Molecular Cell,Vol.6,517–526,September,2000,Copyright ?2000by Cell Press
A Regulatory Cascade of the Nuclear Receptors FXR,SHP-1,and LRH-1Represses Bile Acid Biosynthesis
of the nuclear receptor superfamily of ligand-activated transcription factors (Chiang,1998;Gustafsson,1999;Russell,1999).Recently,two nuclear receptors,the liver X receptor ?(LXR ?;NR1H3)(Apfel et al.,1994;Willy et al.,1995)and farnesoid X receptor (FXR;NR1H4)Bryan Goodwin,*Stacey A.Jones,*Roger R.Price,*Michael A.Watson,*David D.McKee,*
Linda B.Moore,*Cristin Galardi,*Joan G.Wilson,?Michael C.Lewis,?Matthew E.Roth,§
Patrick R.Maloney,?Timothy M.Willson,?(Forman et al.,1995;Seol et al.,1995),were implicated and Steven A.Kliewer*
in the feedforward and feedback regulation of CYP7A1,*Department of Molecular Endocrinology respectively (Peet et al.,1998;Russell,1999).Both LXR ??Department of Metabolic Diseases and FXR are abundantly expressed in the liver and ?Department of Medicinal Chemistry
bind to their cognate hormone response elements as Glaxo Wellcome Research and Development heterodimers with the 9-cis retinoic acid receptor Research Triangle Park,North Carolina 27709RXR (Mangelsdorf and Evans,1995).LXR ?is activated §CuraGen Corporation
by the cholesterol derivative 24,25(S )-epoxycholesterol New Haven,Connecticut 06511
and binds to a response element in the CYP7A1pro-moter (Lehmann et al.,1997).Mice lacking LXR ?do not induce CYP7A1expression in response to cholesterol Summary
feeding (Peet et al.,1998).Moreover,these animals ac-cumulate massive amounts of cholesterol in their livers when fed a high cholesterol diet.These data establish Bile acids repress the transcription of cytochrome LXR ?as the cholesterol sensor responsible for feedfor-P4507A1(CYP7A1),which catalyzes the rate-limiting ward regulation of CYP7A1expression.
step in bile acid biosynthesis.Although bile acids acti-Bile acids stimulate the expression of genes involved vate the farnesoid X receptor (FXR),the mechanism in bile acid transport,such as the intestinal bile acid–underlying bile acid–mediated repression of CYP7A1binding protein (I-BABP ),and repress CYP7A1and other remained unclear.We have used a potent,nonsteroi-genes encoding enzymes involved in bile acid biosyn-dal FXR ligand to show that FXR induces expression thesis,such as CYP8B1,which converts chenodeoxy-of small heterodimer partner 1(SHP-1),an atypical cholic acid (CDCA)to cholic acid,and CYP27,which member of the nuclear receptor family that lacks a catalyzes the first step in the alternative,“acidic”path-DNA-binding domain.SHP-1represses expression of way for bile acid synthesis (Russell and Setchell,1992;CYP7A1by inhibiting the activity of liver receptor ho-Javitt,1994;Russell,1999).Recently,FXR was shown molog 1(LRH-1),an orphan nuclear receptor that is to be a bile acid receptor (Wang et al.,1996;Makishima known to regulate CYP7A1expression positively.This et al.,1999;Parks et al.,1999).Several different bile bile acid–activated regulatory cascade provides a acids,including CDCA and its glycine and taurine conju-molecular basis for the coordinate suppression of gates,bind and activate FXR at physiologic concentra-CYP7A1and other genes involved in bile acid biosyn-tions.Moreover,FXR response elements (FXREs)were identified in both the mouse and human I-BABP promot-thesis.ers (Grober et al.,1999;Makishima et al.,1999),which provided strong evidence that FXR mediates the posi-Introduction
tive effects of bile acids on I-BABP expression.Notably,the rank order of bile acids that activate FXR correlates Cholesterol is essential for a number of cellular func-with that for repression of CYP7A1in a hepatocyte-tions,including membrane biogenesis and steroid hor-derived cell line (Makishima et al.,1999).These data mone and bile acid biosynthesis.However,in excess,suggested that FXR also has a role in the negative ef-cholesterol can contribute to disease processes such fects of bile acids on gene expression.However,since as atherosclerosis and gallstone formation.Therefore,the region of the CYP7A1promoter that is necessary cholesterol biosynthesis and catabolism must be coor-for bile acid–mediated repression lacks a strong FXR-dinately regulated.The metabolism of cholesterol to bile binding site (Chiang and Stroup,1994;Chiang et al.,acids represents a major pathway for its elimination 2000),it seemed unlikely that this repression was a di-from the body,accounting for approximately half of daily rect effect of FXR.Thus,the molecular mechanism for excretion.Cytochrome P4507A (CYP7A1)is a liver-spe-bile acid–mediated repression of CYP7A1remained cific enzyme that catalyzes the first and rate-limiting in question.
step in one of the two pathways for bile acid biosynthesis In this report,we have used a potent,nonsteroidal FXR (Chiang,1998;Russell and Setchell,1992).The gene ligand to demonstrate that FXR regulates the hepatic encoding CYP7A1is regulated by a variety of small,expression of small heterodimer partner 1(SHP-1;lipophilic molecules,including steroid and thyroid hor-NR0B2),an atypical,orphan member of the nuclear re-mones,cholesterol,and bile acids.Notably,CYP7A1ceptor family that lacks a DNA-binding domain (Seol et expression is stimulated by cholesterol feeding and re-al.,1996).SHP-1has been shown to bind to other nu-pressed by bile acids.Thus,CYP7A1is under both feed-clear receptors and to repress their transcriptional activ-forward and feedback regulation.
ities (Seol et al.,1996;Masuda et al.,1997;Johansson CYP7A1expression is regulated by several members
et al.,1999;Lee et al.,2000).We show that SHP-1re-presses the CYP7A1promoter through interaction with liver receptor homolog 1(LRH-1;NR5A2),an orphan To whom correspondence should be addressed (e-mail:sak15922@
https://www.doczj.com/doc/be4711190.html,).
nuclear receptor that binds as a monomer to a response
Molecular Cell
518
element in the CYP7A1promoter and activates tran-
scription(Becker-Andre et al.,1993;Galarneau et al.,
1996;Nitta et al.,1999).LRH-1is a mammalian homolog
of the Drosophila fushi tarazu F1gene product,which
regulates Drosophila metamorphosis(Lavorgna et al.,
1991;Broadus et al.,1999).Our findings define a novel
regulatory cascade of three orphan nuclear receptors
that provides a molecular basis for the coordinate re-
pression of gene expression by bile acids.
Results
Identification of GW4064as a Potent,
Selective FXR Activator
FXR was recently shown to be a receptor for CDCA as
well as other bile acids(Makishima et al.,1999;Parks et
al.,1999;Wang et al.,1999).However,these compounds
bind to FXR with only micromolar affinities and at these
concentrations also interact with other proteins,includ-
ing bile acid–binding proteins and transporters.We
sought to identify a potent,selective FXR ligand for use
as a chemical tool in elucidating the genes regulated
by https://www.doczj.com/doc/be4711190.html,binatorial libraries of compounds were
screened using a ligand-sensing fluorescence reso-
nance energy transfer assay that detects interactions
between FXR and a peptide derived from the steroid
receptor coactivator1(SRC-1)as previously described
(Parks et al.,1999).Among the compounds that pro-
moted an interaction between FXR and SRC-1was the
isoxazole GW4064(Figure1A),which bound to FXR with
a half-maximal effective concentration(EC50)of15nM
(Maloney et al.,2000).GW4064activated mouse and
human FXR with EC50values of80and90nM,respec-
tively,in CV-1cells transfected with FXR expression
vectors and a reporter plasmid containing two copies
of an established FXR response element(FXRE)derived
from the Drosophila heat shock protein27(hsp27)pro-
Figure1.GW4064Is a Potent,Selective Activator of FXR
moter(Forman et al.,1995)(Figure1B).Thus,GW4064
(A)Chemical structure of GW4064.
is?1000-fold more potent than CDCA in activating FXR
(B)CV-1cells were transfected with expression plasmids for human in CV-1cells(Figure1B).
or mouse FXR and the(hsp70EcRE)2-tk-LUC reporter plasmid con-GW4064was tested for selectivity against a panel
taining two copies of the hsp70ecdysone response element up-of nuclear receptors.CV-1cells were transfected with stream of the thymidine kinase(tk)promoter and luciferase gene.
expression plasmids for various nuclear receptor–GAL4Transfected cells were treated with the indicated concentrations of chimeras and the reporter plasmid(UAS)5-tk-CAT as either GW4064or CDCA.Open circles,mouse FXR and GW4064; previously described(Parks et al.,1999).GW4064acti-
open triangles,human FXR and GW4064;closed circles,mouse FXR vated only the FXR-GAL4chimera(Figure1C).Thus,
and CDCA;closed triangles,human FXR and CDCA.Data points
represent the mean of assays performed in triplicate.
GW4064is a highly selective activator of FXR.
(C)CV-1cells were transfected with expression vectors for various
GAL4–nuclear receptor ligand-binding domain chimeras and the FXR Regulates SHP-1Expression in the Liver
reporter plasmid(UAS)5-tk-CAT.Transfected cells were treated with GW4064was exploited as a chemical tool to identify1?M GW4064.Data represent the mean of assays performed in the genes regulated by FXR in the liver.Male Fisher rats triplicate?S.D.
were treated for7days with GW4064or vehicle alone
(methyl cellulose).Following treatment,RNA was pre-
pared from the livers of GW4064-and vehicle-treated CYP7A1as part of a regulatory feedback loop that con-animals,and genes that were either induced or re-
trols the rate of their biosynthesis from cholesterol pressed by GW4064treatment were determined using(Russell and Setchell,1992;Russell,1999).Two recent CuraGen GeneCalling?differential gene expression
studies implicate FXR in the repression of CYP7A1 technology(Shimkets et al.,1999).A comprehensive list(Makishima et al.,1999;Wang et al.,1999),although the of the liver genes regulated by GW4064will be published
molecular mechanisms have remained unclear since the elsewhere.Interestingly,the gene that was most strongly CYP7A1promoter does not contain a consensus FXRE induced by GW4064treatment was that encoding the
(Chiang et al.,2000).In parallel with our analysis of orphan nuclear receptor SHP-1.Northern analysis showed SHP-1expression,we examined whether GW4064treat-that SHP-1expression was increased?6-fold in the
ment resulted in decreased CYP7A1expression in male livers of GW4064-treated rats relative to vehicle-treated Fisher rats.Rats treated with GW4064showed a sub-animals(Figure2A).
stantial decrease in CYP7A1mRNA levels(?4-fold,Fig-Bile acids are known to repress the expression of ure2A).Thus,GW4064mimics the well documented
A Regulatory Cascade of Nuclear Receptors 519
Figure3.Identification of FXR Binding Sites in the Human,Rat,and
Mouse SHP-1Promoters
(A)Alignment of the proximal regions of the human,rat,and mouse
SHP-1promoters.The conserved IR1FXR binding site is boxed.
Conserved nucleotides are indicated by asterisks.
Figure2.FXR Ligands Induce SHP-1and Repress CYP7A1Ex-(B)Electrophoretic mobility-shift assays were performed with in vitro pression synthesized human FXR and/or human RXR?as indicated and[32P]-(A)Total RNA was prepared from the livers of male Fisher rats treated labeled oligonucleotides containing the IR1motif from the rat, for7days with GW4064or vehicle alone.Northern analysis was mouse,or human SHP-1promoters or the mouse or human I-BABP performed using probes for rat SHP-1and CYP7A1.Data represent promoters.The positions of the shifted FXR/RXR?complex and free the mean(n?3)?standard error of the means.The asterisk denotes probes are indicated.
a statistically significant difference between vehicle-and GW4064-(C)Electrophoretic mobility-shift assays were performed with in vitro treated animals;P?0.05.synthesized human FXR and/or human RXR?,a[32P]-labeled oligo-(B)Total RNA was prepared from primary rat or human hepatocytes nucleotide containing the human I-BABP FXRE,and either a5-,25-, treated for48hr with the indicated concentrations of GW4064or or75-fold excess of unlabeled oligonucleotides containing the IR1 vehicle alone.Northern analysis was performed using probes for motifs from the human I-BABP promoter,the mouse,rat,or human rat or human SHP-1,CYP7A1,or?-actin.SHP-1promoters,or a mutated derivative of the mouse SHP-1IR1 (C)Total RNA was prepared from primary human hepatocytes motif(mSHPmut).The position of the shifted FXR/RXR?complex treated for48hr with the indicated concentrations of CDCA.North-is indicated.
ern analysis was performed using probes for human SHP-1,
CYP7A1,or?-actin.
