 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hulzebos, C. V. (2004). The enterohepatic circulation of bile salts in health and disease: a kinetic approach Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 01-06-2018
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University of Groningen
The enterohepatic circulation of bile salts in health and diseaseHulzebos, Christian Victor
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.
Document VersionPublisher's PDF, also known as Version of record
Publication date:2004
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Hulzebos, C. V. (2004). The enterohepatic circulation of bile salts in health and disease: a kinetic approachGroningen: s.n.
CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.
Values are expressed as means ± SD (n = 6 mice per group). *Signifi cant difference
between wild-type and Fxr (+/-) or Fxr
(-/-) mice.
biliary bile salt output rates were plotted against bile fl ow for the individual mice of
the three groups, the classical linear relationship between these parameters was
observed (Figure 2). This strongly indicates that the bile formation process itself
is not affected by FXR defi ciency and that the higher bile fl ow rate in Fxr (-/-) mice
is caused by the higher bile salt output.
Analysis of biliary bile salt composition (Table 3) revealed that, in all three groups,
cholate constituted the major fraction of biliary bile salts and that this fraction
was higher in Fxr (-/-) mice than in the other two groups. Accordingly, the relative
contents of α-muricholate and chenodeoxycholate were signifi cantly decreased
in Fxr (-/-) mice. Thin-layer chromatography revealed that essentially all biliary
cholate was conjugated to taurine in wild-type as well as in Fxr (-/-) mice (data not
shown). Despite the fact that bile salt-conjugated enzymes have recently been
identifi ed as FXR target genes34, unconjugated bile salts were undetectable by this
procedure in bile of wild-type and Fxr (-/-) mice.
Steady-state mRNA levels of genes involved in bile salt synthesis and bile
formation
Real-time quantitative PCR was used to evaluate hepatic expression of specifi c
genes as infl uenced by FXR defi ciency (Figure 3). As expected, expression of
Shp tended to be lower in Fxr (-/-) mice. Cyp7a1 was clearly increased in Fxr
(-/-)
mice, whereas the expression levels of Cyp27 and Cyp8b1 were not signifi cantly
affected (Figure 3A). The mRNA levels of the gene encoding the canalicular
bile salt transporter Bsep, a well-known FXR target gene14,15, were signifi cantly
decreased in Fxr (-/-) mice (Figure 3B). Expression of other transporters genes
relevant to bile formation, such as the phospholipid translocator Mdr2, Ntcp,
and the putative cholesterol transporters Abcg5/g8, was not changed by FXR
defi ciency. Likewise, no effects on expression of Oatp1, Mrp2, and Mrp3 were
observed.
Effects of FXR defi ciency on kinetic parameters of cholate metabolism
To evaluate the physiological consequences of the observed changes in expression
of bile salt synthesis and transporter genes, kinetic parameters of the enterohepatic
circulation of cholate were determined by stable isotope dilution21. Because of the
small differences between the heterozygotes and wild-type mice with respect to
94
Chapter 5
Figure 4. Decay of intravenously administered [2H4]-cholate in wild-type and Fxr
(-/-) mice.
A dose of 240 µg of [2H4]-cholate was intravenously injected into wild-type (open symbols)
and Fxr (-/-) (closed symbols) mice, and blood samples were collected at 24, 36, 48 and 60
h after injection for determination of plasma cholate enrichments by GC-MS as described
under Experimental Procedures. Values are expressed in a logarithmic fashion, and the pool
size (y-intercept), fractional turnover rate (slope of the curve), and synthesis rate (pool size
x fractional turnover rate) were calculated for individual mice. Data are means ± standard
deviation of n = 5 mice per group.
Figure 3. Steady-state mRNA levels of genes involved in bile salt synthesis and transport in
livers of wild-type, Fxr (+/-) and Fxr
(-/-) mice. Total mRNA was isolated form the livers of wild-
type (white bars), Fxr (+/-) (grey bars) and Fxr
(-/-) (black bars) mice, transcribed into cDNA,
and subjected to real-time PCR as described in Experimental Procedures. (A) Hepatic mRNA
levels of Fxr, Shp, Cyp7a1, Cyp27 and Cyp8b1. (B) Hepatic mRNA levels of Bsep, Mdr2,
Abcg5, Abcg8, Ntcp, Oatp1, Mrp2 and Mrp3. n = 5 for all groups. *Signifi cant difference
between wild-type and Fxr (+/-) or Fxr
(-/-) mice.
