Use of primary human hepatocytes for the elucidation of bile acid synthesis
Author: Ellis, Ewa C S
Date: 2003-01-24
Location: R64, plan 6, Huddinge Universitetssjukhus
Time: 13.30
Department: Institutionen för medicin, Huddinge Sjukhus / Department of Medicine at Huddinge University Hospital
Abstract
In the present investigation we cultured primary human hepatocytes on matrigel under serum-free conditions and studied bile acid formation. The following observations were made:
· The major steroid products excreted from primary human hepatocytes into the culture medium were cholic acid, CA (70%) and chenodeoxycholic acid, CDCA (25%) conjugated with glycine or taurine. This composition is similar to that formed in man under in vivo conditions.
· A new alternative pathway from cholesterol to cholic acid was defined in the hepatocytes including 7alpha, 12alpha, 27-trihydroxy-4-cholesten-3 -one, 7alpha,12alpha-dihydroxy-3-oxo-4-cholestenoic acid and 7alpha,12alpha-dihydroxy-3oxo-5beta-cholanoic acid as intermediates.
· The oxysterol 24S-hydroxycholesterol (formed in brain) was taken up by hepatocytes and partly metabolised into cholic acid, chenodeoxycholic acid and 5- cholestene-3beta,24S,27-triol.
· 27-Hydroxycholesterol, the first product in the acidic pathway was found to be converted into bile acids less efficiently than 7alphahydroxycholesterol. Cheriodeoxycholic acid was found to be the major product. The findings suggest that the acidic pathway is responsible for a minor part of bile acid synthesis in human hepatocytes.
· Glycine-conjugated and free bile acids suppressed bile acid synthesis and mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) in the order CDCA > DCA > CA > UDCA. mRNA levels of sterol 12alpha-hydroxylase (CYP81B1) and especially sterol 27-hydroxylase (CYP27A1) were suppressed to a much lower degree than CYP7A1.
· T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of both CYP7A 1 and CYP8B 1. CA formation was inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA / CDCA ratio.
The results are in agreement with a number of previous in vivo studies in humans and emphasize that human hepatocytes cultured as above are suitable for studies on human bile acid formation.
· The major steroid products excreted from primary human hepatocytes into the culture medium were cholic acid, CA (70%) and chenodeoxycholic acid, CDCA (25%) conjugated with glycine or taurine. This composition is similar to that formed in man under in vivo conditions.
· A new alternative pathway from cholesterol to cholic acid was defined in the hepatocytes including 7alpha, 12alpha, 27-trihydroxy-4-cholesten-3 -one, 7alpha,12alpha-dihydroxy-3-oxo-4-cholestenoic acid and 7alpha,12alpha-dihydroxy-3oxo-5beta-cholanoic acid as intermediates.
· The oxysterol 24S-hydroxycholesterol (formed in brain) was taken up by hepatocytes and partly metabolised into cholic acid, chenodeoxycholic acid and 5- cholestene-3beta,24S,27-triol.
· 27-Hydroxycholesterol, the first product in the acidic pathway was found to be converted into bile acids less efficiently than 7alphahydroxycholesterol. Cheriodeoxycholic acid was found to be the major product. The findings suggest that the acidic pathway is responsible for a minor part of bile acid synthesis in human hepatocytes.
· Glycine-conjugated and free bile acids suppressed bile acid synthesis and mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) in the order CDCA > DCA > CA > UDCA. mRNA levels of sterol 12alpha-hydroxylase (CYP81B1) and especially sterol 27-hydroxylase (CYP27A1) were suppressed to a much lower degree than CYP7A1.
· T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of both CYP7A 1 and CYP8B 1. CA formation was inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA / CDCA ratio.
The results are in agreement with a number of previous in vivo studies in humans and emphasize that human hepatocytes cultured as above are suitable for studies on human bile acid formation.
List of papers:
I. Ellis E, Goodwin B, Abrahamsson A, Liddle C, Mode A, Rudling M, Bjorkhem I, Einarsson C (1998). Bile acid synthesis in primary cultures of rat and human hepatocytes. Hepatology. 27(2): 615-20.
Pubmed
II. Axelson M, Ellis E, Mork B, Garmark K, Abrahamsson A, Bjorkhem I, Ericzon BG, Einarsson C (2000). Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid. Hepatology. 31(6): 1305-12.
Pubmed
III. Bjorkhem I, Andersson U, Ellis E, Alvelius G, Ellegard L, Diczfalusy U, Sjovall J, Einarsson C (2001). From brain to bile. Evidence that conjugation and omega-hydroxylation are important for elimination of 24S-hydroxycholesterol (cerebrosterol) in humans. J Biol Chem. 276(40): 37004-10.
Pubmed
IV. von Bahr S, Ellis E, Andersson U, Diczfalusy U, Einarsson C, Bjorkhem I (2002). Studies on the acid pathway for formation of bile acids in human primary hepatocytes - evidence that chenodeoxycholic acid is the predominant product. [Manuscript]
V. Ellis E, Axelson M, Abrahamsson A, Eggertsen G, Thorne A, Nowak G, Ericzon BG, Bjorkhem I, Einarsson C (2002). Feedback regulation of bile acid synthesis in primary human hepatocytes. Evidence that CDCA is the strongest inhibitor. [Submitted]
VI. Ellis ECS (2002). Suppresion of bile acid synthesis by thyroid hormone in primary human hepatocytes. [Submitted]
I. Ellis E, Goodwin B, Abrahamsson A, Liddle C, Mode A, Rudling M, Bjorkhem I, Einarsson C (1998). Bile acid synthesis in primary cultures of rat and human hepatocytes. Hepatology. 27(2): 615-20.
Pubmed
II. Axelson M, Ellis E, Mork B, Garmark K, Abrahamsson A, Bjorkhem I, Ericzon BG, Einarsson C (2000). Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid. Hepatology. 31(6): 1305-12.
Pubmed
III. Bjorkhem I, Andersson U, Ellis E, Alvelius G, Ellegard L, Diczfalusy U, Sjovall J, Einarsson C (2001). From brain to bile. Evidence that conjugation and omega-hydroxylation are important for elimination of 24S-hydroxycholesterol (cerebrosterol) in humans. J Biol Chem. 276(40): 37004-10.
Pubmed
IV. von Bahr S, Ellis E, Andersson U, Diczfalusy U, Einarsson C, Bjorkhem I (2002). Studies on the acid pathway for formation of bile acids in human primary hepatocytes - evidence that chenodeoxycholic acid is the predominant product. [Manuscript]
V. Ellis E, Axelson M, Abrahamsson A, Eggertsen G, Thorne A, Nowak G, Ericzon BG, Bjorkhem I, Einarsson C (2002). Feedback regulation of bile acid synthesis in primary human hepatocytes. Evidence that CDCA is the strongest inhibitor. [Submitted]
VI. Ellis ECS (2002). Suppresion of bile acid synthesis by thyroid hormone in primary human hepatocytes. [Submitted]
Issue date: 2003-01-03
Publication year: 2003
ISBN: 91-7349-424-0
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