examined by Northern blot analysis.GW4064treatment
markedly increased SHP-1expression and decreased
CYP7A1expression in hepatocytes from both species effects of naturally occurring FXR ligands,namely bile
acids,on CYP7A1expression.This observation pro-in a dose-dependent fashion(Figure2B).Similar results
were obtained in human hepatocytes treated with the vides compelling evidence that FXR mediates feedback
repression of CYP7A1by bile acids.natural FXR ligand CDCA(Figure2C).As expected,
CDCA was less potent than GW4064in its effects on To substantiate the in vivo data and extend them to
human hepatocytes,we examined whether SHP-1and gene expression(compare Figures2B and2C).These
data strongly suggest that FXR regulates SHP-1and CYP7A1expression were regulated by FXR in primary
cultures of rat and human hepatocytes.Hepatocytes CYP7A1expression in both human and rodent hepato-
cytes.Notably,there was a striking reciprocal relation-were treated with increasing concentrations of GW4064,
and the levels of SHP-1and CYP7A1expression were ship between the regulation of SHP-1and CYP7A1
Molecular Cell
520
Figure4.FXR Activates the Rat and Human
SHP-1Promoters
HepG2cells were transfected with the human
FXR expression plasmid and luciferase re-
porter plasmids containing the proximal pro-
moters of the rat([A],nucleotides?441to
?19)or human([B],nucleotides?572to?10)
SHP-1genes or the corresponding reporter
plasmids in which the IR1elements had been
mutated(?IR1).Following transfection,cells
were treated for48hr with GW4064(1?M)
or CDCA(100?M).Data represent the
mean?S.D.of six individual transfections.
expression:GW4064and CDCA repressed CYP7A1ex-human SHP-1promoters.GW4064treatment of cells pression at the same concentrations that were required
transfected with the FXR expression plasmid and either to induce SHP-1expression(Figures2B and2C).Since promoter construct resulted in a marked induction of
reporter activity(Figures4A and4B).Based on Northern SHP-1is known to heterodimerize with several other
members of the nuclear receptor superfamily and to blot analysis of SHP-1expression(Figure2B),the mag-repress their transcriptional activity(Seol et al.,1996;
nitude of the response from the rat(7-fold)and human Masuda et al.,1997;Johansson et al.,1999),these data(3-fold)SHP-1promoters was somewhat lower than ex-raised the intriguing possibility that FXR-mediated in-
pected and it is possible that other promoter or enhancer duction of SHP-1might underlie the repression of elements contribute to the regulation of SHP-1expres-CYP7A1expression(see below).
sion.Alternately,additional factors present in well differ-
entiated cultures of rat hepatocytes but not HepG2cells
may be required for maximal FXR responsiveness.In FXR Binds and Activates SHP-1Promoters
the absence of exogenously expressed FXR,the rat and We next sought to determine whether SHP-1expression
human SHP-1promoters exhibited a modest(?1.5-fold) is directly regulated by FXR.FXR preferentially binds as
induction on exposure to GW4064,which is most likely a heterodimer with RXR to FXREs composed of two
due to endogenous FXR in HepG2cells(data not shown). nuclear receptor half-sites of consensus AG(G/T)TCA
FXR responsiveness was eliminated when mutations organized as an inverted repeat and separated by a
were introduced into the IR1motifs in either the rat or single nucleotide(IR1)(Forman et al.,1995).IR1-type
human SHP-1promoters(Figures4A and4B).These FXREs have been identified in the human and mouse
data provide strong evidence that SHP-1expression I-BABP promoters(Grober et al.,1999;Makishima et
is regulated directly by the FXR/RXR heterodimer in al.,1999).The mouse,rat,and human SHP-1promoters
multiple species.
were examined for IR1motifs.A highly conserved IR1-
like element was identified?300nucleotides upstream
of the transcription initiation site in the SHP-1promoter SHP-1Interacts with Orphan Nuclear
of all three species(Figure3A).Electrophoretic mobility-
Receptor LRH-1
shift analyses demonstrated that the FXR/RXR complex The finding that SHP-1expression is regulated by FXR binds efficiently to the IR1element from the SHP-1pro-
together with the reciprocal relationship between SHP-1 moter of each species(Figure3B).In agreement with and CYP7A1regulation(Figure2)suggested that SHP-1 earlier observations(Grober et al.,1999),the FXR/RXR
might play a pivotal role in bile acid–mediated repression heterodimer also bound to the mouse and human of CYP7A1expression.Regulation of the CYP7A1pro-I-BABP FXREs(Figure3B).Competition binding analy-
moter is complex and involves numerous transcription ses showed that these interactions were specific:no factors,including nuclear receptors with known ligands competition was seen with a mutated derivative of the
such as the thyroid hormone receptor(TR),retinoic acid IR1motif derived from the mouse SHP-1promoter(Fig-receptor(RAR),RXR and LXR?,and the orphan recep-
tors COUP-TFII,HNF4?,and LRH-1(Lehmann et al., ure3C).
The presence of an FXR/RXR binding site suggested1997;Stroup et al.,1997;Chiang,1998;Peet et al.,1998; that the SHP-1gene is directly regulated by FXR.To
Nitta et al.,1999;Russell,1999;Stroup and Chiang, test this hypothesis,HepG2cells were transfected with2000).SHP-1has previously been show to bind to and an FXR expression plasmid and reporter plasmids ex-
repress the transcriptional activities of TR,RAR,and pressing luciferase under the control of either the rat or RXR in the presence of their ligands and HNF4?in the
A Regulatory Cascade of Nuclear Receptors
521
FXR,COUP-TFII,HNF4?,RAR?,or TR?in our mamma-
lian two-hybrid assay(Figure5A).The lack of a stronger
interaction between SHP-1and either TR?,RAR?,or
HNF4?was surprising in light of the previous results of
others(Seol et al.,1996;Masuda et al.,1997)and may
reflect differences in the assay systems used.Notably,
strong reporter activity was detected when GAL4-SHP-1
was expressed with VP16-human LRH-1or VP16-mouse
LRH-1(?14-fold activation for both human and mouse).
This activity was completely dependent on the presence
of GAL4-SHP-1(data not shown).These data demon-
strate that SHP-1can interact with LRH-1in cells.Inter-
estingly,little or no interaction was detected between
SHP-1and steroidogenic factor1(SF-1)(Figure5A),a
closely related orphan receptor that shares?60%amino
acid identity with LRH-1in the ligand-binding domain
(Tsukiyama et al.,1992;Honda et al.,1993;Ikeda et al.,
1993).
Using a glutathione S–transferase(GST)pull-down
assay,we examined whether SHP-1binds directly to
LRH-1.SHP-1was expressed in E.coli as a fusion pro-
tein with GST,and[35S]-labeled LRH-1was synthesized
in vitro.Glutathione–Sepharose beads efficiently copre-
cipitated[35S]-labeled LRH-1in the presence of GST-
SHP-1but not in its absence(Figure5B).In parallel
incubations,GST-SHP-1interacted strongly with[35S]-
labeled human RXR?in the presence of9-cis retinoic
acid(Figure5B).These data are in close agreement with
those derived from mammalian two-hybrid experiments
(Figure5A).Thus,SHP-1interacts directly with LRH-1. Figure5.SHP-1Interacts with the Orphan Nuclear Receptor LRH-1
SHP-1Represses Expression of CYP7A1
(A)Mammalian two-hybrid experiments were performed in CV-1
Does SHP-1have a role in the repression of CYP7A1 cells cotransfected with expression plasmids for the GAL4-human
expression by FXR ligands?We addressed this question SHP-1chimera and various VP16–nuclear receptor ligand-binding
domain chimeras.Transfection assays containing the LXR?-,FXR-,by performing cotransfection experiments with a rat RAR?-,TR?-,ER?-,and RXR?-GAL4chimeras were performed in CYP7A1luciferase reporter plasmid(pGL3-rCYP7A1 the absence or presence of the indicated ligands[respectively:EPC,[?1573/?36])containing nucleotides?1573to?36of 24(S),25-epoxycholesterol(10?M),GW4064(1?M);RA,all-trans the rat CYP7A1promoter,which includes a conserved retinoic acid(0.1?M);T3,triiodothyronine(0.1?M);E2,estradiol(0.1LRH-1binding site(Nitta et al.,1999).In the absence of ?M);9-cis RA,9-cis retinoic acid(0.1?M)].Data are expressed as
exogenously expressed LRH-1,the activity of the pGL3-fold activation over cells transfected with the(UAS)5-tk-CAT reporter
rCYP7A1(?1573/?36)reporter was low when transiently alone and represent the mean of assays(n?8)?S.D.
transfected into HepG2cells(data not shown).Cotrans-(B)GST pull-down assays were performed with[35S]-labeled LRH-1
fection of increasing amounts of an LRH-1expression or RXR?in the presence of GST or GST-SHP-1as indicated.9-cis
retinoic acid(9-cis RA)was added to the binding reaction to a final plasmid resulted in a dose-dependent increase in re-concentration of10?M.porter activity(Figure6).This LRH-1-dependent reporter
activity was completely blocked by the cotransfection
of SHP-1expression plasmid(Figure6).These data sug-
gest that interactions between SHP-1and LRH-1repre-absence of any exogenous ligand(Seol et al.,1996;
sent a basis for bile acid–mediated repression of Masuda et al.,1997).Using a mammalian two-hybrid
CYP7A1expression.
approach,we examined whether SHP-1interacts with
these and other nuclear receptors that have been impli-
Discussion
cated in the regulation of CYP7A1.CV-1cells were trans-
fected with an expression plasmid for a GAL4-SHP-1
chimera,the(UAS)5-tk-CAT reporter,and expression The recent discovery that FXR is a bile acid receptor plasmids for chimeras between the strong transcrip-
provided a great deal of insight into the molecular mech-tional activation domain of VP16and the isolated ligand-anisms underlying bile acid signaling.In particular,these binding domains of a panel of nuclear receptors(Figure
studies uncovered the mechanism whereby bile acids 5A).When transfected alone,the GAL4-SHP-1chimera stimulate the transcription of genes,such as I-BABP, caused a minor reduction(?0.3-fold)in reporter activity
involved in bile acid transport.High-affinity binding sites (Figure5A).However,reporter activity was strongly in-for the FXR/RXR heterodimer have been identified in duced when GAL4-SHP-1was coexpressed with VP16-
both the human and mouse I-BABP promoters(Grober RXR?(?44-fold)or VP16-estrogen receptor?(ER?,et al.,1999;Makishima et al.,1999).By contrast,the ?11-fold)in the presence of9-cis retinoic acid and estra-
mechanism underlying bile acid–mediated repression of diol,respectively(Figure5A).These interactions were CYP7A1expression remained a puzzle,since an FXRE strongly dependent on the presence of ligand.Little or
had not been identified in the bile acid response ele-no interaction was detected between SHP-1and LXR?,ments of this gene(Chiang and Stroup,1994;Chiang et
Molecular Cell
522
DNA-binding domain typically found in members of the
nuclear receptor family.SHP-1was originally cloned in
yeast two-hybrid experiments using the orphan nuclear
receptors CAR or PPAR?as bait,but it interacts with a
number of additional nuclear receptors,including ER?
and ER?,RAR,RXR,and TR(Seol et al.,1996;Masuda
et al.,1997;Seol et al.,1998;Johansson et al.,1999).
In each case,SHP-1represses the ligand-induced tran-
scriptional activity of these receptors.How does SHP-1
repress transcription of the CYP7A1promoter?Our data
indicate that SHP-1exerts much of its effect through
interaction with the orphan nuclear receptor LRH-1.
SHP-1interacted strongly with LRH-1in both a mamma-
lian two-hybrid assay and an in vitro pull-down assay.
Moreover,SHP-1efficiently repressed LRH-1-depen-
dent activation of the rat CYP7A1promoter.LRH-1was
recently shown to activate the human CYP7A1promoter
by binding to an extended nuclear receptor half-site Figure6.SHP-1Represses LRH-1-Dependent Activation of the Rat sequence that is conserved in the mouse,rat,and ham-CYP7A1Promoter
ster CYP7A1promoters(Nitta et al.,1999).Earlier stud-HepG2cells were transfected with the rat CYP7A1reporter plasmid,ies had defined DNA response elements in the CYP7A1 pGL3-rCYP7A1(?1573/?36),and the indicated amounts of LRH-1
and CYP8B1gene promoters that conferred repression and/or SHP-1expression plasmids.Data represent the mean of
in response to bile acids(Chiang and Stroup,1994; assays performed in triplicate?S.D.
Chiang et al.,2000;del Castillo-Olivares and Gil,2000).
Notably,each of these negative bile acid response ele-
ments contains an LRH-1binding site.Consistent with al.,2000).We now present evidence that FXR does not
these data,CYP8B1expression was repressed3-fold repress CYP7A1expression directly,but rather through
in Fisher rats treated with GW4064(S.A.J.,unpublished induction of the gene encoding the orphan nuclear re-
data).Thus,interactions between SHP-1and LRH-1are ceptor SHP-1,which,in turn,represses CYP7A1expres-
likely to be important for the coordinate repression of sion.Similar findings have been reported by Lu et al.
a number of genes by bile acids.Among the genes that (2000[this issue of Mol.Cell]).Consistent with this
may be regulated by the interaction between SHP-1and model,it was recently shown that SHP-1expression is
LRH-1is SHP-1itself.An LRH-1-responsive region of markedly lower and not inducible by cholic acid in the
the murine SHP-1gene has been identified(Lee et al., livers of mice lacking FXR(Sinal et al.,2000).Taken
1999).Thus,SHP-1is likely to regulate its own expres-together,these data provide a molecular explanation
sion.This feedback regulation may provide a mecha-for the coordinate suppression of gene expression by
nism for attenuating the bile acid–mediated repression bile acids.
of genes by SHP-1.A model for bile acid–mediated re-
pression of gene expression via increased SHP-1levels SHP-1Represses CYP7A1Expression
is shown in Figure7.