95
Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice
bile formation and gene expression patterns, kinetic studies were conducted in
wild-type and Fxr (-/-) mice only. Analysis of plasma cholate enrichments over time
(Figure 4) demonstrated that the cholate pool size, calculated from the y-intercept
of the linear regression line shown in Figure 4, was larger in Fxr (-/-) mice than
in wild-type mice (Figure 5A: 42 ± 8 µmol/100g vs. 23 ± 3 µmol/100g, Fxr (-/-)
mice vs. wild-type; p < 0.0001). The percentage of cholate in hepatic bile, as
determined by gas chromatographic analysis in individual mice (Table 3), was
used to calculate total bile salt pool sizes. Under the assumption that all bile salt
species displayed a similar cycling frequency, the calculated total pool sizes of
non-cholate bile salts were similar (22 ± 4 mmol/100g vs. 23 ± 9 mmol/100g,
Fxr (-/-) vs. wild-type mice, respectively, NS), leading to calculated total bile salt
pool sizes of 64 ± 12 and 46 ± 13 mmol/100g in Fxr (-/-) and wild-type mice,
respectively (p < 0.05). Deuterated cholate disappeared from plasma at the same
rate in Fxr (-/-) and wild-type mice (Figure 4). The fractional turnover rate of
cholate, calculated from the slope of the linear regression curve, was similar in
Figure 5. Effects of FXR defi ciency on pool size (A), fractional turnover rate (B), synthesis
rate (C), cycling time (D), and daily intestinal reabsorption (E) of cholate as derived from
[2H4]-cholate isotope enrichment measurements in plasma of wild-type and Fxr (-/-) mice.
The pool size, fractional turnover rate (FTR), synthesis rate, cycling time and daily intesti-
nal reabsorption were calculated in wild-type (white bars) and Fxr (-/-) (black bars) mice as
described under Experimental Procedures. Data are means ± standard deviation of n = 5
mice per group. *Signifi cant difference between wild-type and Fxr (-/-) mice.
96
Chapter 5
both groups of mice. (Figure 5B: 0.5 ± 0.1 per day vs. 0.5 ± 0.2 per day, Fxr (-/-)
vs. wild-type, respectively, NS).
In the Fxr (-/-) mice, the calculated cholate synthesis rate (Figure 5C) was two
times increased compared to the wild-type mice (22 ± 2 mmol/100g/day vs.
11 ± 3 mmol/100g/day, Fxr (-/-) vs. wild-type mice; p < 0.001). In accordance with
the increased cholate synthesis rate determined by stable isotope dilution, fecal
loss of bile salts was increased by ~ 70 % (4.1 ± 1.1 mmol/day vs. 2.3 ± 0.7
mmol/day, Fxr (-/-) vs. wild-type mice; p < 0.05). The calculated cholate cycling
time (Figure 5D) was not affected by FXR defi ciency (4.4 ± 1.3 h vs. 4.3 ± 0.7 h,
Fxr (-/-) mice vs. wild-type mice; NS). The calculated absolute amount of cholate
reabsorbed in the intestines of Fxr (-/-) mice (Figure 5E) was ~ 2 fold larger than
that in the intestines of wild-type mice (227 ± 81 mmol/100g/day vs. 121 ± 11
mmol/100g/day, Fxr (-/-) mice vs. wild-type mice; p < 0.05).
Figure 6. mRNA and protein levels of intestinal bile salt transporters in wild-type and Fxr (-/-)
mice. (A) Steady-state mRNA levels of Asbt, Ibabp, t-Asbt, and Fic-1 in the ilea of wild-
type (white bars) and Fxr (-/-) (black bars) mice. Total mRNA was isolated from the ileum,
transcribed into cDNA, and subjected to real-time PCR as described under Experimental Pro-
cedures. *Signifi cant difference between wild-type and Fxr (-/-) mice. (B) Western blot analy-
sis of Asbt and Ibabp on brush-border membranes and liver homogenates, respectively,
from the ilea of wild-type and Fxr (-/-) mice. Apparent molecular masses are indicated to the
right.
