Bile acid mediated changes after bariatric surgery: what can we learn from mouse models?

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1 Accepted Manuscript Bile acid mediated changes after bariatric surgery: what can we learn from mouse models? Emma Rose McGlone, Tricia Tan, Stephen R. Bloom, Julian R.F. Walters PII: S (19) DOI: Reference: YGAST To appear in: Gastroenterology Accepted Date: 28 February 2019 Please cite this article as: McGlone ER, Tan T, Bloom SR, Walters JRF, Bile acid mediated changes after bariatric surgery: what can we learn from mouse models?, Gastroenterology (2019), doi: doi.org/ /j.gastro This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Title page Title: Bile acid mediated changes after bariatric surgery: what can we learn from mouse models? Short title: Bile acids and bariatric surgery Authors: Emma Rose McGlone, 1 Tricia Tan, 1 Stephen R. Bloom, 1 Julian R.F. Walters 2 Institution: 1 Section of Endocrinology and Investigative Medicine, 2 Division of Digestive Diseases, Imperial College London, Hammersmith Hospital, London W12 0NN, U.K. Article type: Invited Commentary Grant support: The Section of Endocrinology and Investigative Medicine is funded by grants from the MRC, BBSRC, NIHR, an Integrative Mammalian Biology (IMB) Capacity Building Award, an FP7- HEALTH EuroCHIP grant and is supported by the NIHR Biomedical Research Centre Funding Scheme. The views expressed are those of the authors and not necessarily those of the funders, the NHS, the NIHR or the Department of Health. ER McGlone is also funded by a Clinical Research Training Fellowship from the MRC. Abbreviations: CDCA Chenodeoxycholic acid DIO Diet induced obesity FGF19 Fibroblast growth factor 19 FXR Farnesoid X receptor

3 GB-IL GLP-1 Bile diversion from the gallbladder to the distal ileum Glucagon-like peptide-1 LCA RYGB T-αMCA T-ßMCA Lithocholic acid Roux-en-Y gastric bypass Tauro-α-muricholic acid Tauro-beta-muricholic acid TGR5 Takeda G-protein coupled receptor 5 Correspondence: Professor Julian Walters Division of Digestive Diseases Imperial College London Hammersmith Hospital London W12 0NN Telephone: julian.walters@imperial.ac.uk Disclosures: The authors have no potential conflicts of interest relevant to this manuscript Author contributions: All authors contributed to paper concept and design, and to writing and editing the manuscript.

4 Main body of text: There is mounting evidence that change in bile delivery to the intestine after bariatric surgery is a key mediator of improved metabolic health. Studying this pathway in humans, however, can be challenging. In this issue of Gastroenterology, Albaugh et al present a model of bariatric surgery in lean mice, in which bile is diverted to the distal ileum, and demonstrate that this leads to a dramatic increase in circulating bile acids, increased gut hormone secretion, changes in gut microbiota and improved glucose tolerance 1. We consider to what extent data from mouse experiments are consistent with clinical findings following bariatric surgery in humans; and review the similarities and differences between humans and mice regarding bile acid metabolism. Bile acid diversion in humans and mice: Fasting and post-prandial bile acids increase in the blood after Roux-en-Y gastric bypass (RYGB) in patients with and without diabetes (reviewed in 2 ). In 2015 Albaugh et al. demonstrated that the rise in fasting bile acids after RYGB persists as long as two years post-surgery 3. Using liquid chromatography mass spectrometry, they found no long-term change in the relative proportion of individual bile acid species, with rises in both major primary bile acids (cholic and chenodeoxycholic) as well as secondary bile acids (e.g. deoxycholic and its glycine conjugate). This non-specific rise in circulating bile acids, with no particular change in the composition, is consistent with the findings of several other groups who have looked at bile acids in patients post RYGB 2. The increase in serum bile acids after RYGB occurs in association with improved glucose tolerance and lipid metabolism 2 ; but since there are multiple major changes in physiology following RYGB, it has been difficult to demonstrate a causative effect of bile acid diversion on improved metabolic health. To address this question, Albaugh et al. developed the experimental technique of bile diversion from the gallbladder to the distal ileum (GB-IL) in a mouse model, publishing their first results in (Figure). This model excludes other anatomical

