Jonathan D. Nolan

Potent stimulation of fibroblast growth factor 19 expression in the human ileum by bile acids

Fibroblast growth factor 19 (FGF19) is proposed to be a negative feedback regulator of hepatic bile acid (BA) synthesis. We aimed to clarify the distribution of FGF19 expression in human intestine and to investigate induction in a novel explant system. Ileal and colonic mucosal biopsies were obtained at endoscopy and analyzed for FGF19 transcript expression. Primary explants were incubated with physiological concentrations of various BA for up to 6 h, and expression of FGF19 and other genes was determined. FGF19 transcripts were detected in ileum but were unquantifiable in colon. No loss of FGF19 mRNA occurred as a consequence of the explant system. Ileal FGF19 transcript expression was induced 350-fold by 50 μM chenodeoxycholate (CDCA, n = 24, P < 0.0001) and 161-fold by 50 μM glycochenodeoxycholate (GCDCA, n = 12, P = 0.0005). The responses of other genes to CDCA or GCDCA (50 μM) were smaller: median increases of ileal bile acid binding protein, organic solute transporter-α and -β, and short heterodimer partner were 2.4- to 4.0-fold; apical membrane sodium bile acid transporter and farnesoid X receptor (FXR) showed little change. The EC50 for FGF19 transcript induction by CDCA was 20 μM. FGF19 protein concentrations were significantly higher in the culture fluid from BA-stimulated explants. FGF19 induction with cholate was 81% of that found with CDCA, but deoxycholate (40%) and lithocholate (4%) were significantly less potent. The synthetic FXR agonist obeticholic acid was much more potent than CDCA with a 70-fold FGF19 stimulation at 1 μM. We concluded that FGF19 expression in human ileum is very highly responsive to BA. Changes in FGF19 induction are a potential mechanism involved in disorders of BA homeostasis.

New Insights into Bile Acid Malabsorption

Bile acid malabsorption occurs when there is impaired absorption of bile acids in the terminal ileum, so interrupting the normal enterohepatic circulation. The excess bile acids in the colon cause diarrhea, and treatment with bile acid sequestrants is beneficial. The condition can be diagnosed with difficulty by measuring fecal bile acids, or more easily by retention of selenohomocholyltaurine (SeHCAT), where this is available. Chronic diarrhea caused by primary bile acid diarrhea appears to be common, but is under-recognized where SeHCAT testing is not performed. Measuring excessive bile acid synthesis with 7α-hydroxy-4-cholesten-3-one may be an alternative means of diagnosis. It appears that there is no absorption defect in primary bile acid diarrhea but, instead, an overproduction of bile acids. Fibroblast growth factor 19 (FGF19) inhibits hepatic bile acid synthesis. Defective production of FGF19 from the ileum may be the cause of primary bile acid diarrhea.

Characterizing Factors Associated With Differences in FGF19 Blood Levels and Synthesis in Patients With Primary Bile Acid Diarrhea

OBJECTIVES:Chronic diarrhea caused by primary bile acid diarrhea (PBAD) is a common condition. We have previously shown PBAD is associated with low fasting serum levels of the ileal hormone, fibroblast growth factor 19 (FGF19). FGF19 is a negative regulator of hepatic bile acid synthesis and is stimulated by farnesoid X receptor agonists, which produce symptomatic improvement in PBAD. We aimed to assess possible causes for low serum FGF19 in patients with PBAD.METHODS:Patients with PBAD, defined by reduced 75Se-labelled homocholic acid taurine (SeHCAT) retention, and idiopathic diarrhea controls had measurements of fasting lipids and fasting/post-prandial FGF19 serum profiles. Specific functional variants in candidate genes were investigated in exploratory studies. In further groups, basal and bile acid-stimulated transcript expression was determined in ileal biopsies and explant cultures by quantitative PCR.RESULTS:FGF19 profiles in PBAD patients included low fasting and meal-stimulated responses, which were both strongly correlated with SeHCAT. A subgroup of 30% of PBAD patients had fasting hypertriglyceridemia and higher FGF19. No clear significant differences were found for any genetic variant but there were borderline associations with FGFR4 and KLB. SeHCAT retention significantly correlated with the basal ileal transcript expression of FGF19 (rs=0.59, P=0.03) and apical sodium-dependent bile acid transporter (ASBT) (rs=0.49, P=0.04), and also with the degree of stimulation by chenodeoxycholic acid at 6 h for transcripts of FGF19 (median 184-fold, rs=0.50, P=0.02) and ileal bile acid binding protein (IBABP) (median 2.2-fold, rs=0.47, P=0.04). Median stimulation of FGF19 was lower in patients with SeHCAT retention <10% (P=0.01).CONCLUSIONS:These studies demonstrate a complex, multifactorial etiology of PBAD, including impairments in ileal FGF19 expression and responsiveness.

