Supplementary MaterialsFile S1: Cohort descriptions and array parameters; AMY-ACC probeset correlations. Supplementary MaterialsFile S1: Cohort descriptions and array parameters; AMY-ACC probeset correlations.

Supplementary Materials Supplemental Data supp_56_2_319__index. of herb sterols was increased in and mice, but not in mice. Reverse cholesterol transport, as assessed from your portion of intravenously administered 3H-cholesterol that Rabbit Polyclonal to IKK-gamma appeared in feces, was reduced in mice. Thus, G5G8 expression in both the liver and intestine protects animals from sterol accumulation, and intestinal G5G8 contributes to extrahepatic cholesterol efflux in mice. mice (17). The rate of excretion of biliary cholesterol is usually directly related to the level of hepatic G5G8 expression (17). These experiments have strongly established that hepatic G5G8 transports neutral sterols order Phloridzin into bile. The role of G5G8 in the intestine has been more difficult to define. Humans with sitosterolemia have increased fractional absorption of dietary cholesterol and herb sterols (10). mice also have an increased fractional absorption of dietary phytosterols, but not of cholesterol (11). Based on these observations, it has been proposed that intestinal G5G8 exports dietary sterols into the gut lumen after they are transported into enterocytes via the sterol transporter Niemann-Pick C1-like 1 (NPC1L1) (18). However, direct support for this hypothesis is usually lacking. Biliary sterols compete with dietary sterols for uptake in the gut (19). It is possible that this increased fractional absorption of dietary phytosterols in mice is usually a consequence of reduced biliary cholesterol levels due to absence of hepatic G5G8. Several experimental models have been used to determine the relative functions of the intestine and liver in sterol excretion. Biliary secretion of cholesterol also requires ABCB4, which transports phospholipids into bile (13, 20). mice have levels of biliary cholesterol as low as those seen in mice (13). Surprisingly, fecal sterol excretion is not reduced in the mice (13, 21). These results are not consistent with the premise that this biliary system is the major source of fecal sterols and order Phloridzin thus the major pathway for neutral sterol excretion (22, 23). Evidence from several sources has implicated the intestine as a major conduit for sterol excretion in mice (24, 25). This process, which has been referred to as transintestinal cholesterol excretion (26), requires the transit of sterols order Phloridzin from your blood circulation through enterocytes into the order Phloridzin gut lumen. Intestinal G5G8 may play a role in excretion of endogenous cholesterol rather than, or in addition to, dietary cholesterol (27). A recent case statement provided the first insights into the relative functions of intestinal and hepatic G5G8. Liver transplantation in a sitosterolemic individual with cirrhosis reduced his plasma herb sterols to near normal levels (28). Because the transplant presumably restored G5G8 activity in liver, but not intestine, the authors concluded that the liver functions as the predominant organ for maintaining noncholesterol sterol balance. Although the study was limited to a single case and the patient had severe liver disease of unknown etiology, these observations raise fundamental questions regarding the functional significance of intestinal G5G8. To determine the relative functions of hepatic and intestinal G5G8, we established mice in which G5G8 was inactivated selectively in the liver (mice) or in the intestine mice). Sterol trafficking and regulation in these two new mouse models was compared with that observed in WT mice and mice expressing no order Phloridzin G5G8 (mice). MATERIALS AND METHODS Chemicals Deuterated sterols were purchased from Medical Isotopes Inc. Radiolabeled sterols, including [3H]cholesterol, [3H]cholesteryl oleate, and [14C]cholesterol, were purchased from PerkinElmer Inc. Bile acid levels and phospholipids were measured using packages from Crystal Chem Inc. and Wako Chemicals USA Inc., respectively. Abs against mouse G5 and G8 were developed as previously explained (11); Abs against ABCA1 were purchased from Pierce Biotechnology Inc. All other Abdominal muscles, including polyclonal Abdominal muscles against low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and monoclonal Abdominal muscles against sterol-regulatory element binding protein (SREBP) 1 and SREBP-2 were gifts from Guosheng Liang (University or college of Texas Southwestern Medical Center). Generation of tissue-specific mice The strategy was used to inactivate G5 and G8 in either liver or intestine. sites were launched into intron 2 of ((sites prior to being transduced into C57Bl/6J mouse embryonic stem cells by electroporation. Homologous recombination occurred in 7 out of 352 clones and resulted in insertion of a site and the cassette in intron 2 of and a site in intron 1 of (supplementary Fig. 1A). Both genes were inactivated subsequently by cre recombinase, which removed the first two exons of and the first exon of (supplementary Fig. 1A). The map of the targeted allele was confirmed by genomic blotting, using using the oligonucleotide primers provided in supplementary Table 1. Three recombinant clones were injected into blastocysts of C57BL/6J mice. On day 2.5, the blastocysts were transferred into pseudopregnant recipient mice. A total of 17 (out of 72) of the offspring were chimeric (30C100%). Eleven mice transmitted the.