Bile Acid Reabsorption Research Articles

Effect of a Farnesoid X-receptor Agonist on Postprandial Lipemia in Rats Fed a Supraphysiological Fat Dozes

Introduction. One of the most progressive directions of the modern stage of development of biology is the deepening of knowledge about the mechanisms of regulation of metabolic processes, in particular about signal molecules that transmit information to the cell through ion channels and nuclear receptors associated with G-protein or with enzymatic activity. The nuclear Farnesoid X receptor (FXR) is mainly expressed in the liver and intestines, it regulates key genes that provide the processes of synthesis, transport and reabsorption of bile acids, and is also involved in the metabolism of lipids and carbohydrates.Aim. To evaluate the effect of a farnesoid X receptor agonist on postprandial lipemia in rats fed a supraphysiological fat diet.Materials and methods. An experimental, prospective, controlled, unblinded, randomized study was conducted to study the effect of a farnesoid X receptor agonist (obeticholic acid) on postprandial lipemia in rats receiving a diet containing a supraphysiological dose of fats.Results and discussion. It has been shown that when assessing postprandial lipemia, an oral test for tolerance to supraphysiological doses of fat with the determination of the initial lipid profile parameters and 4 hours after exercise has a sufficiently high information content. It was found that in animals that received a diet containing an increased amount of fat for 28 days, there was an imbalance in lipid metabolism with activation of their absorption in the intestine, but a "slow" reaction of the mechanisms of intermediate lipid metabolism, which was accompanied by the accumulation of triglycerides and cholesterol in the blood of hungry rats, chylomicrons and LDL. At 4 hours post-feeding, these animals showed abnormal increases in triglycerides and cholesterol.Conclusion. The use of obeticholic acid harmonizes lipid metabolism against the background of alimentary fat load, due to the activation of farnesoid X-receptors of the intestine and liver, which is manifested by a simultaneous increase in the intensity of lipid absorption processes and their intermediate metabolism. As a result, the risk of hyperchylomicronemia, hypercholesterolemia and hypertriglyceridemia is eliminated, the likelihood of developing secondary hyperlipedemia, insulin tolerance and functional overload (or pathology) of the liver is reduced.

Dietary Isoquercetin Reduces Hepatic Cholesterol and Triglyceride in NAFLD Mice by Modulating Bile Acid Metabolism via Intestinal FXR-FGF15 Signaling

Isoquercetin, a monosaccharide flavonoid, was recently reported to have significant amelioration effects on high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) of mice. However, the underlying mechanism of hepatic cholesterol and triglyceride improvement in mice fed HFD by isoquercetin remains unclear. Here, a combination of 16S rRNA gene sequencing, targeted quantification of bile acids (BAs), and biological assays was employed to investigate the beneficial effects of isoquercetin on NAFLD in mice. The results showed that dietary isoquercetin markedly modulated the BAs profiling in various samples such as liver, serum, intestine, and feces. We found that dietary isoquercetin promoted BA biosynthesis via the activation of alternative pathways and inhibition of intestinal FXR-Fgf15 signaling, thus reducing 13.2% hepatic cholesterol and 16.05% triglyceride in NAFLD mice. Dietary isoquercetin also regulated a series of receptors mediating correspondent processes of BA transportation, reabsorption, and excretion. Of particular note, dietary isoquercetin significantly modulated cross-talk between BAs and specific gut bacteria of NAFLD mice. These findings revealed that long-term intake of isoquercetin plays beneficial roles in the prevention or intervention of fatty liver disease.