97
Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice
Intestinal expression of genes involved in bile salt transport
To provide an explanation for the high bile salt absorption rate in Fxr (-/-) mice,
the mRNA levels of several genes considered to be involved in intestinal bile salt
absorption were determined in the terminal ileum. These studies showed that
expression of Ibabp, a well known FXR target gene8,18 thought to be involved
in intracellular bile salt traffi cking and active bile salt reabsorption8,9, was very
strongly decreased at the mRNA level in Fxr (-/-) mice (Figure 6A). FXR defi ciency
did not affect expression of transporter protein-encoding genes Asbt, responsible
for the major part of active ileal bile salt reabsorption, and of t-Asbt, putatively
involved in basolateral bile salt effl ux. Expression of Fic1 (Atp8b1), a P-type
ATPase proposed to function as an aminophospholipid translocator and essential
for normal bile salt metabolism35, also did not differ at the mRNA level between
wild-type and Fxr (-/-) mice. Western blot experiments on brush-border membrane
fractions and homogenates of the terminal part of the ileum (Figure 6B) showed
that Asbt protein levels were similar in wild-type and Fxr (-/-) mice, whereas the
protein levels of Ibabp were essentially non-detectable in the Fxr (-/-) mice.
DISCUSSION
This study has established the physiological consequences of FXR defi ciency on
bile formation and on the kinetics of enterohepatic bile salt circulation employing
an FXR-null mouse model generated by homologous recombination. A microscale
stable isotope dilution technique21 was used to quantify important parameters
of bile salt metabolism. Data show that the bile formation process per se was
not affected by FXR defi ciency and that effects on bile fl ow seen in Fxr (-/-) mice
were secondary to alterations in bile salt metabolism. In accordance with current
concepts of the role of FXR in control of bile salt synthesis36,37, hepatic Cyp7a1
mRNA levels were signifi cantly increased in Fxr (-/-) mice and were associated with
an increased cholate synthesis rate. Enhanced bile salt synthesis was confi rmed
by increased fecal bile salt loss in Fxr (-/-) mice, although the absolute difference
was somewhat less pronounced among the strains using this methodology. We
attribute the discrepancy between outcome of fecal excretion and the isotope
dilution method to the fact that no stool marker has been applied, which is
required to correct for fecal balance measurements. Furthermore, the intrinsic
diffi culties of quantitative fecal bile salt analysis have been extensively reviewed
by Setchell et al.38. As a consequence of defective feedback inhibition of hepatic
bile salt synthesis, Fxr (-/-) mice developed an increased bile salt pool size, which
implies that potential adaptive responses of intestinal bile salt reabsorption were
not effective in maintenance of the bile salt pool size. No change in intestinal Asbt
mRNA and protein levels was found in FXR-defi cient mice. By contrast, the well
known FXR target gene Ibabp was not expressed at all in the terminal ileum of
Fxr (-/-) mice. Despite the absence of Ibabp, our kinetic study revealed that the
absolute amount of bile salts reabsorbed from the intestine was not reduced, but
was actually enhanced by 2-fold in Fxr (-/-) mice. These fi ndings suggest that Ibabp
may not function as a ‘facilitator’9, but rather as a negative regulator of intestinal
bile salt absorption under physiological conditions in the mouse.
FXR has been shown to be involved in control of various steps of bile
98
Chapter 5
salt metabolism, i.e., synthesis and transport13,36,37,39, as well as in regulation
of plasma lipoprotein metabolism2,3,13,40. FXR-defi cient mice have been very
informative in elucidation of the various functions of this nuclear bile salt-activated
receptor. Studies by Sinal et al.13 and Lambert et al.40 were performed with
FXR-defi cient mice (C57BL/6J-SV129 background) that were generated by Cre-
mediated deletion of a fragment containing the last exon of the Fxr gene, encoding
the ligand-binding/dimerization domain, and the 3’-untranslated region of the
Fxr mRNA. In theory, a truncated protein containing the DNA-binding domain
could be formed that might affect expression of FXR target genes. In this study,
we used an FXR knockout model (C57BL/6J-129/OlaHsd background) generated
by homologous recombination, in which 292 bp of exon 2, encoding a part of
the DNA-binding domain, were deleted. These mice showed plasma HDL and
triglyceride levels (Elzinga et al., unpublished) that were elevated to a similar
extent as reported earlier13. In our study, liver function parameters were found
to be unaffected in Fxr (-/-) mice. Plasma bile salt concentrations were only slightly
increased in Fxr (-/-) mice compared with wild-type mice, in marked contrast to the
8-fold increase in plasma bile salt concentration in Fxr (-/-) mice reported by Sinal et
al.13. These strongly elevated plasma bile salt concentrations have been attributed
to defective hepatobiliary bile salt transport due to down-regulation of the major
canalicular bile salt export pump (Bsep)13. However, we have shown that a similar
or even more pronounced down-regulation of Bsep expression in mice was not
associated with impaired biliary bile salt secretion41. Furthermore, the more than
4-fold increase in biliary bile salt secretion during bile salt feeding in mice42 is
accommodated by a very modest increase in hepatic Bsep expression. These
data have been interpreted to indicate that Bsep at normal expression levels has
a marked overcapacity in mice. In fact, in this study, biliary bile salt secretion
was more than 2-fold increased despite a 40% reduction of Bsep expression.