We encountered the orphan nuclear receptor SHP-1as
Two recent reports showed that SHP-1represses the part of a comprehensive,unbiased effort to identify FXR
transcriptional activation of ER?and ER?,RXR,and the target genes in the liver.SHP-1expression was strongly
orphan receptor HNF4?by competing with coactivator induced in the livers of rats treated with the potent,
binding to these receptors(Johansson et al.,1999;Lee nonsteroidal FXR ligand GW4064.SHP-1expression
et al.,2000).In addition,SHP-1contains a strong tran-was also markedly induced by GW4064in primary cul-
scriptional repressor domain in its C terminus(Lee et tures of human and rat hepatocytes,whereas CYP7A1
al.,2000).Furthermore,SHP-1has been shown to inhibit expression was suppressed under the same conditions.
DNA binding of RAR-RXR heterodimers(Seol et al., The reciprocal relationship between SHP-1and CYP7A1
1996).Taken together,these studies suggest that SHP-1 regulation,together with the established inhibitory ef-
inhibits the transcriptional activity of nuclear receptors fects of SHP-1on nuclear receptor activity,suggested
through multiple mechanisms.To date,we have been that SHP-1might repress CYP7A1expression.Indeed,
unable to demonstrate inhibition of LRH-1binding to its expression of SHP-1repressed the activity of the rat
response element in the CYP7A1promoter by SHP-1 CYP7A1promoter in HepG2cells.
SHP-1is unusual in that it lacks the highly conserved(data not shown).Thus,the mechanism by which SHP-1
Figure7.Model for the Feedforward and
Feedback Regulatory Effects of Bile Acids on
Gene Expression
Activation of FXR by bile acids results in the
induction of I-BABP and SHP-1expression.
SHP-1,in turn,interacts with LRH-1and re-
presses expression of CYP7A1and CYP8B1.
SHP-1may also repress expression of its own
gene.
A Regulatory Cascade of Nuclear Receptors
523
inhibits LRH-1-mediated transactivation of the CYP7A1enigma.Through the use of a potent,nonsteroidal FXR promoter remains unresolved.ligand,we have identified SHP-1as an FXR target gene In addition to the interactions between SHP-1and in the liver of humans and rodents.Furthermore,we LRH-1,other mechanisms may play a role in bile acid–have demonstrated that SHP-1can interact with LRH-1 mediated repression of CYP7A1expression.First,SHP-1and efficiently repress expression of CYP7A1.Thus,bile binds to and represses the transcriptional activity of acid–induced repression of CYP7A1is mediated by a other nuclear receptors that regulate CYP7A1,including novel regulatory cascade of three nuclear receptors. RXR and TR(Seol et al.,1996;Masuda et al.,1997).Since both the CYP7A1and CYP8B1gene promoters These interactions may also contribute to bile acid–contain LRH-1binding sites,the SHP-1/LRH-1partner-mediated repression of CYP7A1expression.Second,ship is likely to have broad implications in bile acid ligand-bound FXR was reported to repress LXR?activity signaling.Both SHP-1and LRH-1are orphan receptors, on an LXR?response element(Wang et al.,1999),al-which raises the possibility that bile acid biosynthesis though the mechanism for this trans-repression is not will be regulated by additional,unidentified hormones. clear.Since LXR?stimulates rodent CYP7A1expression Regardless of whether SHP-1and LRH-1have natural in response to oxysterols,repression of LXR?activity ligands,pharmacologic modulation of their interaction may contribute to the overall repression of CYP7A1.represents an exciting new opportunity for the discovery Thus,SHP-1/LRH-1interactions may be one of several of drugs that regulate cholesterol homeostasis. mechanisms whereby bile acids repress expression of
CYP7A1and other genes.Experimental Procedures
Parallels between SHP-1/LRH-1and Other
Materials
Nuclear Receptor Pairs
The synthesis of GW4064will be described elsewhere(Maloney et
al.,2000).CDCA,dexamethasone,estradiol,all-trans retinoic acid, Intriguing parallels exist between the SHP-1/LRH-1in-
9-cis retinoic acid,and charcoal-stripped,delipidated calf serum teraction and another pair of nuclear receptors.LRH-1
were acquired from the Sigma Chemical Co.(St.Louis,MO). is most closely related to the orphan receptor SF-1,
24(S),25-epoxycholesterol was synthesized in-house.DNA-modi-which regulates the expression of enzymes required for
fying enzymes,polymerases,and restriction endonucleases were steroid hormone biosynthesis(Parker,1998;Hammer
provided by Roche Molecular Biochemicals(Indianapolis,IN).Char-and Ingraham,1999).SF-1and LRH-1are?85%identi-coal/dextran-treated fetal bovine serum(FBS)was purchased from cal in the amino acid sequences of their DNA-binding Hyclone Laboratories Inc.(Logan,UT).The human hepatocellular domains,and both bind as monomers to the same ex-carcinoma cell line HepG2was obtained from the American Type tended nuclear receptor half-site sequence.Notably,the
Culture Collection(ATCC number HB-8065,Manassas,VA).Matrigel transcriptional activity of SF-1is repressed by binding
was provided by Becton Dickinson Labware(Bedford,MA).All other
tissue culture reagents were obtained from Life Technologies Inc. to DAX-1(dosage-sensitive sex-reversal adrenal hypo-
(Gaithersburg,MD).
plasia congenital region on the X chromosome,region
1;NR0B1),an orphan nuclear receptor most closely
Animals
related to SHP-1that also lacks the DNA-binding domain
Male Fisher rats were obtained from Charles River Laboratories Inc. characteristic of nuclear receptors(Zanaria et al.,1994;
(Raleigh,NC)and maintained on a12hr light/12hr dark cycle. Hammer and Ingraham,1999).Thus,both SF-1and
Animals were allowed food and chow ad libitum.GW4064(30mg/ LRH-1are negatively regulated in a trans-dominant fash-
kg)was administered by gavage twice a day for7days and the ion by heterodimerization with orphan receptors lacking animals sacrificed by cervical dislocation4hr after the final treat-
DNA-binding domains.Since SHP-1expression is stim-ment.Livers were excised and snap-frozen in liquid nitrogen.Differ-ulated by bile acids,it will be interesting to determine ential gene expression analysis was performed by CuraGen Corp. whether DAX-1expression is also regulated by hor-(New Haven,CT).
mones.
A second nuclear receptor pair with similarities to Plasmid Constructs
SHP-1/LRH-1occurs in Drosophila.Hormonal activation
Expression plasmids for the human nuclear receptor–GAL4chime-of the ecdysone receptor(EcR)during the third larval
ras were prepared by inserting amplified cDNAs encoding the li-
gand-binding domains into a modified pSG5expression vector instar phase of Drosophila metamorphosis results in
(Stratagene,La Jolla,CA)containing the GAL4DNA-binding domain an increase in the expression of two orphan nuclear
(amino acids1–147)and the Simian virus40(SV40)large T antigen receptors,DHR3,which has a functional DNA-binding
nuclear localization signal(APKKKRKVG).The(UAS)5-tk-CAT and domain,and E75B,which does not.E75B binds to DHR3
(hsp27EcRE)2-tk-LUC reporter constructs have been previously de-and represses its transcriptional activity(Thummel,
scribed(Forman et al.,1995;Parks et al.,1999).p?-actin-SPAP,an 1997;White et al.,1997).This interaction is critical for expression vector containing the human secreted placental alkaline
determining the temporal progression of metamorpho-phosphatase(SPAP)cDNA under the control of?-actin promoter, sis.The EcR/E75/DHR3and FXR/SHP-1/LRH-1regula-was used as an internal control in all transfections.The expression tory cascades are remarkably similar in that hormone-plasmids for human and mouse FXR(pSG5-hFXR and pSG5-mFXR, mediated activation of a nuclear receptor(either FXR or
respectively)and human SRC-1are described elsewhere(Kliewer EcR)induces expression of a second nuclear receptor,
et al.,1998;Parks et al.,1999).The full-length coding regions for
human LRH-1(GenBank Accession Number AB019246)and human which,in turn,binds to and represses the activity of a
SHP-1(GenBank Accession Number L76571)were amplified by PCR third nuclear receptor.The similarities in these genetic
and cloned into pSG5,creating pSG5-hLRH-1and pSG5-hSHP-1, hierarchies across evolution suggest that repression via
respectively.A consensus Kozak sequence was created during heterodimerization may represent an important para-
amplification.The rat(bases?441to?19,GenBank Accession digm for the modulation of orphan receptor activity.
Number D86745)(Masuda et al.,1997)and human(bases?572to
?10,GenBank Accession Number AF044316)(Lee et al.,1998) Conclusions SHP-1promoters were amplified by PCR using the following primer The mechanism whereby FXR represses expression of pairs:Rat,5?-gggtgtgcgagatctCCCTGGCTGGCTCCTTGGCTCTGT-3?CYP7A1and other genes has until now remained an
(sense)and5?-gggtgtgcgagatctCCTGTTTCTTCCTGGCTCTGT
Molecular Cell
524
GGC-3?(antisense);and human,5?-gggtgtgcgagatctTCCTAGACT Subsequently,blots were stripped and reprobed with a radiolabeled
?-actin cDNA(CLONTECH Laboratories). GGACAGTGGGCAAAG-3?(sense)and5?-gggtgtgcgagatctCTTCC
AGCTCTCTGGCTCTGTGTT-3?(antisense).The resultant fragments
were inserted into the Bgl II site of pGL3-Basic,a promoter-less
Electrophoretic Mobility-Shift Assays
luciferase reporter vector(Promega,Madison,WI).Site-directed
Electrophoretic mobility-shift assays(EMSA)were performed essen-mutagenesis of putative FXREs in the rat and human SHP-1promot-
tially as described elsewhere(Lehmann et al.,1997).hFXR and ers was performed using the Transformer mutagenesis system
hRXR?were synthesized from pSG5-hFXR and pSG5-hRXR?ex-(CLONTECH Laboratories,Palo Alto,CA)with the?ratIR1(bases
pression vectors,respectively,using the TNT T7Coupled Reticulo-?321to?287,5?-CCTGGTACAGCCTGGaaTAATAtaaCTGTTTATAC-3?)
cyte System(Promega).Unprogrammed lysate was prepared using and?humanIR1(bases?304to?270,5?-CCTGGTACAGCCTGA
the pSG5expression vector(Stratagene).Binding reactions con-aaTAATGtaCTTGTTTATCC-3?)primers.Mutated constructs were
tained10mM HEPES(pH7.8),60mM KCl,0.2%Nonidet P-40, verified to be free of nonspecific base changes by sequencing.
6%glycerol,2mM dithiothreitol(DTT),2?g of poly(dI-dC)?poly(dI-pGL3-rCYP7A1(?1573/?36)contains bases?1573to?36of the rat
dC),and1?l each of synthesized hFXR or hRXR?.Control incuba-CYP7A1promoter(GenBank Accession Number Z14108)inserted
tions received unprogrammed lysate alone.Reactions were pre-into the Nhe I site of pGL3-Basic.VP16-nuclear receptor chimeras
incubated on ice for10min prior to the addition of[32P]-labeled contain the80aa Herpes virus VP16transactivation domain linked
double-stranded oligonucleotide probe(0.2pmol).Competitor oli-to the ligand-binding domain of the following nuclear receptors in
gonucleotides were added to the preincubation at5-,25-,and75-a modified pSG5expression vector:human COUP-TFII,ER?,LRH-1,
fold molar excess.Samples were held on ice for a further20min, LXR?,RAR?,and TR?;mouse FXR,LRH-1,RXR?,and SF-1;and
and the protein–DNA complexes resolved on a pre-electrophoresed rat HNF4?.
5%polyacrylamide gel in0.5?TBE(45mM Tris-borate,1mM EDTA)
at room temperature.Gels were dried and autoradiographed at Transient Transfection Assays?70?C for1–2hr.The following double-stranded oligonucleotides Transient transfection of CV-1cells was performed exactly as de-were used as probes and competitors in EMSA:rSHP,5?-gatcCCTG scribed elsewhere(Jones et al.,2000).Typically,transfection mixes GGTTAATAACCCTGT-3?;mSHP,5?-gatcCCTGGGTTAATGACCC contained2–5ng of receptor expression vector,20ng of reporter TGT-3?;hSHP,5?-gatcCCTGAGTTAATGACCTTGT-3?;mI-BABP, construct,and8ng of p?-actin-SPAP.The amount of DNA used5?-gatcTTAAGGTGAATAACCTTGG-3?;hI-BABP,5?-gatcCCAGGT in each transfection was adjusted to80ng with carrier plasmid GAATAACCTCGG-3?(Grober et al.,1999);and mSHPmut5?-gatcCC (pBluescript,Stratagene).Mammalian two-hybrid experiments uti-TGGaaTAATGttCCTGT-3?.
