Endogenous Bile Acids Research Articles

Structural basis for selective inhibition of human serine hydroxymethyltransferase by secondary bile acid conjugate

SummaryBile acids are metabolites of cholesterol that facilitate lipid digestion and absorption in the small bowel. Bile acids work as agonists of receptors to regulate their own metabolism. Bile acids also regulate other biological systems such as sugar metabolism, intestinal multidrug resistance, and adaptive immunity. However, numerous physiological roles of bile acids remain undetermined. In this study, we solved the crystal structure of human serine hydroxymethyltransferase (hSHMT) in complex with an endogenous secondary bile acid glycine conjugate. The specific interaction between hSHMT and the ligand was demonstrated using mutational analyses, biophysical measurements, and structure-activity relationship studies, suggesting that secondary bile acid conjugates may act as modulators of SHMT activity.

Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids

AbstractCytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3’-phosphoadenosine 5’-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3’,5’-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species.

Tauroursodeoxycholic acid (TUDCA) is neuroprotective in a chronic mouse model of Parkinson’s disease

ABSTRACT Objective: Parkinson’s disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp. Methods: Markers for dopaminergic function, neuroinflammation, oxidative stress and autophagy were assessed using high performance liquid chromatography (HPLC), immunohistochemistry (IHC) and western blot (WB) methods. Locomotion was measured before and after treatments. Results: MPTPp decreased the expression of dopamine transporters (DAT) and tyrosine hydroxylase (TH), indicating dopaminergic damage, and induced microglial and astroglial activation as demonstrated by IHC analysis. MPTPp also decreased DA and its metabolites as demonstrated by HPLC analysis. Further, MPTPp-induced protein oxidation; increased LAMP-1 expression indicated autophagy and the promotion of alpha-synuclein (α-SYN) aggregation.. Discussion: Pretreatment with TUDCA protected against dopaminergic neuronal damage, prevented the microglial and astroglial activation, as well as the DA and DOPAC reductions caused by MPTPp. TUDCA by itself did not produce any significant change, with data similar to the negative control group. Pretreatment with TUDCA prevented protein oxidation and autophagy, in addition to inhibiting α-SYN aggregation. Although TUDCA pretreatment did not significantly affect locomotion, only acute treatment effects were measured, indicating more extensive assessments may be necessary to reveal potential therapeutic effects on behavior. Together, these results suggest that autophagy may be involved in the progression of PD and that TUDCA may attenuate these effects. The efficacy of TUDCA as a novel therapy in patients with PD clearly warrants further study.

Bariatric surgery reveals a gut-restricted TGR5 agonist with anti-diabetic effects.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone GLP-1 and changes in levels of circulating bile acids. The levels of individual bile acids in the GI tract following surgery, however, have remained largely unstudied. Using UPLC-MS-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a TGR5 agonist that increases Tgr5 expression and induces GLP-1 secretion. Further, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut-restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into circulation. By studying changes in individual metabolites following surgery, this study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

TUDCA inhibits HSV-1 replication by the modulating unfolded protein response pathway.

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, was used to protect liver function through antiapoptosis or reducing endoplasmic reticulum stress (ER stress). Previous studies showed that ER stress was modulated by herpes simplex virus types 1 (HSV-1) infection to facilitate viral replication. Here, we investigated the effect of TUDCA on HSV-1 infection of HEC-1-A cells and showed that both replication and multiplication of the virus were inhibited by TUDCA in a dose dependent manner. Unfolded protein response was induced to deliver stress signals from ER to nucleus. We found that TUDCA alleviated activating transcription factor 6 branch inhibition, partially enhanced protein kinase RNA-like ER kinase pathway activation, and repressed inositol-requiring protein 1α arm activation significantly in infected cells. The findings of this study suggest that TUDCA inhibits HSV-1 replication through ER stress pathway, which may provide a potential therapeutic strategy for HSV-1 infection.