Altered enterohepatic circulation of bile acids in Crohn's disease and their clinical significance: a new perspective

The role of bile acids (BA) extends far beyond lipid digestion and cholesterol metabolism. The transcriptional regulation of multiple genes within the liver and intestine are under their influence. BA exert these effects through binding and activating receptors in much the same way as endocrine hormones. The farnesoid X receptor (FXR) is the intracellular transcription factor for BA; TGR5 is the cell-surface receptor. The main target genes of FXR are those involved in BA and cholesterol metabolism. Yet more recently, FXR has also been shown to influence and promote certain protective pathways within the liver. These pathways are being harnessed by semisynthetic BAs in Phase II and III clinical trials. FXR activation within the intestine is also associated with similar protective pathways. This article examines the consequences of altered FXR activation in the context of BA malabsorption in Crohn’s disease and the potential benefits of FXR agonists in Crohn’s disease.

Tu1262 Obeticholic Acid, a Farnesoid X Receptor Agonist, Reduces Bile Acid Synthesis in Patients With Primary Bile Acid Diarrhea

ACKNOWLEDGMENTS & CORRESPONDENCE IJ and JN were supported by the Bardhan Research and Education Trust. Obeticholic acid was donated by Intercept Pharmaceuticals. Disclosures: DS is employed by Intercept Pharmaceuticals. JW has received consulting, speaking and teaching fees from GE Healthcare, Intercept, Novartis, NGMBio, Sanofi and Pendopharm. Email: julian.walters@imperial.ac.uk; 1Claire Vassie, 1Ian M Johnston, 1Jonathan D Nolan, 2David Shapiro, 1Julian RF Walters

Diet1, bile acid diarrhea, and FGF15/19: mouse model and human genetic variants

Diet1 modulates intestinal production of the hormone, fibroblast growth factor (FGF)15, which signals in liver to regulate bile acid synthesis. C57BL/6ByJ mice with a spontaneous Diet1-null mutation are resistant to hypercholesterolemia compared with wild-type C57BL/6J mice through enhanced cholesterol conversion to bile acids. To further characterize the role of Diet1 in metabolism, we generated Diet1−/− mice on the C57BL/6J genetic background. C57BL/6J Diet1−/− mice had elevated bile acid levels, reduced Fgf15 expression, and increased gastrointestinal motility and intestinal luminal water content, which are symptoms of bile acid diarrhea (BAD) in humans. Natural genetic variation in Diet1 mRNA expression levels across 76 inbred mouse strains correlated positively with Ffg15 mRNA and negatively with serum bile acid levels. This led us to investigate the role of DIET1 genetic variation in primary BAD patients. We identified a DIET1 coding variant (rs12256835) that had skewed prevalence between BAD cases and controls. This variant causes an H1721Q amino acid substitution that increases the levels of FGF19 protein secreted from cultured cells. We propose that genetic variation in DIET1 may be a determinant of FGF19 secretion levels, and may affect bile acid metabolism in both physiological and pathological conditions.