lized transfection mixes containing20ng of VP16nuclear receptor
ligand-binding domain expression vector,5ng of pSG5-GAL4-
GST Pull-Down Assays
SHP-1,15ng of(UAS)5-tk-CAT,and8ng of p?-actin-SPAP.Cells
GST-SHP-1fusion protein was expressed in BL21(DE3)plysS cells were maintained for24hr in the presence of drug(added as a
and bacterial extracts prepared by one cycle of freeze-thaw of the 1000?stock in dimethyl sulfoxide)in DMEM/F-12nutrient mixture
cells in protein lysis buffer containing50mM Tris(pH8.0),250 containing10%charcoal-stripped,delipidated calf serum.An ali-
mM KCl,1%Triton X-100,10mM DTT and1?Complete Protease quot of medium was assayed for SPAP activity,and the cells were
Inhibitor(Roche Molecular Biochemical)followed by centrifugation lysed prior to determination of luciferase expression.Luciferase
at40,000?g for30min.Glycerol was added to the resultant super-activities were normalized to SPAP.HepG2cells were maintained
natant to a final concentration of10%.Lysates were stored at?80?C in DMEM/F-12supplemented with10%heat-inactivated FBS(Life
until use.[35S]-labeled human LRH-1or human RXR?was generated Technologies Inc.).Plasmid DNA was transfected into HepG2cells
using TNT T7Coupled Reticulocyte System(Promega)in the pres-using the FuGENE6transfection reagent according to the manufac-
ence of Pro-Mix(Amersham Pharmacia Biotech Inc.).Coprecipita-turer’s instructions(Roche Molecular Biochemicals).Thus,24-well
tion reactions included25?l of lysate containing GST-SHP-1fusion culture plates(15mm diameter)were inoculated with7?105cells
protein or control GST;25?l of incubation buffer(50mM KCl,40 24hr prior to transfection.Cells were transfected overnight in serum-
mM HEPES[pH7.5],5mM?-mercaptoethanol,0.1%Tween20and free DMEM/F-12with100ng of reporter construct,32ng of p?-
1%nonfat dry milk);and5?l of[35S]-labeled LRH-1or RXR?.The actin-SPAP,and0–400ng of receptor expression vectors(adjusted
mixtures were,incubated for25min with gentle rocking at4?C to400ng with carrier plasmid).Following transfection,the medium
prior to the addition of20?l of glutathione-Sepharose4B beads was aspirated and the cells were cultured for a further48hr in
(Amersham Pharmacia Biotech Inc.)that had been extensively DMEM/F-12supplemented with10%heat-inactivated FBS.SPAP
washed in protein lysis buffer.Reactions were incubated at4?C with and luciferase values were determined as described above.
gentle rocking for a further20min.The beads were pelleted at3000
rpm in a microfuge and washed four times with protein incubation Primary Culture of Human and Rat Hepatocytes buffer.Following the final wash,the beads were resuspended in25 and Northern Blot Analysis?l of2?SDS-PAGE sample buffer containing50mM DTT.Samples Primary human hepatocytes were obtained from Dr.Steve Strom were heated to100?C for5min and resolved on a10%acrylamide (University of Pittsburgh).Rat hepatocytes were isolated as de-gel.Autoradiography was performed overnight.
scribed elsewhere(LeCluyse et al.,1996).Cells(1.5?106)were
cultured on Matrigel-coated6-well plates in serum-free Williams’
Statistical Analyses
E medium supplemented with100nM dexamethasone,100U/ml
Unless otherwise stated,data are expressed as mean?standard penicillin G,100?g/ml streptomycin,and insulin-transferrin-sele-
deviation(S.D.).The significance of differences in SHP-1and nium(ITS-G,Life Technologies Inc.).Twenty-four hours after isola-
CYP7A1expression between vehicle-and GW4064-treated animals tion,hepatocytes were treated with either GW4064(0.1–10?M)or
were analyzed using an unpaired Student’s t-test.
CDCA(1–100?M),which were added to the culture medium as
1000?stocks in dimethyl sulfoxide.Control cultures received vehi-
cle alone.Cells were cultured for a further48hr prior to harvest,
Acknowledgments
and total RNA was isolated using a commercially available reagent
(Trizol,Life Technologies Inc.)according to the manufacturer’s in-
We thank Dr.Traci Mansfield(CuraGen Corp.,New Haven,CT)for structions.Total RNA(10?g)was resolved on a1%agarose/2.2M
assistance with the CuraGen data analysis,Dr.Geraldine Hamilton formaldehyde denaturing gel and transferred to a nylon membrane
(University of North Carolina,Chapel Hill)for preparation of the rat (Hybond N?,Amersham Pharmacia Biotech Inc.,Piscataway,NJ).
hepatocytes,James Way for advice on statistical analyses,Dr.Scott Blots were hybridized with32P-labeled cDNAs corresponding to hu-
Sundseth for providing the rat CYP7A1cDNA,and Drs.Rich Buck-man SHP-1(GenBank Accession Number L76571),human CYP7A1
holz and Catherine Stoltz for comments on the manuscript. (bases99–1564,GenBank Accession Number M93133),mouse
SHP-1(bases30–783,GenBank Accession Number L76567),or rat
CYP7A1(bases235–460,GenBank Accession Number J05460).Received May23,2000;revised July18,2000.
A Regulatory Cascade of Nuclear Receptors
525
References Lavorgna,G.,Ueda,H.,Clos,J.,and Wu,C.(1991).FTZ-F1,a steroid
hormone receptor–like protein implicated in the activation of fushi
tarazu.Science252,848–851.
Apfel,R.,Benbrook,D.,Lernhardt,E.,Ortiz,M.A.,Salbert,G.,and
Pfahl,M.(1994).A novel orphan receptor specific for a subset of LeCluyse,E.,Bullock,P.,Parkinson,A.,and Hochman,J.H.(1996). thyroid hormone–responsive elements and its interaction with the Cultured rat hepatocytes.In Model Systems for Biopharmaceutical retinoid/thyroid hormone receptor subfamily.Mol.Cell.Biol.14,Assessment of Drug Absorption and Metabolism,R.T.Borchardt, 7025–7035.G.Wilson,and P.Smith,eds.(New York:Plenum),pp.121–159. Becker-Andre,M.,Andre,E.,and DeLamarter,J.F.(1993).Identifica-Lee,H.K.,Lee,Y.K.,Park,S.H.,Kim,Y.S.,Lee,J.W.,Kwon,H.B., tion of nuclear receptor mRNAs by RT–PCR amplification of con-Soh,J.,Moore,D.D.,and Choi,H.S.(1998).Structure and expression served zinc-finger motif https://www.doczj.com/doc/be4711190.html,-of the orphan nuclear receptor SHP gene.J.Biol.Chem.273,14398–mun.194,1371–1379.14402.
Broadus,J.,McCabe,J.R.,Endrizzi,B.,Thummel,C.S.,and Wood-Lee,Y.K.,Dell,H.,Dowhan,D.H.,Hadzopoulou-Cladaras,M.,and ard,C.T.(1999).The Drosophila?FTZ-F1orphan nuclear receptor Moore,D.D.(2000).The orphan nuclear receptor SHP inhibits hepa-provides competence for stage-specific responses to the steroid tocyte nuclear factor4and retinoid X receptor transactivation:two hormone ecdysone.Mol.Cell3,143–149.mechanisms for repression.Mol.Cell.Biol.20,187–195. Chiang,J.Y.,and Stroup,D.(1994).Identification and characteriza-Lee,Y.K.,Parker,K.L.,Choi,H.S.,and Moore,D.D.(1999).Activation tion of a putative bile acid–responsive element in cholesterol7of the promoter of the orphan receptor SHP by orphan receptors alpha-hydroxylase gene promoter.J.Biol.Chem.269,17502–17507.that bind DNA as monomers.J.Biol.Chem.274,20869–20873. Chiang,J.Y.L.(1998).Regulation of bile acid synthesis.Front.Biosci.Lehmann,J.M.,Kliewer,S.A.,Moore,L.B.,Smith-Oliver,T.A.,Oliver, 3,176–193. B.B.,Su,J.L.,Sundseth,S.S.,Winegar,D.A.,Blanchard,D.E.,Spen-
cer,T.A.,and Willson,T.M.(1997).Activation of the nuclear receptor Chiang,J.Y.L.,Kimmel,R.,Weinberger,C.,and Stroup,D.(2000).
LXR by oxysterols defines a new hormone response pathway.J. Farnesoid X receptor responds to bile acids and represses choles-
Biol.Chem.272,3137–3140.
terol7?-hydroxylase gene(CYP7A1)transcription.J.Biol.Chem.
275,10918–10924.Lehmann,J.M.,McKee,D.D.,Watson,M.A.,Willson,T.M.,Moore,
J.T.,and Kliewer,S.A.(1997).The human orphan nuclear receptor del Castillo-Olivares,A.,and Gil,G.(2000).?-Fetoprotein transcrip-
PXR is activated by compounds that regulate CYP3A4gene expres-tion factor is required for the expression of sterol12a-hydroxylase,
sion and cause drug interactions.J.Clin.Invest.102,1016–1023. the specific enzyme for cholic acid synthesis.J.Biol.Chem.,275,
Lu,T.T.,Makishama,M.,Repa,J.J.,Schoonjans,K.,Kerr,T.A.,Au-17793–17799.
werx,J.,and Mangelsdorf,D.J.(2000).Molecular basis for feedback Forman,B.M.,Goode,E.,Chen,J.,Oro,A.E.,Bradley,D.J.,Perl-
regulation of bile acid synthesis by nuclear receptors.Mol.Cell6, mann,T.,Noonan,D.J.,Burka,L.T.,McMorris,T.,Lamph,W.W.,et
this issue,507–515.
al.(1995).Identification of a nuclear receptor that is activated by
Makishima,M.,Okamoto,A.Y.,Repa,J.J.,Tu,H.,Learned,R.M., farnesol metabolites.Cell81,687–693.
Luk,A.,Hull,M.V.,Lustig,K.D.,Mangelsdorf,D.J.,and Shan,B. Galarneau,L.,Pare,J.F.,Allard,D.,Hamel,D.,Levesque,L.,Tug-
(1999).Identification of a nuclear receptor for bile acids.Science wood,J.D.,Green,S.,and Belanger,L.(1996).The alpha1-fetopro-
284,1362–1365.
tein locus is activated by a nuclear receptor of the Drosophila FTZ-
Maloney,P.R.,Parks,D.J.,Haffner,C.D.,Fivush,A.M.,Chandra,G., F1family.Mol.Cell.Biol.16,3853–3865.
Plunket,K.D.,Creech,K.L.,Moore,L.B.,Wilson,J.G.,Lewis,M.C., Grober,J.,Zaghini,I.,Fujii,H.,Jones,S.A.,Kliewer,S.A.,Willson,
et al.(2000).Identification of a chemical tool for the orphan nuclear T.M.,Ono,T.,and Besnard,P.(1999).Identification of a bile acid–
receptor FXR.J.Med.Chem.43,2971–2974.
responsive element in the human ileal bile acid–binding protein
Mangelsdorf,D.J.,and Evans,R.M.(1995).The RXR heterodimers gene.Involvement of the farnesoid X receptor/9-cis-retinoic acid
and orphan receptors.Cell83,841–850.
receptor heterodimer.J.Biol.Chem.274,29749–29754.
Masuda,N.,Yasumo,H.,Tamura,T.,Hashiguchi,N.,Furusawa,T., Gustafsson,J.A.(1999).Seeking ligands for lonely orphan receptors.
Tsukamoto,T.,Sadano,H.,and Osumi,T.(1997).An orphan nuclear Science284,1285–1286.
receptor lacking a zinc-finger DNA-binding domain:interaction with Hammer,G.D.,and Ingraham,H.A.(1999).Steroidogenic factor-1:
several nuclear receptors.Biochim.Biophys.Acta1,27–32.
its role in endocrine organ development and differentiation.Front.
Nitta,M.,Ku,S.,Brown,C.,Okamoto,A.Y.,and Shan,B.(1999). Neuroendocrinol.20,199–223.
CPF:an orphan nuclear receptor that regulates liver-specific expres-Honda,S.,Morohashi,K.,Nomura,M.,Takeya,H.,Kitajima,M.,and
sion of the human cholesterol7alpha-hydroxylase gene.Proc.Natl. Omura,T.(1993).Ad4BP regulating steroidogenic P-450gene is a
https://www.doczj.com/doc/be4711190.html,A96,6660–6665.
member of steroid hormone receptor superfamily.J.Biol.Chem.
Parker,K.L.(1998).The roles of steroidogenic factor1in endocrine 268,7494–7502.
development and function.Mol.Cell.Endocrinol.145,15–20. Ikeda,Y.,Lala,D.S.,Luo,X.,Kim,E.,Moisan,M.P.,and Parker,K.L.
Parks,D.J.,Blanchard,S.G.,Bledsoe,R.K.,Chandra,G.,Consler, (1993).Characterization of the mouse FTZ-F1gene,which encodes
T.G.,Kliewer,S.A.,Stimmel,J.B.,Willson,T.M.,Zavacki, A.M., a key regulator of steroid hydroxylase gene expression.Mol.Endo-
Moore,D.D.,and Lehmann,J.M.(1999).Bile acids:natural ligands crinol.7,852–860.
for an orphan nuclear receptor.Science284,1365–1368.
Javitt,N.B.(1994).Bile acid synthesis from cholesterol:regulatory
Peet,D.J.,Janowski,B.A.,and Mangelsdorf,D.J.(1998).The LXRs:a and auxiliary pathways.FASEB J.8,1308–1311.
new class of oxysterol receptors.Curr.Opin.Genet.Dev.8,571–575. Johansson,L.,Thomsen,J.S.,Damdimopoulos,A.E.,Spyrou,G.,
Peet,D.J.,Turley,S.D.,Ma,W.,Janowski,B.A.,Lobaccaro,J.M., Gustafsson,J.A.,and Treuter,E.(1999).The orphan nuclear receptor
Hammer,R.E.,and Mangelsdorf,D.J.(1998).Cholesterol and bile SHP inhibits agonist-dependent transcriptional activity of estrogen
acid metabolism are impaired in mice lacking the nuclear oxysterol receptors ER?and ER?.J.Biol.Chem.274,345–353.
receptor LXR?.Cell93,693–704.