5 effects of RYGB which may independently affect metabolism: these include reduction of gastric volume; loss of coordinated emptying of gastric contents via a functional pylorus; and diversion of pancreatic enzymes as well as bile acids (Figure). There is no equivalent operation routinely performed in humans. In mice with diet-induced obesity (DIO), Flynn et al observed that GB-IL reduced weight and improved glucose tolerance to a similar degree as RYGB 4, suggesting that the bile acid diversion component of RYGB could be key to its beneficial metabolic effects. 5,6 As in humans following RYGB, a dramatic increase in circulating bile acids is observed in mice following GB-IL 1,4, and RYGB 5,6. Unlike humans, however, this rise can be attributed to an increase in individual species of bile acids, namely conjugated muricholic acids, and in particular, tauro-beta-muricholic acid (T-ßMCA) 1,4,5,6. Following RYGB, weight loss reduces insulin resistance and improves glucose tolerance: this makes any independent contribution of bile acid diversion difficult to assess. Scant data exist regarding the outcomes of RYGB in non-obese humans. In their most recent study, Albaugh et al performed GB-IL in lean mice, with the aim of excluding weight loss as a potential confounder 1. Beyond the first post-operative week, GB-IL did not cause weight loss in lean mice but nonetheless was associated with durable improvements in glucose tolerance 1. Taken together, these studies suggest that bile acid diversion has a beneficial impact on glucose tolerance, independent of the accompanying anatomical changes of RYGB, and of ensuing weight loss. The gut microbiome plays a critical role in biotransformation of bile acids, thereby influencing the relative amounts of different bile acid species; bile acids themselves also influence the relative abundance of different gut bacteria species 7. After bile acid diversion in mice, as well as after RYGB, there is an increase in abundance of Akkermansia mucinophilia 1,4,6,8. This species of the phylum Verrucomicrobia is associated with a healthy metabolic phenotype in both mice 9 and humans 10. Other bacteria that increase in prevalence after bile acid diversion in mice are Escherichia and Klebsiella 4,8. Although overall murine and human gut flora have major

6 compositional differences 11, an increase in prevalence of Akkermansia, Escherichia and Klebsiella has also been observed in humans following RYGB (e.g. 12 ). Bile acids stimulate secretion of several hormones, including fibroblast growth factor 19 (FGF19) from enterocytes, and glucagon-like peptide-1 (GLP-1) from enteroendocrine cells; these hormones increase after RYGB 13. GLP-1 is an incretin hormone with diverse positive metabolic effects 7 ; we and others have shown that it contributes to the improvements in metabolic phenotype seen after RYGB in humans 14. Albaugh et al investigated whether the beneficial effects of GB-IL in mice might be mediated by GLP-1 and observed the following: firstly, that in mice following GB-IL there was increased ileal secretion of GLP-1 into mesenteric lymph, and secondly, that blockade of GLP-1 receptor decreased the beneficial effects on glucose tolerance 1. These findings suggest that the effects of GB-IL are mediated at least in part by GLP-1. It might also be interesting to consider the role of other gut hormones which also increase after bariatric surgery, such as peptide tyrosine tyrosine and oxyntomodulin in this pathway; the latter also works through the GLP-1 receptor. Importantly, in this paper, Albaugh et al investigated the signaling pathway of bile acids by performing GB-IL in DIO mice lacking one of the two bile acid receptors, Takeda G-protein coupled receptor 5 (TGR5; in the whole mouse) and Farnesoid X receptor (FXR; in the intestine only) 1. They demonstrated that GB-IL improves glucose tolerance and reduces weight in TGR5 knockout mice but not in intestinal FXR knock out mice 1. These findings together suggest that the glucose lowering effects of bile acid diversion are mediated by an intestinal FXR-GLP-1 pathway. This apparent dependence of the pathway on intestinal FXR is at first glance surprising since previous studies agree that TGR5 is the major receptor mediating bile acid stimulated incretin hormone secretion 2, 7. There is evidence however, that intestinal FXR may in fact repress secretion of GLP-1, and that inhibition of the receptor can release this brake: FXR activation has been shown to cause a decrease in proglucagon transcription (the