Bile acid dysmetabolism in inflammatory bowel diseases

Aim: To summarize the state-of-the-art data on the molecular mechanisms of bile acid (BA) synthesis and absorption, their impaired absorption and receptor-dependent signaling, as well as on the effects of the gut microbiota on BA metabolism in inflammatory bowel diseases (IBD).
 Key messages: BA malabsorption is one of the relevant mechanisms in the development of diarrhea in IBD. It may occur due to various disorders of the ileum, such as terminal ileitis, ileocolitis or ileocecal resection in Crohn's disease and ileoanal reservoir in ulcerative colitis. Molecular mechanisms of BA malabsorption in IBD are related to a defect in the BA uptake by the apical sodium dependent bile acid transporter (ASBT), as well as to a decrease in the expression of pregnane X receptor (PXR) and farnesoid X receptor (FXR), whose activation by glucocorticoids results in an increase in the BA reabsorption in the ileum and a decrease in hologenic diarrhea. The metabolic profile of luminal BA in IBD is characterized by an increased content of conjugated and 3-OH-sulfated BA and reduced levels of secondary BA. The decrease in the relative abundance of the Lachnospiraceae and Oscillospiraceae spp. in IBD patients leads to a decrease in the efficiency of microbial biotransformation of BA. Changes in the BA metabolic profile in IBD affect the gut microbiota, and impaired interaction with the FXR, PXR, G protein-coupled bile acid receptor (GPBAR1), retinoid-related orphan receptors (RORs) and vitamin D receptor (VDR) results in a pro-inflammatory response and increased intestinal permeability, bacterial translocation, and IBD progression. BA metabolism in IBD-associated primary sclerosing cholangitis (PSC-IBD) is characterized by a significant decrease in the luminal BA pool, and the microbiota composition is remarkable for an increase in the relative abundance of Fusobacterium and Ruminococcus spp., and a decrease in Veillonella, Dorea, Blautia, Lachnospira and Roseburia.
 Conclusion: Disordered synergistic interplay of BA with intestinal microbiota results in disruption of the ligand-receptor interaction and BA metabolic transformation, which contributes to the activation of the immune system, formation of a vicious circle of chronic inflammation and IBD progression. Further studies into mutual influence of the gut microbiota, BA metabolism and receptor signaling may promote the development of new methods for the diagnosis and treatment of IBD.

Dietary cholesterol drives the development of nonalcoholic steatohepatitis by altering gut microbiota mediated bile acid metabolism in high-fat diet fed mice

Nonalcoholic fatty liver disease (NAFLD) is the most widespread chronic liver disorder globally. Unraveling the pathogenesis of simple fatty liver to nonalcoholic steatohepatitis (NASH) has important clinical significance for improving the prognosis of NAFLD. Here, we explored the role of a high-fat diet alone or combined with high cholesterol in causing NASH progression. Our results demonstrated that high dietary cholesterol intakes accelerate the progression of spontaneous NAFLD and induces liver inflammation in mice. An elevation of hydrophobic unconjugated bile acids cholic acid (CA), deoxycholic acid (DCA), muricholic acid and chenodeoxycholic acid, was observed in high-fat and high-cholesterol diet fed mice. Full-length sequencing of the 16S rDNA gene of gut microbiota revealed a significant increase in the abundance of Bacteroides, Clostridium, and Lactobacillus that possess bile salt hydrolase activity. Furthermore, the relative abundance of these bacterial species was positively correlated with content of unconjugated bile acids in liver. Moreover, the expression of genes related to bile acid reabsorption (organic anion-transporting polypeptides, Na+-taurocholic acid cotransporting polypeptide, apical sodium dependent bile acid transporter and organic solute transporter β) was found to be increased in mice with a high-cholesterol diet. Lastly, we observed that hydrophobic bile acids CA and DCA induce an inflammatory response in free fatty acids-induced steatotic HepG2 cells. In conclusion, high dietary cholesterol promotes the development of NASH by altering gut microbiota composition and abundance and thereby influencing with bile acid metabolism.

A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Effects of Atorvastatin on Bile Acid Metabolism in High-fat Diet-fed ApoE -/- Mice.

Statins are considered as the cornerstone of the prevention and treatment of atherosclerotic cardiovascular disease, where pleiotropic effects are thought to contribute greatly in addition to the lipid-lowering effect. Bile acid metabolism has been gradually reported to be involved in the antihyperlipidemic and antiatherosclerotic effects of statins, but with inconsistent results and few studies carried out on animal models of atherosclerosis. The study aimed to examine the possible role of bile acid metabolism in the lipid-lowering and antiatherosclerotic effects of atorvastatin (ATO) in high-fat diet-fed ApoE -/- mice. The results showed that the levels of liver and faecal TC as well as ileal and faecal TBA were significantly increased in mice of the model group after 20 weeks of high-fat diet feeding compared with the control group, with significantly downregulated mRNA expression of liver LXR-α, CYP7A1, BSEP, and NTCP. ATO treatment further increased the levels of ileal and faecal TBA and faecal TC, but no obvious effect was observed on serum and liver TBA. In addition, ATO significantly reversed the mRNA levels of liver CYP7A1 and NTCP, and no obvious changes were observed in the expression of LXR-α and BSEP. Our study suggested that statins may enhance the synthesis of bile acids and facilitate the reabsorption of bile acids from the ileum via portal into the liver, possibly through the upregulation of the expression of CYP7A1 and NTCP. The results are helpful in enriching the theoretical basis for the clinical use of statins and have good translational value.

