Insulin suppresses bile acid synthesis in cultured rat hepatocytes by down-regulation of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase gene transcription.
Evidence from in vivo studies indicates that the bile acid pool and bile acid excretion are increased in humans with diabetes mellitus and in experimental diabetic animals, and that both parameters return to normal levels after administration of insulin. To investigate the biochemical background of these changes, the effects of insulin on bile acid synthesis and cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids was dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L) and a maximal decrease at 140 nmol/L (-65%). The decrease of bile acid synthesis correlated well with the suppression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7 alpha-hydroxylase, examined in more detail, was dose dependently diminished on incubation of hepatocytes with various concentrations of insulin, reaching maximal reduction at 14 nmol/L of insulin. Maximal decrease of the enzyme activity was seen after 8 hours of incubation (-70%). Insulin strongly reduced the rise in cholesterol 7 alpha-hydroxylase activity induced by incubation with dexamethasone. Sterol 27-hydroxylase activity was inhibited up to -58% after 24 hours of incubation with 140 nmol/L insulin. To study the mechanism of suppression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messenger RNA (mRNA) and gene transcription were assessed. The decrease in enzyme activities could be explained by a concomitant reduction in the cholesterol 7 alpha-hydroxylase (-76%) and sterol 27-hydroxylase (-62%) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7 alpha-hydroxylase and -75% for sterol 27-hydroxylase. Transient expression experiments using a construct containing the proximal 348 basepairs of the cholesterol 7 alpha-hydroxylase promoter fused to the chloramphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significant reduction of transcriptional activity (-64%) with insulin, indicating that a sequence important for an insulin-induced transcriptional response is located within the first 348 basepairs, preceding the transcription start of the cholesterol 7 alpha-hydroxylase promoter.(ABSTRACT TRUNCATED AT 400 WORDS)
Gaubius Laboratory Netherlands Organization for Applied Scientific Research-Prevention and Health, Leiden.
J
J
Twisk
M F
MF
Hoekman
E M
EM
Lehmann
P
P
Meijer
W H
WH
Mager
H M
HM
Princen
D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase; D053493:Cholestanetriol 26-Monooxygenase; D002790:Cholesterol 7-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology*; D009687:Nuclear Proteins / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D001647:Bile Acids and Salts; D007328:Insulin; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D015500:Chloramphenicol O-Acetyltransferase