Metabolic Control Analysis

今年实验室的工作主要集中在酮体代谢影响的四个方面。 酮体单酯的生产和应用 在本文报告的所有研究中使用的所有酮体单酯，D--羟基丁酸酯R 1，3丁二醇单酯，均由托德金使用本文设计的方法在本实验室制备。 Robert Pawlosky博士使用本文设计的色谱分离原理和质谱方法分析了酯的纯度及其光学性质。 我们在牛津大学的同事对这些酮酯在人类受试者中的药代动力学进行了进一步测试（1）。 酮酯具有GRAS状态，所有人体使用均符合牛津大学IRB批准的方案要求。 我们向其提供酮酯的10个合作者的列表随附于本报告。 酮体代谢增强生理和认知能力 1969年描述了氧化还原状态与磷酸化电位的关系（Krebs HA &Veech RL，巴里研讨会）然而，需要10年来确定磷酸化电位的能量的精确关系，所述磷酸化电位需要测定细胞内游离Mg 2 +（Veloso D等人JBC 248：4811-119，1973）和由此产生的含磷酸盐代谢反应的平衡常数的变化，以能够确定ATP水解的能量，能量代谢的中心反应（Veech RL和Krebs，HA 1979 JBC 254：6538-47）。 酮体代谢克服胰岛素抗性的能力和ATP水解G的增加首先在工作灌注心脏中确定（Sato，N等FASEB J 9：651-58，1995）。 今年，我们将这项工作扩展到人类受试者，Clarke博士和她的团队表明酮酯的代谢改善了精英运动员的身体表现（2），从而履行了我们对DARPA的义务，DARPA最初为我们的酮体工作提供资金。 这篇论文发表在Cell Metabolism上，并被引用为该杂志2016年最佳出版物之一。 在与来自剑桥和牛津的工作人员合作完成的该领域的第二篇论文中，研究表明，喂食酮酯饮食的大鼠表现出增强的认知能力（3） 酮体改变转录因子的定位和活性 与西南大学医学院的研究人员合作，在肝细胞中证明了酮体D-羟基丁酸酯改变了转录因子碳水化合物反应元件结合蛋白ChREBP的核定位，从而抑制脂肪合成（4）。 在本报告的后面部分中，D--羟基丁酸对转录的另一种作用被描述为组蛋白脱乙酰酶的抑制剂，从而增加抗氧化酶的活性。 酮酯的代谢通过胰岛素敏感性的丧失。补充Krebs循环代谢缺陷导致阿尔茨海默病 描述和审查的原因，造成损失的胰岛素激活大脑丙酮酸脱氢酶和治疗所产生的阿尔茨海默病与酮体（5）。Pawlosky博士和他的合作者比较了长期喂食酮酯饮食的3XTgAD小鼠大脑额叶皮质和海马区的样本，发现海马区克雷布斯循环中间体的缺乏在阿尔茨海默病模型中得到了逆转。 这些发现对阿尔茨海默病治疗策略发展的重要性是《神经化学杂志》（Puchowicz M.A.等人，J Neurochem. 141：162-4，2017）。 酮体代谢降低游离NADP+ /NADPH比值，从而减轻氧自由基损伤 在1969年，我们已经表明，游离NADP+ /NADPH比率，具有-0.42V的氧化还原电位，是体内最负的（Krebs，H.A和Veech RL，1969）。 1995年，我们发现酮体的代谢减少NADP对。 我们现在回顾了氧化还原活性剂的使用，显示了为什么使用单一氧化还原活性剂（如抗坏血酸等）在体内没有明显的影响，但由于细胞内氧化还原对的连接，酮体的代谢可以减轻氧自由基损伤（7）。 已向核生物恐怖主义和培训小组、核辐射委员会、辐射标准机构间指导委员会和NIAID对策方案介绍了酮体减轻流氓核装置电离辐射损害的能力。 酮体模拟热量限制的寿命延长特性。 热量限制长期以来一直被认为可以延长从线虫到小鼠的许多物种的寿命。 喂食酮可以减少胰岛素/胰岛素生长因子受体的信号传导，这是延长寿命的遗传途径中的一个主要因素。D-羟基丁酸是组蛋白脱乙酰酶的天然抑制剂，其抑制FOXO基因的转录，从而增加抗氧化酶的转录。 因此，酮症减少胰岛素信号传导，增加抗氧化酶，并增加减少ROS损伤的还原潜力，这在延长寿命中起着重要作用（8）

The work of the laboratory for this year was centered on four aspects of the effects of ketone body metabolism. Manufacture and Use of Ketone Body Monoesters All the ketone body monoesters, D--hydroxybutyrate R 1,3 butanediol monoester, used in all the studies reported here were made in this laboratory by Todd King using methods devised here. The purity of the ester and its optical properties were analyzed by Dr Robert Pawlosky using chromatographic resolution principles and mass spec methods devised here. Further tests of the pharmacokinetics of these ketones esters in human subjects were performed by our colleagues at Oxford (1). The ketone esters have GRAS status and all use in humans complied with protocol requirements approved by the Oxford University IRB. The list of 10 collaborators to which we have supplied ketone ester is appended to this report. Ketone Body Metabolism Enhances Physiological and Cognitive Performance The relationship of redox states to phosphorylation potential was described in 1969 (Krebs HA &Veech RL, Bari Symposium) However it required 10 years to determine the precise relationship of the energy of the phosphorylation potential required determination of the intracellular free Mg2+ (Veloso D et al JBC 248: 4811-119, 1973) and the resultant changes in the equilibrium constants of phosphate containing metabolic reactions to be able to determine the energy of the hydrolysis of ATP , the central reaction of energy metabolism ( Veech RL and Krebs, HA 1979 JBC 254: 6538-47) . The ability of ketone body metabolism to overcome insulin resistance and the increase in the G of ATP hydrolysis was first determined in working perfused heart (Sato, N et al FASEB J 9: 651-58, 1995). This year we extended this work to human subjects where Dr Clarke and her group showed that the metabolism of ketone esters improved the physical performance in elite athletes (2) thus fulfilling our obligation to DARPA which originally provided the funds for our work on ketone bodies. This paper was published in Cell Metabolism and was cited as among the best publications in that journal for 2016. In a second paper in this area, done in collaboration with workers from both Cambridge and Oxford, it was shown that rats fed a ketone ester diet showed enhanced cognitive performance (3) Ketone Bodies Alter the Localization and Activity of Transcription Factors In collaboration with investigators at Southwestern U Medical school, it was demonstrated in hepatic cells that the ketone body D--hydroxybutyrate altered the nuclear localization of the transcription factor carbohydrate-response element binding protein, ChREBP, thereby inhibiting fat synthesis (4). In a later section of this report, another effect of D--hydroxybutyrate on transcription is described as an inhibitor of histone deacetylase thus increasing the activity of antioxidant enzymes. The Metabolism of Ketone Esters By Passes the Loss of Insulin Sensitivity. Causing Alzheimers Disease By Replenishing A Deficiency of Krebs Cycle Metabolites The description and review of the causes, resulting from a loss of insulin activation of cerebral pyruvate dehydrogenase and the treatment of resultant Alzheimers disease with ketone bodies is presented (5). Comparing samples from the frontal cortex to those from hippocampal regions from the brains of 3XTgAD mice chronically fed a ketone ester diet, Dr Pawlosky and his collaborators showed (6) that the deficiency in Krebs cycle intermediates in the hippocampal region was reversed in this model of Alzheimers disease. The importance of these findings toward the development of a therapeutic strategy for the treatment of Alzheimers disease was the topic of a special article in the Journal of Neurochemistry (Puchowicz M.A. et al, J Neurochem. 141: 162-4, 2017). The Metabolism of Ketone Bodies Reduces the Free NADP+ /NADPH Ratio Thus Mitigating Oxygen Free Radical Damage In 1969 we had shown that the free NADP+ /NADPH ratio, with a redox potential of -0.42V was the most negative in the body (Krebs,H.A and Veech RL, 1969). In 1995, we showed that the metabolism of ketone bodies reduce the NADP couple. We now reviewed the use of redox active agents, showing why the use of single redox active agents such as ascorbic acid and the like were without demonstrable in vivo effects but that the metabolism of ketone bodies due to the linkage of the intracellular redox couple could mitigate oxygen free radical damage (7). The ability of ketone bodies to mitigate the damage of ionizing radiation from rogue nuclear devices has been presented to the Nuclear Bioterrorism and Training Group, the Nuclear Radiation Commission, the Interagency Steering Committee on Radiation Standards and the NIAID Countermeasures Program. Ketone Bodies Mimic the Life Span Extending Properties of Caloric Restriction. Caloric restriction has long been known to extend life span in many species from nematodes to mice. Feeding ketones can decrease signaling in the insulin/ insulin grow factor receptor, a major factor in the genetic pathway of life span extension. D--hydroxybutyrate is the natural inhibitor of the histone deacetylase which represses the transcription of the FOXO gene thus increasing the transcription of antioxidant enzymes. Thus ketosis decreases insulin signaling, increases antioxidant enzymes and increases the reducing potential to diminish ROS damage which plays an important role in prolonging life span (8)