Active enterohepatic cycling is not required for the choleretic actions of 24-norUrsodeoxycholic acid in mice.

The pronounced choleretic properties of 24-norUrsodeoxycholic acid (norUDCA) to induce bicarbonate-rich bile secretion have been attributed to its ability to undergo cholehepatic shunting. The goal of this study was to identify the mechanisms underlying the choleretic actions of norUDCA and the role of the bile acid transporters. Here, we show that the apical sodium-dependent bile acid transporter (ASBT), organic solute transporter-α (OSTα), and organic anion transporting polypeptide 1a/1b (OATP1a/1b) transporters are dispensable for the norUDCA stimulation of bile flow and biliary bicarbonate secretion. Chloride channels in biliary epithelial cells provide the driving force for biliary secretion. In mouse large cholangiocytes, norUDCA potently stimulated chloride currents that were blocked by siRNA silencing and pharmacological inhibition of calcium-activated chloride channel transmembrane member 16A (TMEM16A) but unaffected by ASBT inhibition. In agreement, blocking intestinal bile acid reabsorption by coadministration of an ASBT inhibitor or bile acid sequestrant did not impact norUDCA stimulation of bile flow in WT mice. The results indicate that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

Bile acid metabolism disorder mediates hepatotoxicity of Nafion by-product 2 and perfluorooctane sulfonate in male PPARα-KO mice

Perfluorooctane sulfonate (PFOS) and Nafion by-product 2 (H-PFMO2OSA) induce hepatotoxicity in male mice via activation of the peroxisome proliferator-activated receptor α (PPARα) pathway; however, accumulating evidence suggests that PPARα-independent pathways also play a vital role in hepatotoxicity after exposure to per- and polyfluoroalkyl substances (PFASs). Thus, to assess the hepatotoxicity of PFOS and H-PFMO2OSA more comprehensively, adult male wild-type (WT) and PPARα knockout (PPARα-KO) mice were exposed to PFOS and H-PFMO2OSA (1 or 5 mg/kg/d) for 28 d via oral gavage. Results showed that although elevations in alanine transaminase (ALT) and aspartate aminotransferase (AST) were alleviated in PPARα-KO mice, liver injury, including liver enlargement and necrosis, was still observed after PFOS and H-PFMO2OSA exposure. Liver transcriptome analysis identified fewer differentially expressed genes (DEGs) in the PPARα-KO mice than in the WT mice, but more DEGs associated with the bile acid secretion pathway after PFOS and H-PFMO2OSA treatment. Total bile acid content in the liver was increased in the 1 and 5 mg/kg/d PFOS-exposed and 5 mg/kg/d H-PFMO2OSA-exposed PPARα-KO mice. Furthermore, in PPARα-KO mice, proteins showing changes in transcription and translation levels after PFOS and H-PFMO2OSA exposure were involved in the synthesis, transportation, reabsorption, and excretion of bile acids. Thus, exposure to PFOS and H-PFMO2OSA in male PPARα-KO mice may disturb bile acid metabolism, which is not under the control of PPARα.

Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis.

