Ileal Bile Acid Binding Protein Research Articles

Ursodeoxycholic Acid-Decorated Zwitterionic Nanoparticles for Orally Liver-Targeted Close-Looped Insulin Delivery.

Oral insulin therapies targeting the liver and further simulating close-looped secretion face significant challenges due to multiple trans-epithelial barriers. Herein, ursodeoxycholic acid (UDCA)-decorated zwitterionic nanoparticles (NPs) (UC-CMs@ins) are designed to overcome these barriers, target the liver, and respond to glycemia, thereby achieving oral one-time-per-day therapy. UC-CMs@ins show excellent mucus permeability through the introduction of zwitterion (carboxy betaine, CB). Furthermore, UC-CMs@ins possess superior cellular internalization via proton-assisted amino acid transporter 1 (PAT1, CB-receptor) and apical sodium-dependent bile acid transporter (ASBT, UDCA-receptor) pathways. Moreover, UC-CMs@ins exhibit excellent endolysosomal escape ability and improve the basolateral release of insulin into the bloodstream via the ileal bile acid-binding protein and the heteromeric organic solute transporter (OSTα- OSTβ) routes compared with non-UDCA-decorated C-CMs@ins. Therefore, CB and UDCA jointly overcome mucus and intestinal barriers. Additionally, UC-CMs@ins prevent insulin degradation in the gastrointestinal tract for crosslinked structure, improve insulin accumulation in the liver for UDCA introduction, and effectively regulate glycemia for "closed-loop" glucose control. Surprisingly, oral ingestion of UC-CMs@ins shows a superior effect on glycemia (≈22h, normoglycemia) and improves postprandial glycemic levels in diabetic mice, illustrating the enormous potential of the prepared NPs as a platform for oral insulin administration in diabetes treatment.

Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis

Bile acids (BAs) have increasingly been implicated in the onset and progression of necrotizing enterocolitis (NEC); multiple findings have demonstrated their ability to induce damage to the intestinal epithelium, thereby exacerbating disease severity. Although we previously showed that melatonin was able to treat NEC by correcting the Treg/Th17 imbalance, the modulatory effect of melatonin on BAs remains unclear. In this study, we conducted transcriptome analysis on intestinal tissues from patients with NEC and validated these findings. Subsequently, we treated mice with melatonin alone or in combination with an agonist/inhibitor of Sirtuin 1 (SIRT1) to assess faecal and serum BA levels, the expression levels of BA transporters and regulators, and the extent of intestinal injury. Our transcriptome results indicated dysregulation of BA metabolism and abnormal expression of BA transporters in patients with NEC, which were also observed in our NEC mouse model. Furthermore, exogenous BAs were found to aggravate NEC severity in mice. Notably, melatonin effectively restored the aberrant expression of BA transporters, such as apical membrane sodium-dependent bile acid transporters (ASBT), ileal bile acid-binding protein (IBABP), and organic solute transporter-alpha (OST-α), by upregulating SIRT1 expression while reducing farnesoid X receptor (FXR) acetylation, consequently leading to decreased serum and faecal BA levels and mitigated NEC severity. Thus, we propose a potential mechanism through which melatonin reduces BA levels via the SIRT1/FXR signalling axis in an NEC mouse model. Collectively, these results highlight that melatonin holds promise for reducing BA levels and represents a promising therapeutic strategy for treating NEC.

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling βD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Enhanced Oral Absorption and Liver Distribution of Polymeric Nanoparticles through Traveling the Enterohepatic Circulation Pathways of Bile Acid.