Bile Acids Signal via TGR5 to Activate Intestinal Stem Cells and Epithelial Regeneration

Renewal and patterning of the intestinal epithelium is coordinated by intestinal stem cells (ISCs); dietary and metabolic factors provide signals to the niche that control ISC activity. Bile acids (BAs), metabolites in the gut, signal nutrient availability by activating the G protein-coupled bile acid receptor 1 (GPBAR1, also called TGR5). TGR5 is expressed in the intestinal epithelium, but it is not clearhowitsactivation affects ISCs and regeneration of the intestinal epithelium. We studied the role of BAs and TGR5 inintestinal renewal, and regulation of ISC function in mice and intestinal organoids. We derived intestinal organoids from wild-type mice and Tgr5-/- mice, incubated them with BAs or the TGR5 agonist INT-777, and monitored ISC function by morphologic analyses and colony-forming assays. We disrupted Tgr5 specifically in Lgr5-positive ISCs in mice (Tgr5ISC-/- mice) and analyzed ISC number, proliferation, and differentiation by flow cytometry, immunofluorescence, and organoid assays. Tgr5ISC-/- mice were given cholecystokinin; we measured the effects of BA release into the intestinal lumen and on cell renewal. We induced colitis in Tgr5ISC-/- mice by administration of dextran sulfate sodium; disease severity was determined based on body weight, colon length, and histopathology analysis of colon biopsies. BAs and TGR5 agonists promoted growth of intestinal organoids. Administration of cholecystokinin to mice resulted in acute release of BAs into the intestinal lumen and increased proliferation of the intestinal epithelium. BAs and Tgr5 expression in ISCs were required for homeostatic intestinal epithelial renewal and fate specification, and for regeneration after colitis induction. Tgr5ISC-/- mice developed more severe colitis than mice without Tgr5 disruption in ISCs. ISCs incubated with INT-777 increased activation of yes-associated protein 1 (YAP1) andofits upstream regulator SRC. Inhibitors of YAP1 and SRC prevented organoid growth induced by TGR5 activation. BAs promote regeneration of the intestinal epithelium via activation of TGR5 in ISCs, resulting in activation of SRC and YAP and activation of their target genes.Release of endogenous BAs in the intestinal lumenissufficient to promote ISC renewal and drives regeneration in response to injury.

Reprogramming of Lipid Metabolism as a New Driving Force Behind Tauroursodeoxycholic Acid-Induced Neural Stem Cell Proliferation.

Recent evidence suggests that neural stem cell (NSC) fate is highly dependent on mitochondrial bioenergetics. Tauroursodeoxycholic acid (TUDCA), an endogenous neuroprotective bile acid and a metabolic regulator, stimulates NSC proliferation and enhances adult NSC pool in vitro and in vivo. In this study, we dissected the mechanism triggered by this proliferation-inducing molecule, namely in mediating metabolic reprogramming. Liquid chromatography coupled with mass spectrometry (LC-MS) based detection of differential proteomics revealed that TUDCA reduces the mitochondrial levels of the long-chain acyl-CoA dehydrogenase (LCAD), an enzyme crucial for β-oxidation of long-chain fatty acids (FA). TUDCA impact on NSC mitochondrial proteome was further confirmed, including in neurogenic regions of adult rats. We show that LCAD raises throughout NSC differentiation, while its silencing promotes NSC proliferation. In contrast, nuclear levels of sterol regulatory element-binding protein (SREBP-1), a major transcription factor of lipid biosynthesis, changes in the opposite manner of LCAD, being upregulated by TUDCA. In addition, alterations in some metabolic intermediates, such as palmitic acid, also supported the TUDCA-induced de novo lipogenesis. More interestingly, a metabolic shift from FA to glucose catabolism appears to occur in TUDCA-treated NSCs, since mitochondrial levels of pyruvate dehydrogenase E1-α (PDHE1-α) were significant enhanced by TUDCA. At last, the mitochondria-nucleus translocation of PDHE1-α was potentiated by TUDCA, associated with an increase of H3-histones and acetylated forms. In conclusion, TUDCA-induced proliferation of NSCs involves metabolic plasticity and mitochondria-nucleus crosstalk, in which nuclear PDHE1-α might be required to assure pyruvate-derived acetyl-CoA for histone acetylation and NSC cycle progression.