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10mg/kg s.c., the mice model of ICP) and labetalol (10mg/kg s.c.) for 5days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Fucoidan alleviates the hepatorenal syndrome through inhibition organic solute transporter α/β to reduce bile acids reabsorption

The high levels of bile acids are a critical factor in hepatorenal syndrome. Organic solute transporter α/β (Ostα/β) participate in bile acids reabsorption in the kidney. Fucoidan has the great potential in protecting against liver and kidney injury. However, whether Ostα/β increase bile acids reabsorption in bile duct ligature (BDL)-induced hepatorenal syndrome and the blockade of fucoidan are still not clear. Male mice that received BDL were given to fucoidan (at 12.5, 25 and 50 ​mg/kg) through intraperitoneal injection once daily for three weeks. The serum, liver and kidney samples of these experimental mice were collected to carry out biochemical, pathological and Western blot analysis. In this study, fucoidan significantly lowered serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), decreased serum levels of uric acid, creatinine and uric nitrogen, restored the deregulation of the renal urate transporter 1 (URAT1), organic anion transporter 1 (OAT1), and organic cation/carnitine transporter 1/2 (OCTN1/2), consistence with alleviation BDL-induced liver and kidney dysfunction, inflammation and fibrosis in mice. Furthermore, fucoidan significantly hampered Ostα/β and reduced bile acids reabsorption in BDL-induced mice, protected against AML12 and HK-2 ​cells injury in vitro. These results demonstrate that fucoidan alleviates BDL-induced hepatorenal syndrome through inhibition Ostα/β to reduce bile acids reabsorption in mice. Therefore, suppression of Ostα/β by fucoidan may be a novel strategy for attenuating hepatorenal syndrome.

Lead and copper influenced bile acid metabolism by changing intestinal microbiota and activating farnesoid X receptor in Bufo gargarizans

Lead (Pb) and copper (Cu) are ubiquitous metal contaminants and can pose a threat to ecosystem and human health. Bile acids have recently received considerable attention for their role in the maintenance of health. However, there were few studies on whether Pb and Cu affect bile acid metabolism in amphibians. In this study, a combination approach of histological analysis, targeted metabolomics, 16S rDNA sequencing and qPCR was used to explore the impacts of Pb, Cu and their mixture (Mix) on bile acid in Bufo gargarizans tadpoles. The results showed that Pb, Cu, and Mix resulted in intestinal damage and altered the bile acid profiles. Specifically, Pb and Mix exposure decreased total bile acid concentrations while increased toxic bile acid levels; in contrast, Cu exposure increased total bile acid levels. And hydrophilic bile acids were reduced in all treated tadpoles. Moreover, Pb and/or Cu changed the composition of intestinal microbiota, especially Clostridia, Bacteroides and Eubacterium involved in bile acid biotransformation. qPCR revealed that the decreased total bile acid concentrations in Pb- and Mix-treated tadpoles were most likely attributed to the activation of intestinal farnesoid X receptor (Fxr), which suppressed bile acid synthesis and reabsorption. While activated fxr in the Cu treatment group may be a regulatory mechanism in response to increased bile excretion, which is a detoxification route of tadpoles under Cu stress. Collectively, Pb, Cu and Mix changed bile acid profiles by affecting intestinal microbial composition and activating Fxr signaling. This study provided insight into the impacts of Pb and Cu on bile acid metabolism and contributed to the assessment of the potential ecotoxicity of heavy metals on amphibians.

Calculus Bovis Sativus alleviates estrogen cholestasis-induced gut and liver injury in rats by regulating inflammation, oxidative stress, apoptosis, and bile acid profiles

Ethnopharmacological relevanceNatural Calculus Bovis (NCB) is a traditional Chinese medicine used for anti-inflammation, treating fever, pain, sedation, and recovering hepatobiliary function. Calculus Bovis Sativus (CBS), produced from in vitro artificial cultivation by bioengineering techniques, acts as an ideal substitute for NCB when treating various diseases.Aim of the study: Gut-liver injury is an important pathological feature of several cholestatic liver diseases, including estrogen-induced cholestasis (EIC). The strong link between cholestatic liver injury and intestinal damage emphasizes the need of considering gut-liver integrity during treatment. The purpose of this study is to look into the pharmacological activities of CBS on EIC-induced gut and liver damage. Materials and methodsEIC-induced cholestatic rats were given oral gavage daily for five days with or without CBS (150 mg/kg). The liver/body weight, serum biochemistry, and tissue histopathology were then evaluated. Quantitative real-time PCR, Western blot analyses, and immunofluorescence were used to determine the gene expression associated with pathological alterations of the liver and intestine in EIC-induced cholestatic rats. Bile acid profiles within enterohepatic circulation were detected by liquid chromatography-mass spectrometry. ResultsCBS significantly reduced relative liver weight, restored serum biochemistry levels, and improved the hepatic and intestinal pathological damage in EIC model rats. CBS reduced EIC-induced hepatic inflammation by inactivation of the NF-κB signaling and inhibition of TNFα, IL-1β, and IL-6 expression. CBS alleviated EIC-induced hepatic and intestinal oxidative stress by regulating Nrf2-GCLM/GCLC and Nrf2-HO-1 pathways, respectively. CBS treatment upregulated Bcl-2 and downregulated Bax and cleaved caspase3 to improve EIC-induced hepatic and intestinal cell apoptosis. Additionally, CBS reversed the disorders of bile acid profiles in the enterohepatic circulation by reducing bile acid accumulation in the liver and plasma and increasing bile excretion and intestinal reabsorption of bile acids. ConclusionCBS alleviates EIC-induced hepatic and intestinal injury through regulating inflammation, oxidative stress, apoptosis, and bile acid profiles. These results suggest that CBS or drugs targeting the gut-liver axis may be effective therapeutic agents for cholestasis.

