UNLABELLED: 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 μM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 μg/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 μg/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.
UNLABELLED: 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 μM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 μg/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 μg/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.
Authors: Esther M Verhaag; Manon Buist-Homan; Martijn Koehorst; Albert K Groen; Han Moshage; Klaas Nico Faber Journal: PLoS One Date: 2016-03-07 Impact factor: 3.240