7 prohormone of GLP-1) in both mouse and human ex vivo intestinal biopsies; and FXR deficiency in mice increases GLP-1 secretion 2. Depletion of intestinal FXR is associated with a beneficial metabolic phenotype in mice 15. In this context, it is interesting to note that the T-ßMCA and tauro-α-muricholic acid (T-αMCA) bile acid species, which massively increase after GB-IL, are FXR antagonists: both decrease FXR activation in the presence of chenodeoxycholic acid (CDCA) in vitro, in a dose-dependent manner 16. Potentially then, GB-IL in mice via an increase in conjugated muricholic acids leads to intestinal FXR inhibition which increases GLP-1 secretion, contributing to improved glucose tolerance. At present, however, we do not have direct evidence that intestinal FXR activation represses incretin secretion in humans. Even in mice the finding is controversial, as the gut-specific FXR agonist fexaramine actually increases GLP-1 secretion and improves glucose tolerance in DIO mice 17. Fexaramine has not been demonstrated to have effects in human intestinal organoids, and none of the major human bile acids have been identified as FXR antagonists. In 2018, however, glycine-ursodeoxycholic acid (G-UDCA) and tauroursodeoxycholic acid (T-UDCA), which occur naturally in small amounts in humans, were reported to act as competitive antagonists for the FXR receptor in the presence of CDCA in vitro and in mice 18. In patients with obesity, it has been observed that treatment with UDCA, such that both UDCA and its conjugates are enriched in circulating blood, leads to hepatic FXR suppression and reduced circulating FGF19 a hormone that is released in response to intestinal FXR activation 19. Furthermore chronic T-UDCA treatment is associated with small improvements in hepatic and muscle insulin sensitivity in obese volunteers 20. After RYGB in humans, however, although there may be a transient increase in UDCA and its glycine and taurine conjugates at one month 3, there is no evidence of a longer-term increase in these putative FXR antagonists. It is unlikely, therefore, that intestinal FXR inhibition, mediated by an increase in bile acids that antagonize FXR, would explain long term improvements in glucose tolerance seen after RYGB; but they could play a role in the short term. This field certainly merits further investigation.

8 Differences in bile acid metabolism in mice and man: Generalizability of findings from murine bile acid diversion experiments to humans is limited by differences in bile acid homeostasis between the two species (Table). It is important to be aware of such differences when interpreting mouse experiment data. Bile acid composition: Primary bile acids are synthesized in the liver from cholesterol: in man these comprise cholate (CA) and CDCA. In mice the major primary bile acids are T-ßMCA and T-αMCA, which are formed from the 6-ß hydroxylation of CDCA and UDCA respectively, via the enzyme Cyp2c This pathway indirectly decreases the amount of lithocholic acid (LCA) in the circulation, because it reduces the amount of CDCA entering the large bowel LCA is formed from the action of bacterial enzymes in the colon. It has been proposed that 6-ß hydroxylation of primary bile acids has evolved in mice as a way of limiting the amount of hepatotoxic LCA in circulation 21. Muricholic acids are FXR antagonists, whereas CDCA is a potent FXR agonist: this difference in primary bile acid composition may well therefore engender metabolic differences between the two species. Bile acid conjugation: In humans, bile acids may be sulfated by sulfotransferase enzymes but this pathway is virtually absent in mice 22. Sulfation is an alternative way of handling toxic bile acids, since a large proportion of LCA in human bile in present in the less toxic, sulfated form 22. Sulfation markedly decreases the absorption of bile acids from the small bowel, and also decreases their extraction from the liver into systemic blood. When concentration is high however, sulfated bile acids spillover into the systemic circulation, from where they are very efficiently excreted in the urine by the kidneys. Desulfation occurs in the large bowel, but not small bowel, affecting active concentrations at receptors. it would be interesting therefore to know whether the presence of a sulfate moiety