Bile acid metabolism is altered in multiple sclerosis and supplementation ameliorates neuroinflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the CNS. Bile acids are cholesterol metabolites that can signal through receptors on cells throughout the body, including in the CNS and the immune system. Whether bile acid metabolism is abnormal in MS is unknown. Using global and targeted metabolomic profiling, we identified lower levels of circulating bile acid metabolites in multiple cohorts of adult and pediatric patients with MS compared with controls. In white matter lesions from MS brain tissue, we noted the presence of bile acid receptors on immune and glial cells. To mechanistically examine the implications of lower levels of bile acids in MS, we studied the in vitro effects of an endogenous bile acid, tauroursodeoxycholic acid (TUDCA), on astrocyte and microglial polarization. TUDCA prevented neurotoxic (A1) polarization of astrocytes and proinflammatory polarization of microglia in a dose-dependent manner. TUDCA supplementation in experimental autoimmune encephalomyelitis reduced the severity of disease through its effects on G protein-coupled bile acid receptor 1 (GPBAR1). We demonstrate that bile acid metabolism was altered in MS and that bile acid supplementation prevented polarization of astrocytes and microglia to neurotoxic phenotypes and ameliorated neuropathology in an animal model of MS. These findings identify dysregulated bile acid metabolism as a potential therapeutic target in MS.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 μm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

Research Note: Evaluation of deoxycholic acid for antihistomonal activity

Deoxycholic acid (DCA) is a naturally occurring secondary bile acid that originates from intestinal bacterial metabolic conversion of cholate, a primary bile acid. Deoxycholic acid was shown to have antihistomonal properties in vitro, leading to our hypothesis that DCA inclusion within the feed might prevent histomoniasis. Selected dietary concentrations of DCA were evaluated for effects on body weight gain (BWG), lesions, and mortality of turkeys challenged with wild-type Histomonas meleagridis (WTH). Treatments consisted of non-challenged control (NC; basal diet), 0.25% DCA diet + challenge, 0.5% DCA diet + challenge, 1% DCA diet + challenge, and a positive-challenged control (PC; basal diet). All groups were fed a basal starter diet until day 7, at which time DCA diets were administered to the respective groups. On day 14, 2 × 105 WTH cells/turkey were intracloacally administered. H. meleagridis-related lesions were evaluated on day 13 post-challenge. Pre-challenge day 0 to 14 BWG was higher (P ≤ 0.05) in the 0.25% DCA group than in the 1% DCA group. There were no significant differences in pre-challenge day 0 to 14 BWG between any of the other groups. No significant differences in mortalities from histomoniasis occurred in the DCA groups as compared to the PC group. No H. meleagridis lesions or mortalities were observed at any time in the NC group. Presence of H. meleagridis-related liver lesions was higher (P ≤ 0.05) in the 0.5% DCA group as compared to the PC group. Using the same controls and experimental timeline, an additional group was included to evaluate a biliogenic diet formulated with 20% whole egg powder to encourage endogenous bile acid production. The biliogenic diet had no statistical impact on pre-challenge day 0 to 14 BWG, but did not reduce H. meleagridis-related mortalities or lesions after the challenge. Taken together, these data suggest that DCA inclusion within the feed at these concentrations and under these experimental conditions does not prevent histomoniasis.