Jones,S.A.,Moore,L.B.,Shenk,J.L.,Wisely,G.B.,Hamilton,G.A.,
Russell,D.W.(1999).Nuclear orphan receptors control cholesterol McKee,D.D.,Tomkinson,N.C.,LeCluyse,E.L.,Lambert,M.H.,Will-
catabolism.Cell97,539–542.
son,T.M.,et al.(2000).The pregnane X receptor:a promiscuous
Russell,D.W.,and Setchell,K.D.(1992).Bile acid biosynthesis.Bio-xenobiotic receptor that has diverged during evolution.Mol.Endo-
chemistry31,4737–4749.
crinol.14,27–39.
Seol,W.,Choi,H.S.,and Moore,D.D.(1995).Isolation of proteins Kliewer,S.A.,Moore,J.T.,Wade,L.,Staudinger,J.L.,Watson,M.A.,
that interact specifically with the retinoid X receptor:two novel Jones,S.A.,McKee,D.D.,Oliver,B.B.,Willson,T.M.,Zetterstrom,
orphan receptors.Mol.Endocrinol.9,72–85.
R.H.,et al.(1998).An orphan nuclear receptor activated by preg-
nanes defines a novel steroid signaling pathway.Cell92,73–82.Seol,W.,Choi,H.S.,and Moore,D.D.(1996).An orphan nuclear
Molecular Cell
526
hormone receptor that lacks a DNA binding domain and heterodi-merizes with other receptors.Science272,1336–1339.
Seol,W.,Hanstein,B.,Brown,M.,and Moore,D.D.(1998).Inhibition of estrogen receptor action by the orphan receptor SHP(short heterodimer partner).Mol.Endocrinol.12,1551–1557. Shimkets,R.A.,Lowe,D.G.,Tai,J.T.,Sehl,P.,Jin,H.,Yang,R., Predki,P.F.,Rothberg,B.E.,Murtha,M.T.,Roth,M.E.,et al.(1999). Gene expression analysis by transcript profiling coupled to a gene database query.Nat.Biotechnol.17,798–803.
Sinal,C.J.,Tohkin,M.,Miyata,M.,Ward,J.M.,Lambert,G.,and Gonzalez,F.J.(2000).Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell102,731–744. Stroup,D.,and Chiang,J.Y.(2000).HNF4and COUP-TFII interact to modulate transcription of the cholesterol7alpha-hydroxylase gene (CYP7A1).J.Lipid Res.41,1–11.
Stroup,D.,Crestani,M.,and Chiang,J.Y.(1997).Orphan receptors chicken ovalbumin upstream promoter transcription factor II(COUP-TFII)and retinoid X receptor(RXR)activate and bind the rat choles-terol7alpha-hydroxylase gene(CYP7A).J.Biol.Chem.272,9833–9839.
Thummel,C.S.(1997).Dueling orphans—interacting nuclear recep-tors coordinate Drosophila metamorphosis.Bioessays19,669–672. Tsukiyama,T.,Ueda,H.,Hirose,S.,and Niwa,O.(1992).Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-F1,a member of the steroid receptor superfamily.Mol.Cell.Biol. 12,1286–1291.
Wang,D.P.,Stroup,D.,Marrapodi,M.,Crestani,M.,Galli,G.,and Chiang,J.Y.(1996).Transcriptional regulation of the human choles-terol7alpha-hydroxylase gene(CYP7A)in HepG2cells.J.Lipid Res.37,1831–1841.
Wang,H.,Chen,J.,Hollister,K.,Sowers,L.C.,and Forman,B.M. (1999).Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol.Cell3,543–553.
White,K.P.,Hurban,P.,Watanabe,T.,and Hogness,D.S.(1997). Coordination of Drosophila metamorphosis by two ecdysone-induced nuclear receptors.Science276,114–117.
Willy,P.J.,Umesono,K.,Ong,E.S.,Evans,R.M.,Heyman,R.A.,and Mangelsdorf,D.J.(1995).LXR,a nuclear receptor that defines a distinct retinoid response pathway.Genes Dev.9,1033–1045. Zanaria,E.,Muscatelli,F.,Bardoni,B.,Strom,T.M.,Guioli,S.,Guo, W.,Lalli,E.,Moser,C.,Walker,A.P.,McCabe,E.R.,et al.(1994). An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita.Nature372, 635–641.
氨测定试剂盒（循环酶法）适用范围：用于体外定量测定人血浆中氨的浓度。 1.1规格 a）试剂1：2×45ml，试剂2：2×15ml； b）试剂1：2×60ml，试剂2：2×20ml； c）试剂1：2×30ml，试剂2：2×10ml； d）试剂1：1×30ml，试剂2：1×10ml； e）试剂1：2×16.8ml，试剂2：2×5.6ml； f）试剂1：1×45ml，试剂2：1×15ml； g）试剂1：3×60ml，试剂2：1×60ml。 1.2 组成 试剂主要组分见表1： 表1 试剂主要组分 2.1 净含量 应不低于试剂瓶标示装量。 2.2 外观
试剂1：无色或淡黄色透明溶液，试剂2：无色或淡黄色透明溶液。 2.3 试剂空白 在450nm处测定试剂空白吸光度，应不超过1.2。 2.4 分析灵敏度 测试350μg/100ml（206μmol/L）的被测物时，吸光度变化（ΔA）应不低于0.03。 2.5 准确度 回收率应在85%～115% 范围内。 2.6 重复性 变异系数（CV）应不超过5%。 2.7 线性 2.7.1在[3,400]μg/100ml（[2,235]μmol/L）范围内，线性回归的相关系数应不低于0.990； 2.7.2测试浓度(60,400]μg/100ml（(35,235]μmol/L）的样品，相对偏差应不超过±15%；测试浓度[3,60]μg/100ml（[2,35]μmol/L）的样品，绝对偏差应不超过±15μg/100ml（9μmol/L）。 2.8 批间差 相对极差应小于10%。 2.9 稳定性 取在2℃～8℃条件下贮存达到18个月后的试剂进行检测，应符合本技术要求2.2、2.3、2.4、2.5、2.6、2.7之规定。
总胆汁酸测定试剂盒（酶循环法） 适用范围：本试剂用于体外定量测定人体血清中总胆汁酸的含量。 1.1 包装规格 a) 试剂1：2×45 mL，试剂2：2×15 mL； b) 试剂1：4×60 mL，试剂2：4×20mL； c) 试剂1：2×60mL，试剂2：2×20mL。 1.2主要组成成分 1.2.1试剂1主要组分 磷酸氢二钠-柠檬酸缓冲液100mmol/L 氧化型硫代辅酶(THIO-NAD) 950mg/L 还原型辅酶(NADH) 6g/L 聚氧乙烯油醇醚适量 1.2.2试剂2主要组分 Tris缓冲液100mmol/L 3α-羟基类固醇脱氢酶(3α-HSDH) 12.5KU/L 聚氧乙烯油醇醚适量 2.1外观 试剂1：浅黄绿色澄清液体；试剂2：无色澄清液体。 2.2试剂装量 应不低于试剂瓶标示装量。 2.3 试剂空白 2.3.1试剂空白吸光度：在405nm处测定试剂空白吸光度，应≤0.8。
2.3.2试剂空白吸光度变化率：在405nm处测定空白吸光度变化率|△A/min|≤0.05。 2.4分析灵敏度 测定TBA含量为10 μmol/L样本时，其|△A/min|应≥0.01。 2.5线性范围 2.5.1测试浓度在[0，180] μmol/LL范围内，线性回归的相关系数（r）应不低于0.990； 2.5.2测试浓度在[0，20] μmol/L范围内，线性绝对偏差应不超过±2μmol/L；测试浓度在(20，180] μmol/L范围内，线性相对偏差应不超过±10%。 2.6 测量精密度 2.6.1重复性：重复测试三个水平的样本，所得结果的变异系数（CV）应不大于5％。 2.6.2批间差：抽取3个不同批号的试剂，对同一份样本进行重复测定，相对极差≤10％。 2.7准确度 比对试验：相关系数r≥0.975，各个浓度点中≤20 μmol /L的绝对偏差不超过±2μmol /L。各个浓度点中＞20 μmol /L 的相对偏差不超过±10%。 2.8 稳定性 取在2℃～8℃条件下贮存达到18个月的试剂进行检测，应符合2.1、2.3、2.4、2.5、2.6.1、2.7的要求。
胆汁酸测定试剂盒（循环酶法）标准化操作规程 1 目的 规范实验室操作，保证检验工作顺利有效进行特制定此规程。 2 授权操作人经培训且考核通过的实验室检验人员。 3 适用范围本试剂适用于体外定量检测人血清或血浆中胆汁酸的含量。 4 检验方法 本试剂盒采用循环酶法测定胆汁酸的含量。 5 检验原理 本试剂采用酶分析法测定血清总胆汁酸。3α-羟基类固醇脱氢酶特异性作用于3α-羟甾族化合物，使之转化为相应的酮类固醇，反应中thio-NAD被还原为thio-NADH。通过在405nm波长处测定单位时间内吸光度的变化值，可以计算出样品中总胆汁酸的含量。 6 标本要求 6.1 样本为空腹患者血清。样本应及时离心分离，不得使用溶血或被污染的样本。 6.2 样本应避免微生物污染，在2℃～8℃可稳定数月。 7 试剂及配套品 7.1试剂来源 长春迪瑞医疗科技股份有限公司胆汁酸试剂盒（循环酶法） 7.2 7.3试剂的稳定性与贮存： 7.3.1 试剂在2℃~8℃条件下，干燥、避光、密封贮存，有效期为12个月。 7.3.2 试剂开封后在2℃~8℃条件下可稳定30天。 8 实验仪器及性能指标 8.1 实验仪器 迪瑞CS系列全自动生化分析仪 8.2试剂性能指标 8.2.1 试剂空白：
试剂空白吸光度：A≤0.800。 试剂空白吸光度变化率：△A/min≤0.040。 8.2.2 分析灵敏度：测试1μmol/L被测物时，吸光度变化率（△A/min）＞0.0002。 8.2.3 线性范围：1μmol/L～180μmol/L；线性相关系数r≥0.9900；[1,36]μmol/L区间内，线性绝对偏差应不超过±7.2μmol/L；（36,180]μmol/L区间内，相对偏差不超过±15％。 8.2.4 准确度：比对试验：r≥0.9900；[1，36]μmol/L区间内，绝对偏差不超过±7.2μmol/L，（36，180]μmol/L区间内，相对偏差不超过±15%。 8.2.5 测量精密度： 重复性：CV≤5.0%。 批间差：R≤6.0%。 9 校准程序 9.1校准品来源 长春迪瑞医疗科技股份有限公司生产的临床化学校准血清 9.2校准品的组成：人血清 9.3校准品使用 9.3.1 小心打开瓶盖，避免内容物的任何损失； 9.3.2在20-25℃的室温下，准确量取5ml蒸馏水复溶1瓶校准血清； 9.3.3拧紧瓶盖，避光放置30分钟，使之完全溶解； 9.3.4轻轻混匀，确保溶液均一性。勿摇晃小瓶，避免泡沫产生； 9.3.5使用时，移取所需的体积后，把校准血清盖紧瓶盖放回冰箱中保存； 9.3.6复溶后的校准血清可用于手工测试，也可用于全自动生化分析仪 9.4校准品使用注意事项 9.4.1 若该复溶血清受细菌污染，将会降低许多成分的稳定性。 9.4.2 不同批号的校准血清不能交叉使用，因为批号于批号之间的赋值不同。9.4.3 请勿用嘴直接吸取试剂，避免接触皮肤、眼睛及粘膜，一旦接触，应立即用大量水冲洗。 9.5 校准程序 建议使用试剂盒配套的校准品，以纯化水和校准品进行2点校准测定。测定后仪器自动拟合成校准曲线。当试剂批号更换或质控失控时，需要重新校准。10 质量控制 10.1质控品来源 长春迪瑞医疗科技股份有限公司生产的临床化学质控血清(水平I和水平II) 10.2质控品组成：人血清