9 affects the binding of bile acids to small bowel bile acid receptors FXR and TGR5. Both sulfated and unsulfated primary bile acids are amidated/ conjugated by BA-CoA:amino acid N-acyltransferase before leaving the liver: in mice bile acids are almost exclusively conjugated with taurine, whilst in humans they are predominately conjugated with glycine with a small proportion conjugated with taurine 23. Conjugation in this way increases the solubility from the unconjugated state, taurine to a greater extent than glycine 22. Gut microbiota in mice and humans: Gut bacteria are responsible for desulfating, deamidating, dehydroxylating and oxidating primary bile acids: in germ free mice, which lack normal gut flora, one consequence is the accumulation of T-ßMCA 24, which leads to a reduction in FXR signaling and increased bile acid synthesis. The gut bacteria present in mice and man have coevolved with the bile acid composition in the two species: for example, human gut microbiota desulfate bile acids, whereas mouse gut microbiota do not 24. However, in germ-free mice colonized with a human microbiota, total bile acids are reduced to a similar extent as in those colonized with a mouse microbiota 24. suggesting that despite differences in bacterial genera, overall they are broadly similar in terms of phyla and function. Conclusion: Unpicking the relative contribution of bile acid changes to the extensive clinical changes seen after RYGB is difficult. Using mice to model the situation in humans has advantages in terms of homogeneity, closer and more standardized nutritional manipulation, latitude in terms of surgical models, and the availability of transgenic knockouts to probe specific details of physiology. In experimental rodent work reductionist models of human bariatric surgery can be pursued to help distinguish the relevance of different components. There are several similarities observed following bile acid diversion in mice and RYGB in man: an increase in circulating bile acids, improvements in weight, glucose intolerance and other metabolic outcomes, and an increase in specific gut flora, notably Akkermansia. However, there are also differences, perhaps the most significant of which is that

10 there is no evidence that the composition of bile acids changes in humans, while in mice after GB-IL there is a preponderance of the FXR antagonist, T-ßMCA. The presence of muricholic acids in mice is one of several difference between human and mouse bile acid metabolism, outlined above. Even more generally, we know that there are key details of gut hormone metabolism which differ between rodents and humans, for example the thermogenic effects of GLP-1 in mice but not humans 25. Mice, therefore, are therefore useful hypothesisgenerating models, but not all lessons from these animal experiments are transferrable, and there remains still a role for experimental medicine studies to definitively answer our questions about the effects of bariatric surgery in humans.

11 Figure: Comparison of anatomical features and main physiological effects of gallbladder diversion to the ileum (GB-IL) in mice with Roux-en-Y gastric bypass (RYGB) in humans Abbreviations: DIO: diet induced obesity; T-ßMCA: tauro-beta-muricholic acid Table: Differences in bile acid metabolism between humans and mice Bile acids synthesized in liver (Primary bile acids) Cholate (CA) Human Chenodeoxycholate (CDCA) Mouse α-muricholate (αmca) [from CDCA] β-muricholate (βmca) [from UDCA] Cholate Liver enzymes Cyp2c70 responsible for 6 β- hydroxylation to form MCAs Conjugation of bile acids in liver Glycine and taurine Essentially only taurine Bile acids resulting from microbial action (Secondary bile acids) Deoxycholate (DCA), Lithocholate (LCA) [by 7-dehydroxylation] Ursodeoxycholate (UDCA) [by 7-epimerization] Further BA metabolism Sulfated BA common Deoxycholate [by 7-dehydroxylation] Ursodeoxycholate (UDCA) [by 7-epimerization] ω-muricholate (ω-mca) [by 6β-epimerization] Behavioural differences Unabsorbed BA lost in feces Coprophagia is common resulting in recycling of

12 secondary BA from feces Bile acid effects on FXR Most bile acids are strong or Tauro-β-MCA & tauro-α-mca weak FXR agonists G-UDCA / T-UDCA may be competitive FXR antagonists Gut microbiome: Most prominent phyla Phyla change in obesity Highly abundant genera References Bacteriodetes and firmicutes Higher B: F ratio Prevotella, Faecalibacterium, Ruminococcus Clostridium, Bacteroides, Blautia are FXR antagonists Bacteriodetes and firmicutes Higher B: F ratio Lactobacillus, Alistipes, Turicibacter Clostridium, Bacteroides, Blautia 1. Albaugh VL, Banan B, Antoun J, et al. Role of Bile Acids and GLP-1 in Mediating the Metabolic Improvements of Bariatric Surgery. Gastroenterology Chávez-Talavera O, Tailleux A, Lefebvre P, et al. Bile Acid Control of Metabolism and Inflammation in Obesity, Type 2 Diabetes, Dyslipidemia, and Nonalcoholic Fatty Liver Disease. Gastroenterology 2017;152: e3.

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