Therefore, it appears that the livers of Fxr (-/-) mice are well able to handle the
(increased) bile salt load. The discrepancy between both strains with respect to
control of plasma bile salt concentrations remains unexplained at the moment.
FXR defi ciency was associated with an enhanced bile fl ow, as determined
during a 30 min period of bile collection. Biliary bile salt concentrations and
secretion rates were clearly enhanced in Fxr (-/-) mice, reinforcing the issue that
decreased Bsep expression levels do not necessarily correlate with defective bile
salt transport. The increase in bile fl ow in Fxr (-/-) mice appeared to be exclusively
due to the higher bile salt output, as is evident from the linear relationship
between bile fl ow and biliary bile salt output that was obtained when data from
wild-type, Fxr (+/-) mice and Fxr
(-/-) mice were combined (Figure 2). The fact that
values of individual mice from the three groups fi tted well to this relationship
indicates that the actual bile formation process was not affected by FXR defi ciency.
The value found for the choleretic activity (8 µl/µmol) of biliary bile salts is similar
to that reported earlier in rodents43. The bile salt-independent fraction of bile
fl ow (3 µl/min/100g body weight) was unaffected by FXR defi ciency, which is
in accordance with unchanged expression of Mrp2. Mrp2 is crucially involved in
hepatobiliary transport of glutathione44, which represents the major driving force
for the generation of bile salt-independent fl ow in rodents43,45. Although Mrp2 has
been identifi ed as an FXR-target gene46, Fxr (-/-) mice did not show reduced Mrp2
99
Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice
mRNA levels in this study or in a study by Schuetz et al.47. Biliary secretion rates
of both cholesterol and phospholipids were slightly enhanced in Fxr (-/-) mice as
compared to wild-type mice. Secretion of cholesterol and phospholipids into bile
is coupled to that of bile salts33. Cholesterol secretion appears to involve the
activity of Abcg5/Abcg8 dimers48,49, although the exact role of this twin transporter
remains to be defi ned50, whereas phospholipid secretion critically depends on the
activity of the Mdr2 P-glycoprotein51,52. Because expression of Abcg5/Abcg8 as
well as of Mdr2 was unaffected in Fxr (-/-) mice (Figure 3B), it is plausible to ascribe
the slight stimulation of biliary lipid secretion entirely to the enhanced bile salt
secretion. It should be noted that Lambert et al.40 did report reduced hepatic
Abcg5/Abcg8 expression in their strain of Fxr (-/-) mice, but this was found to
be associated with increased biliary cholesterol output rates. The reason for the
discrepancy in hepatic Abcg5/Abcg8 expression between both strains of mice is
not clear.
We have focused on the effects of FXR defi ciency on the kinetics of cholate
metabolism. For this purpose, we used a novel microscale isotope dilution
technique, applicable in unanesthetized animals21. FXR defi ciency was associated
with an increased cholate synthesis rate, in accordance with increased hepatic
Cyp7a1 mRNA levels in Fxr (-/-) mice. Although FXR has been advocated as the
major regulator of hepatic bile salt synthesis36,39, the effects of FXR defi ciency
on the basal expression of Cyp7a1 (~ +150%) and cholate synthesis (~ +67%)
were relatively modest. This fi nding underscores the importance of the recently
described FXR/SHP-independent mechanisms of regulation. Recent studies in bile
salt-fed SHP knockout mice53,54 have clearly demonstrated the existence of FXR/
SHP-independent repression of Cyp7a1 expression. Cyp27 and Cyp8b1 expression
levels were not signifi cantly affected in Fxr (-/-) mice, although the latter showed
tendency to increase, in accordance with an increase in the fractional contribution
of cholate in the bile salt pool of Fxr (-/-) mice. The fecal loss of bile salts was
increased by ~70 % in Fxr (-/-) mice. Because the mass of bile salts excreted
into feces is, by defi nition, directly proportional to the amount synthesized in
the liver36, the data on fecal loss confi rm a generalized derepression of bile salt
synthesis in FXR-defi cient mice. This again is at variance with the study of Sinal et
al.13, who reported increased expression of Cyp7a1, but decreased fecal bile salt
loss in Fxr (-/-) mice.