The intestinal epithelium is known to be a main hindrance to oral delivery of nanoparticles. Even though surface ligand modification can enhance cellular uptake of nanoparticles, the "easy entry and hard across" was frequently observed for many active targeting nanoparticles. Here, we fabricated polymeric nanoparticles relayed by bile acid transporters with monomethoxy poly(ethylene glycol)-poly(D,l-lactide) and deoxycholic acid-conjugated poly(2-ethyl-2-oxazoline)-poly(D,l-lactide) based on structural characteristics of intestine epithelium and the absorption characteristics of endogenous substances. As anticipated, deoxycholic acid-modified polymeric nanoparticles featuring good stability in simulated gastrointestinal fluid could notably promote the internalization of their payload by Caco-2 cells through mediation of apical sodium-dependent bile acid transporter (ASBT) and transmembrane transport of the nanoparticles across Caco-2 cell monolayers via relay-guide of ASBT, ileal bile acid-binding protein, and the heteromeric organic solute transporter (OSTα-OSTβ) along with multidrug resistance-associated protein 3 (MRP3) evidenced by competitive inhibition and fluorescence immunoassay, which was further visually confirmed by the stronger fluorescence from C6-labeled nanoparticles inside enterocytes and the basal side of the intestinal epithelium of mice. The transcellular transport of deoxycholic acid-modified nanoparticles in an intact form was mediated by caveolin/lipid rafts and clathrin with intracellular trafficking trace of endosome-lysosome-ER-Golgi apparatus and bile acid transport route. Furthermore, the increased uptake by HepG2 cells compared with unmodified nanoparticles evidenced the target ability of deoxycholic acid-modified nanoparticles to the liver, which was further supported by ex vivo imaging of excised major organs of mice. Thus, this study provided a feasible and potential strategy to further enhance transepithelial transport efficiency and liver-targeted ability of nanoparticles by means of the specific enterohepatic circulation pathways of bile acid.

L-Arabinose improves hypercholesterolemia via regulating bile acid metabolism in high-fat-high-sucrose diet-fed mice

BackgroundHypercholesterolemia is closely associated with an increased risk of cardiovascular diseases. l-Arabinose exhibited hypocholesterolemia properties, but underlying mechanisms have not been sufficiently investigated. This study aimed to elucidate the mechanisms of l-arabinose on hypocholesterolemia involving the enterohepatic circulation of bile acids.MethodsThirty six-week-old male mice were randomly divided into three groups: the control group and the high-fat-high-sucrose diet (HFHSD)-fed group were gavaged with distilled water, and the l-arabinose-treated group were fed HFHSD and received 400 mg/kg/day l-arabinose for 12 weeks. Serum and liver biochemical parameters, serum and fecal bile acid, cholesterol and bile acid metabolism-related gene and protein expressions in the liver and small intestine were analyzed.Resultsl-Arabinose supplementation significantly reduced body weight gain, lowered circulating low-density lipoprotein cholesterol (LDL-C) while increasing high-density lipoprotein cholesterol (HDL-C) levels, and efficiently alleviated hepatic inflammation and lipid accumulations in HFHSD-fed mice. l-Arabinose inhibited cholesterol synthesis via downregulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Additionally, l-arabinose might facilitate reverse cholesterol transport, evidenced by the increased mRNA expressions of low-density lipoprotein receptor (LDL-R) and scavenger receptor class B type 1 (SR-B1). Furthermore, l-arabinose modulated ileal reabsorption of bile acids mainly through downregulation of ileal bile acid-binding protein (I-BABP) and apical sodium-dependent bile acid transporter (ASBT), resulting in the promotion of hepatic synthesis of bile acids via upregulation of cholesterol-7α-hydroxylase (CYP7A1).Conclusionsl-Arabinose supplementation exhibits hypocholesterolemic effects in HFHSD-fed mice primarily due to regulation of bile acid metabolism-related pathways.

