Fructose Induced Regulation of Profibrogenic Factors in Hepatic Nonparenchymal Cells

项目摘要 NAFLD在当今社会越来越普遍，影响成人和儿童。高 果糖玉米糖浆的消费量与糖尿病、肥胖和非酒精性脂肪 肝病（NAFLD）。NAFLD患者的果糖消耗量高出2至3倍 与对照组相比，增加了小叶炎症和纤维化的存在。主要 小叶炎症和纤维化的贡献者是活化的肝非实质细胞（NPC）， 其由肝星状细胞（HSC）和枯否细胞组成。果糖代谢一直是 主要在肝细胞中研究，已知其诱导脂肪生成和氧化应激。这些 肝细胞中的事件已经显示出影响枯否细胞功能，将细胞推到 炎症表型然而，果糖代谢对NPC表型和 NAFLD的功能尚不清楚。这个项目的广泛和长期目标是确定新的机制 果糖过量导致NAFLD的发展，通过揭示代谢途径， 影响NPC功能的果糖调节。初步数据显示， 细胞和HSC表达KHK和果糖补充剂，增加组织抑制因子， 与补充葡萄糖的饮食相比，Kupffer细胞中的金属蛋白酶（TIMP 1）表达 或饮用水中不含单糖。在体外，果糖处理增加了细胞内的 乙酸盐和甲酸盐。根据这些观察，该项目的具体目标 1）在体外确定果糖驱动促纤维化表型的机制， Kupffer细胞和2）表征高浓度下Kupffer细胞的表型和果糖代谢 在正常和NAFLD肝脏条件下的果糖消耗。我们的研究设计同时使用了 体内和体外系统，研究提出的目标。我们整合了核磁共振 和液相色谱质谱法在体外（Aim 1）和体内（Aim 2）追踪13 C果糖 在枯否细胞中，并鉴定调节纤维化基因和蛋白质的果糖产生的代谢物 在NPC中的表达。为了研究TIMP-1和醋酸盐在枯否细胞中的作用，小干扰 将在体外使用RNA（siRNA）转染（目标1）。预计从本提案中获得的数据 为NPC中碳水化合物代谢的新机制奠定基础，并提供关键的 对预防和治疗NAFLD的治疗靶点的潜在开发的见解。

PROJECT SUMMARY NAFLD has become progressively more common in today's society impacting adults and children. High fructose corn syrup consumption is strongly correlated with diabetes, obesity and nonalcoholic fatty liver disease (NAFLD). Fructose consumption is two to three times higher in patients with NAFLD compared to controls and increases the presence of lobular inflammation and fibrosis. The major contributors of lobular inflammation and fibrosis are activated hepatic nonparenchymal cells (NPCs), which consist of hepatic stellate cells (HSCs), and Kupffer cells. Fructose metabolism has been investigated primarily in hepatocytes and is known to induce lipogenesis and oxidative stress. These events in hepatocytes have been shown to impact Kupffer cell function pushing the cells to an inflammatory phenotype. However, the distinct role of fructose metabolism on NPCs phenotype and function in NAFLD is unclear. The broad, long term goals of this project are to identify new mechanism by which fructose excess leads to the development of NAFLD, by uncovering metabolic pathways regulated by fructose that impact NPCs function. Preliminary data demonstrate that primary Kupffer cells and HSCs express KHK and fructose supplementation in vivo, increases tissue inhibitor of metalloproteinase (TIMP1) expression in Kupffer cells compared to diets supplemented with glucose or no monosaccharides in drinking water. In vitro, fructose treatment increases intracellular levels of acetate and formate in macrophages. Based on these observations, the specific aims of this project are: 1) to determine in vitro the mechanism by which fructose drives a profibrogenic phenotype in Kupffer cells and 2) characterize the phenotype and fructose metabolism in Kupffer cells under high fructose consumption during normal and NAFLD liver conditions. Our research design uses both in vivo and in vitro systems, to investigate the proposed aims. We integrate nuclear magnetic resonance and liquid chromatography mass spectrometry to trace 13C fructose in vitro (Aim 1) and in vivo (Aim 2) in Kupffer cells and to identify fructose generated metabolites that regulate fibrogenic gene and protein expression in NPCs. To investigate the role of TIMP-1 and acetate in Kupffer cells, small interfering RNA (siRNA) transfections (Aim 1) will be used in vitro. Data obtained from this proposal are expected to establish a foundation for new mechanisms of carbohydrate metabolism in NPCs and provide critical insight for the potential development of therapeutic targets for the prevention and treatment of NAFLD.