Glyoxalase 1 and its Role in Metabolic Syndrome

摘要 这项提案的总体目标是确定谷胱甘肽酶1（GLO 1）在肥胖症发病机制中的作用， 2型糖尿病（T2 D）和非酒精性脂肪肝（NAFLD）。超过3400万美国人 糖尿病，另有8800万被认为是糖尿病前期。这在很大程度上是由于 肥胖，目前有72%的美国成年人被归类为超重或肥胖。合并症中 与T2 D相关，超过70%的患者患有NAFLD。流行病学研究表明高果糖 与肥胖、T2 D和NAFLD相关。该建议将GLO 1鉴定为促NAFLD/致肥胖基因。 使用CRISPR-Cas9，我们已经产生了GLOl敲除小鼠。当喂食高脂肪高蔗糖饮食时， 小鼠表现出显著的减缓的体重增加、恢复的葡萄糖耐量和减少的肝脂肪变性 与野生型对应物相比。GLO 1是一种普遍表达的酶，通过以下方式对糖酵解进行解毒- 产物甲基乙二醛（MGO）。当GLO 1活性被破坏时，MGO水平增加，导致长寿命的 蛋白质翻译后修饰。我们已经表明，MGO作为营养通量的代谢传感器， 调节糖酵解输出和对糖的转录反应。因此，我们假设GLO 1是一个亲- NAFLD/致肥胖基因，减少MGO并消除代谢制动。我们将检验这一假设 通过解决以下三个具体目标：在具体目标1中，我们将量化Glo 1对整体的影响- 使用16周普通饲料或高脂肪高蔗糖饮食的身体能量学和肝脏脂质代谢。我们将 量化瘦与脂肪质量、能量消耗和总活动。肝脂肪酸氧化与线粒体 将在原代肝细胞中定量呼吸。最后，将使用RNA-seq评估脂肪生成基因。 在具体目标2中，我们将确定Glo 1对肠道果糖代谢的影响。肠道是一个 果糖代谢的主要部位。通过13 C6-果糖经口灌胃的稳定同位素标记将用于定量 肠、肝、肌肉、脂肪和循环果糖代谢物。这种方法将定量 确定Glo 1如何调节肠道碳水化合物代谢，这是一个以前未探索的研究领域。 最后，在具体目标3中，我们将证实MGO衍生的组蛋白PTM调节转录反应， 体内代谢将在每个组织/队列中定量位点特异性典型和MGO衍生的PTM。 将鉴定MG-H1组蛋白修饰的推定阅读器结构域。最后，肝脏，肠，肌肉， 脂肪和胰腺将经受RNA-seq和ATAC-seq。这种方法将决定全球 在肥胖、T2 D和NAFLD的生理学相关模型中跨多个组织的表观基因组景观。 总的来说，这项建议将联合收割机机械生物化学与多组学方法相结合，以确定 GLO 1传播疾病进展的机制。

Abstract The overall goal of this proposal is to determine the role of glyoxalase 1 (GLO1) in the pathogenesis of obesity, Type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). Greater than 34 million Americans have diabetes, and another 88 million are considered pre-diabetic. This is largely attributed to the prevalence of obesity, with 72% of American adults currently classified as overweight or obese. Among the comorbidities associated with T2D, over 70% of patients have NAFLD. Epidemiological studies have linked high fructose consumption with obesity, T2D, and NAFLD. This proposal identifies GLO1 as pro-NAFLD/obesogenic gene. Using CRISPR-Cas9, we have generated GLO1 knockout mice. When fed a high-fat high sucrose diet, these mice display significantly blunted weight gain, restored glucose tolerance, and reduced hepatic steatosis compared to wild-type counterparts. GLO1 is a ubiquitously expressed enzyme that detoxifies the glycolytic by- product, methylglyoxal (MGO). When GLO1 activity is disrupted, MGO levels increase, resulting in long-lived protein post-translational modifications. We have shown that MGO serves as a metabolic sensor for nutrient flux, regulating glycolytic output and transcriptional responses to sugar. Thus, we hypothesize that GLO1 is a pro- NAFLD/obesogenic gene, reducing MGO and removing the brakes on metabolism. We will test this hypothesis by addressing the following three Specific Aims: In Specific Aim 1 we will quantify the impact of Glo1 on whole- body energetics and hepatic lipid metabolism using a 16-week chow- or high-fat high-sucrose diet. We will quantify lean vs. fat mass, energy expenditure, and total activity. Hepatic fatty acid oxidation and mitochondrial respiration will be quantified in primary hepatocytes. Lastly, lipogeneic genes will be assessed using RNA-seq. In Specific Aim 2 we will determine the impact of Glo1 on intestinal fructose metabolism. The intestine is a primary site of fructose metabolism. Stable isotope labeling via 13C6-fructose oral gavage will be used to quantify intestinal, hepatic, muscle, adipose, and circulating fructose metabolites. This approach will quantitatively determine how Glo1 regulates intestinal carbohydrate metabolism, a previously unexplored area of research. Finally, in Specific Aim 3, we will confirm that MGO-derived histone PTMs regulate transcriptional responses to metabolism in vivo. Site-specific canonical and MGO-derived PTMs will be quantified in each tissue/cohort. Putative reader domains will be identified for MG-H1 histone modifications. Lastly, liver, intestine, muscle, adipose, and pancreas will be subjected to RNA-seq and ATAC-seq. This approach will determine the global epigenomic landscape across multiple tissues in a physiologically relevant model for obesity, T2D and NAFLD. Collectively, this proposal will combine mechanistic biochemistry with a multi-omics approach to determine the mechanisms by which GLO1 propagates disease progression.