Exposure to the mycotoxin deoxynivalenol reduces the transport of conjugated bile acids by intestinal Caco-2 cells

Conjugated bile acids are synthesized in liver and subsequently secreted into the intestinal lumen from which they are actively reabsorbed and transported back to liver. The efficient enterohepatic circulation of conjugated bile acids is important to maintain homeostasis. The mycotoxin deoxynivalenol (DON) is a fungal secondary metabolite that contaminates cereal food. Upon human exposure, it can cause intestinal dysfunction. We explored the effects of DON exposure on the intestinal absorption of conjugated bile acids and the expression of bile acid transporters using an in vitro model based on Caco-2 cell layers grown in transwells. Our study shows that the transport rate of taurocholic acid (TCA) is decreased after 48-h pre-exposure of the Caco-2 cells to 2 µM DON, which is a realistic intestinal DON concentration. Exposure to DON downregulates expression of the genes coding for the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP) and the organic solute transporter α (OSTα), and it counteracts the agonist activity of Farnesoid X receptor (FXR) agonist GW4064 on these genes. In addition, the transport of ten taurine or glycine-conjugated bile acids in a physiological relevant mixture by the intestinal Caco-2 cell layers was decreased after pre-exposure of the cells to DON, pointing at a potential for DON-mediated accumulation of the conjugated bile acids at the intestinal luminal side. Together the results reveal that DON inhibits intestinal bile acid reabsorption by reducing the expression of bile acid transporters thereby affecting bile acid intestinal kinetics, leading to bile acid malabsorption in the intestine. Our study provides new insights into the hazards of DON exposure.

Lactobacillus delbrueckii Interfere With Bile Acid Enterohepatic Circulation to Regulate Cholesterol Metabolism of Growing-Finishing Pigs via Its Bile Salt Hydrolase Activity.

Microbiota-targeted therapies for hypercholesterolemia get more and more attention and are recognized as an effective strategy for preventing and treating cardiovascular disease. The experiment was conducted to investigate the cholesterol-lowering mechanism of Lactobacillus delbrueckii in a pig model. Twelve barrows (38.70 ± 5.33 kg) were randomly allocated to two groups and fed corn–soybean meal diets with either 0% (Con) or 0.1% Lactobacillus delbrueckii (Con + LD) for 28 days. L. delbrueckii–fed pigs had lower serum contents of total cholesterol (TC), total bile acids (TBAs), and triglyceride, but higher fecal TC and TBA excretion. L. delbrueckii treatment increased ileal Lactobacillus abundance and bile acid (BA) deconjugation and affected serum and hepatic BA composition. Dietary L. delbrueckii downregulated the gene expression of ileal apical sodium-dependent bile acid transporter (ASBT) and ileal bile acid binding protein (IBABP), and hepatic farnesoid X receptor (FXR), fibroblast growth factor (FGF19), and small heterodimer partner (SHP), but upregulated hepatic high-density lipoprotein receptor (HDLR), low-density lipoprotein receptor (LDLR), sterol regulatory element binding protein-2 (SREBP-2), and cholesterol-7α hydroxylase (CYP7A1) expression. Our results provided in vivo evidence that L. delbrueckii promote ileal BA deconjugation with subsequent fecal TC and TBA extraction by modifying ileal microbiota composition and induce hepatic BA neosynthesis via regulating gut–liver FXR–FGF19 axis.

Effect of Zanthoxylum alkylamides on lipid metabolism and its mechanism in rats fed with a high-fat diet.

