Unconjugated Bile Salts Shuttle Through Hepatocyte Peroxisomes for Taurine Conjugation

Krzysztof P. Rembacz, Jannes Woudenberg, Mark Hoekstra, Elles Z. Jonkers, Fiona A. J. van den Heuvel, Manon Buist-Homan, Titia E. Woudenberg-Vrenken, Jana Rohacova, M. Luisa Marin, Miguel A. Miranda, Han Moshage, Frans Stellaard, Klaas Nico Faber*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

15 Citations (Scopus)

Abstract

Bile acid-CoA.amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation However, the subcellular location of BAAT remains a topic of debate The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes Primary rat hepatocytes were incubated with deuterium-labeled cholic acid (D(4)CA) Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS) Subcellular accumulation of D(4)-labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 mu M D(4)CA to D(4)TCA and D(4)GCA The relative amounts of D4TCA and D4GCA produced were dependent on the presence of glycine or taurine in the medium Treatment of D(4)CA-exposed hepatocytes with 30-150 mu g/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker) Full release of D(4)TCA, catalase, and BAAT was only observed at 500 mu g/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. Conclusion We established a novel assay to study conjugation and intra and transcellular transport of bile salts Using this assay, we show that cholic acid shuttles through peroxisomes for taurine-conjugation (HEPATOLOGY 2010,52 2167-2176)

Original languageEnglish
Pages (from-to)2167-2176
Number of pages10
JournalHepatology
Volume52
Issue number6
DOIs
Publication statusPublished - Dec-2010

Keywords

  • ACID N-ACYLTRANSFERASE
  • PROTECTS RAT HEPATOCYTES
  • INDUCED APOPTOSIS
  • MASS-SPECTROMETRY
  • HUMAN LIVER
  • IN-VITRO
  • TRANSPORT
  • MEMBRANE
  • ACTIVATION
  • EXPRESSION

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