Elucidating Molecular Mechanisms Linking Fructose to Cholesterol Metabolism

项目总结 摄入果糖不仅是发展为非酒精性脂肪性肝病(NAFLD)的主要风险因素， 但也会促进人类和啮齿动物的高胆固醇血症和动脉粥样硬化。身份识别 将NAFLD与心血管疾病(CVD)联系起来的机制仍然知之甚少。这项提案将重点放在 确定饮食中果糖对胆固醇合成和代谢的影响。使用鼠标 含糖饮料消费模型，候选人显示果糖代谢增加 柠檬酸、乙酰辅酶A和肝脏胆固醇水平。此外，候选人还表现出果糖的减少 线粒体脂肪酸转运蛋白肉碱棕榈酰转移酶1a(CPT1a)的蛋白表达。 此外，条件性CPT1A基因敲除小鼠表现出与喂食果糖的小鼠相似的血脂紊乱。因此， 目的1利用双稳定同位素技术结合核磁共振和质谱学对胆固醇进行定量 男性和女性胆固醇生物合成途径中的合成和富含果糖的碳 老鼠。来自小鼠的肝脏将用于乙酰蛋白质组学，以描绘连接果糖的潜在机制 胆固醇的生物合成。目的2确定CPT1A的转录调控如何改变果糖诱导的 体外和体内联合应用抑制脂肪酸氧化和促进胆固醇合成 接近了。这一目标的目的是揭示CPT1A在协调 调节两个脂肪信号通路(脂肪酸氧化和胆固醇合成)对果糖的反应。 完成这些目标将产生将饮食糖代谢与高胆固醇血症联系起来的机制洞察力。 拟议研究的新颖性是综合双稳定同位素方法与 测量胆固醇合成和果糖衍生碳浓缩到甾醇中的分析技术 在同一群动物中的合成途径。此外，拟议的研究揭示了几个创新之处 尚未探索的机制，包括胆固醇合成酶的乙酰化和调控 CPT1a通过转录机制表达。代谢组学核心在 肯塔基大学，巴克衰老研究所的质谱学核心，以及科学 咨询委员会成员确保候选人成功完成拟议的研究。这 研究得到了职业发展计划的赞扬，在该计划中，候选人将学习新的实验性知识 稳定同位素代谢组学方法学，通过参加研讨会和 他经常参加各种会议，并培养自己的沟通技巧，以便成为一名独立的调查员。 这一K01奖项将使他实现他的长期目标，即建立一个资金充足的实验室研究 心脏代谢性疾病的饮食机制。

PROJECT SUMMARY Fructose consumption is not only a major risk factor for development of non-alcoholic fatty liver disease (NAFLD), but also promotes hypercholesterolemia and atherosclerosis in humans and rodents. Identification of the mechanisms linking NAFLD to cardiovascular disease (CVD) remains poorly understand. This proposal focuses on identifying the influence dietary fructose has on synthesis and metabolism of cholesterol. Using a mouse model of sugar-sweetened beverage consumption, the candidate shows that fructose metabolism increases citrate, acetyl-CoA, and hepatic cholesterol levels. In addition, the candidate demonstrates fructose decreases the protein expression of carnitine palmitoyltransferase 1a (Cpt1a), a mitochondrial fatty acid transport protein. Moreover, conditional CPT1a knockout mice exhibit similar lipid perturbations as mice fed fructose. Therefore, aim 1 utilizes dual stable isotope techniques coupled with NMR and mass spectrometry to quantify cholesterol synthesis and fructose-derived carbon enrichment into the cholesterol biosynthetic pathway in male and female mice. Livers from the mice will be used for acetyl-proteomics to delineate potential mechanisms linking fructose to cholesterol biosynthesis. Aim 2 determines how transcriptional regulation of Cpt1a alters fructose-induced suppression of fatty acid oxidation and enhanced cholesterol synthesis using both in-vitro and in-vivo approaches. The purpose of this aim is to uncover a previous unrecognized role of Cpt1a in coordinating the regulation of both lipid-signaling pathways (fatty acid oxidation and cholesterol synthesis) in response to fructose. Completion of these aims will yield mechanistic insight linking dietary sugar metabolism to hypercholesterolemia. The novelty of the proposed research is the comprehensive dual-stable isotope approach in conjunction with analytical techniques to measure cholesterol synthesis and fructose-derived carbon enrichment into the sterol synthesis pathway in the same cohort of animals. In addition, the proposed research reveals several innovative mechanisms that have yet to be explored, including acetylation of cholesterol synthesis enzymes and regulation of Cpt1a through transcriptional mechanisms. Strong collaborations among the Metabolomics Core at the University of Kentucky, Mass Spectrometry Core at the Buck Institute for Research on Aging, and scientific advisory committee members ensure successful completion of the proposed research by the candidate. This research is complimented by a career development plan in which the candidate will learn new experimental methodology in stable isotope metabolomics, broaden his scientific network through attending workshops and conferences, and develop his communication skills so that he is poised to become an independent investigator. This K01 award will allow him to reach his long-term goals of establishing a well-funded laboratory studying dietary mechanisms in cardiometabolic disease.