The fractional turnover rate of cholate was similar in wild-type and Fxr (-/-)
mice, whereas the cholate pool size was increased 2-fold in Fxr (-/-) mice, implying
enhanced intestinal cholate reabsorption in Fxr (-/-) mice. The calculated total bile
salt pool size was increased by ~40% in Fxr (-/-) mice. This is in contrast to the
situation reported by Sinal et al.13, i.e., a reduction of the total bile salt pool
size by ~50% in Fxr (-/-) mice fed a chow diet. In this case, bile salt pool size
was measured in homogenates of gallbladder, the liver immediately surrounding
the gall bladder, and the entire small intestine harvested after termination of
the animals. The bile salt contents of the homogenates, which were extracted
into ethanol, were determined colorimetrically. Whether methodological or strain-
differences underlie the deviating results between both studies in not clear: the
stable isotope dilution method is a well established procedure to quantify bile salt
kinetics in humans55 and in laboratory animals21.
100
Chapter 5
Maintenance of bile salt pool size can theoretically be regulated at the level
of the intestine by controlled reabsorption in the terminal ileum. Asbt had been
identifi ed as the major transporter involved in this process. However, expression
of Asbt was not different between wild-type and Fxr (-/-) mice. In contrast, Chen et
al.17 reported an increase in Asbt protein levels in Fxr (-/-) mice, in accordance with
the presence of LRH-1 sites in the promoter of the murine Asbt gene identifi ed by
these authors. t-Abst and Fic1 expression was also not changed at the mRNA level
between wild-type and Fxr (-/-) mice and therefore does not seem to play a role
in enhanced bile salt reabsorption effi ciency. Ibabp, a well known FXR gene, was
drastically down-regulated at mRNA and protein level in the ilea of Fxr (-/-) mice, in
accordance with earlier studies13,17. Ibabp is thought to be involved in intracellular
bile salt traffi cking and to facilitate reuptake of bile salts in the small intestine8,9.
Yet despite the complete absence of Ibabp protein in the ilea of the Fxr (-/-) mice,
daily intestinal cholate reabsorption was much higher than in wild-type mice. This
suggests that, under physiological conditions, Ibabp functions as a negative rather
than as a positive regulator of intestinal bile salt reabsorption in the mouse.
In conclusion, this work shows that the absence of FXR in vivo in mice is
associated with defective feedback inhibition of hepatic cholate synthesis, which
leads to an enlarged circulating cholate pool with an unaltered fractional turnover
rate. The absence of Ibabp does not negatively interfere with the enterohepatic
circulation of cholate in mice.
ACKNOWLEDGMENTS
We thank Sara Berdy, Theo Boer, Renze Boverhof, Anke ter Harmsel, Bert Hellinga,
Laura Hoffmann, Karen Siegler and Fjodor van der Sluijs for excellent technical
assistance. This work was supported by grant 902-23-191 from The Netherlands
Organization for Scientifi c Research (NWO).
101
Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice
REFERENCES
1. Hofmann AF. Bile Acids: The Good, the Bad, and the Ugly. News Physiol Sci 1999; 14:24-9.
2. Claudel T, Sturm E, Duez H, Torra IP, Sirvent A, Kosykh V, Fruchart JC, Dallongeville J, Hum DW, Kuipers F, Staels B. Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element. J Clin Invest 2002; 109:961-71.
3. Kast HR, Nguyen CM, Sinal CJ, Jones SA, Laffi tte BA, Reue K, Gonzalez FJ, Willson TM, Edwards PA. Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids. Mol Endocrinol 2001; 15:1720-8.
