Elucidating Molecular Mechanisms Linking Fructose to Cholesterol Metabolism

项目摘要 果糖摄入不仅是非酒精性脂肪肝（NAFLD）发展的主要危险因素， 而且还促进人和啮齿动物的高胆固醇血症和动脉粥样硬化。识别 NAFLD与心血管疾病（CVD）的联系机制仍然知之甚少。该提案重点 确定膳食果糖对胆固醇合成和代谢的影响。使用鼠标 模型的含糖饮料消费，候选人表明，果糖代谢增加， 柠檬酸盐、乙酰辅酶A和肝脏胆固醇水平。此外，候选人证明果糖减少 线粒体脂肪酸转运蛋白肉毒碱棕榈酰转移酶1a（Cpt1a）的蛋白表达。 此外，条件性CPT 1a基因敲除小鼠表现出与喂食果糖的小鼠相似的脂质扰动。因此，我们认为， 目的1利用双稳定同位素技术结合核磁共振和质谱法来定量胆固醇 合成和果糖衍生的碳富集到男性和女性的胆固醇生物合成途径中 小鼠小鼠的肝脏将用于乙酰蛋白质组学，以描绘果糖与 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细胞凋亡。 使用体外和体内两种方法抑制脂肪酸氧化和增强胆固醇合成 接近。这一目的的目的是揭示以前未被认识到的Cpt1a在协调 调节两种脂质信号通路（脂肪酸氧化和胆固醇合成）对果糖的反应。 这些目标的完成将产生联系膳食糖代谢与高胆固醇血症的机制见解。 拟议研究的新奇在于综合性双稳定同位素方法， 测量胆固醇合成和果糖衍生碳富集到甾醇中的分析技术 在同一组动物中的合成途径。此外，该研究还揭示了几个创新点。 尚未探索的机制，包括胆固醇合成酶的乙酰化和调节 Cpt1a通过转录机制。代谢组学核心之间的强有力合作， 肯塔基州大学，巴克衰老研究所质谱核心，以及科学 咨询委员会成员确保候选人顺利完成拟议的研究。这 研究是由一个职业发展计划，其中候选人将学习新的实验补充 稳定同位素代谢组学的方法，通过参加研讨会扩大他的科学网络， 会议，并发展他的沟通技巧，使他准备成为一个独立的调查员。 这个K01奖项将使他能够实现建立一个资金充足的实验室研究的长期目标 心脏代谢疾病的饮食机制

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.