Novel associations of bile acid diarrhoea with fatty liver disease and gallstones: a cohort retrospective analysis

Background Bile acid diarrhoea (BAD) is a common cause of chronic diarrhoea with a population prevalence of primary BAD around 1%. Previous studies have identified associations with low levels of the ileal hormone fibroblast growth factor 19 (FGF19), obesity and hypertriglyceridaemia. The aim of this study was to identify further associations of BAD. Methods A cohort of patients with chronic diarrhoea who underwent 75selenohomocholic acid taurate (SeHCAT) testing for BAD was further analysed retrospectively. Additional clinical details available from the electronic patient record, including imaging, colonoscopy, chemistry and histopathology reports were used to calculate the prevalence of fatty liver disease, gallstones, colonic neoplasia and microscopic colitis, which was compared for BAD, the primary BAD subset and control patients with diarrhoea. Findings Of 578 patients, 303 (52%) had BAD, defined as a SeHCAT 7d retention value <15%, with 179 (31%) having primary BAD. 425 had an alanine aminotransferase (ALT) recorded, 184 had liver imaging and 176 had both. Overall, SeHCAT values were negatively associated with ALT (rs=−0.19, p<0.0001). Patients with BAD had an OR of 3.1 for an ALT >31 ng/mL with imaging showing fatty liver (p<0.001); similar figures occurred in the primary BAD group. FGF19 was not significantly related to fatty liver but low levels were predictive of ALT >40 IU/L. In 176 subjects with gallbladder imaging, 27% had gallstones, 7% had a prior cholecystectomy and 34% either of these. The median SeHCAT values were lower in those with gallstones (3.8%, p<0.0001), or gallstones/cholecystectomy (7.2%, p<0.001), compared with normal gallbladder imaging (14%). Overall, BAD had an OR of 2.0 for gallstones/cholecystectomy (p<0.05). BAD was not significantly associated with colonic adenoma/carcinoma or with microscopic colitis. Interpretation The diagnosis of BAD is associated with fatty liver disease and with gallstones. The reasons for these associations require further investigation into potential metabolic causes.

PTH-251 Bile acid diarrhoea is associated with gallstones

Introduction Bile acid diarrhoea (BAD) is a common disorder which in the primary, idiopathic form may result from low serum fibroblast growth factor 19 (FGF19) producing impaired regulation of bile acid synthesis. 75Selenium Homocholic Acid Taurate (SeHCAT) 7day retention is used in diagnosis, predicting faecal bile acid loss and correlating with fasting serum FGF19. Several groups have shown that overweight and obesity are associated with BAD and low FGF19. Reduced FXR target gene expression, of which FGF19 is one, is associated with gallstones. We hypothesised that the two conditions might share pathophysiologic mechanisms and commonly coexist. Method SeHCAT 7 day retention values were collected between 2003–2014 in patients being investigated for chronic diarrhoea. Other conditions known to be associated with BAD such as cholecystectomy or Crohn’s disease were recorded. A subset of these patients had fasting FGF19 measured. Retrospectively, gallbladder imaging was added to database following cross-referencing with the NHS trust’s PACS system. Where multiple investigations had been performed, the test nearest to the date of the SeHCAT test was recorded. Imaging reporting gallbladder polyps without gallstones were excluded from the final analysis. Results Of 578 SeHCAT values on the database, 303 (52%) were positive with a value <15%. 183 had imaging that reported upon the gallbladder, of which 7 were excluded due to the presence of polyps, leaving 176. Of these 103 (59%) had a positive SeHCAT scan, 47 (27%) had gallstones, 12 (7%) had a cholecystectomy, so 59 (34%) had either gallstones or cholecystectomy. There was no significant difference in the rate of gallstones or cholecystectomy by gender (64% v 72%, p = 0.2), but mean age in the gallstones/cholecystectomy group was higher (50 v 57, p < 0.005). Median SeHCAT for the whole cohort was 11.7%. The median SeHCAT for those with gallstones or cholecystectomy was significantly lower than those without (7.2% v 14%, p < 0.001). With cholecystectomy excluded, the median SeHCAT was still lower for those with gallstones (3.8% v 14%, p < 0.0001). Overall, a SeHCAT value of <15% conferred an increased risk of gallstones or cholecystectomy (OR=2.0, 95% CI 1.04–3.919, p < 0.05) and the presence of primary bile acid diarrhoea was associated with gallstones, although this did not reach statistical significance (OR=1.98, 95% CI 0.65–5.99, p = 0.23). FGF19 was measured in 60 patients with imaging, of whom 15 had gallstones or cholecystectomy. There was no significant difference in the median serum FGF19 between those with gallstones or cholecystectomy and those without (224 v 227 pg/ml, p = 0.7). Conclusion Bile acid diarrhoea is associated with gallstones, even when adjusted for cholecystectomy which is a known risk factor for BAD. Bile acid diarrhoea and gallstones may share a pathophysiological mechanism. Disclosure of interest None Declared.

