Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture.
Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.
Gaubius Institute TNO, Leiden, The Netherlands.
J
J
Kwekkeboom
H M
HM
Princen
E M
EM
van Voorthuizen
H J
HJ
Kempen
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000833:Animals, Suckling; D001647:Bile Acids and Salts / Q000096:biosynthesis* / Q000302:isolation & purification; D001769:Blood; D002478:Cells, Cultured; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D002849:Chromatography, Gas; D003907:Dexamethasone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine; D014886:Weaning
D001647:Bile Acids and Salts; D007328:Insulin; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase