Bile acid synthesis in primary cultures of rat and human hepatocytes

Abstract

The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7␣-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 ؎ 24 ng/mL (67% cholic acid) on day 1 to 55 ؎ 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, ␣-muricholic acid, and ␤-muricholic acid were formed. Treatment with triidothyronine (T 3 ) or dexamethasone alone had no significant effect on bile acid production. A combination of T 3 and dexamethasone significantly increased the total bile acid production on day 4 (224 ؎ 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12␣-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7␣-hydroxylase activity of 3.3 ؎ 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T 3 had an activity of 16.4 ؎ 3.6 pmol/mg protein/min. The cholesterol 7␣-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 ؎ 0.9 cpm/ g total nucleic acids (tNAs). T 3-or dexamethasone-treated cells did not differ from control cells, whereas the combination of T 3 and dexamethasone increased the mRNA levels to 20.6 ؎ 2.8 cpm/ g tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 ؎ 79 ng/mL on day 2 to 1490 ؎ 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T 3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7␣-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T 3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3-to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids. (HEPATOLOGY 1998;27: 615-620.)Bile acid synthesis is localized within the liver and represents an important pathway for cholesterol elimination from the body. The major bile acids, primarily f...

Dexamethasone Exposure of Neonatal Rats Modulates Biliary Lipid Secretion and Hepatic Expression of Genes Controlling Bile Acid Metabolism in Adulthood Without Interfering With Primary Bile Acid Kinetics

“…GC acutely enhances hepatic gene expression of Cyp7a1 (27,28) and Cyp27 (12) in cultured hepatocytes. Surprisingly, neither Cyp7a1 gene expression nor activity was decreased upon adrenalectomy in rats (29).…”

Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965)

“…Bile acids from cell culture media were measured as described previously (Ellis et al, 1998). Trimethylsilyl ether derivatives were analyzed by gas chromatography/mass spectrometry (Column HP-1, 6890N GC System, 5973 Mass Selective Detector; Agilent Technologies).…”

Addition of Dexamethasone Alters the Bile Acid Composition by Inducing CYP8B1 in Primary Cultures of Human Hepatocytes

“…11,12 We have found that primary human and rat hepatocytes in culture exhibit significant species differences in both types of bile acids formed and, more importantly, in the regulation of bile acids homeostasis. 13 We have also shown that primary human hepatocytes conjugate all secreted CA and CDCA by amidation to glycine or taurine 13,14 and thus, no unconjugated bile acids are found in the media.…”

“…13 Briefly, 500 ng of deuterium-labeled CA and CDCA was added to 1 ml of cell culture medium. The mixture was hydrolyzed in 1 M KOH at 120 8C overnight.…”