同型半胱氨酸测定试剂盒（酶循环法） 适用范围：本试剂盒用于体外定量测定人血清中同型半胱氨酸的含量。 1.1产品型号/规格及其划分说明 1.2主要组成成分
2.1外观 2.1.1 试剂1（R1）为无色或淡黄色液体，无混浊，无未溶解物； 2.1.2 试剂2（R2）为无色或淡黄色液体，无混浊，无未溶解物； 2.1.3 校准液应为无色澄清液体，无混浊，无未溶解物； 2.1.4 试剂盒标签标识清晰，外包装完整无损。 2.2 净含量 试剂1（R1）、试剂2（R2）和校准液的净含量不少于标示值。 2.3 试剂空白 在主波长340nm、副波长405nm处（光径1cm），试剂空白吸光度A ≥1.0。 在主波长340nm、副波长405nm处（光径1cm），试剂空白吸光度变化率ΔA/min≤0.05。 2.4分析灵敏度 测量1μmol/L的被测物时，吸光度变化率？A/min≥0.001。 2.5 线性范围 在[3，45]μmol/L线性范围内，线性回归的相关系数r≥0.995。 在[3，10]μmol/L范围内，绝对偏差不超过±1μmol /L；在(10，45]μmol/L范围内，相对偏差不超过±10%。 2.6 精密度 2.6.1 重复性
重复测定(10±2)μmol/L的样品，变异系数CV≤5%；重复测定(20±4)μmol/L的样品，变异系数CV≤3%。 2.6.2 批间差 相对极差≤10%。 2.7 准确度 测定SRM1955，测定值与靶值的相对偏差不超过±15%。 2.8 稳定性 原包装的HCY试剂盒在2℃～8℃避光保存，有效期为18个月。 试剂盒在规定的储存条件下保存至有效期满后，检测2.3、2.4、2.5、2.6.1、2.7项，结果应符合各项目的要求。 2.9 校准液溯源性 按GB/T21415-2008《体外诊断医疗器械生物样品中量的测量校准品和控制物质赋值的计量学溯源性》的要求，试剂盒校准液溯源至NIST SRM1955。
Hans Journal of Biomedicine 生物医学, 2018, 8(4), 62-68 Published Online October 2018 in Hans. https://www.doczj.com/doc/be4711190.html,/journal/hjbm https://https://www.doczj.com/doc/be4711190.html,/10.12677/hjbm.2018.84008 Advanced Progression in the Mechanism of Bile Acid Metabolism Targeting FXR Xiuli Yang1, Sicong Tian1, Bo Pang1, Baolong Li2, Yujuan Shan1* 1Department of Food Science and Engineering, Harbin Institute of Technology, Harbin Heilongjiang 2Center of Drug Safety Evaluation, Heilongjiang University of Chinese Medicine, Harbin Heilongjiang Received: Oct. 4th, 2018; accepted: Oct. 19th, 2018; published: Oct. 26th, 2018 Abstract Bile acids are important physiological factors that facilitate the digestion & absorption of dietary lipids and fat-soluble vitamins in the gut. In addition, they also act as signaling molecules to regu-late glucose homeostasis, lipid metabolism and energy expenditure. Disorders of bile acid meta-bolism can lead to a series of diseases. The nuclear receptor farnesoid X receptor (FXR) is a spe-cific bile acid receptor which plays an important role in the metabolism of bile acids through the regulation of multiple metabolic pathways and of corresponding target genes. Consequently, FXR is targeted to be a new drug for the therapy of disorders related to bile acid metabolism. This ar-ticle reviews the recent progressions of FXR in regulating bile acid metabolism and its mechanism, which aims to provide scientific strategies for the prevention/treatment of bile acid metabolic disorders, and new drugs exploration. Keywords Bile Acids, Farnesoid X Receptor, Bile Acid Metabolism 以FXR为核心的胆汁酸代谢机制研究进展 杨修利1，田思聪1，庞博1，李宝龙2，单毓娟1* 1哈尔滨工业大学食品科学与工程系，黑龙江哈尔滨 2黑龙江中医药大学药物安全性评价中心，黑龙江哈尔滨 收稿日期：2018年10月4日；录用日期：2018年10月19日；发布日期：2018年10月26日 *通讯作者。
总胆汁酸TBA（Total Bile Acids） 胆汁酸是肝细胞以胆固醇为原料合成的。正常人肝脏合成的胆汁酸有胆酸（CA）、鹅脱氧胆酸(CDCA)和代谢中产生的脱氧胆酸（DCA)还有少量石胆酸（LCA）和微量熊脱氧胆酸(UDCA)，合称总胆汁酸(TBA)。总胆汁酸(TBA)是在肝脏内合成，与甘氨酸或牛磺酸结合成为结合型胆汁酸，然后被肝细胞分泌入胆汁，随胆汁至肠道后，在肠道内细菌作用下被水解成游离型胆汁酸，有97%被肠道重新吸收后回到肝脏。如此循环不息。这样能使总胆汁酸发挥最大生理效应。更可防止总胆汁酸大量进入循环中对其它组织细胞的毒害。
健康人的周围血液中血清胆汁酸含量极微，当肝细胞损害或肝内、外阻塞时，胆汁酸代谢就会出现异常，总胆汁酸就会升高。 因此，总胆汁酸测定是一项比较敏感和有效的肝功能试验之一。 血清总胆汁酸在医学上的测定： 正常参考值： 血清总胆汁酸(TBA)<10μmol/L 血清氨胆酸(CG)<2.6mg/L 鹅脱氧胆酸(CDCA)<1.61μmol/L 临床意义 1.正常人的血清总胆汁酸（TBA）是0 ～10 μmol/L的含量。 2.总胆汁酸（TBA）>10μmol/L提示肝细胞发生病变，血液中胆汁酸含量升高。急性肝炎、慢性活动性肝炎、肝硬化、肝癌时胆汁酸明显升高。特别是肝硬化、肝癌时总胆汁酸的升高率>（95%），也大于丙氨酸转氨基酶（ALT）20%。 3.当肝脏实质损害时，肝细胞对胆酸合成降低，鹅脱氧胆酸的合成绝对升高。 4.阻塞性黄疸时CA/CDCA比值大于1.0。 5.肝实质细胞损伤时，CA/CDCA比值小于1.0。 6..当幽门功能不全时，胆酸会反流到胃内，同胃酸一起造成对胃粘膜的损伤，并引起胃痛等不适症状。 7.鹅脱氧胆酸(CDCA)增高见于急慢性病毒性肝炎、胆汁瘀滞、慢性乙醇中毒、肝硬化、原发性肝癌、胆道梗塞等。
第三节胆汁酸的代谢 一、胆汁与胆汁酸 1．胆汁 述：胆汁是由肝细胞分泌的一种黄色或棕色液体，通过胆道系统流入胆囊，循总胆管入十二指肠。 2．胆汁分泌量：成人为500～1000ml/d 3．肝分泌胆道系统胆囊浓缩 （肝胆汁）（胆囊胆汁） 4．主要有机成分：胆汁酸盐（含量最高）、多种酶类（如脂肪酶、 磷脂酶等）、胆色素、磷脂、脂肪、胆固醇等。5．主要功能 ＊胆汁酸盐的功能：促进脂质物质的吸收，以及抑制胆汁中胆固醇 的析出，防止胆石生成。 ＊胆汁中酶的功能：消化酶、胆汁中的其他成分多属排泄物 二、胆汁酸的代谢 ※胆汁酸(bile acids)的概念 胆汁酸是存在于胆汁中一大类胆烷酸的总称，以钠盐或钾盐的形式存在，即胆汁酸盐，简称胆盐(bile salts)。 述：胆汁酸盐是胆汁的重要成分，在脂类物质消化吸收及调节胆固醇代谢方面起重要的作用。 ※胆汁酸的种类 ⑴按结构分为两类 ①游离型胆汁酸：包括胆酸、脱氧胆酸、鹅脱氧胆酸、石胆酸； ②结合型胆汁酸：主要有甘氨胆酸、牛黄胆酸、甘氨鹅脱氧胆酸、 牛黄鹅脱氧胆酸。
⑵按来源不同又可将胆汁酸分为两类 ①初级胆汁酸：指在肝内由胆固醇直接生成的胆汁酸，包括 胆酸、鹅脱氧胆酸及与甘氨酸或牛磺酸的结合物； ②次级胆汁酸：由初级胆汁酸在肠道细菌作用下转变而成的， 包括脱氧胆酸和石胆酸。 （一）初级胆汁酸的生成 述：在肝细胞内由胆固醇生成初级胆汁酸的过程很复杂，需经过许多酶促反应才能完成。胆固醇转化成胆汁酸是其在体内代谢的主要去路 1．部位：肝细胞的胞液和微粒体中 2．原料：胆固醇 3．限速酶：胆固醇7α-羟化酶 述：胆固醇7α-羟化酶受产物-胆汁酸的反馈抑制，维生素C、生长激素、糖皮质激素等可提高此酶的活性。此外，甲状腺素可促进肝细胞合成胆汁酸。 4．过程 胆固醇(27C) ↓7α-羟化酶 7α-羟化胆固醇→→初级胆汁酸(24C) ↓ 结合型初级胆汁酸 5．临床意义 述：口服消胆胺药或进食大量纤维素食物，使肠道胆汁酸重吸收减少，胆汁酸对7α-羟化胆固醇的反馈抑制减弱，有利于肝内胆固醇转化为胆汁酸，而降低血胆固醇含量。
血清总胆汁酸循环酶法测定法 1. 实验原理 胆汁酸会被3α-羟基类固醇脱氢酶（3α-HSD）以及β-硫代烟酰胺腺嘌呤二核苷酸氧化型（Thio-NAD）特异性地氧化，生成3-酮类固醇以及β-硫代烟酰胺腺嘌呤二核苷酸还原型（Thio-NADH）。生成的3-酮类固醇在3α-羟基类固醇脱氢酶（3α-HSD）及β-硫代烟酰胺腺嘌呤二核苷酸还原型（Thio-NADH）存在下，生成胆汁酸及β-硫代烟酰胺腺嘌呤二核苷酸氧化型（Thio-NAD）。如上所述，依据循环酶而放大微量的胆汁酸量，测定单位时间内生成的β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）在405nm处的吸光度变化，以求得胆汁酸的浓度。 （胆汁酸会被3α-羟基类固醇脱氢酶及Thio-NAD 特异性地氧化，生成3-酮类固醇及Thio-NADH，此外，生成的3-酮类固醇在3α-羟基类固醇脱氢酶及NADH 存在下，和NAD。如上所述，循环往复从而放大微量的胆汁酸量，测定生成的Thio-NADH的吸光度变化，以求得胆汁酸的浓度。） 2. 标本： 2.1 病人准备：标本以空腹血清为宜，餐后胆汁酸会
升高，应注意采血时间。 2.2类型：血清、肝素或EDTA处理的血浆。应使用新鲜标本。标本必须避光保存。 3. 标本存放4℃保存可稳定7天，-20℃保存可稳定3个月。 4. 标本运输常温条件下避光保存运输。 5. 标本拒收标准细菌污染的标本。 6. 实验材料 6.1 试剂威特曼总胆汁酸测定试剂盒 Good’s缓冲液120mmol/L 3a-羟基类固醇脱氢酶(3a-HSD) 12.5 KU/L 硫代辅酶(Thio-NAD) 6.1g/L 6.1.2 试剂准备：试剂为即用式。 6.1.3 试剂稳定性与贮存：试剂避光保存于2～8℃，若无污染，可稳定至失效期。试剂不可冰冻，试剂2必须避光保存。 6.1.4 变质指示：当试剂有看得见的微生物生长，有浊度，或者未开盖的液体有沉淀时，表明试剂已变质，不能继续使用。
第十六章肝的生物化学 一、内容提要 肝是体内重要的代谢器官之一，具有多种生物化学功能。本章主要介绍肝除了与其他组织器官相同的功能外还具有一些重要功能，如物质代谢功能、生物转化功能和排泄功能等。 （一）肝的物质代谢功能 1．肝在糖、脂类、蛋白质代谢作用中的特点 （1）糖代谢肝通过糖原合成、分解与糖异生作用调节血糖水平，维持血糖浓度的相对恒定。 （2）脂类代谢肝在脂类的消化、吸收、合成、分解及运输等过程中均起着重要作用。如肝将胆固醇转化为胆汁酸，协助脂类的消化吸收；肝是体内合成磷脂、胆固醇、脂肪酸的重要器官，并能以脂蛋白的形式转运出去；肝是体内合成酮体的主要器官。 （3）蛋白质代谢肝对蛋白质代谢极为活跃，除γ-球蛋白外，几乎所有的血浆蛋白质均来自肝；肝是除支链氨基酸外所有氨基酸分解代谢的重要器官，是处理氨基酸分解代谢产物的重要场所，如氨主要在肝中合成尿素。 2．肝在维生素和激素代谢作用中的特点 （1）维生素代谢肝在维生素的吸收、贮存、运输及代谢中起重要作用， 肝是人体内含维生素A、K、B 1、B 2 、B 6 、B 12 、泛酸与叶酸最多的器官，且多种维 生素在肝中转化为辅酶的组成成分。 （2）激素代谢许多激素在发挥其调节作用后，主要在肝内被分解转化，从而降低或失去其活性，此灭活过程对于激素作用时间的长短及强度具有调控作用。 （二）肝的生物转化作用 1．生物转化的概念非营养物质经过氧化、还原、水解和结合反应，使其极性增加或活性改变，而易于排出体外的这一过程称为生物转化作用。 2．生物转化的物质生物转化的内源性非营养物质有体内代谢过程中生成