Nuciferine Protects Against High-Fat Diet-Induced Hepatic Steatosis via Modulation of Gut Microbiota and Bile Acid Metabolism in Rats.

Our previous study showed that nuciferine (NF) attenuated non-alcoholic fatty liver disease (NAFLD), which is attributed to a high-fat diet (HFD) through reinforcing intestinal barrier functions, regulating lipid metabolism, and improving inflammation. To clarify whether other mechanisms contribute to the anti-NAFLD efficacy of NF, the present study investigated the influence of NF on bile acid (BA) metabolism and gut microbiota in HFD-fed rats. The data demonstrated that NF changed the composition of colonic BA, particularly elevating conjugated BA and non-12OH BA levels. As shown by downregulated protein levels of FXR, FGF15, FGFR4, and ASBT and upregulated protein levels of CYP7A1 and CYP27A1, NF inhibited ileal FXR signaling, promoted BA synthesis, suppressed BA reabsorption, and facilitated fecal BA excretion. NF might affect hepatic FXR signaling, BA conjugation, and enterohepatic circulation by the changed mRNA levels of Fxr, Shp, Baat, Bacs, Bsep, Ntcp, Ibabp, and Ostα/β. Meanwhile, NF regulated the gut microbiota, characterized by decreased BSH-producing genus, 7α-dehydroxylation genus, and increased taurine metabolism-related genus. Spearman rank correlation analysis implied that Colidextribacter, Adlercreutzia, Family_XIII_AD3011_group, Lachnospiraceae_UCG-010, Eisenbergiella, and UCG-005 were robustly associated with particular BA monomers. In conclusion, our experiment results suggested that NF could exert a mitigating effect on NAFLD via regulating BA metabolism and modulating the gut microbiota.

Rationale and design of a multicenter, single-group, open-label trial aiming at investigating the effectiveness of elobixibat for loss of defecation desire in patients with chronic constipation

BackgroundApproximately 60% of patients with chronic constipation (CC) have a significantly higher rate of loss of defecation desire (LODD). Bile acids are expected to have a restorative effect on defecation desire (DD) because they lower the rectal sensory threshold, which is an objective index of DD. Elobixibat (EXB) specifically inhibits the ileal bile acid transporter/apical sodium-dependent bile acid transporter, which is a transporter involved in the reabsorption of bile acids in the terminal ileum. This study aims to investigate the LODD improvement rate in patients with CC after 4 weeks of EXB treatment. MethodsA total of 40 adult patients with CC who meet the eligibility criteria will be enrolled. Patients will receive oral EXB (10 mg/day) for 4 weeks. A patient diary will be provided daily at 4 weeks after treatment. The primary endpoint will be the percentage LODD improvement at week 4 of the treatment period from week 2 of the observation period using questionnaires. Ethics and disseminationEthical approval was obtained from the Yokohama City University Certified Institutional Review Board prior to participant enrolment (approval number: CRB21-008). The results of this study will be submitted for publication in international peer-reviewed journals, and key findings will be presented at international scientific conferences. Participants desiring the results of this study will be directly contacted for data dissemination. Trial registrationThis trial was registered at ClinicalTrials.gov (NCT05165199). Protocol version1.0, September 21, 2021.

