Twisk J, Hoekman M F, Lehmann E M, Meijer P, Mager W H, Princen H M
Gaubius Laboratory Netherlands Organization for Applied Scientific Research-Prevention and Health, Leiden.
Hepatology. 1995 Feb;21(2):501-10.
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)
体内研究的证据表明，糖尿病患者和实验性糖尿病动物的胆汁酸池和胆汁酸排泄量增加，且在给予胰岛素后这两个参数均恢复至正常水平。为了探究这些变化的生化背景，在培养的大鼠肝细胞中研究了胰岛素对胆汁酸合成以及胆固醇7α-羟化酶和甾醇27-羟化酶（胆固醇转化为胆汁酸过程中的两种关键酶）的影响。胆汁酸的大量生成呈剂量依赖性减少，在该激素的生理浓度（1.4至14 nmol/L）下显著降低（-33%至-53%），在140 nmol/L时最大降幅为-65%。胆汁酸合成的减少与胆固醇7α-羟化酶和甾醇27-羟化酶活性的抑制密切相关。更详细地检测胆固醇7α-羟化酶的酶活性，发现随着肝细胞与不同浓度胰岛素孵育，其呈剂量依赖性降低，在胰岛素浓度为14 nmol/L时达到最大降幅。孵育8小时后酶活性出现最大降幅（-70%）。胰岛素强烈降低了与地塞米松孵育诱导的胆固醇7α-羟化酶活性的升高。与140 nmol/L胰岛素孵育24小时后，甾醇27-羟化酶活性被抑制高达-58%。为了研究胆固醇7α-羟化酶和甾醇27-羟化酶活性受抑制的机制，评估了胰岛素对它们各自信使核糖核酸（mRNA）水平和基因转录的影响。酶活性的降低可以通过胆固醇7α-羟化酶（-76%）和甾醇27-羟化酶（-62%）mRNA水平的相应降低来解释。通过核转录分析评估的转录活性也降低到相同程度，即胆固醇7α-羟化酶降低60%，甾醇27-羟化酶降低75%。使用含有与氯霉素乙酰转移酶（CAT）基因融合的胆固醇7α-羟化酶启动子近端348个碱基对的构建体（-348Rcat）进行的瞬时表达实验表明，胰岛素可使转录活性显著降低（-64%），这表明胰岛素诱导转录反应的重要序列位于胆固醇7α-羟化酶启动子转录起始之前的前348个碱基对之内。（摘要截断于400字）