This research aimed at exploring the effect of Zanthoxylum alkylamides on lipid metabolism and its potential mechanisms using high-fat diet rat model. Treatment with Zanthoxylum alkylamides for 6weeks, food efficiency and atherogenic index of the low, medium, and high doses of Zanthoxylum alkylamides-treated groups were significantly reduced. Meanwhile, the histopathological structure of the livers showed that hepatic steatosis in the groups treated with Zanthoxylum alkylamides was reduced, particularly the HD group. Moreover, the related genes were studied, such as, liver X receptor (LXR), cholesterol 7 alpha-hydroxylase (CYP7A1), hepatic 3-hydroxyl-2-methylglutaryl CoA (HMG-CoA) reductase, sterol regulatory element-binding protein 2 (SREBP-2), ileal bile acid-binding protein (IBABP), sodium-dependent bile acid transporter (ASBT), and transient receptor potential vanilloid subtype1 (TRPV1). These results demonstrated that Zanthoxylum alkylamides could ameliorate abnormal lipid metabolism in rats fed with a high-fat diet. The underlying mechanism may be the downregulation of the expression levels of cholesterol synthesis and bile acid reabsorption-related genes, reduction of endogenous cholesterol synthesis, and increase in bile acid and neutral sterol excretion. PRACTICAL APPLICATIONS: High-energy diet is a potential risk of lipid metabolic disorder. Many studies have shown that hyperlipidemia can lead to atherosclerosis and even hemangioma, cerebral thrombosis, coronary heart disease, and other diseases, which seriously threaten human health. Therefore, seeking an effective and safe way to prevent the obesity-related disease is necessary. This research found that Zanthoxylum alkylamide could ameliorate abnormal lipid metabolism in rats fed with a high-fat diet. The underlying mechanism may be the downregulation of the expression levels of cholesterol synthesis and ileal absorption of bile acid genes, reduction of endogenous cholesterol synthesis, and increase in bile acid and neutral sterol excretion. Therefore, Zanthoxylum alkylamide has the potential for preventing or alleviating high-energy intake-related obesity.

A Villin-Driven Fxr Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an n-6-Enriched High-Fat Diet.

A diet high in n-6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by n-6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low n-6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (n-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the Fxrα1 isoform under the control of the intestine-specific Villin promoter (Fxrα1TG) were fed the CNTL or n-6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the n-6HFD supports higher weight gain in both WT and FxrαTG littermates; increases the expression of Fxrα1/2, and peroxisome proliferator-activated receptor-γ1 (Pparγ1) in the small intestine, Fxrα1/2 in the colon, and cytochrome P4507A1 (Cyp7a1) and small heterodimer protein (Shp) in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and β-muricholic (βMCA) acid in the cecum. Intestinal overexpression of the Fxra1TG augments expression of Shp and ileal bile acid-binding protein (Ibabp) in the small intestine and Ibabp in the proximal colon. Conversely, it antagonizes n-6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of Cyp7a1; and expression of Pparγ in the small intestine. We conclude that intestinal Fxrα1 overexpression represses hepatic de novo BA synthesis and protects against n-6HFD-induced accumulation of human-specific primary bile acids in the cecum.

Serum Cholesterol-Lowering Activity of β-Sitosterol Laurate Is Attributed to the Reduction of Both Cholesterol Absorption and Bile Acids Reabsorption in Hamsters.

The research was performed to delineate how β-sitosterol laurate (β-SLE) consumption influenced serum and hepatic lipids. The results showed that 220 mg/5 mL oil/kg body weight of β-SLE robustly reduced serum total triglyceride and cholesterol levels and the epididymal adipocyte size, and efficiently protected hepatic polyunsaturated fatty acids against lipid peroxidation through superoxide dismutase and glutathione transferase activity enhancement and malondialdehyde level reduction. Based on the changes of fecal cholesterol contents, fecal and hepatic bile acid (BAs) levels, and related protein expression, it was concluded that the mechanisms for lowering serum cholesterol by β-SLE involved (i) the enhanced excretion of fecal cholesterol via down-regulation of intestinal Niemann-Pick C1-like 1 protein; (ii) the increased conversion from cholesterol to primary BAs via up-regulation of cholesterol-7α-hydroxylase and sterol 27-hydroxylase, which was induced by the reduced BAs reabsorption through up-regulating ileal apical sodium-dependent bile acid transporter and ileal bile acid-binding protein.

