Expression Of DBP Is Involved In The Increase The Bile Acids Synthesis By LXR In The Mice

Objective: It is a main pathway for maintaining the balanceof cholesterol metabolism to converse cholesterol to bile acids inliver. There are two major bile acid biosynthetic pathway in liver:classic(or neutral) and alternative(or acidic) pathway.Cholesterol is catalyzed to 7α-hydroxycholesterol by CYP7A1or conversed into 27-hydroxycholesterol by CYP27A1. The 7α-,or 27-hydroxycholesterol is hydroxylated and links to HsCoA.Then the tri-and dihydroxycholestanoyl-CoA (THC-CoA andDHC-CoA) enter into peroxisome for beta-oxidation and areconverted to cholic acid (CA) or chenodeoxycholic acid (CDCA).CYP7A1 is the rate-limiting enzyme in bile acid biosynthesis. Itsactivity and expression limit synthesis of bile acid. Inperoxisome, hydration and dehydrogenation reactions werecatalyzed by D-3-Hydroxyacyl-CoA dehydratase /D-3-Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP).DBP is a necessary enzyme for bile acid synthesis. But it'sunclear how the DBP expression and activity are regulated.The homeostasis of cholesterol metabolism and synthesis ofbile acid are regulated accurately by many factors. Manynuclear receptors like LXR (liver X-activated receptor)areresponsible for this regulation. The ligand-activated LXRidentifies the specific cis element (LXR response element,LXRE)and regulates the expression of target genes involved incholesterol metabolism, such as CYP7A1,ATP-binding cassettetransporter ABCG5, ABCA1,ABCG1,ABCG8 and so no, tomaintain cholesterol metabolism balance. LXR agonist 24(S)-Hydroxycholesterol or T0901317 can increase the expression ofCYP7A1 and promote the conversion of cholesterol to bile acidsin CV-1 cell or in C57BL/6J mice liver. However it is unknowthat whether the LXR ligands also activate expression of DBPalong the increase in CYP7A1 expression and bile acidssynthesis by avtivated-LXR.Recently,we found that high cholesterol diet can increasethe expression of hepatic CYP7A1 and DBP in rat (Basicmedical sciences and clinics, in pressing ). If this is due tooxidation of cholesterols into Hydroxycholesterol then the latteractivated LXR?In preseant experiment, we provided the normal miceswith synthetic LXR agonist T0901317 to enhance thetranscriptional activity of LXR target genes: CYP7A1,ABCG5,ABCG8. And the expression and activity of DBP, the totalcholesterol level in serum and bile were assayed to prove that notonly CYP7A1 but also DBP are involved in induced synthesis ofbile acids by activatied-LXR in liver. The regulation of DBPexpression was dicussed.Methods1 Animals and groupsMale C57BL/6J mice aged 8-10 weeks were dividedrandomly into control group(n=10)and agonist group(n=15).Control group (control) and agonist group (TO) wereadministeed orally with vehicle solution and LXR agongistT0901317 at 20mg/kg/day, respectively for 1week. All animalswere sacrified at day 8. After choledochuses were shutted for 5minutes, the gallbladders were extracted and the bile wascollected for cholesterol assay; blood were collected for serumtotal cholesterol assay;liver was immediately submerged in liquidNitrogen then stored at -70℃for the extraction of total RNAand measurement of DBP activity.2 Total cholesterol assay of serum and bileTotal cholesterol of serum and bile were measured withShangHai RongSheng-biotech company kit by CHOD-PAPmethod.3 Preparation of liver homogenate0.25g of liver were quickly homogenized in 1ml ofhomogenate buffer(50mmol/LKPB, pH7.4 ,1mmol/LBenzamidine , 1mmol/LPMSF, 0.1% Tween-20 ,0.5mol/L NaCl , 1mmol/L EDTANa3 β-Mercaptoethanol).Homogenate was centrifuged at 10000g (10min, 4 ℃),thesupernatant were used for the measurement of DBP activity andprotein concentration.4 DBP activity assayThe activity of D-3-hydoxyacyl-CoA dehydrogenase ofDBP was assayed by following change in absorbance at 303nmas described by Jiang LL. One enzyme unit was defined as theamount of the enzyme converting 1umol of substrate per minunder the assay conditions.5 Extraction of liver total RNATotal RNA was isolated by using the guanidiumthiocyanate-phenol-chloroform method of Chomczynski andSacchi.6 The expression of mRNA assayThe relative mRNA content were measured by RT-PCRusing β-actin as inner standard.Results1 The expression of ABCG5 and ABCG8 mRNAThe relative expression of ABCG5 (0.688 ±0.084)andABCG8(0.667±0.055)of the TO group is significantly higherthan ABCG5(0.496±0.084)and ABCG8(0.523±0.081)in controlgroup p<0.01.2 The changes of total cholesterol in bileThe cholesterol concent in bile of To group(1.395±0.207)was signficantly higher than that in control group(0.956±0.165)p<0.01.These results above indicated that the increased expressionof ABCG5 and ABCG8 by LXR enhanced the secretion ofcholesterol into bile.3 The relative expression of CYP7A1 mRNA in liverThe expression of CYP7A1 mRNA in TO group(0.764±0.067)is significantly higher than that in control group...