Kidney International, Vol. HMG-CoA reductase, cholesterol 7-hydroxylase, LDL receptor, SR-B1, and ACAT in diet-induced syndrome X. Role and classification of cholesterol-lowering. Regulation of direct transintestinal cholesterol excretion in mice. Animals and diets. Male FVB mice (2–4 mo) were purchased from Harlan (Horst, the Netherlands). The mice were fed a semipurified reference diet . Abdiets, Woerden, The Netherlands; 2. Abdiets; 2. 0% wt/wt casein, 1. Abdiets; 2. 4% casein, 2. The amount of energy delivered by fat in reference diet, Western- type diet, and the high- fat diet, are 1. Although there was no cholesterol added to the reference diet, it contained trace amounts of cholesterol: 0. There was no difference in energy intake between these diets. Intestinal perfusions were also performed on chow (RM3, Special Diet Services, Witham, UK, 4% wt/wt fat, no cholesterol added)- fed Sr- B1. To study the effect of luminal modifications on TICE, mice were used that received chow diet . Modest cholesterol diet is 11. SR-BI Acetyl-CoA Cholesterol.Food and water were supplied ad libitum. Mice were maintained on a 2. All experiments were performed with the approval of the local Ethical Committee for Animal Experiments. Cholesterol intake and output measurements.
![]() Mice were housed in normal mouse cages (3 mice per cage), to mimic their natural situation as much as possible. Every day, mice and remaining chow pellets were weighed and feces was collected. Fecal neutral sterols were determined as described below. Intestine perfusion procedures. Mice were anesthetized by intraperitoneal injection with 0. FFD . The bile duct was cannulated via the gallbladder, and bile was collected in 1. Bile flow was determined gravimetrically assuming a density of 1 mg/ml. The first fraction was used to measure biliary cholesterol secretion. Proximal small intestines (first 1. TICE takes place. Perfusions were performed as previously described (2. At the end of the perfusion period, blood was collected by cardiac puncture. The perfused intestinal segments were isolated for gene expression analysis. Perfusion fluid composition. Perfusions were carried out with a modified Krebs solution (1. M Na. Cl, 4. 8 m. M KCl, 1. 2 m. M KH2. PO4, 1. 2 m. M Mg. SO4. Bile salt- phospholipid mixtures were made as follows: taurocholate (TC; Sigma, Zwijndrecht, The Netherlands), tauroursodeoxycholate (TUDC; Calbiochem, Amsterdam, The Netherlands) or taurodeoxycholate (TDC; Sigma, Zwijndrecht, The Netherlands) dissolved in methanol and egg yolk l- . After evaporation, films were lyophilized overnight. Lyophilized samples were stored under nitrogen gas at . Before the start of the intestine perfusions, the films were dissolved in perfusion buffer. Determination of m. RNA levels. Total RNA was isolated by using the Trizol reagent according to the manufacturer's protocol (Invitrogen, Breda, The Netherlands). Purified RNA was treated with RQ1 RNase- free DNase (1 unit/2 . Gene expression analysis was performed on a Bio- Rad Myi. Q Single- Color Real- Time PCR Detection System by using the Bio- Rad i. Q SYBRgreen Supermix (Bio- Rad). PCR primers were designed on the basis of Primer Express 1. Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) and validated for identical efficiencies. Hypoxanthine- guanine phosphoribosyl transferase (HPRT), cyclophilin, and acidic ribosomal phosphoprotein P0 (3. B4) were used as standard housekeeping genes. Western blotting. Lysis buffer containing phosphate buffered saline, 1% Triton X- 1. SDS, and complete protease inhibitor was added to the intestinal tissue. Homogenates were made by sonication. The protein concentrations of the lysates were determined by the BCA assay (1. Equal amounts of protein (4. Membranes were probed using a mouse anti- Sr- B1 (p. Ab anti- SRB1 NB4. Novus Biologicals, Littleton, CO), diluted 1: 1,0. Lumi- Light Western blotting substrate (Roche, Woerden, The Netherlands). In selected cases, the membranes were stripped for 3. M 2- mercaptoethanol, 2% (wt/vol) SDS, and 6. M Tris. Protein abundance was calculated by densitometry using Lumi. Analyst 3. 1 software (Roche). Analytical procedures. Perfusate and biliary lipids were extracted by the Bligh and Dyer method (4). Biliary and perfusate cholesterol concentrations were measured by a fluorescent method as described previously (7). To verify whether this method indeed measures only cholesterol in the perfusate samples, in some experiments cholesterol in the perfusate was also measured by gas chromatography (GC) as described below. Good agreement (coefficient of variation < 6%) was observed. Total cholesterol concentrations of serum samples were determined using the Cholesterol RTU assay (Biomerieux, Marcy l'Etoile, France). For fecal neutral sterol analysis, 1- day fecal samples were collected, lyophilized, weighed, and ground. Fecal neutral sterols were measured as follows: 5. A second portion of 5. Release of neutral sterols was achieved by addition of 1 ml methanol- 1 M Na. OH 3: 1 vol/vol. After cooling to room temperature sterols were extracted three times with 3 ml of petroleumether (6. Thereafter the sterols were derivatized to the trimethylsilyl derivative after addition of 1. After evaporation to dryness the residue was taken up in 1 ml of hexane and the sample was shaken, sonicated, and centrifuged. The supernatant was transferred to a 2- ml GC vial. Calibration samples were prepared containing 0–5 . Group means for TICE, as depicted in the figures, were calculated by averaging the outcomes of all mice that got the same treatment. The values for the individual mice, used for the calculation of the group mean, were obtained by averaging the data of the last three perfusion time points. Differences between different groups were determined by one- way ANOVA. Outcomes of P < 0. Analysis was performed using SPSS.
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