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Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

ABSTRACT: Bile acid reflux is known to be associated with the development of Barrett's esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient's refluxates induced expansion of multi-layered glandular structures and development of columnar metaplasia at the squamo-columnar junction. The glycine conjugated bile acids induced an intestinal type of metaplasia more typical for Barrett's esophagus. Through lineage tracing, we excluded the involvement of K5+, DCLK1+, and LGR5+ progenitor cells as the primary source in the development of the glandular metaplastic epithelium. We show that the mechanism behind development of metaplasia involves crypt fission and may be independent of stem cell proliferation. Our findings support the hypothesis that in humans, BE arises from non-squamous cells residing in submucosal gland ducts and that induction of intestinal type of metaplasia is most effectively induced by glycine-conjugated bile acids. These novel insights may lead to more effective strategies to prevent development of Barrett's esophagus and esophageal adenocarcinoma.

SUBMITTER: Straub D

PROVIDER: S-EPMC6660124 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

Straub Danielle D Oude Elferink Ronald P J RPJ Jansen Peter L M PLM Bergman Jacques J G H M JJGHM Parikh Kaushal K Krishnadath Kausilia K KK

PloS one 20190726 7

Bile acid reflux is known to be associated with the development of Barrett's esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient's refluxates induced expansion of multi-layered glandular str ...[more]

PMID: 31348796

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Project description:BackgroundSignificantly elevated serum and hepatic bile acid (BA) concentrations have been known to occur in patients with liver fibrosis. However, the roles of different BA species in liver fibrogenesis are not fully understood.MethodsWe quantitatively measured blood BA concentrations in nonalcoholic steatohepatitis (NASH) patients with liver fibrosis and healthy controls. We characterized BA composition in three mouse models induced by carbon tetrachloride (CCl4), streptozotocin-high fat diet (STZ-HFD), and long term HFD, respectively. The molecular mechanisms underlying the fibrosis-promoting effects of BAs were investigated in cell line models, a 3D co-culture system, and a Tgr5 (HSC-specific) KO mouse model.FindingsWe found that a group of conjugated 12α-hydroxylated (12α-OH) BAs, such as taurodeoxycholate (TDCA) and glycodeoxycholate (GDCA), significantly increased in NASH patients and liver fibrosis mouse models. 12α-OH BAs significantly increased HSC proliferation and protein expression of fibrosis-related markers. Administration of TDCA and GDCA directly activated HSCs and promoted liver fibrogenesis in mouse models. Blockade of BA binding to TGR5 or inhibition of ERK1/2 and p38 MAPK signaling both significantly attenuated the BA-induced fibrogenesis. Liver fibrosis was attenuated in mice with Tgr5 depletion.InterpretationIncreased hepatic concentrations of conjugated 12α-OH BAs significantly contributed to liver fibrosis via TGR5 mediated p38MAPK and ERK1/2 signaling. Strategies to antagonize TGR5 or inhibit ERK1/2 and p38 MAPK signaling may effectively prevent or reverse liver fibrosis.FundingsThis study was supported by the National Institutes of Health/National Cancer Institute Grant 1U01CA188387-01A1, the National Key Research and Development Program of China (2017YFC0906800); the State Key Program of National Natural Science Foundation (81430062); the National Natural Science Foundation of China (81974073, 81774196), China Postdoctoral Science Foundation funded project, China (2016T90381), and E-institutes of Shanghai Municipal Education Commission, China (E03008).

Project description:1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40-58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15-22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55-62%. 2. The conjugated pigment has red-brown fluorescence and an absorption maximum at 450nm with in(mM) 59.8cm(-1). Diazotization by the Malloy-Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with in(total) 28.4x10(3)lmol(-1)cm(-1) at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb(2+) but not by Ba(2+), Ca(2+) or Zn(2+). 3. At pH7.0 and 37 degrees C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by beta-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2-3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [(14)C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [(14)C]bilirubin into normal or Gunn rats, 79-91% was excreted in bile and 2-7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30-41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.

Dataset Information

Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

Publications

Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

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