Carbohydrate Regulation of Hepatic Gene Expression

描述（由申请人提供）：进化压力倾向于允许身体在食物充足时有效地以脂肪形式储存营养物质的机制，以防止偶尔的饥荒。随着现代生活方式的巨大变化，包括高碳水化合物和高脂肪食物的消费，这些机制现在可能是导致美国肥胖流行的主要原因，美国大多数人口都超重。葡萄糖不仅是所有哺乳动物组织的主要燃料，也是脂肪和蛋白质合成碳的来源。肝脏是负责将过量的膳食碳水化合物转化为甘油三酯的主要器官。摄入高碳水化合物饮食可诱导超过15个基因的转录，这些基因与葡萄糖转化为储存脂肪有关。该实验室首次发现了一种从大鼠肝脏中纯化出来的转录因子，该转录因子具有碳水化合物反应元件（ChRE）的特异性，该转录因子存在于脂肪生成所需的多个基因的启动子中，并表现出适当的饮食反应调节，被称为“碳水化合物反应元件结合蛋白，ChREBP”。ChREBP被激活以响应过量碳水化合物，以诱导脂肪生成酶基因的转录，然后被关闭的完整过程尚不完全清楚。我们已经证明，葡萄糖和cAMP在调节脂肪生成方面具有相反的活性，部分是通过ChREBP上多个位点的去磷酸化/磷酸化。葡萄糖通过激活xu5p刺激的蛋白磷酸酶Xu5P-PP2A来刺激至少一些位点的去磷酸化。我们最近发现，ChREBP与14-3-3的相互作用是调控ChREBP核定位的重要步骤之一。ChREBP磷酸化激活其与14-3-3的结合，是饥饿条件下ChREBP输出细胞核的必要条件。此外，我们最近发现肝脏中的一种特定代谢物促进了ChREBP与14-3-3的相互作用。为了进一步表征代谢物，我们建议：(1)以纯形式分离代谢物并确定其结构；(2)确定其激活ChREBP与14-3-3相互作用的机制；(3)确定代谢物是如何合成和降解的，哪些酶负责这些反应以及如何调节这些反应。(4)研究其代谢物是否可能是调节ChREBP亚细胞定位的信号化合物，以应对营养和饥饿；(5)利用x射线晶体学阐明Xu5P如何激活ChREBP和双功能酶Fru6P， 2kinase/ fru2,6 bisPase。

DESCRIPTION (provided by applicant): Evolutionary pressure has favored mechanisms that allow the body to efficiently store nutrients as fat when food is abundant as a safeguard against occasional famine. With the dramatic changes in modern lifestyle including consumption of high carbohydrate and high fat foods, these mechanisms may now be contributing to a major epidemic of obesity in the US where the majority of the population is overweight. Glucose is not only a major fuel of all mammalian tissues but also a source of carbon for fat and protein synthesis. The liver is the principal organ responsible for the conversion of excess dietary carbohydrate into triglycerides. Ingestion of a high carbohydrate diet induces transcription of more than 15 genes involve in the conversion of glucose to storage fat. A transcription factor, purified from rat liver, with specificity for carbohydrate responsive elements (ChRE) found in the promoters of multiple genes required for lipogenesis and which displayed appropriate dietary responsive regulation was first identified in this laboratory and termed "carbohydrate response element binding protein, ChREBP". The complete process by which ChREBP is activated in response to excess carbohydrate in order to induce the transcription of lipogenesis enzyme genes, and then is turned off, is not yet fully understood. We have shown that glucose and cAMP have opposing activities in the regulation of lipogenesis, in part through dephosphorylation/phosphorylation of multiple sites on ChREBP. Glucose stimulates dephosphorylation of at least some of these sites by activating a Xu5P-stimulated protein phosphatase, Xu5P-PP2A. We recently found that the interaction of ChREBP with 14-3-3 is one of the most important steps regulating the nuclear localization of ChREBP. Phosphorylation of ChREBP activates its binding to 14-3-3 and is essential for ChREBP export out of the nucleus under starvation conditions. In addition, we found more recently that a specific metabolite in liver promotes the interaction of ChREBP with 14-3-3. To characterize the metabolite further we propose to (1) isolate the metabolite in pure form and determine the structure, (2) determine the mechanism by which it activates the interaction between ChREBP and 14-3-3, (3) determine how the metabolite is synthesized and degraded, what enzymes are responsible for these reactions and how they are regulated, (4) investigate whether the metabolite might be a signaling compound for regulation of the subcellular localization of ChREBP in response to nutrients and starvation, and (5) elucidate how Xu5P activates ChREBP and the bifunctional enzyme, Fru6P, 2kinase/Fru2,6 bisPase, using X-ray crystallography. PUBLIC HEALTH RELEVANCE: Obesity and its associated diseases, diabetes, hypertension, heart disease and some cancers are among the most serious health problems now facing the US. The efficient conversion of excess carbohydrates to fat for long-term storage, which until modern times it would appear was an evolutionary advantage in adapting to uncertain food supplies, now appears to contribute significantly to development of obesity. Carbohydrate response element binding protein (ChREBP) is a transcription factor that becomes active when carbohydrates are eaten and increases the expression of genes required for synthesis of fatty acids. The results of the work proposed in this application will elucidate the mechanisms involved both in activation and termination of ChREBP dependent increases in enzymes needed to make fatty acids. This knowledge will provide information that may be useful in developing strategies for the prevention and treatment of obesity.