Department of Endocrinology and Metabolism, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan; Department of Cell Biology, Jiangsu Key Laboratory of Stem Cell Research, Medical College of Soochow University, Ren Ai Road 199, Suzhou, 215123, China.
Department of Endocrinology and Metabolism, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
Mol Metab. 2020 Apr;34:16-26. doi: 10.1016/j.molmet.2019.12.015. Epub 2020 Jan 7.
Adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) influences hepatic cholesterol transportation. Accumulation of hepatic cholesterol leads to fatty liver disease, which is improved by glucagon-like peptide 1 (GLP-1) in diabetes. Therefore, we analyzed the molecular mechanism in the regulation of hepatic ABCA1 by GLP-1 analogue exendin-4.
Hepatic ABCA1 expression and transcription were checked by western blotting, real-time polymerase chain reaction (PCR), and luciferase assay in HepG2 cells. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were employed to determine transcriptional regulation of the ABCA1 gene. Prolactin regulatory element-binding (PREB)-transgenic mice were generated to access the effect of exendin-4 on improving lipid accumulation caused by a high-fat diet (HFD).
Exendin-4 stimulated hepatic ABCA1 expression and transcription via the Ca/calmodulin (CaM)-dependent protein kinase kinase/CaM-dependent protein kinase IV (CaMKK/CaMKIV) pathway, whereas GLP-1 receptor antagonist exendin9-39 cancelled this effect. Therefore, exendin-4 decreased hepatic lipid content. ChIP showed that PREB could directly bind to the ABCA1 promoter, which was enhanced by exendin-4. Moreover, PREB stimulated ABCA1 promoter activity, and mutation of PREB-binding site in ABCA1 promoter cancelled exendin-4-enhanced ABCA1 promoter activity. Silencing of PREB attenuated the effect of exendin-4 and induced hepatic cholesterol accumulation. Blockade of CaMKK by STO-609 or siRNA cancelled the upregulation of ABCA1 and PREB induced by exendin-4. In vivo, exendin-4 or overexpression of PREB increased hepatic ABCA1 expression and decreased hepatic lipid accumulation and high plasma cholesterol caused by a HFD.
Our data shows that exendin-4 stimulates hepatic ABCA1 expression and decreases lipid accumulation by the CaMKK/CaMKIV/PREB pathway, suggesting that ABCA1 and PREB might be the therapeutic targets in fatty liver disease.
三磷酸腺苷（ATP）结合盒转运体 A1（ABCA1）影响肝脏胆固醇转运。肝脏胆固醇积累会导致脂肪肝疾病，而在糖尿病中，胰高血糖素样肽 1（GLP-1）可以改善这种疾病。因此，我们分析了 GLP-1 类似物 exendin-4 调节肝脏 ABCA1 的分子机制。
通过 Western blot、实时聚合酶链反应（PCR）和荧光素酶测定法检测 HepG2 细胞中肝 ABCA1 的表达和转录。采用染色质免疫沉淀（ChIP）和定点突变来确定 ABCA1 基因的转录调节。生成催乳素调节元件结合（PREB）-转基因小鼠以评估 exendin-4 对改善高脂肪饮食（HFD）引起的脂质积累的影响。
exendin-4 通过钙/钙调蛋白（CaM）依赖性蛋白激酶激酶/CaM 依赖性蛋白激酶 IV（CaMKK/CaMKIV）途径刺激肝 ABCA1 的表达和转录，而 GLP-1 受体拮抗剂 exendin9-39 则取消了这种作用。因此，exendin-4 降低了肝内脂质含量。ChIP 显示，PREB 可以直接与 ABCA1 启动子结合，而 exendin-4 可以增强这种结合。此外，PREB 刺激 ABCA1 启动子活性，而 ABCA1 启动子中 PREB 结合位点的突变则取消了 exendin-4 增强的 ABCA1 启动子活性。PREB 的沉默减弱了 exendin-4 的作用，并诱导肝内胆固醇积累。用 STO-609 或 siRNA 阻断 CaMKK 取消了 exendin-4 诱导的 ABCA1 和 PREB 的上调。在体内，exendin-4 或 PREB 的过表达增加了肝 ABCA1 的表达，并减少了 HFD 引起的肝内脂质积累和高血浆胆固醇。
我们的数据表明，exendin-4 通过 CaMKK/CaMKIV/PREB 途径刺激肝 ABCA1 的表达并减少脂质积累，提示 ABCA1 和 PREB 可能是脂肪肝疾病的治疗靶点。