4. Lin Y, Havinga R, Verkade HJ, Moshage H, Slooff MJ, Vonk RJ, Kuipers F. Bile acids suppress the secretion of very-low-density lipoprotein by human hepatocytes in primary culture. Hepatology 1996; 23:218-28.
5. Duane WC. Abnormal bile acid absorption in familial hypertriglyceridemia. J Lipid Res 1995; 36:96-107.
6. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, Meier PJ. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998; 273:10046-50.
7. Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol 2002; 64:635-61.8. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD,
Mangelsdorf DJ, Shan B. Identifi cation of a nuclear receptor for bile acids. Science 1999; 284:1362-5.
9. Landrier JF, Grober J, Zaghini I, Besnard P. Regulation of the ileal bile acid-binding protein gene: an approach to determine its physiological function(s). Mol Cell Biochem 2002; 239:149-55.
10. Kullak-Ublick GA, Stieger B, Hagenbuch B, Meier PJ. Hepatic transport of bile salts. Semin Liver Dis 2000; 20:273-92.
11. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 2000; 6:517-26.
12. Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry 1992; 31:4737-49.13. Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption
of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 2000; 102:731-44.
14. Ananthanarayanan M, Balasubramanian N, Makishima M, Mangelsdorf DJ, Suchy FJ. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem 2001; 276:28857-65.
15. Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PLM, Müller M. Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002; 35:589-96.
16. Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, Karpen SJ. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001; 121:140-7.
17. Chen F, Ma L, Dawson PA, Sinal CJ, Sehayek E, Gonzalez FJ, Breslow J, Ananthanarayanan M, Shneider BL. Liver receptor homologue-1 mediates species- and cell line-specifi c bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J Biol Chem 2003; 278:19909-16.
18. Grober J, Zaghini I, Fujii H, Jones SA, Kliewer SA, Willson TM, Ono T, Besnard P. Identifi cation of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer. J Biol Chem 1999; 274:29749-54.
19. Li-Hawkins J, Gafvels M, Olin M, Lund EG, Andersson U, Schuster G, Bjorkhem I, Russell DW, Eggertsen G. Cholic acid mediates negative feedback regulation of bile acid synthesis in mice. J Clin Invest 2002; 110:1191-200.
20. Miyake JH, Doung XD, Strauss W, Moore GL, Castellani LW, Curtiss LK, Taylor JM, Davis RA. Increased production of apolipoprotein B-containing lipoproteins in the absence
102
Chapter 5
of hyperlipidemia in transgenic mice expressing cholesterol 7alpha-hydroxylase. J Biol Chem 2001; 276:23304-11.
21. Hulzebos CV, Renfurm L, Bandsma RHJ, Verkade HJ, Boer T, Boverhof R, Tanaka H, Mierau I, Sauer PJJ, Kuipers F, Stellaard F. Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans. J Lipid Res 2001; 42:1923-9.
22. Kuipers F, van Ree JM, Hofker MH, Wolters H, In ‘t Veld G, Havinga R, Vonk RJ, Princen HM, Havekes LM. Altered lipid metabolism in apolipoprotein E-defi cient mice does not affect cholesterol balance across the liver. Hepatology 1996; 24:241-7.
23. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6:986-94.
24. Hulzebos CV, Wolters H, Plösch T, Kramer W, Stengelin S, Stellaard F, Sauer PJJ, Verkade HJ, Kuipers F. Cyclosporin A and enterohepatic circulation of bile salts in rats: decreased cholate synthesis but increased intestinal reabsorption. J Pharmacol Exp Ther 2003; 304:356-63.
25. Plösch T, Kok T, Bloks VW, Smit MJ, Havinga R, Chimini G, Groen AK, Kuipers F. Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1. J Biol Chem 2002; 277:33870-7.
26. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purifi cation of the human intestinal brush border membrane. Biochim Biophys Acta 1973; 323:98-112.
27. Lowry OH, Rosebrough AL, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-75.
28. Labonte ED, Li Q, Kay CM, Agellon LB. The relative ligand binding preference of the murine ileal lipid binding protein. Protein Expr Purif 2003; 28:25-33.
29. Torchia EC, Stolz A, Agellon LB. Differential modulation of cellular death and survival pathways by conjugated bile acids. BMC Biochem 2001; 2:11.