Bile acid transporter-mediated oral drug delivery

Bile acids are synthesized in the liver, stored in the gallbladder, and secreted into the duodenum at meals. Apical sodium-dependent bile acid transporter (ASBT), an ileal Na+-dependent transporter, plays the leading role of bile acid absorption into enterocytes, where bile acids are delivered to basolateral side by ileal bile acid binding protein (IBABP) and then released by organic solute transporter OSTα/β. The absorbed bile acids are delivered to the liver via portal vein. In this process called “enterohepatic recycling”, only 5% of the bile acid pool (~3 g in human) is excreted in feces, indicating the large recycling capacity and high transport efficacy of ASBT-mediated absorption. Therefore, bile acid transporter-mediated oral drug delivery has been regarded as a feasible and potential strategy to improve the oral bioavailability. This review introduces the key factors in enterohepatic recycling, especially the mechanism of bile acid uptake by ASBT, and the development of bile acid-based oral drug delivery for ASBT-targeting, including bile acid-based prodrugs, bile acid/drug electrostatic complexation and bile acid-containing nanocarriers. Furthermore, the specific transport pathways of bile acid in enterocytes are described and the recent finding of lymphatic delivery of bile acid-containing nanocarriers is discussed.

Increased ileal bile acid binding protein and galectin-9 are associated with mild cognitive impairment and Alzheimer's disease

Enterocyte damage and subsequent microbial translocation drive neuroinflammation in the pathogenesis of Alzheimer's disease (AD). Human ileal bile acid binding protein (I-BABP) and intestinal fatty acid binding proteins (I-FABP) are the indicators of enterocyte damage. Lipopolysaccharide-binding protein (LBP) is an indirect marker of microbial translocation. The activation of peripheral innate immune cells plays a crucial role in modulating AD progression. Galectin-9 is a versatile immunomodulatory molecule. The purpose of this study was to determine I-FABP, I-BABP, LBP, and galectin-9 levels in MCI and AD and investigate the relationship between I-FABP, I-BABP, LBP and galectin-9. In this study, I-FABP, I-BABP, LBP, and galectin-9 levels were measured using ELISA assay in 115 AD patients, 115 MCI patients, and 115 non-demented control subjects. Increased I-BABP and galectin-9 were observed in MCI and AD patients. Furthermore, AD patients had higher I-BABP and galectin-9 levels compared with MCI patients. However, I-FABP and LBP in three groups had no difference. I-BABP levels were positively correlated with galectin-9, after adjusting confounding factors (r = 0.409, p < 0.001). In addition, multivariate analysis revealed that increased I-BABP and galectin-9 levels were significantly associated with reduced mini-mental state examination (MMSE) score. In conclusion, galectin-9 is correlated with I-BABP after adjusting confounding covariates. Moreover, increased I-BABP and galectin-9 in MCI and AD are significant factors for reduced MMSE score. Further studies are needed.

The Role of Gastrointestinal-Related Fatty Acid-Binding Proteins as Biomarkers in Gastrointestinal Diseases.

The fatty acid-binding proteins play a major role in intracellular transportation of long-chain fatty acids. Nine fatty acid-binding proteins have been identified, with each having individual tissue-specific functions in addition to regulation of fatty acids. This review focuses on the three fatty acid-binding proteins found in the gastrointestinal tract and discusses their role as diagnostic or disease monitoring markers in neonatal necrotizing enterocolitis, acute mesenteric ischemia, celiac disease, and inflammatory bowel disease. Of these three fatty acid-binding proteins, intestinal fatty acid-binding protein is of the most interest due to its exclusive expression in the gastrointestinal tract. The elevation of intestinal fatty acid-binding protein in blood and urine reflects enterocyte damage, regardless of the underlying cause. The short half-life of intestinal fatty acid-binding protein also means it is a relatively sensitive marker. In contrast, there is currently less evidence to support liver fatty acid-binding protein and ileal bile acid-binding protein as sensitive biomarkers in these conditions. More extensive studies with specific endpoints are required to validate the roles of these fatty acid-binding proteins in gastrointestinal diseases.