Using targeted LC‐MS/MS‐based metabolomics to measure a broad constellation of bile acids/salts in disorders of human health

BackgroundBile is an important liver secretion which aids the digestion of fats in the intestine. Bile acids are the major organic solute component of bile and are synthesized from cholesterol in the liver and stored in the gallbladder. After secretion in the intestine, bile acids are re‐absorbed in the distal small intestine (ileum). Imbalances in the reabsorption of bile acids can lead to bile acid diarrhea when bile acid malabsorption occurs. Conversely, when there is an increase in bile acid absorption hepatic disorders such as nonalcoholic fatty liver disease can arise. A wide array of distinct bile acid structures have been identified in vertebrates. Developing and refining experimental tools to identify the role of bile acids in health and disease will enhance our ability to treat complex disorders impacting multiple organ systems.Methods & ResultsWe have expanded our existing reverse‐phase liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐based targeted metabolomics method to accommodate a simple‐to‐use standards kit that includes reference standards for 16 bile acids/salts and their corresponding deuterated isotopologs as internal standards. LC‐MS/MS method enhancements include scheduled Selected‐Reaction Monitoring (sSRM) to reduce instrument duty cycle, and carefully tailored chromatographic conditions to resolve the following groups of structurally similar or isobaric bile acids/salts: i) Glycocholic Acid (GCA) and Taurocholic Acid (TCA); ii) Lithocholic Acid (LCA), Glycolithocholic Acid (GLCA), and Taurolithocholic Acid (TLCA); iii) isobaric Cholic Acid (CA) and beta‐Muricholic Acid (b‐MCA); iv) isobaric Ursodeoxycholic Acid (UDCA), Chenodeoxycholic Acid (CDCA), and Deoxycholic Acid (DCA); v) isobaric Glycoursodeoxycholic Acid (GUDCA), Glycochenodeoxycholic Acid (GCDCA), and Glycodeoxycholic Acid (GDCA); and vi) isobaric Tauroursodeoxycholic Acid (TUDCA), Taurochenodeoxycholic Acid (TCDCA), and Taurodeoxycholic Acid (TDCA). Our tissue processing and metabolite extraction procedures have been optimized for hepatic and fecal tissues. Individuals with inactivating mutations in the molecular motor, Myosin Vb, have severe diarrhea and often present with liver cholestasis. We used an animal model of loss of Myosin Vb to determine alterations in bile acid composition in the distal small intestine and liver. Whole segments of the ileum and liver were processed in methanol and analyzed by LC‐MS/MS.ConclusionWe have developed a targeted metabolomics method to accurately identify and quantify bile acids by LC‐MS/MS and this technique can be used to investigate changes in bile acid composition in gastrointestinal disorders.

Modulation Mechanism of Proanthocyanidins from Leaves of Bayberry (Myrica rubra Sieb. Et Zucc.) on Hyperlipemic Rats Induced by High-fat-diet

Aim: To investigate the lipid-lowering effect of proanthocyanidins from Bayberry leaf (BLP) in rat model of hyperlipidemia and to clarify the underlying molecular mechanism.
 Study Design: The study includes in-vitro and in-vivo experiments in rat model.
 Place and Duration of Study: College of Life Sciences, China Jiliang University, Zhejiang Province, China, between March 2021 to December 2021.
 Methodology: BLP were preliminarily characterized. The adsorption rate of BLP was determined based on in-vitro binding to bile acids. The effect of BLP on membrane transport of bile acids was examined through Caco-2 mono-layer in trans-well. The effect of BLP on serum enzyme activity and lipid metabolism genes expression were also investigated in a high-fat diet rat model.
 Results: Total poly-phenol and proanthocyanidins contents were 97.93% and 82.25% respectively, using gallic acid and Epigallocatechin-3-O-Gallate (EGCG) as equivalent. The total flavonoid content was 12.05% (quercetin as equivalent) and the average polymerization degree of BLP was determined as 4.51. And the in-vitro average binding rates of BLP to bile acids were all higher than 80%. Meanwhile, the membrane transport of bile acids in Caco-2 monolayer cells were significantly obstructed by BLP. Furthermore, the total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) concentrations in rat serum were markedly decreased after 28-day of BLP treatment. And the hepatic steatosis was significantly ameliorated in high-dose BLP treatment group compared with high fat group. High-dose administration of BLP significantly reduced the levels of 3-hydroxy-3-3methylglutary-CoA (HMG-CoA) reductase, while the levels of ATP binding cassette transporters (ABCG-5), liver X receptor-α (LXR-α) and cholesterol-7α-hydroxylase (CYP7A1) were significantly increased.
 Conclusion: BLP can mediate the serum lipid metabolism via preventing bile acids re-absorption, reducing oxidative stress and regulating the expression of lipid metabolism-related genes.