FXR regulates metabolic function of fat depots during obesity

Obesity is a major epidemic and is associated with excess calorie intake leading to adipose tissue expansion and metabolic dysfunction. Farnesoid X receptor (FXR) is a nuclear receptor that transcriptionally regulates lipid and glucose metabolism. Despite several studies revealing FXR expression in the adipose tissue, its exact role in this tissue remains elusive. We determined that both primary pre‐ and post‐differentiated adipocytes from inguinal white (WAT) and brown (BAT) adipose tissue express Fxr transcripts. Notably, adipose‐specific Fxr knockout (Ad‐FxrKO) mice recently generated in our laboratory exhibit glucose intolerance, increased fat accumulation and concomitant reduction in the expression of lipolytic genes in both WAT and BAT when challenged with a high‐fat diet. These data suggest that adipose‐specific FXR may play a crucial role in regulating fat accumulation and subsequent glucose homeostasis during diet‐induced obesity. Since BAT is specialized for non‐shivering thermogenesis by generating heat through uncoupled mitochondrial respiration, we investigated the role for FXR in brown adipocyte mitochondrial function. We found that when Fxr is deleted, genes involved in BAT mitochondrial β‐oxidation, biosynthesis and respiratory chain complex are down‐regulated. Conversely, FXR activation by its endogenous bile acid ligand, chenodeoxycholic acid increases mitochondrial membrane potential of primary differentiated brown adipocytes in vitro, suggesting a key role for FXR in controlling brown adipocyte mitochondrial function and a potential role in thermogenesis. Overall, we demonstrate that FXR can modulate fat break down and regulate mitochondrial functions in the adipose tissue.Support or Funding InformationSupported by R01 DK113080 from NIDDK, and UIUC Start Up Funds.

Ursodeoxycholic acid enriches intestinal bile salt hydrolase-expressing Bacteroidetes in cholestatic pregnancy

Ursodeoxycholic acid (UDCA) treatment can reduce itch and lower endogenous serum bile acids in intrahepatic cholestasis of pregnancy (ICP). We sought to determine how it could influence the gut environment in ICP to alter enterohepatic signalling. The gut microbiota and bile acid content were determined in faeces from 35 pregnant women (14 with uncomplicated pregnancies and 21 with ICP, 17 receiving UDCA). Faecal bile salt hydrolase activity was measured using a precipitation assay. Serum fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) concentrations were measured following a standardised diet for 21 hours. Women with a high ratio of Bacteroidetes to Firmicutes were more likely to be treated with UDCA (Fisher’s exact test p = 0.0178) than those with a lower ratio. Bile salt hydrolase activity was reduced in women with low Bacteroidetes:Firmicutes. Women taking UDCA had higher faecal lithocholic acid (p < 0.0001), with more unconjugated bile acids than women with untreated ICP or uncomplicated pregnancy. UDCA-treatment increased serum FGF19, and reduced C4 (reflecting lower bile acid synthesis). During ICP, UDCA treatment can be associated with enrichment of the gut microbiota with Bacteroidetes. These demonstrate high bile salt hydrolase activity, which deconjugates bile acids enabling secondary modification to FXR agonists, enhancing enterohepatic feedback via FGF19.

Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct-Ligated Mouse.

Background & AimsInflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study.MethodsGene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1–/– (Casp1–/–) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids.ResultsCaspase-1, NLRP1, NLRP3 and IL-1β were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1β (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1–/– BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1–/– BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1–/– BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1–/– BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300μM TCA), neither IL-1β induction nor procaspase-1 cleavage were detected.ConclusionsThe inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.

A second-generation micro/nano capsules of an endogenous primary un-metabolised bile acid, stabilized by Eudragit-alginate complex with antioxidant compounds

Bile acids (BAs) are amphiphilic compounds and of recently have demonstrated wide range of formulation stabilizing effects. A recent study showed that primary un-metabolised bile acids (PUBAs) have β-cell protective effects, and synergistic antidiabetic effects when combined with antioxidant and anti-inflammatory drugs, such as probucol (PB). Thus, this study aimed to design and test microcapsules containing a PUBA incorporated with PB and an alginate-Eudragit matrix.Six types of microcapsules were developed without (control) or with (test) PUBA, and tested for internal and external features and β-cell protective effects.The incorporation of PB-alginate-Eudragit with PUBA produced stable microcapsules but did not exert consistent positive effects on cell viability in the hyperglycaemic state, which suggests that PUBA in alginate-Eudragit matrices did not exhibit synergistic effects with PB nor exerted antidiabetic effects.

Bile acids in glucose metabolism and insulin signalling - mechanisms and research needs.