的氨、胺、胆色素、激素等物质。外源性非营养物质有摄入体内的药物、毒物、食品防腐剂、色素等。 3．生物转化的反应类型主要有两相反应。第一相反应包括氧化、还原和水解反应，其中最重要的是存在于微粒体的加单氧酶系，其特点是可被诱导生成，生理意义是参与药物和毒物的转化；第二相反应是结合反应，结合反应是体内重要的生物转化方式，主要与葡萄糖醛酸（供体UDPGA）、硫酸（PAPS）和乙酰基（乙酰CoA）等结合，尤以葡萄糖醛酸结合反应最为普遍。 4．生物转化的作用特点①连续性，非营养物质在肝内进行的生物转化是在一系列酶的催化下连续进行的化学反应，最终将这些物质清除至体外。②多样性，在连续的化学反应中，非营养物质有的经过第一相反应可以清除，有的还要经过第二相反应才能清除。③失活与活化双重性，经过生物转化，有的非营养物质的活性基团被遮蔽而失去活性；有的却获得活性基团而被活化，表现出解毒与致毒双重性。 5．生物转化的生理意义对体内生物活性物质进行灭活，同时有利于排除废物及异物，具有保护机体的作用。对外源物质的生物转化，有时反而出现毒性或致畸等作用，如3,4-苯骈芘转化后生成致癌性物质，但易于排出体外。 （三）胆汁酸与血红素的代谢 1．胆汁酸的代谢胆汁酸的生成及分类胆汁酸是胆汁中存在的一大类胆烷酸的总称。按其来源分为初级胆汁酸或次级胆汁酸；按其是否与甘氨酸和牛磺酸结合又分为结合胆汁酸和游离胆汁酸。 （1）初级胆汁酸初级胆汁酸在肝细胞内由胆固醇转化而来，包括游离型（胆酸和鹅脱氧胆酸）及结合型（胆酸和鹅脱氧胆酸分别与甘氨酸或牛磺酸的结合物）。7-α-羟化酶是胆汁酸合成的限速酶，受胆汁酸的负反馈调节。 （2）次级胆汁酸初级胆汁酸在肠道细菌的作用下，进行7位脱羟基反应，生成脱氧胆酸和石胆酸，即游离型次级胆汁酸。游离型次级胆汁酸分别与甘氨酸或牛磺酸结合生成次级结合胆汁酸，以脱氧胆酸的结合物为主。 （3）胆汁酸肠肝循环由肠道重吸收的各类胆汁酸经门静脉入肝，游离型又转变成结合型胆汁酸，并同新合成的结合型胆汁酸一起再次排入肠道的过程，称胆汁酸的肠肝循环。除石胆酸外，95%的胆汁酸经“肠肝循环”而再被反复利
如对您有帮助，可购买打赏，谢谢总胆汁酸偏高的原因有哪些 导语：随着社会的越来越越发展，人们的生活也越来越富裕，在吃的食物上，可以说是山珍海味应有尽有了，这样便对健康也就有了新的理解，总胆汁酸偏 随着社会的越来越越发展，人们的生活也越来越富裕，在吃的食物上，可以说是山珍海味应有尽有了，这样便对健康也就有了新的理解，总胆汁酸偏高这个名词我们都不太了解，或许跟胃液的酸多，差不多，胆汁在我们的消化系统中发挥着不可替代的作用，可是总胆汁酸偏高的原因是什么，该怎么治疗呢。 胆汁是胆囊中棕色稠厚的液体，胆汁中主要成分就是胆汁酸.肝脏是合成，并从门静脉摄取胆汁酸的唯一场所，胆道是胆汁酸排泄最主要的途径，因此，血清胆汁酸水平与肝胆疾病密切相关，胆汁酸增高时主要见于肝胆疾病，总胆汁酸生理性升高：可见于进食后可一过性增高，病理性升高见于各种原因引起的肝细胞损害、胆道梗阻、门脉分流术后，胆汁酸增高时，可出现急性肝炎，慢性肝炎，黄疸，胆囊炎，胆道梗阻等， 总胆汁酸是由肝脏合成，主要包括胆酸、鹅脱氧胆酸、少量的石胆酸等，总胆汁酸与甘氨酸或者牛磺酸结合以后，形成了结核性胆汁酸，然后被肝细胞分泌到胆汁中，在肠道中的细菌的作用下，水解成游离的胆汁酸，大部分又被吸收回肝脏中。所以一般情况下，健康的人的血液中胆汁酸的含量是非常的低的。那么总胆汁酸偏高什么意思呢？》》总胆汁酸偏高的危害总胆汁酸升高提示肠肝循环被破坏，胆汁酸不能重复利用，它可影响脂类的消化吸收，另外胆汁中胆固醇含量相对增高，处于饱和状态，极易形成胆固醇结石。总胆汁酸偏高的原因一般指的是病理性的原因，如多种肝病：肝细胞损伤，肝纤维化，肝癌， 预防疾病常识分享，对您有帮助可购买打赏
关于胆汁酸与血糖、血脂及能量代谢关系的研究进展 [摘要] 过去胆汁酸仅仅被作为肝脏中来源于胆固醇的两亲性分子，可促进胆固醇、脂溶性维生素和脂质的吸收。近几十年的研究显示——胆汁酸涉及多种代谢过程（血糖代谢、血脂代谢、能量代谢），与这些过程中的某些基因表达及细胞信号通路的调节有关。此外，血糖也可以调节胆汁酸的合成过程。胆汁酸成为代谢综合征、胰岛素抵抗、肥胖的全新研究视角。 近年来，大量的研究显示胆汁酸除了参与食物来源脂质吸收及体内胆固醇的溶解，还作为信号分子在多种代谢过程（包括血糖、血脂及能量代谢）中发挥作用。本文就胆汁酸的这些新作用及可能涉及的机制作一综述。 1.胆汁酸的概述 胆汁酸是胆汁的主要成分，胆汁产生于肝脏而储存于胆囊，经释放进入小肠发挥作用。作为两性分子，胆汁酸内既含有亲水性的羟基及羧基或磺酸基，又含有疏水性烃核和甲基。胆汁酸具有较强的界面活性，能降低油水两相间的表面张力，促进脂类乳化；同时扩大脂肪和脂肪酶的接触面，加速脂类的消化。 胆汁酸按结构可以分为两类：一类为游离型胆汁酸，包括胆酸、脱氧胆酸、鹅脱氧胆酸和少量的石胆酸；另一类是上述游离胆汁酸与甘氨酸或牛磺酸结合的产物，称结合型胆汁酸。从来源上分类可分为初级胆汁酸和次级胆汁酸。肝细胞内，以胆固醇为原料直接合成的胆汁酸称为初级胆汁酸，包括胆酸和鹅脱氧胆酸。胆固醇主要的排泄途径即合成胆汁酸，此反应的限速酶是7α-羟化酶（CYP7A1）。初级胆汁酸在肠道中受细菌作用，进行7α脱羟作用生成的胆汁酸，称为次级胆汁酸，包括脱氧胆酸和石胆酸。 肠道中的各种胆汁酸平均有95%被肠壁重吸收，其余的随粪便排出。由肠道重吸收的胆汁酸(包括初级和次级胆汁酸；结合型和游离型胆汁酸)均由门静脉进入肝脏，在肝脏中游离型胆汁酸再转变为结合型胆汁酸，再随胆汁排入肠腔。此过程称为“胆汁酸的肠肝循环”，其生理意义在于使有限的胆汁酸重复利用，促进脂类的消化与吸收。 最近20年来，胆汁酸不断被人们重新认识，作为调节分子而活化肝脏和肠道中存在的核受体（FXR、PXR及VDR）、G蛋白偶联受体TGR5及细胞信号通路（JNK1/2、AKT、ERK1/2）。这些核受体及信号通路的活化继而改变了许多调节过程（如胆汁酸、血糖、脂肪酸、脂蛋白的合成、代谢、转运过程及能量代谢）中涉及的酶和蛋白质编码基因的表达[1]。 2.胆汁酸与糖代谢 作为糖异生和糖原合成的场所，肝脏在调节血糖水平上起重要作用。PEPCK，即磷酸烯醇式丙酮酸羧激酶，G6Pase,即葡萄糖-6-磷酸酶，以及FBP1，即果糖-1,6-双磷酸酶均是糖异生过程中的关键酶。糖原合成中的关键酶是糖原合酶，肝糖原的分解是补充血糖、维持血糖水平的一个途径。
酶循环法及其在酶法分析中的应用 杨昌国，许叶（宁波市医学科学研究所，浙江宁波315010） 关键词：酶法分析；酶循环法 中图分类号：R461.1 文献标识码：A 由于酶法测定的特异性好，检测简便，反应温和，无污染，且灵敏度较高(10～100umol/L)，已能满足体液中大部分物质的测定，因此在临床化学测定中已广为应用。随着临床化学的发展，体液中某些微量物质的准确测定逐渐显得重要。酶循环法是利用酶的底物特异性来放大靶物质（被测物）的测定方法。此法仅循环靶物质，减少了样品中存在的其它物质对测定的干扰，因此不需要对样品进行预处理或队靶物质的提取，而且该法不需要专门的设备，是一种前景广阔的测定技术。日本旭化成（Asahi Chemical ）公司发展了这一技术，笔者参考旭化成公司的有关资料，将这一技术介绍给国内的同行。 1 酶循环法的原理 物质A 在酶a 和底物a(Sa)的存在下转化为物质B ，同时生成产物a(Pa)；这物质B 又在酶b(Eb)和底物b(Sb)的存在下转化为物质A ，同时生成产物b(Pb)。上述反应每循环一次，将消耗与物质A 等量的Sa 和Sb ，同时生成等当量的Pa 和Pb 。因此在一定时间内，循环反应的次数亦就是Sa 和Sb 消耗或Pa 和Pb 生成相当于物质A(或物质B)的倍数，通过检测Sa 或Sb 的减少或Pa 或Pb 的生成就可以提供检测物质A(或物质 B)的n 倍灵敏度。见图1。 底物a(Sa) 产物a(Pa) 酶a(Ea) 物质A 物质B 酶b(Eb) 产物b(Pb) 底物b(Sb) 图1 酶循环法原理 1.1 条件 1.1.1 物质A 和B 的浓度与酶a 和酶b 对底物Sa 和Sb 的Km 值相比较，应相当小，即[A]或[B]《K m Sa (或 K m Sa )。 1.1.2 酶a 和酶b 对底物Sa 和Sb 的Km 值应相当小，对底物应有高亲和力。 1.2 原理 米氏方程 v ＝V ?[S]/( [S]+Km)，v ＝K ?[S]，(K ＝V /Km )。当t ＝0、[A]=[A 0]、[B]=0时，[A]＋[B A 0]；当t ≥0并假设反应已达到平衡状态，那么，Ea 的速度是Vb ＝Kb[A]。Eb 的速度是Vb ＝Kb[B]。在平衡状态时，va ＝vb 因此，[A]:[B]=Kb:Ka 。循环反应速度vc ＝va ＝vb(固定的)，假设vc ＝Kc[A 0](Kc=循环速率常数)，那么从Kc ＝Ka ?Kb/(Ka+Kb)推导，如果Ka+Kb ＝常数，当Ka ＝Kb 时，Kc 是最大的。 1.3 测定灵敏度 [][][]000 t A Kc Vcdt Pb Pa t ===?。 2 酶循环反应的灵敏度 可通过产物的循环常数(Kc)和反应时间(t)来确定；Kc 又可通过两种酶的量来确定；酶量又取决于酶的特异活性和Km 值，循环反应是在相同的条件(如温度、离子强度等)下进行，两种酶的反应常数亦相同(由于Va/Kma=Vb/Kmb ，因此Kc ＝Ka ＝Kb)。既然循环反应的灵敏度可通过Kc ×t(min)来确定，故可用调整加入酶量或反应时间来随意控制。选择Km 小的酶，用较小的酶就能得到较高的灵敏度。 3 循环反应的类型 Ka 循环 Kb
血清总胆汁酸循环酶法测定法 1.实验原理 胆汁酸会被3α-羟甾醇脱氢（3α-HSD）以及β-硫代烟酰胺脲嘌呤二核苷酸氧化型（Thio-NAD）特异性地氧化，生成3-酮类固醇以及β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）。此外生成的3-酮类固醇在3α-羟甾醇脱氢（3α-HSD）及β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）存在下，生成胆汁酸及β-硫代烟酰胺脲嘌呤二核苷酸氧化型（Thio-NAD）。如上所述，依据循环酶而放大微量的胆汁酸量，测定单位时间内生成的β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）在405nm处的吸光度变化，以求得胆汁酸的浓度。2.标本： 2.1病人准备：标本以空腹血清为宜，餐后胆汁酸会升高，应注意采血时间。 2.2类型：血清、肝素或EDTA处理的血浆。应使用新鲜标本。标本必须避光保存。 3.标本存放4℃保存可稳定7天，-20℃保存可稳定3个月。 4.标本运输常温条件下避光保存运输。 5.标本拒收标准细菌污染的标本。 6.实验材料 6.1试剂南京波因特总胆汁酸测定试剂盒（试剂1：2×60ml试剂2：2×20ml） 6.1.1试剂组成
试剂1（R1）： β-硫代烟酰胺脲嘌呤二核苷酸氧化型(Thio-NAD)2mmol/L Tris缓冲液30mmol/L 试剂2（R2）： 脲嘌呤二核苷酸氧化型(NAD)2mmol/L 3a-羟甾醇脱氢酶(3a-HSD)5U/L 6.1.2试剂准备：试剂为即用式。 6.1.3试剂稳定性与贮存：试剂避光保存于2～8℃，若无污染，可稳定至失效期。试剂不可冰冻，试剂2必须避光保存。 6.1.4变质指示：当试剂有看得见的微生物生长，有浊度，或者未开盖的液体有沉淀时，表明试剂已变质，不能继续使用。 6.1.5注意事项：不可入口！避免接触皮肤及粘膜。应采取必要的预防措施使用试剂 6.2校准品：使用南京波因特公司提供的TBA校准品对自动分析仪进行校准，具体参见生化检验校准品和质控品.SOP文件。 6.3质控品：具体参见生化检验校准品和质控品.SOP文件。 7.仪器：奥林巴斯AU1000生化分析仪 8.操作步骤: 8.1项目基本参数：参见AU1000生化分析仪项目测定参数.SOP文件 8.2仪器操作步骤：参见AU1000生化分析仪操作规程.SOP文件 9.检验结果的判断与分析 10.质量控制：在每一批标本中都应把非定值血清水平I与II质控做
总胆汁酸(TBA) 概述:胆汁酸是胆汁的重要成分，在脂肪代谢中起着重要作用。胆汁酸主要存在于肠肝循环系统，并通过再循环起一定的保护作用。只有一少部分胆汁酸进入外围循环。促进胆汁酸肠肝循环的动力是肝细胞的转运系统，吸收胆汁酸并将其分泌入胆汁、缩胆囊素诱导的胆囊收缩、小肠的推进蠕动，回肠黏膜的主动运输及血液向门静脉的流入。新合成及再循环的胆汁酸被分泌至胆管以防止肝内高浓度梯度的胆汁淤积。胆汁酸的主动运输是调节胆汁酸形成及流动的一个重要因素。 正常人肝脏合成的胆汁酸有胆酸（CA）、鹅脱氧胆酸（CDCA）和代谢中产生的脱氧胆酸（DCA）还有少量石胆酸（LCA）和微量熊脱氧胆酸（UDCA），合称总胆汁酸（TBA）。胆汁酸的类型:胆汁酸按结构分：游离胆汁酸（CA.CDCA.DCA.LCA）结合胆汁酸（与甘氨酸或牛磺酸结合） 胆汁酸按来源分：初级胆汁酸（CA.CDCA) 次级胆汁酸（DCA.LCA) 胆汁酸的合成机理:胆汁酸主要在肝细胞内由转化而来，其合成分3个阶段:①初级胆汁酸的合成。在肝细胞内质网微粒体酶系作用下，其中胆固醇7α－羟化酶为限速酶，胆固醇转化为7α－羟胆固醇，而后经过羟化、加氢及侧链断裂和加辅酶A等多步反应，最终形成具有24碳的初级胆酸（胆酸和鹅脱氧胆酸）。②次级胆汁酸的合成。初级胆汁酸随胆汁进入小肠参与脂类的消化吸收后，在回肠末端，约有95％-98％的胆汁酸经门静脉被重新吸收，经门静脉回流的胆汁酸80％以上被肝细胞的窦膜有效摄取。大约有1/4的结合型初级胆汁酸在空肠、回肠及结肠上段，在细菌酶的催化下，发生7-位脱羟基，生成脱氧胆酸、石胆酸、熊脱氧胆酸等次级胆酸，石胆酸具有毒性，可以导致肝脏实质细胞的损伤，多数石胆酸随粪便排出体外。③三级胆汁酸的合成。肝细胞合成的胆汁酸与甘氨酸或牛磺酸结合，形成的结合型胆汁酸。 胆汁酸的代谢过程：进食后，胆汁酸进入小肠，乳化脂肪。乳化的脂肪被水解为甘油和脂肪酸后，胆汁酸与脂肪酸形成的脂肪酸－胆汁酸复合物被小肠绒毛膜吸收，然后与脂肪酸分离，经门静脉入肝。肝细胞将摄取的游离型胆汁酸再合成结合型胆汁酸，并同新合成的结合型胆汁酸一起重新进入肠道，这一过程为胆汁酸的肠肝循环。 胆汁酸的代谢过程：肝肠循环使胆汁酸发挥最大限度的乳化作用，以保证脂类物质正常的消化和吸收。排入肠道的胆汁酸约有95％以上被重新吸收，其余随粪便排除，因此，每次肝肠循环胆汁酸的损失率约为5％左右。肠道内脂类物质的消化每日需12-32g胆汁酸，肝脏合成胆汁酸的能力仅为0.4-0.6g，仅仅依靠肝脏每天的合成是不够的，胆汁酸的肝肠循环则可以弥补肝脏胆汁酸合成的不促。 据统计，胆汁酸的肠肝循环每天要进行6-15次，对发挥胆汁酸的乳化作用，促进脂类正常消化吸收发挥了重要作用。经过胃酸和小肠蠕动得到初步乳化的脂肪进入小肠后，小肠黏膜细胞释放胆囊收缩素（CCK）到血液里，刺激胆囊释放胆汁，胰腺分泌胰脂肪酶。脂肪在胆汁酸盐的乳化作用下，形成脂肪微粒；胰脂肪酶将脂肪分解为甘油、游离脂肪酸、甘油一酯、甘油二酯。低于12个碳原子的短链脂肪酸直接被小肠粘膜内壁吸收通过门静脉入肝进入血液循环。长链脂肪酸在肠黏膜细胞内与甘油一酯重新合成甘油三酯，然后与胆固醇、脂蛋白、磷脂结合，形成乳糜微粒进入淋巴系统，最后进入血液，运送到身体各个组织。TBA的吸收的主要有肠道吸收与肝脏吸收：（1）在肠道中的吸收：在肠道中，各种形式的胆汁酸充分发挥各自的生理功能，并再次决定了自身的命运。肠道上段胆汁酸与脂类的消化吸收有关。肠道下段（即回肠及近侧结肠）胆汁酸自身发生变化：在肠内细菌作用下发生转化，并在肠黏膜中大部分以原来的或转化的形式按主动运输或被动运输机理被重新吸收。只有一小部分随食物残渣排出体外。在末端回肠pH条件下，六种主要胆汁酸盐都是可溶的，因此均为游离态酸。当与吸收表面接触时，这些复合物全部被吸收。但是，石胆酸及其复合