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.

Flaxseed-derived peptides ameliorate hepatic cholesterol metabolism in Sprague-Dawley rats fed a high-cholesterol and high-fat diet.

Plant peptides have been reported to have cholesterol-lowering activities. Previous research has found that ≤1 kDa flaxseed peptide (FP5 ) reduces cholesterol absorption and synthesis in vitro. In this research, we investigated the cholesterol-lowering activity of FP5 in Sprague-Dawley (SD) rats fed a high-cholesterol and high-fat diet. In addition, amino acid sequences of FP5 were determined by high-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. FP5 supplement significantly decreased the serum and hepatic cholesterol levels and modulated the hepatic gene and protein expression of cholesterol metabolism-related enzymes or regulators (3-hydroxy-3-methylglutaryl coenzyme A reductase, Low-Density Lipoprotein Receptor (LDLR), Cholesterol 7 α-hydroxylase, Niemann-Pick C1-like 1, ATP-binding cassette transporters G5 and G8). Eleven peptides were identified from FP5 . These peptides were characterized as hydrophobic amino acids such as leucine (L), proline (P), glycine (G), isoleucine (I) and continuous sequences, including LP, LL, LG and II, with low molecular weights. FP5 has a certain cholesterol-lowering activity in SD rats fed a high-cholesterol and high-fat diet. The possible mechanism for ameliorating hepatic cholesterol metabolism of FP5 includes inhibiting hepatic cholesterol de novo synthesis, promoting the synthesis and excretion of bile acids, and inhibiting the reabsorption of bile acids during enterohepatic circulation. © 2022 Society of Chemical Industry.

Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairment of Bile Acid Metabolomics in Rats.

Multidrug resistance-associated protein 2 (Mrp2) mediates biliary secretion of anionic endobiotics and xenobiotics. Genetic alteration of Mrp2 leads to conjugated hyperbilirubinemia and predisposes to the development of intrahepatic cholestasis of pregnancy (ICP), characterized by increased plasma bile acids (BAs) due to mechanisms that are incompletely understood. Therefore, this study aimed to characterize BA metabolomics during experimental Mrp2 deficiency and ICP. ICP was modeled by ethinylestradiol (EE) administration to Mrp2-deficient (TR) rats and their wild-type (WT) controls. Spectra of BAs were analyzed in plasma, bile, and stool using an advanced liquid chromatography–mass spectrometry (LC–MS) method. Changes in BA-related genes and proteins were analyzed in the liver and intestine. Vehicle-administered TR rats demonstrated higher plasma BA concentrations consistent with reduced BA biliary secretion and increased BA efflux from hepatocytes to blood via upregulated multidrug resistance-associated protein 3 (Mrp3) and multidrug resistance-associated protein 4 (Mrp4) transporters. TR rats also showed a decrease in intestinal BA reabsorption due to reduced ileal sodium/bile acid cotransporter (Asbt) expression. Analysis of regulatory mechanisms indicated that activation of the hepatic constitutive androstane receptor (CAR)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by accumulating bilirubin may be responsible for changes in BA metabolomics in TR rats. Ethinylestradiol administration to TR rats further increased plasma BA concentrations as a result of reduced BA uptake and increased efflux via reduced Slco1a1 and upregulated Mrp4 transporters. These results demonstrate that Mrp2-deficient organism is more sensitive to estrogen-induced cholestasis. Inherited deficiency in Mrp2 is associated with activation of Mrp3 and Mrp4 proteins, which is further accentuated by increased estrogen. Bile acid monitoring is therefore highly desirable in pregnant women with conjugated hyperbilirubinemia for early detection of intrahepatic cholestasis.

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.