P315 The role of FGF19 in the assessment of diarrhoea and disease activity in non-ileal resected Crohn's disease

P315 The role of FGF19 in the assessment of diarrhoea and disease activity in non-ileal resected Crohn’s disease J. Nolan1 *, I. Johnston1, J.H. Zhang1, T. Dew2, P. Dixon3, C. Williamson3, J.R. Walters1. 1Imperial College London, Gastroenterology, London, United Kingdom, 2King’s College Hospital, Biochemistry, London, United Kingdom, 3Imperial College, Institute of Reproductive and Developmental Biology, London, United Kingdom

OC-027 Fgf19 Levels In Subjects With Primary Bile Acid Diarrhoea And Elevated Triglycerides

Introduction There is evidence that primary bile acid diarrhoea (PBAD) is caused by disordered bile acid homeostasis. Most patients with severe PBAD have low fasting serum FGF19 which fails to rise above 300 pg/ml postprandially. Different patterns of postprandial FGF19 response have been demonstrated, with some resembling those of healthy individuals. Others have shown that serum triglyceride levels reflect expression of the luminal bile acid transporter ASBT. It is hypothesised that a subset of individuals with hypertriglyceridaemia have different fasting FGF19 levels and postprandial FGF19 response. Methods Study 1: 162 patients with chronic diarrhoea were recruited prospectively. All patients underwent routine testing to exclude other causes of diarrhoea and had SeHCAT tests. Patients were classified as having PBAD, or unexplained chronic diarrhoea (CD). Other diagnoses were excluded. Fasting blood samples were taken and processed for triglycerides and FGF19. Subjects with either diagnosis were also analysed within 2 subgroups according to triglyceride level (cut off 2.30 mmol/l). Study 2: 18 subjects took part in a study to examine FGF19 levels over the course of 6 h. After an overnight fast, blood was sampled every 90 min for 6 h. Meals were provided at 9 am and 12 noon. Serum FGF19 was quantified by ELISA using a commercially available kit. Triglycerides were quantified by standard colorimetric technique. Mann-Whitney and Spearman rank correlation tests were used in analyses. Results Study 1: Overall subjects with elevated triglycerides (n = 18) have significantly lower SeHCAT retention (median 7.95 vs. 19.5% p = 0.01). Subjects with severe BAD with elevated triglycerides had higher fasting FGF19 levels (241 vs 101 pg/l p = 0.02). Study 2: There was no significant difference in fasting triglycerides between different phenotypes of FGF19 response (previously presented work). The percentage increase in FGF19 from fasting to 90 min after breakfast correlates with fasting serum triglyceride level (R = 0.59, p < 0.005). Conclusion We have identified a subset of PBAD subjects with high triglycerides and fasting FGF19 levels comparable to healthy individuals. The post prandial rise in FGF19 suggests no defect in the response of FGF19 synthesis in this subset. It may instead be caused by impaired BA absorption due to reduced ASBT expression which is also manifested as high serum triglycerides. PBAD may be a heterogenous condition with more than one underlying key abnormality. Disclosure of Interest None Declared.