Biomarkers of intestinal barrier function in multiple sclerosis are associated with disease activity

Background: Recent evidence suggests a role for the gut–brain axis in the pathophysiology of multiple sclerosis (MS). Materials and methods: We studied biomarkers of intestinal permeability in 126 people with MS (57 relapsing-remitting multiple sclerosis (RRMS) and 69 progressive MS) and in a group of healthy controls for comparison. Serum/plasma concentrations of zonulin (a regulator of enterocyte tight junctions), tight junction proteins (ZO-1 and occludin), intestinal fatty acid binding protein (IFABP)/ileal bile acid binding protein (IBABP), D-lactate, and lipopolysaccharide (LPS) binding protein were measured. Results: Zonulin concentrations were significantly higher when a concurrent magnetic resonance imaging (MRI) confirmed the presence of blood–brain barrier (BBB) disruption (Gad+ RRMS) and were correlated with tight junction proteins. IBABP and D-lactate were elevated in people with RRMS compared to controls, but did not discriminate between Gad+ and Gad– subgroups. Baseline zonulin concentrations were associated with 1-year disease progression in progressive MS. Conclusions: People with MS have altered biomarkers of intestinal barrier integrity. Zonulin concentrations are associated with 1-year disease progression in progressive MS and closely mirror BBB breakdown in RRMS. Zonulin may mediate breakdown of both the intestinal barrier and the BBB in gut dysbiosis through the regulation of tight junctions. This could explain how the gut–brain axis modulates neuroinflammation in MS.

HgS and Zuotai differ from HgCl2 and methyl mercury in intestinal Hg absorption, transporter expression and gut microbiome in mice

Mercury (Hg) is generally considered as a toxic metal; yet the biological outcomes of Hg-containing compounds are highly dependent upon their chemical forms. We hypothesize that mercury sulfide (HgS) is different from HgCl2 and methylmercury (MeHg) in producing intestinal Hg absorption and disruption of gut microbiome. To test this hypothesis, mice were given orally with HgS (α-HgS, 30 mg/kg), Zuotai (β-HgS, 30 mg/kg), HgCl2 (33.6 mg/kg, equivalent Hg as HgS), or MeHg (3.1 mg/kg, 1/10 Hg as HgS) for 7 days. Accumulation of Hg in the duodenum and ileum after HgCl2 (30–40 fold) and MeHg (10–15 fold) was higher than HgS and Zuotai (~2-fold). HgCl2 and MeHg decreased intestinal intake peptide transporter-1 and Ost-β, and increased ileal bile acid binding protein and equilibrative nucleoside transporter-1. The efflux transporters ATP-binding cassette sub-family C member-4 (Abcc4), Abcg2, Abcg5/8, and Abcb1b were increased by HgCl2 and to a lesser extent by MeHg, while HgS and Zuotai had minimal effects. Bacterial DNA was extracted and subjected to 16S rDNA sequencing. Operational taxonomic unit (OTU) results showed that among the 10 phyla, HgS increased Firmicutes, Proteobacteria, while HgCl2 increased Bacteroidetes, Cyanobacteria and decreased Firmicutes; among the 79 families, HgS increased Rikenellaceae, Lactobacillaceae, Helicobacteraceae, and decreased Prevotellaceae, while HgCl2 increased Odoribacteraceae, Porphyromonadaceae, and decreased Lactobacillaceae; among the 232 genus/species, HgS and Zuotai affected gut microbiome quite differently from HgCl2 and MeHg. qPCR analysis with 16S rRNA confirmed sequencing results. Thus, chemical forms of mercury are a major determinant for intestinal Hg accumulation, alterations in transporters and disruption of microbiome.

