Hormonal Control of Hepatic Gluconeogenesis/Glycolysis

描述（由申请人提供）：双功能酶，6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶在葡萄糖代谢中起关键作用。它是负责合成和降解果糖2，6-二磷酸的唯一酶，果糖2，6-二磷酸是肝碳通量的有效细胞内调节剂。在肝脏中，胰高血糖素通过cAMP依赖性蛋白激酶的代谢作用由细胞内果糖-2，6-二磷酸浓度介导。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-激酶并抑制促糖酵解酶果糖-1，6-二磷酸酶，从而调节碳通量的方向。在缺乏循环胰岛素的小鼠实验性（链脲佐菌素诱导的）糖尿病中，已显示果糖-2，6-二磷酸以拟胰岛素方式适当调节葡萄糖激酶（增加）和葡萄糖-6-磷酸酶（抑制）基因表达。在所有形式的糖尿病中，肝脏过度产生葡萄糖是高血糖症的主要原因，高血糖症导致与疾病相关的主要问题。果糖-2，6-二磷酸在控制肝葡萄糖产生和利用以及基因调节中的核心作用表明，针对增加果糖-2，6-二磷酸水平的治疗将对糖尿病患者有益。拟议的研究使用两种方法来靶向双功能酶的双磷酸酶结构域进行抑制，从而增加果糖-2，6二磷酸水平。首先，正在研究通过腺病毒介导的双功能酶的过表达来操纵果糖-2，6-二磷酸的代谢和基因表达效果。双磷酸酶或激酶结构域缺陷的双功能酶的过表达或表达分别产生高或低水平的肝果糖-2，6-二磷酸。使用上述相同的论点，抑制双功能酶的激酶活性应该对肝葡萄糖输出具有相反的作用，并且应该增加血糖水平。第二，使用NMR光谱的物理研究，导致表征的反应机制和结构的双磷酸酶域正在进行中。这些研究将确定水解反应过程中活性位点氨基酸残基的功能作用。对肝促生/糖酵解通量的这一重要组成部分的了解为肝6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶的二磷酸酶活性的特异性抑制剂的合理设计提供了基础。

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.