30. Mashige F, Imai K, Osuga T. A simple and sensitive assay of total serum bile acids. Clin Chim Acta 1976; 70:79-86.
31. Kuipers F, Havinga R, Bosschieter H, Toorop GP, Hindriks FR, Vonk RJ. Enterohepatic circulation in the rat. Gastroenterology 1985; 88:403-11.
32. Parmentier G, Eyssen H. Thin-layer chromatography of bile salt sulphates. Journal of Chromatography 1978; 152:285-9.
33. Verkade HJ, Vonk RJ, Kuipers F. New insights into the mechanism of bile acid-induced biliary lipid secretion. Hepatology 1995; 21:1174-89.
35. Bull LN, van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, Liao M, Klomp LW, Lomri N, Berger R, Scharschmidt BF, Knisely AS, Houwen RH, Freimer NB. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998; 18:219-24.
36. Chiang JY. Bile Acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev 2002; 23:443-63.
37. Russell DW. The Enzymes, Regulation, and Genetics of Bile Acid Synthesis. Annu Rev Biochem 2003; 72:137-74.
38. Setchell KD, Street JM, Sjövall J. The bile acids: Chemistry, Physiology,and Metabolism. Plenum Publishing Corp, New York. 1988: 457-62.
39. Lu TT, Repa JJ, Mangelsdorf DJ. Orphan nuclear receptors as eLiXiRs and FiXeRs of sterol metabolism. J Biol Chem 2001; 276:37735-8.
40. Lambert G, Amar MJ, Guo G, Brewer HB, Gonzalez FJ, Sinal CJ. The farnesoid X-receptor is an essential regulator of cholesterol homeostasis. J Biol Chem 2003; 278:2563-70.
41. Kok T, Bloks VW, Wolters H, Havinga R, Jansen PLM, Staels B, Kuipers F. Peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated regulation of multidrug resistance 2 (Mdr2) expression and function in mice. Biochem J 2003; 369:539-47.
42. Wolters H, Elzinga BM, Baller JF, Boverhof R, Schwarz M, Stieger B, Verkade HJ, Kuipers F. Effects of bile salt fl ux variations on the expression of hepatic bile salt transporters in vivo in mice. J Hepatol 2002; 37:556-63.
43. Kuipers F, Enserink M, Havinga R, van der Steen AB, Hardonk MJ, Fevery J, Vonk RJ. Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in
103
Enterohepatic circulation of bile salts in farnesoid X receptor-defi cient mice
the rat. J Clin Invest 1988; 81:1593-9.44. Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, Scheper RJ,
Borst P, Oude Elferink RPJ. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 1996; 271:1126-8.
45. Ballatori N, Truong AT. Glutathione as a primary osmotic driving force in hepatic bile formation. Am J Physiol 1992; 263:G617-G624.
46. Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, Edwards PA. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem 2002; 277:2908-15.
47. Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C, Lamba J, Kim RB, Ramachandran V, Komoroski BJ, Venkataramanan R, Cai H, Sinal CJ, Gonzalez FJ, Schuetz JD. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J Biol Chem 2001; 276:39411-8.
48. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, Hobbs HH. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest 2002; 110:671-80.
49. Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A 2002; 99:16237-42.
50. Kosters A, Frijters RJ, Schaap FG, Vink E, Plösch T, Ottenhoff R, Jirsa M, De Cuyper IM, Kuipers F, Groen AK. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice. J Hepatol 2003; 38:710-6.
51. Smit JJ, Schinkel AH, Oude Elferink RPJ, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993; 75:451-62.
52. Oude Elferink RPJ, Ottenhoff R, van Wijland M, Smit JJ, Schinkel AH, Groen AK. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest 1995; 95:31-8.
53. Kerr TA, Saeki S, Schneider M, Schaefer K, Berdy S, Redder T, Shan B, Russell DW, Schwarz M. Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis. Dev Cell 2002; 2:713-20.
54. Wang L, Lee YK, Bundman D, Han Y, Thevananther S, Kim CS, Chua SS, Wei P, Heyman RA, Karin M, Moore DD. Redundant pathways for negative feedback regulation of bile acid production. Dev Cell 2002; 2:721-31.
55. Stellaard F, Sackmann M, Sauerbruch T, Paumgartner G. Simultaneous determination of cholic acid and chenodeoxycholic acid pool sizes and fractional turnover rates in human serum using 13C-labeled bile acids. J Lipid Res 1984; 25:1313-9.