Ections stained with lead citrate and platinum blue had been imaged at 120 kV using a Tecnai G 2 FEI microscope (FEI, Eindhoven, The Netherlands) equipped having a Gatan ultrascan 1000 CCD camera. 2.7. Energy Metabolism In Vivo Energy intake and power expenditure had been assessed applying a climate-controlled indirect calorimetry program (TSE Systems, Terrible Homburg, Germany) as Florfenicol amine medchemexpress described [14]. WTD-fed WT and LAL-KO mice have been housed in automatic metabolic cages at area temperature in a typical light-dark cycle (12 h light, 12 h dark) with free of charge access to meals and water. Power expenditure was measured every 15 min. two.8. Acute Cholesterol Absorption Acute cholesterol absorption was measured as described previously [30]. Chow dietfed mice have been fasted for 4 h and thereafter gavaged with 200 corn oil containing two i [3 H]cholesterol (ARC Inc., St Louis, MO, USA) and 200 cholesterol. 4 hours postgavage, plasma, liver, and three parts of the smaller intestine (duodenum, jejunum, ileum) had been isolated. Intestinal tissues had been rinsed with PBS to get rid of luminal contents just before all tissues had been lyophilized overnight. Radioactivity in plasma and tissues was analyzed by liquid scintillation counting. 2.9. Basolateral FA Uptake FA uptake from the basolateral side of enterocytes was determined as previously described [32]. Briefly, chow diet-fed mice have been fasted for 4 h and injected intraperitoneally with 100 intralipid (Fresenius Kabi Austria GmbH, Graz, Austria) containing 7 i [9,10-3H(N)]-oleate (Hartmann Analytics, Braunschweig, Germany). Radioactivity in plasma and lyophilized tissues (liver, duodenum, jejunum, ileum) was measured by liquid scintillation counting. two.ten. Fecal Neutral Sterol Measurements Neutral sterols in feces of WT and LAL-KO mice fed a WTD for four weeks had been quantified by GC as described [33,34] applying 5-cholestane as internal normal. 2.11. BA Measurements BA measurements have been performed in WT and LAL-KO mice fed a WTD for four weeks. Biliary BA concentrations had been determined by (U)HPLC-MS/MS coupled to a SCIEX QTRAP 4500 MD triple quadrupole mass spectrometer and quantified applying D4-labeled BA as internal requirements [35]. For fecal BA measurements, BA in dried and grounded feces was methylated and trimethylsilylated before quantification by gas-liquid chromatography working with 5cholanic acid-7,12-diol as internal typical [36]. The hydrophobicity index (HI) was calculated because the sum on the molar fractions of individual BA multiplied by their individual HI values in line with the procedure of Heuman [37]. Hydrophobicity index employed: TCA, 0; T-MCA, -0.84, T-MCA, -0.78; taurohyodeoxycholic acid, -0.37; T-MCA, -0.33; TUDCA, -0.27; TCDCA, 0.46; TDCA, 0.59; TLCA, 1. BA was groupedCells 2021, ten,5 ofinto principal and secondary BA according to prior reports [33,38]. Key BA involves cost-free and conjugated types of CA, CDCA, -MCA, and -MCA, whereas secondary BA incorporates DCA, LCA, -MCA, UDCA, and their conjugates. two.12. Microbiota Analysis Cecal contents of LAL-KO and manage mice fed WTD for 4 weeks were subjected to quantitative 16S rRNA transcript amplifications and microbiota analysis as described earlier [39]. 2.13. Isolation of Major Enterocytes Principal enterocytes from the jejunum of chow diet-fed LAL-KO and control mice had been isolated as lately described [40]. 2.14. Immunohistochemical Hematoxylin and Eosin as well as Oil-Red O (ORO) Diethyl phthalate-d10 Biological Activity Staining Immunohistochemical staining was performed as previously described [30]. Tissues from 12 h-fasted mice.