Development and Characterization of a Mouse Model Overexpressing a Farnesoid-X-Receptor Transgene with Reduced Markers of Intestinal Inflammation (P08-122-19)

Abstract Objectives The farnesoid-X-receptor (FXR) regulates bile acid (BA) homeostasis, protecting against colonic inflammation and cancer. Conversely, increased intestinal levels of BA and expression of cyclooxygenase-2 (COX-2) increase the risk of inflammation and cancer of the colon. Earlier studies from our laboratory show that a diet rich in n-6 linoleic acid (n-6HFD) epigenetically activates Fxr, inducing the expression of downstream factors that regulate BA homeostasis. However, the chronic exposure to n-6HFD induces COX-2 expression through CpG demethylation of the PTSG-2 gene and activates the β-catenin pathway. The objective of this study is to determine in a mouse and cell culture models the influence of an n-6HFD on endpoints of intestinal inflammation and the modifying effects of overexpression of FXR. Methods A mouse line overexpressing FXR in the intestine was developed by injecting an FXRtransgene (FxrTG) construct driven by the villin promoter into FVB zygotes, and then crossed with C57BL6 mice. The small intestine and colon tissue were collected from male founders. Changes in gene expression and DNA methylation were measured by real-time PCR (RT-PCR). Cell culture experiments were performed in colonic human fetal cell (FHC) cells treated with linoleic acid (LA). Results Expression analyses by RT-PCR reveal increased expression of FxrTG in distal small intestine and proximal and distal colon. These changes are paralleled by accumulation of ileal bile acid-binding protein (IBABP) and small heterodimer partner (SHP), two downstream targets of FXR, and a reduction in expression of COX-2. Western blot of colon FHC shows LA induces expression of COX-2. Conclusions We conclude that increased expression of FXR triggers the expression of genes involved in BA homeostasis and downregulates genes involved in inflammation. The FxrTG mouse model is being used to investigate the modifying effects of diets varying in fatty acid level and profile on endpoints of intestinal inflammation and cancer. Funding Sources This work was supported by a grant from NIFA, GRANT12445471, and a predoctoral training grant to M.G.D. from the Cancer Biology Training Grant T32CA009213.

Bile Acid Transporters Are Expressed and Heterogeneously Distributed in Rat Bile Ducts.

Background/AimsCholangiocytes are capable of reabsorbing bile salts from bile, but the pathophysiological significance of this process is unclear. To this end, we detected the expression and distribution of bile acid transport proteins in cholangiocytes from normal rat liver and analyzed the possible pathophysiological significance.MethodsBile duct tissues of Sprague-Dawley rats were isolated by enzymatic digestion and mechanical isolation, and then divided into large and small bile duct tissues. Immunohistochemistry, real-time polymerase chain reaction and Western blotting were used to determine the expression of the apical sodium-dependent bile acid transporter (ASBT), ileal bile acid binding protein (IBABP), and basolateral organic solute transporter α (Ostα) in the biliary tract system of rats. Differences in the expression and distribution of these proteins were analyzed.ResultsIn cholangiocytes, ASBT and IBABP were mainly expressed in cholangiocytes of the large bile ducts, in which the expression of both was significantly higher than that in the small ducts (p<0.05). Ostα was simultaneously expressed in cholangiocytes of both the large and small bile ducts, showing no significant difference in expression between the two groups of bile ducts (p>0.05).ConclusionsBile acid transporters are expressed and heterogeneously distributed in rat bile ducts, indicating that bile acid reabsorption by cholangiocytes might mainly occur in the large bile ducts. These findings may help explore the physiology of bile ducts and the pathogenesis of various cholangiopathies.

Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation

Human ileal bile acid-binding protein (hI-BABP) has a key role in the intracellular transport of bile salts. To explore the role of histidine protonation in the binding process, the pH-dependence of bile salt binding and internal dynamics in hI-BABP was investigated using NMR spectroscopy and biophysical tools. Thermodynamic and kinetic measurements show an increase in the overall binding affinity and the association rate constant of the first binding step below the pKa of the histidines, suggesting that ligand binding is favoured by the protonated state. The overlap between residues exhibiting a high sensitivity to pH in their backbone amide chemical shifts and protein regions undergoing a global ms conformational exchange indicate a connection between the two processes. According to 15N NMR relaxation dispersion analysis, the slow motion is most pronounced at and above the pKa of the histidines. In agreement with the NMR measurements, MD simulations show a stabilization of the protein by histidine protonation. Hydrogen-bonding and van der Waals interactions mediating the flow of information between the C/D- and G/H-turn regions hosting the three histidines, suggest a complex way of pH-governed allosteric regulation of ligand entry involving a transition between a closed and a more open protein state.

N-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer.

The farnesoid-X-receptor (FXR) protects against inflammation and cancer of the colon through maintenance of intestinal bile acid (BA) homeostasis. Conversely, higher levels of BA and cyclooxygenase-2 (COX-2) are risk factors for inflammation and cancer of the colon. In the United States, n-6 linoleic acid (LA) is the most commonly used dietary vegetable fat. Metabolism of n-6 fatty acids has been linked to a higher risk of intestinal cancer. The objectives of this study were to investigate in colonic mucosa the effects of a high-fat diet rich in LA (n-6HFD) on CpG methylation of Fxr and prostaglandin-endoperoxide synthase-2 (Ptsg-2) genes, and the impact on the expression of tumor suppressor adenomatous polyposis Coli (Apc) and proliferative cyclin D1 (Ccnd1) genes. Weaned C57BL/6J male mice were fed for 6 weeks either an n-6HFD containing 44% energy (44%E) from 22% safflower oil (SO, 76% LA by weight) or a 13% energy (13%E) control diet (Control) from SO (5% by weight). Mice fed the n-6HFD had reduced (60%) Fxr promoter CpG methylation and increased (~50%) Fxr mRNA. The expression of FXR-target ileal bile acid-binding protein (Ibabp), small heterodimer protein (Shp), and anti-inflammatory peroxisome proliferator-activated-γ1 genes was increased. The n-6HFD reduced Ptgs-2 CpG methylation, increased the expression of Cox-2, and increased Apc CpG methylation in colonic mucosa. Accordingly, reduced expression of Apc was coupled to accumulation of c-JUN and Ccnd1, respectively cofactor and gene targets for the β-catenin/Wnt signaling pathway. Finally, the n-6HFD reduced the expression of histone deacetylase-1 while favoring the accumulation of acetylated histone 3. We conclude that an n-6HFD epigenetically modifies Fxr, leading to the activation of downstream factors that participate in BA homeostasis. However, epigenetic activation of Ptsg-2 coupled with silencing of Apc and accumulation of C-JUN and Ccnd1 may increase the risk of inflammation and cancer of the colon.

Effects of sanshoamides and capsaicinoids on plasma and liver lipid metabolism in hyperlipidemic rats.

The objective of this study was to determine the effects of sanshoamides and capsaiciniods on plasma and liver lipid levels and the mRNA expression levels of key receptors involved in cholesterol metabolism in hyperlipidemic rats. A total of 56 three-week-old female Sprague-Dawley rats were assigned to 7 treatment groups based on initial body weight (n = 8 rats per group). With certain combinations of sanshoamides and capsaicinoids significantly increased food intake, reduced lipid levels in blood and liver, improved histological characteristics of a fatty liver, down regulated mRNA expression levels of cholesterol 7-alpha-hydroxylase (CYP7A1), 3-hydroxyl-3-methylglutary CoA (HMG-CoA) and Farnesoid X Receptor (FXR) in liver and apical sodium-dependent bile acid transporter, Ileal Bile Acid Binding Protein and FXR in the ileum in hyperlipidemic rats. These results indicated that dietary supplementation with sanshoamides and capsaicinoids reduced blood lipid levels and improved cholesterol metabolism in hyperlipidemic rats.