RNA-seq analysis of livers of mice fed a high fat diet with and without administration of an inhibitor of the ileal bile acid uptake transporter ASBT
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE84994 | GEO | 2016/09/22
SECONDARY ACCESSION(S): PRJNA335841
REPOSITORIES: GEO
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Project description:Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.
| S-EPMC4880014 | biostudies-literature
Project description:The apical Na(+)-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5?-bile alcohols and the skate that utilizes structurally different 5?-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5?-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported (3)H-taurocholic acid (TCA), a modern 5?-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5?-bile alcohols, low affinity for 5?-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5?- and 5?-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.
| S-EPMC3540847 | biostudies-literature
Project description:Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and safe and effective therapies are needed. Bile acids (BAs) and their receptors [including the nuclear receptor for BAs, farnesoid X receptor (FXR)] play integral roles in regulating whole-body metabolism and hepatic lipid homeostasis. We hypothesized that interruption of the enterohepatic BA circulation using a luminally restricted apical sodium-dependent BA transporter (ASBT) inhibitor (ASBTi; SC-435) would modify signaling in the gut-liver axis and reduce steatohepatitis in high-fat diet (HFD)-fed mice. Administration of this ASBTi increased fecal BA excretion and messenger RNA (mRNA) expression of BA synthesis genes in liver and reduced mRNA expression of ileal BA-responsive genes, including the negative feedback regulator of BA synthesis, fibroblast growth factor 15. ASBT inhibition resulted in a marked shift in hepatic BA composition, with a reduction in hydrophilic, FXR antagonistic species and an increase in FXR agonistic BAs. ASBT inhibition restored glucose tolerance, reduced hepatic triglyceride and total cholesterol concentrations, and improved NAFLD activity score in HFD-fed mice. These changes were associated with reduced hepatic expression of lipid synthesis genes (including liver X receptor target genes) and normalized expression of the central lipogenic transcription factor, Srebp1c Accumulation of hepatic lipids and SREBP1 protein were markedly reduced in HFD-fed Asbt(-/-) mice, providing genetic evidence for a protective role mediated by interruption of the enterohepatic BA circulation. Together, these studies suggest that blocking ASBT function with a luminally restricted inhibitor can improve both hepatic and whole body aspects of NAFLD.
| S-EPMC5056562 | biostudies-literature
Project description:Background and aimThe major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity.MethodsSingle nucleotide polymorphisms in SLC10A2 from genomic DNA of ethnically-defined healthy individuals were identified using a polymerase chain reaction (PCR)-based temperature gradient capillary electrophoresis (TGCE) system. A heterologous gene expression system was used to assess transport activity of SLC10A2 nonsynonymous variants and missense mutations. Total and cell surface protein expression of wild-type and variant ASBT was assessed by Western blot analysis and immunofluorescence confocal microscopy. Expression of ASBT mRNA and protein was also measured in human intestinal samples.ResultsThe studies revealed two nonsynonymous SNPs, 292G>A and 431G>A, with partially impaired in vitro taurocholate transport. A novel variant, 790A>G, was also shown to exhibit near complete loss of taurocholate transport, similar to the previously identified ASBT missense mutations. Examination of ASBT protein expression revealed no significant differences in expression or trafficking to the cell surface among variants versus wild-type ASBT. Analysis of ASBT mRNA and protein expression in human intestinal samples revealed modest intersubject variability.ConclusionsGenome sequencing and in vitro studies reveal the presence of multiple functionally relevant variants in SLC10A2 that may influence bile acid homeostasis and physiology.
| S-EPMC3170668 | biostudies-literature
Project description:The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.
| S-EPMC5780253 | biostudies-literature
Project description:Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter ?-? (OST?-OST?). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ost?-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids.Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ost?-/- mice at postnatal days 5, 10, 15, 20, and 30. Ost?-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila.As early as day 5, Ost?-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ost?-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ost?-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ost?-/- mice.Early in postnatal development, loss of Ost? leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ost?-Ost? functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.
| S-EPMC6009794 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below. CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn?s disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn?s disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.
2020-01-10 | GSE143405 | GEO
Project description:Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.
| S-EPMC9460185 | biostudies-literature
Project description:Bile acids act as ligands for several nuclear receptors expressed in the small intestine, especially the terminal ileum. Bile acid pool composition can be influenced by health and disease states. Here, we used RNA-seq to investigate the effects of altering bile acid pool composition on gene expression in ileal organoids.
2020-07-19 | GSE144398 | GEO
Project description:This study was designed to address key questions concerning the use of alternative protein sources for animal feeds and addresses aspects such as their nutrient composition and impact on gut function, the immune system and systemic physiology. We used casein (CAS), partially delactosed whey powder (DWP), spray dried porcine plasma (SDPP), soybean meal (SBM), wheat gluten meal (WGM) and yellow meal worm (YMW) as protein sources. We investigated the effects of feeding mice during a period of four weeks on semi-synthetic diets containing 30% of six different protein sources. Microarrays were used to detail the global gene expression in the ileal mucosae of mice. Male C57BL/6J mice were stratified according to bodyweight and litter of origin into six dietary treatment groups. These mice were fed for four weeks with semi-synthetic diets containing one of the following protein sources. At the end of four weeks, mice were sacrificed by euthenesia and ileal tissue samples (scrapings) were collected for RNA extraction and hybridization on Affymetrix microarrays. Soybean meal (SBM) diet group served as reference to make comparisons with other experimental diets.
2016-08-24 | E-GEOD-84442 | biostudies-arrayexpress