Of all the novel glucoregulatory molecules discovered in the past 20 years, bile acids (BAs) are notable for the fact that they were hiding in plain sight. BAs were well known for their requirement in dietary lipid absorption and biliary cholesterol secretion, due to their micelle-forming properties. However, it was not until 1999 that BAs were discovered to be endogenous ligands for the nuclear receptor FXR. Since that time, BAs have been shown to act through multiple receptors (PXR, VDR, TGR5 and S1PR2), as well as to have receptor-independent mechanisms (membrane dynamics, allosteric modulation of N-acyl phosphatidylethanolamine phospholipase D). We now also have an appreciation of the range of physiological, pathophysiological and therapeutic conditions in which endogenous BAs are altered, raising the possibility that BAs contribute to the effects of these conditions on glycaemia. In this Review, we highlight the mechanisms by which BAs regulate glucose homeostasis and the settings in which endogenous BAs are altered, and provide suggestions for future research.

New treatments/targets for primary biliary cholangitis.

SummaryPrimary biliary cholangitis (PBC) is an autoimmune, cholestatic, chronic liver disease that ultimately progresses towards cirrhosis and liver failure if untreated. While ursodeoxycholic acid has been established as standard of care for PBC in the last few decades, significant advances in second-line treatment options have recently been made and new therapeutic developments are currently under evaluation. The purpose of this article is to provide the clinician with an overview of the current treatment options and future opportunities for patients with PBC.

Obeticholic acid may increase the risk of gallstone formation in susceptible patients

The nuclear farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has been developed for the treatment of liver diseases. We aimed to determine whether OCA treatment increases the risk of gallstone formation. Twenty patients awaiting laparoscopic cholecystectomy were randomized to treatment with OCA (25 mg/day) or placebo for 3 weeks until the day before surgery. Serum bile acids (BAs), the BA synthesis marker C4 (7α-hydroxy-4-cholesten-3-one), and fibroblast growth factor 19 (FGF19) were measured before and after treatment. During surgery, biopsies from the liver and the whole bile-filled gallbladder were collected for analyses of gene expression, biliary lipids and FGF19. In serum, OCA increased FGF19 (from 95.0 ± 8.5 to 234.4 ± 35.6 ng/L) and decreased C4 (from 31.4 ± 22.8 to 2.8 ± 4.0 nmol/L) and endogenous BAs (from 1,312.2 ± 236.2 to 517.7 ± 178.9 nmol/L; all p <0.05). At surgery, BAs in gallbladder bile were lower in patients that received OCA than in controls (OCA, 77.9 ± 53.6 mmol/L; placebo, 196.4 ± 99.3 mmol/L; p <0.01), resulting in a higher cholesterol saturation index (OCA, 2.8 ± 1.1; placebo, 1.8 ± 0.8; p <0.05). In addition, hydrophobic OCA conjugates accounted for 13.6 ± 5.0% of gallbladder BAs after OCA treatment, resulting in a higher hydrophobicity index (OCA, 0.43 ± 0.09; placebo, 0.34 ± 0.07, p <0.05). Gallbladder FGF19 levels were 3-fold higher in OCA patients than in controls (OCA, 40.3 ± 16.5 ng/L; placebo, 13.5 ± 13.1 ng/ml; p <0.005). Gene expression analysis indicated that FGF19 mainly originated from the gallbladder epithelium. Our results show for the first time an enrichment of FGF19 in human bile after OCA treatment. In accordance with its murine homolog FGF15, FGF19 might trigger relaxation and filling of the gallbladder which, in combination with increased cholesterol saturation and BA hydrophobicity, would enhance the risk of gallstone development. Obeticholic acid increased human gallbladder cholesterol saturation and bile acid hydrophobicity, both decreasing cholesterol solubility in bile. Together with increased hepatobiliary levels of fibroblast growth factor 19, our findings suggest that pharmacological activation of the farnesoid X receptor increases the risk of gallstone formation. Clinical trial number: NCT01625026.

