An Integrative Approach to Assessing Effects of a Short-term Western Diet on Gene Expression in Rat Liver

Consumption of a diet rich in saturated fatty acids and carbohydrates contributes to the accumulation of fat in the liver and development of non-alcoholic steatohepatitis (NASH). Herein we investigated the hypothesis that short-term consumption of a high fat/sucrose Western diet (WD) alters the genomic and translatomic profile of the liver in association with changes in signaling through the protein kinase mTORC1, and that such alterations contribute to development of NAFLD. The results identify a plethora of mRNAs that exhibit altered expression and/or translation in the liver of rats consuming a WD compared to a CD. In particular, consumption of a WD altered the abundance and ribosome association of mRNAs involved in lipid and fatty acid metabolism, as well as those involved in glucose metabolism and insulin signaling. Hepatic mTORC1 signaling was enhanced when rats were fasted overnight and then refed in the morning; however, this effect was blunted in rats fed a WD as compared to a CD. Despite similar plasma insulin concentrations, fatty acid content was elevated in the liver of rats fed a WD as compared to a CD. We found that feeding had a significant positive effect on ribosome occupancy of 49 mRNAs associated with hepatic steatosis (e.g., LIPE, LPL), but this effect was blunted in the liver of rats fed a WD. In many cases, changes in ribosome association were independent of alterations in mRNA abundance, suggesting a critical role for diet-induced changes in mRNA translation in the expression of proteins encoded by those mRNAs. Overall, the findings demonstrate that short-term consumption of a WD impacts hepatic gene expression by altering the abundance of many mRNAs, but also causes wide-spread variation in mRNA translation that potentially contribute to development of hepatic steatosis. Overall design: Obesity-prone Sprague Dawley rats (op-SD), initial body weight 160–190 g, were fed either a control diet (CD; TD 08485), or a Western diet (WD; TD 88137) ad libitum for 2 wks. Rats were anesthetized and a sample (~1 g) of the left lobe of the liver was removed and homogenized. Total RNA was extracted from the homogenates using Trizol, and equal amounts of RNA from 3 rats/condition were pooled. Another aliquot of homogenate was subjected to sucrose density gradient centrifugation, and two fractions were collected from each gradient. The first fraction corresponded to the portion of the gradient containing mRNAs associated with three or fewer ribosomes (referred to hereafter as the light fraction), and the second fraction corresponded to the portion of the gradient containing mRNAs associated with four or more ribosomes (referred to as the heavy fraction). RNA was extracted from each fraction using Trizol. 20 µg of RNA from the livers of three rats/condition, i.e., rats fed either the CD or WD, or the light and heavy fractions from sucrose density gradients from three rats/condition, were combined prior RNAseq analysis. RNA quality was assessed using an Agilent 2100 Bioanalyzer.Library preparation from each RNA fraction was performed using a KAPA RNA HyperPrep Kit with RiboErase (Roche Molecular Systems; no. KK8560) according to the manufacturer's instructions. The resulting libraries were amplified, and the quality was assessed by electrophoresis followed by RNAseq analysis using an Illumina Novaseq.

高饱和脂肪酸与碳水化合物富集的膳食摄入，会促进肝脏脂肪蓄积以及非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）的发生发展。本研究针对以下假说展开探究：短期饲喂高脂/高蔗糖西式膳食（Western diet, WD）可改变肝脏的基因组与转录翻译组谱，伴随蛋白激酶mTORC1信号通路的活性变化，且此类改变参与非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）的发生发展。

本研究结果鉴定出大量在西式膳食饲喂大鼠肝脏中，表达水平与/或翻译水平发生改变的信使RNA（messenger RNA, mRNA）。相较于对照膳食（control diet, CD）饲喂组，西式膳食饲喂会改变参与脂质与脂肪酸代谢、葡萄糖代谢以及胰岛素信号通路的mRNA的丰度，及其核糖体结合状态。

当大鼠经一夜禁食后于清晨复饲时，肝脏mTORC1信号通路会被激活，但西式膳食饲喂组的这一效应相较于对照膳食组被显著削弱。尽管两组大鼠血浆胰岛素浓度无显著差异，但西式膳食饲喂大鼠的肝脏脂肪酸含量高于对照膳食组。

我们发现，复饲行为可使49个与肝脏脂肪变性相关的mRNA（例如LIPE、LPL）的核糖体占有率显著升高，但该效应在西式膳食饲喂大鼠的肝脏中被削弱。在多数情况下，核糖体结合状态的变化并不伴随mRNA丰度的改变，这提示膳食诱导的mRNA翻译变化在对应mRNA编码蛋白的表达中发挥关键调控作用。

综上，本研究结果表明，短期西式膳食摄入不仅通过改变大量mRNA的丰度影响肝脏基因表达，还会引发广泛的mRNA翻译变化，此类变化可能参与肝脏脂肪变性的发生发展。

总体实验设计：选取肥胖易感型斯普拉格·道利大鼠（obesity-prone Sprague Dawley, op-SD），初始体重160~190 g，随机分为两组，分别自由饲喂对照膳食（CD；批号TD 08485）或西式膳食（WD；批号TD 88137），持续2周。将大鼠麻醉后，摘取其肝脏左叶约1 g组织并匀浆。采用Trizol试剂从匀浆中提取总RNA，将每个处理组3只大鼠的RNA等量混合。另取一份匀浆样本进行蔗糖密度梯度离心，从每个梯度中收集两个组分：第一组为结合3个及以下核糖体的mRNA组分（以下简称轻组分），第二组为结合4个及以上核糖体的mRNA组分（以下简称重组分）。采用Trizol试剂从各组分中提取RNA。将每个处理组（饲喂CD或WD的大鼠）3只大鼠肝脏的20 μg RNA，或每个蔗糖密度梯度离心组分（来自3只大鼠的轻、重组分）的RNA混合后，进行RNA测序（RNAseq）分析。采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）评估RNA质量。采用带有RiboErase的KAPA RNA HyperPrep试剂盒（Roche分子系统公司；货号KK8560）按照制造商说明书完成各RNA组分的文库构建。对构建完成的文库进行扩增，通过电泳评估质量后，采用Illumina NovaSeq测序平台完成RNA测序分析。