BILE-ACID SYNTHESIS IN PRIMARY CULTURES OF RAT AND HUMAN HEPATOCYTES

The regulation of hepatic bile acid formation is incompletely understo od. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat a nd human hepatocytes in primary culture, we examined bile acid composi tion and the expression of the rate-limiting enzyme of formation, chol esterol 7 alpha-hydroxylase. Control rat hepatocytes showed a declinin g 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 addit ion to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T-3) or dexamethasone alone had no significant effect on bile acid product ion. A combination of T-3 and dexamethasone significantly increased th e 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 al pha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7 alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethason e and T-3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cho lesterol 7 alpha-hydroxylase messenger RNA (mRNA) levels, determined b y solution hybridization after 4 days of culture, showed results simil ar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/m u g total nucleic acids (tNAs). T3- or dexamethasone-treated cells did not differ from control cells, whereas the combination of T-3 and dex amethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/mu g tNAs. In human hepatocytes, isolated from donor liver, bile acid formation inc reased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 a nd then declined slightly, Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined add ition of T-3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7 a lpha-hydroxylase increased severalfold in the human hepatocytes from d ay 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 a cids 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 h igher 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 furt her studies of the synthesis and regulation of bile acids.