1966-P: Manipulating Postprandial Bile Acid Concentrations: Effect on GLP-1 Secretion after Roux-en-Y Gastric Bypass

Enhanced glucagon-like peptide-1 (GLP-1) secretion is important for improved glycemic control after Roux-en-Y gastric bypass (RYGB) and higher concentrations of circulating bile acids (BA) have been suggested to be involved. The BA chenodeoxycholic acid (CDCA) stimulates GLP-1 secretion after RYGB in the absence of nutrients. In this study we investigated the effects of oral intake of CDCA or the BA sequestrant colesevelam (COL) on postprandial GLP-1 secretion in RYGB operated subjects. We hypothesized that CDCA would increase secretion and that COL would decrease secretion by reducing re-absorption of endogenous BA. Twelve participants (BMI 31±2 (mean±SEM) kg/m2, age 43±3 years, time from RYGB 4.3±0.5 years, weight loss after RYGB 34±5 kg) were studied in a single-blinded, randomised study with four experimental days: 1) A mixed meal consisting of solid/semisolid components (1523 kJ, 53E% carb, 33E% fat, 14E% prot) 2) Mixed meal added 1250 mg CDCA 3) Mixed meal added COL 3750 mg 4) Mixed meal with COL administered both as 3750 mg tablets the evening before the experiment and as 3750 mg added to the meal. Oral intake of CDCA increased GLP-1 significantly compared with mixed meal alone (p=0.03), whereas neither single (p=0.74) nor double (p=0.28) dosage of COL affected GLP-1 secretion (Incremental AUC (iAUC) GLP-1: Meal: 2792±288 pmol x min, CDCA: 4362±618, COL: 2703±330, COLx2: 3328±380). Plasma glucose and C-peptide (iAUC) decreased after CDCA but both were unchanged after COL administration vs. meal alone. Beta-cell function (AUC C-peptide/AUC plasma glucose) was equal between all study days. In summary, administration of the exogenous BA CDCA potentiated mixed-meal stimulated GLP-1 secretion after RYGB but did not affect beta-cell function. Administration of the BA sequestrant COL did not affect GLP-1 secretion, glucose concentrations or beta-cell function, suggesting a limited role for endogenous BA for regulation of postprandial glucose metabolism after RYGB. Disclosure M.S. Svane: None. I. Jonsson: None. V. Kristiansen: None. R.E. Kuhre: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. S. Madsbad: None. K.N. Bojsen-Moller: None. Funding European Research Council (695069)

Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer [N-methyl-11C]cholylsarcosine

Introduction[N-methyl-11C]cholylsarcosine ([11C]CSar) is a tracer for imaging and quantitative assessment of intrahepatic cholestatic liver diseases and drug-induced cholestasis by positron emission tomography (PET). The purpose of this study is to determine whole-body biodistribution and dosimetry of [11C]CSar in healthy humans. The results are compared with findings in a patient with primary sclerosing cholangitis (PSC) and a patient with primary biliary cholangitis (PBC) as well as with preclinical findings in pigs. Radiosynthesis and quality control for preparation of [11C]CSar for clinical use are also presented. MethodsRadiosynthesis and quality control of [11C]CSar were set up in compliance with Danish/European regulations. Both healthy participants (3 females, 3 males) and patients underwent whole-body PET/CT to determine the biodistribution of [11C]CSar. The two patients were under treatment with ursodeoxycholic acid at the time of the study. Dosimetry was estimated from the PET data using the Olinda 2.0 software. ResultsThe radiosynthesis provided [11C]CSar in a solution ready for injection. The biodistribution studies revealed that gallbladder wall, small intestine, and liver were critical organs in both healthy participants and patients with the gallbladder wall receiving the highest dose (up to 0.5 mGy/MBq). The gender-averaged (±SD) effective dose for the healthy participants was 6.2 ± 1.4 μSv/MBq. The effective dose for the PSC and the PBC patient was 5.2 and 7.0 μSv/MBq, respectively. ConclusionA radiosynthesis for preparation of [11C]CSar for clinical use was developed and approved by the Danish Medicines Agency. The most critical organ was the gallbladder wall although the amount of [11C]CSar in the gallbladder was found to vary significantly between individuals. The estimated effective dose for humans was comparable to that estimated in anesthetized pigs although the absorbed dose estimates to some organs, such as the stomach, was different. Advances in knowledge and implications for patient care[11C]CSar PET/CT enables detailed quantitative assessment of patients with cholestatic liver disease by tracing the separate hepatobiliary transport steps of endogenous bile acids. The present work offers a radiosynthetic method and dosimetry data suitable for clinical implementation of [11C]CSar.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.