目录 目录 (1) 第1章目的和范围 (2) 1.1 目的 (2) 1.2 适用范围 (2) 第2章操作规程 (2) 2.1 测定原理 (2) 2.2 试剂贮存与稳定性 (2) 2.3 试剂与样本准备 (2) 2.4 测定参数 (2) 2.5 操作步骤 (3) 2.6 校准和质控 (3) 第3章结果计算 (3) 3.1 吸光度变化率的计算 (3) 3.2 活性的计算 (3) 第4章方法学特性 (3) 4.1 正常参考范围 (3) 4.2 线性范围 (3) 第5章临床意义 (4) 第6章安全防护 (4)
第1章目的和范围 1.1目的 为用户使用迈瑞公司TBA试剂时，提供正确的操作流程和操作规范。 1.2适用范围 适用于各级医疗机构的用户。 第2章操作规程 2.1测定原理 γ-GT催化胆汁酸会被3α-羟甾醇脱氢（3α-HSD）以及β-硫代烟酰胺脲嘌呤二核苷酸氧化型（Thio-NAD）特异性地氧化，生成3-酮类固醇以及β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）。此外生成的3-酮类固醇在3α-羟甾醇脱氢（3α-HSD）及β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）存在下，生成胆汁酸及β-硫代烟酰胺脲嘌呤二核苷酸氧化型（Thio-NAD）。如上所述，依据循环酶而放大微量的胆汁酸量，测定单位时间内生成的β-硫代烟酰胺脲嘌呤二核苷酸还原型（Thio-NADH）在405nm处的吸光度变化，以求得胆汁酸的浓度。 2.2试剂贮存与稳定性 试剂贮存在2～8℃环境时，在试剂盒上规定的有效期前可以保持稳定。 2.3试剂与样本准备 本试剂为液体双试剂，取出后可以直接使用。 样本可以为血清、肝素或EDTA抗凝的血浆。尽量使用新鲜标本。 2.4测定参数 测定波长：405nm； 分析类型：连续监测法，扣除试剂空白； 孵育时间：3分钟； 延迟时间：1分钟；
货号：QS1108-25 规格：25管/24样脂肪酸合成酶（fatty acid synthase，FAS）活性试剂盒说明书 紫外分光光度法 注意：正式测定之前选择 2-3 个预期差异大的样本做预测定。 测定意义： FAS是脂肪酸合成关键酶，催化乙酰辅酶A和丙二酰辅酶A而生成长链脂肪酸。FAS普遍表达于各种组织细胞中，在哺乳动物肝、肾、脑、肺和乳腺以及脂肪组织中表达丰富。 测定原理： FAS催化乙酰CoA、丙二酰CoA和NADPH生成长链脂肪酸和NADP+；NADPH在340nm有吸收峰，而NADP+没有；通过测定340nm 光吸收下降速率，计算FAS活性。 自备实验用品及仪器： 研钵、冰、台式离心机、紫外分光光度计、1mL石英比色皿、可调式移液枪和蒸馏水。 试剂组成和配制： 试剂一：液体25mL×1瓶，－20℃保存。用前1 d取出置于4℃充分解冻后混匀。 试剂二：粉剂×1支，4℃保存。临用前加入550 μL试剂四，充分溶解，用不完的试剂分装后-20℃保存，禁止反复冻融。 试剂三：粉剂×1支，4℃避光保存。临用前加入550 μL试剂四，充分溶解，用不完的试剂分装后-20℃保存，禁止反复冻融。 试剂四：液体25mL×1瓶，4℃保存。 试剂五：粉剂×1支，4℃避光保存。临用前加入1050 μL试剂四，充分溶解，用不完的试剂分装后-20℃保存，禁止反复冻融。 粗酶液提取： 1.组织：按照组织质量（g）：试剂一体积(mL)为1：5~10的比例（建议称取约 0.1g组织，加入1mL试剂一）进行冰浴匀浆。12000g，4℃离心40min，取上 清置冰上待测。 2.细菌、真菌：按照细胞数量（104个）：试剂一体积（mL）为500~1000：1的 比例（建议500万细胞加入1mL试剂一），冰浴超声波破碎细胞（功率300w，超声3秒，间隔7秒，总时间3min）；然后12000g，4℃，离心40min，取上清置于冰上待测。 3.血清等液体：直接测定。 FAS测定操作： 1. 分光光度计预热30min，调节波长到340 nm，蒸馏水调零。 2. 试剂四置于40℃水浴中预热30 min。 3. 测定管：在1mL石英比色皿中依次加入100μL上清液、20μL试剂二、20μL 试剂三、820μL试剂四和40μL试剂五，迅速混匀后于340nm处测定吸光值，记录第30s和90s时吸光值，分别记录为A1和A2。△A测=A1-A2。
Abbreviations: ACP, acyl carrier protein; BC, biotin carboxylase domain; BCCP, biotin carboxyl carrier protein; CT, carboxyl transferase domain; DAGAT, diacylglycerol acyltransferase; DAP, day after pollination; DW, dry weight; EM, embryo morphogenesis; ENR, enoyl-ACP reductase; EREBP, ethylene responsive element- binding protein; FAE, fatty acid elongase complex; FAS, fatty acid synthase; HD, hydroxyacyl-ACP dehydratase; HtACCase, heteromeric acetyl-Coenzyme A carboxylase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; LPD, lipoamide dehydrogenase; MAT, malonyltransferase; OPPP, oxidative pentose phosphate pathway; PDC, pyruvate dehydrogenase complex; PDH, pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PKp, plastidic pyruvate kinase; PUFA, polyunsaturated fatty acid; SSP, seed storage protein; RuBisCO, ribulose-1,5- bisphosphate carboxylase/oxygenase; TAG, triacylglycerol; VLCFA, very long-chain fatty acid. 缩写:ACP、酰基载体蛋白;BC,生物素羧化酶域;BCCP,生物素羧基载体蛋白;CT, 羧基转移酶域;DAGAT, 二酰基甘油酰基转移酶,DAP,授粉后天数;DW,干重,EM,胚胎形态发生;ENR, 烯酰-ACP还原酶;EREBP, 乙烯反应元件结合蛋白;FAE,脂肪酸延长酶复合体;FAS,脂肪酸合成酶,HD, 羟烷基-ACP脱水酶;HtACCase,杂聚肽乙酰辅酶A羧化酶;KAR,3-酮脂酰-ACP还原酶;KAS,3-酮脂酰-ACP合成酶;LPD, 硫辛酰胺脱氢酶;MAT，丙二酰转移酶;OPPP,氧化磷酸戊糖途径;PDC,丙酮酸脱氢酶复合体;PDH，丙酮酸脱氢酶;PEP,磷酸烯醇丙酮酸;PKp,质体丙酮酸激酶;PUFA,多不饱和脂肪酸;SSP,种子贮藏蛋白; RuBisCO,核酮糖-1,5 -二磷酸羧化酶/加氧酶;TAG,三酰甘油;VLCFA，极长链脂肪酸。 So far, the small size of A. thaliana seeds has prevented to characterise in a detailed manner the organic compounds delivered to the maturing embryos. In seeds of the related B. napus species, these compounds mainly consist of sucrose, glucose, Gln, Glu, and Ala [21–23].Once transported into embryo cells, incoming sucrose can be cleaved via two distinct pathways involving either invertase or sucrose synthase [24–26]. Cleavage of sucrose generates hexose- phosphates that are metabolised through the oxidative pentose phosphate pathway (OPPP) and the glycolytic pathway, providing precursors for fatty acid production in the form of acetyl-CoA.Glycolysis is considered as the predominant pathway for the production of these precursors. In oilseeds, maturing embryos do have a complete glycolytic sequence in the cytosol and in plastids [27–29]. The extent to which both glycolytic sequences are utilised in the conversion of hexose-phosphates into precursors of fattyacid biosynthesis is still a matter of debate. Sets of ESTs from developing A. thaliana seeds [29] and microarrays displaying seed-expressed genes [30,31] have been produced.The data sets thus generated suggest that a major route for the metabolism of hexose-phosphates involves the cytosolic glycolytic pathway up to phospho- enolpyruvate (PEP), this compound being massively imported into the plastid before conversion to pyruvate [32]Nevertheless, when considering the substrate speci?cities of the two PEP transporters identi?ed in A. thaliana, namely AtPPT1 and AtPPT2 [33], it is tempting to speculate that low amounts of 2-phosphoglycerate and 3-phosphoglycerate may also be transported into the plastids (Fig. 2).T he ADP-dependent conversion of PEP to pyruvate and ATP is catalysed by plastidic pyruvate kinase (PKp).The enzyme prevalent in the plastids of developing seeds likely has a subunit composition of 4a4b1 [34], and the characterisation of Pkp-a–Pkp- b1 double mutants producing wrinkled seeds severely depleted in storage lipids has further established the importance of the plastid route in the conversion of PEP into precursors of fattyacid synthesis [35].The irreversible oxidative decarboxylation of pyruvate to produce acetyl-CoA, NADH, and CO2 is catalysed by the plastid