Transcription factor cascades during fasting amplify gluconeogenesis and instigate a secondary wave of ketogenic gene transcription. [RNA-Seq 2]. Transcription factor cascades during fasting amplify gluconeogenesis and instigate a secondary wave of ketogenic gene transcription. [RNA-Seq 2]

During fasting, bodily homeostasis is maintained due to hepatic production of glucose (via gluconeogenesis) and ketone bodies (via ketogenesis). The major hormones governing hepatic fuel production are glucagon and glucocorticoids who initiate a complex transcriptional program aimed at supporting gluconeogenesis and ketogenesis. Here, we found that in addition to the known metabolic genes induced by glucagon and glucocorticoids, these hormones each induce a set of genes encoding transcription factors (TFs) thereby initiating transcriptional cascades. Glucocorticoids activate the glucocorticoid receptor (GR) that induces the genes encoding two TFs - C/EBPβ and PPARα. Transcriptomic analysis combined with gene silencing revealed the role of C/EBPβ as an amplifier of hormone-induced gluconeogenesis. In contrast, the GR-PPARα cascade initiated a secondary transcriptional wave of genes supporting ketogenesis. We show that a dual treatment of glucocorticoids with a PPARα agonist leads to synergistic induction of ketogenic genes and that this induction is dependent on protein synthesis. Genome-wide analysis of enhancer dynamics revealed numerous enhancers activated by the GR-PPARα cascade. These enhancers are proximal to ketogenic genes, are enriched for the PPARα binding motif and show increased PPARα binding as profiled by ChIP-seq. In summary, this study reveals abundant TF cascades occurring during fasting. These cascades serve two separated purposes: the amplification of the primary gluconeogenic transcriptional program and the induction of a secondary gene program aimed at enhancing ketogenesis. Overall design: Primary hepatocytes were isolated from male, 8-10 weeks-old mice (strain C57BL/6JOlaHsd). Three hours after plating, media was changed to Williams E media. After 18 h, hormones (corticosterone 1 µM, Wy14643 10 µM or a dual treatment) were added for 8h.

在禁食状态下，机体稳态的维持依赖于肝脏通过糖异生（gluconeogenesis）生成葡萄糖，以及通过生酮作用（ketogenesis）生成酮体。调控肝脏燃料生成的主要激素为胰高血糖素与糖皮质激素，二者可启动复杂的转录程序，以支持糖异生与生酮过程。本研究发现，除了已知的胰高血糖素与糖皮质激素诱导的代谢基因外，这两类激素还各自诱导一组编码转录因子（transcription factors, TFs）的基因，从而启动转录级联反应。糖皮质激素可激活糖皮质激素受体（glucocorticoid receptor, GR），进而诱导编码两种转录因子的基因——C/EBPβ与PPARα的表达。结合转录组分析与基因沉默实验，本研究证实C/EBPβ可作为激素诱导的糖异生过程的放大器。与之相反，GR-PPARα级联反应则启动了支持生酮作用的次级转录浪潮。我们发现，糖皮质激素与PPARα激动剂联合处理可协同诱导生酮基因的表达，且该诱导过程依赖于蛋白质合成。通过全基因组增强子动态分析，我们发现GR-PPARα级联反应可激活大量增强子：这些增强子位于生酮基因的邻近区域，富集PPARα结合基序，且经ChIP-seq分析证实其PPARα结合水平有所提升。综上，本研究揭示了禁食过程中存在大量转录因子级联反应。这些级联反应可实现两项独立功能：一是放大初级糖异生转录程序，二是诱导次级基因程序以增强生酮作用。整体实验设计：从8-10周龄雄性C57BL/6JOlaHsd品系小鼠中分离原代肝细胞。铺板3小时后，更换为Williams E培养基。培养18小时后，分别添加激素（1 μM皮质酮、10 μM Wy14643或二者联合处理），继续培养8小时。