Hormonal Control of Hepatic Gluconeogenesis/Glycolysis

描述(申请人提供)：6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase是一种双功能酶，在葡萄糖代谢中起着至关重要的作用。它是唯一负责合成和降解2，6-二磷酸果糖的酶，果糖2，6-二磷酸是一种有效的肝脏碳流量的细胞内调节剂。在肝脏中，胰升糖素的代谢作用通过cAMP依赖的蛋白激酶，由细胞内果糖-2，6-二磷酸的浓度来调节。高水平的果糖-2，6-二磷酸变构激活糖酵解酶6-磷酸果糖-1-激酶，抑制葡萄糖异生酶果糖-1，6-二磷酸酶，从而调节碳流的方向。在缺乏循环胰岛素的实验性(链脲佐菌素诱导的)糖尿病小鼠中，2，6-二磷酸果糖被证明以一种类似胰岛素的方式适当地调节葡萄糖激酶(上调)和葡萄糖-6-磷酸酶(抑制)基因的表达。在所有形式的糖尿病中，肝脏过度产生葡萄糖是导致高血糖的主要因素，高血糖导致与疾病相关的主要问题。果糖-2，6-二磷酸在控制肝脏葡萄糖的产生和利用以及基因调控方面的核心作用表明，旨在提高果糖-2，6-二磷酸水平的治疗将对糖尿病患者有益。建议的研究使用两种方法来针对双功能酶的双磷酸酶结构域进行抑制，从而提高果糖-2，6二磷酸的水平。首先，正在研究腺病毒介导的双功能酶过表达对果糖-2，6-二磷酸的代谢和基因表达的影响。双功能酶的过度表达或表达缺失的双磷酸酶或激酶结构域分别产生高或低水平的肝脏果糖-2，6-二磷酸。根据上面概述的相同论点，抑制双功能酶的激酶活性应该会对肝脏葡萄糖输出产生相反的影响，并应该增加血糖水平。第二，使用核磁共振波谱进行的物理研究正在进行中，这些研究导致了对双磷酸酶结构域的反应机理和结构的表征。这些研究将确定活性部位氨基酸残基在水解反应中的功能作用。了解肝脏糖异生/糖酵解通量的这一重要组成部分，为合理设计肝6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase.双磷酸酶活性的特异性抑制剂提供了基础

DESCRIPTION (provided by applicant): The bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase plays a crucial role in glucose metabolism. It is the sole enzyme responsible for the synthesis and degradation of fructose 2,6-bisphosphate, a potent intracellular modulator of hepatic carbon flux. In liver, the metabolic effects of glucagon, via cAMP-dependent protein kinase, are mediated by the intracellular concentration of fructose-2,6-bisphosphate. High levels of fructose-2,6-bisphosphate allosterically activate the glycolytic enzyme 6-phosphofructo-1-kinase and inhibit the gluconeogenic enzyme fructose-1,6-bisphosphatase, thereby regulating the direction of carbon flux. In experimental (streptozotocin-induced) diabetes in mice, which are devoid of circulating insulin, fructose-2,6-bisphosphate has been shown to appropriately regulate glucokinase (increase) and glucose-6-phosphatase (repress) gene expression in an insulinomimetic manner. In all forms of diabetes, the excessive production of glucose by the liver is a major contributor to hyperglycemia, which leads to the major problems associated with the disease. The central role of fructose-2,6-bisphosphate in control of hepatic glucose production and utilization, as well as gene regulation, suggests that therapies directed toward increasing the fructose-2,6-bisphosphate levels will be beneficial to the diabetic patient. The proposed studies use two approaches to target the bisphosphatase domain of the bifunctional enzyme for inhibition, and thereby, increase the fructose-2,6 bisphosphate levels. First, the metabolic and gene expression effects of manipulation of fructose-2,6-bisphosphate by adenoviral-mediated overexpression of the bifunctional enzyme are being investigated. The overexpression or expression of bifunctional enzyme with deficient bisphosphatase or kinase domains generates high or low levels of hepatic fructose-2,6-bisphosphate, respectively. Using the same arguments outlined above, the inhibition of the kinase activity of the bifunctional enzyme should have opposite effects on hepatic glucose output and should increase blood glucose levels. Second, physical studies using NMR spectroscopy that lead to the characterization of the reaction mechanism and structure of the bisphosphatase domain are underway. These studies will define functional roles of active site amino acid residues during the hydrolysis reaction. Knowledge of this important component of hepatic gluconeogenic/glycolytic flux provides the basis for the rational design of specific inhibitors of the bisphosphatase activity of hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.