BRAIN-1

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。酮症的重要性不仅源于它在生酮饮食中的关键作用，而且还因为它通常频繁发生。它在轻度到中度的快速运动和剧烈运动中迅速发展。例如，禁食36小时通常会导致成人血浆BHB升高7倍，从0.2mM上升到1.5mM，再过12-18小时会进一步上升到2.5mM。也有报道称，在成人中，血浆酮浓度可攀升至8 - 11mM，而葡萄糖则为正常或低正常（3-5mM）。因此，很明显，在酮症中，酮不仅可以补充葡萄糖，还可以成为一种主要的大脑燃料。尽管如此，人们对酮在人脑中的运输和利用知之甚少。研究酮症脑代谢的困难与缺乏一种稳定的放射性酮体示踪剂有关，类似于葡萄糖的FDG。Blomqvist等人使用C-BHB PET报道，人类大脑中酮类的区域摄取可以用一个单室模型来描述，这表明大脑中的酮类库可以忽略不计。然而，考虑到计算出的酮类脑代谢率（CMRkb）值在0.49 - 10.31 nM/ml-min（一室）之间，测量本身容易出现分散。尽管测量酮类的运输参数存在困难，但一些研究表明，酮体氧化对脑总能量代谢有重要贡献。Hasselbalch等人发现，在非禁食志愿者中急性注射BHB后，整体CMRkb从0.011uM/g-min增加到0.081uM/g-min，抵消了整体CMRglc从0.26uM/g-min下降到0.17uM/g-min的影响。这表明酮类替代葡萄糖的程度是相当大的。Owens等人研究了空腹3周的肥胖患者，测量了全球AV差异，发现脑葡萄糖用量下降了50%，剩余的45%的葡萄糖被糖酵解并以乳酸释放。在这些患者中，酮类约占总燃料消耗的65%。然而，酮替代葡萄糖的程度取决于两者在脑代谢和功能中的作用。Magistretti及其同事的假设表明，葡萄糖是神经传递的专性氧化燃料。这与Hertz等人的假设形成对比，Hertz等人对葡萄糖和酮氧化的相互依赖性没有具体的预测。在考虑这些可能性时，通过评估脑氧化率和乳酸水平可以最清楚地评估酮的利用程度。（氧化是葡萄糖和酮的最终能量产生途径。）a .2低碳水化合物饮食和癫痫人们早就知道，生酮饮食（KD，一种80%以上热量来自脂肪的饮食）可以成功地控制难治性癫痫的发作。虽然对于生酮饮食控制癫痫发作的解释提出了许多假设，但代谢作用的证据是最有力的。在两种慢性酮症大鼠模型中，慢性酮症最明显的影响是大脑生物能量储备的增加。Devivo等人报道，慢性酮症患者的总能量储存（糖原、葡萄糖、磷酸肌酸）增加了12%，而Nakazawa等人则报道了ATP浓度的直接增加。虽然很难获得人体数据，但Schwartz等人的研究报告称，接受KD治疗的患者癫痫发作控制较好；这与酮症的实现有关，尽管血浆酮浓度与癫痫发作控制之间没有相关性。最近，Kossoff等人的报告重新开始考虑酮症，因为他们发现即使是低碳水化合物饮食（LCD）也可以改善癫痫发作的控制。在实施这种饮食时，Kossoff等人将受试者维持在饮食的诱导阶段，在此阶段每天使用约10至20克碳水化合物。虽然没有系统的研究对癫痫患者的KD和LCD进行比较，但通过酮替代调节葡萄糖的可用性是这些饮食的一个共同方面。鉴于葡萄糖利用模型将燃料消耗与脑功能活动相结合，我们预计，对酮类如何影响脑代谢的更好理解将提供有关此类饮食如何在临床上有效的信息。众所周知，在禁食、剧烈运动、低碳水化合物饮食以及许多临床疾病的状态下，大脑可以从葡萄糖的主要燃料转换为酮体。然而，人类大脑使用酮的方式并没有很好地定义。此外，考虑到许多大脑功能模型都认为葡萄糖在激活中起着关键作用，大脑如何适应生酮饮食的条件是一个特别有趣的问题。葡萄糖和酮在促进大脑功能活动中所起的独特作用，可能是长期以来人们认为生酮饮食可以显著提高深度癫痫发作频率的原因。最近的数据进一步有力地表明，即使是较温和的生酮饮食（低碳水化合物饮食，如阿特金斯饮食法）也可能同样有效。因此，本项目的目标是更好地了解人脑酮体代谢的代谢生理学。我们将使用磁共振光谱和成像方法，这是一种非侵入性和非放射性的方法，可以从生物化学和生理上评估活体大脑。为了更好地定义酮体在人脑中的生理使用，我们将首先检查酮体的运输，测试空腹诱导单羧酸转运体发生的假设。使用磁共振光谱编辑方法检测羟基丁酸盐和乳酸盐，我们将验证以下假设：在禁食3天的受试者中，脑BHB显著升高，并且脑BHB水平随着血浆酮水平的升高而升高。我们还将测试禁食诱导脑酮体转运蛋白的假设，导致空腹受试者的脑BHB水平高于非禁食志愿者的同等血浆酮水平。通过更好地了解转运体的特征，我们将提高我们对脑酮如何依赖于血浆水平的理解。如果脑酮水平是衡量患者群体中酮症效用的关键变量，那么这种测量对于确定酮症患者和酮症饮食的神经系统功效可能是重要的。目的1：为了更好地了解酮被氧化的代谢区（神经元与星形细胞），我们可以使用C标记（MR可检测）酮。例如，Pellerin和Magistretti所描述的大脑活动模型将神经元燃料描述为主要是乳酸盐，由星形细胞葡萄糖消耗糖水解产生。根据酮的代谢过程（星形细胞或神经元），C标签的分布将展示一种特征模式，可以通过体内C光谱进行评估。因此，我们将检验酮类对氧化代谢的部分贡献很小的假设，并且在更大程度上优先被神经元使用，而不是星形胶质细胞。目的2：作为一种替代燃料，我们的假设是酮可以为神经元提供一种不需要星形细胞加工的燃料来源。这种可能在功能上绕过星形胶质细胞的途径可能是特别有趣的，因为星形胶质细胞功能障碍已被认为是对癫痫过度兴奋性的潜在重要影响。在项目的前两个目标中，我们已经评估了酮在人脑中的代谢过程。然而，在基本水平上，葡萄糖和酮之间的一个重要区别是ATP生成的过程和效率。因此，燃料转换可能会引起能量变化。目的3：我们将通过在超重志愿者中使用低碳水化合物饮食来诱导慢性酮症，以确定神经能量后果。在初步数据中，我们对5名超重志愿者进行了低碳水化合物饮食研究，发现ATP浓度主要在灰质、皮层下核（包括丘脑和下丘脑）中增加。在Aim 3中，我们将完成对低碳水化合物饮食导致大脑能量增加的假设的测试，这是组织特异性的，其中灰质和皮层下结构表现出最大的影响。脑酮症的这种影响可能再次与癫痫特别相关，大量的人类和动物癫痫模型工作已经证明了一个网络过程，即癫痫发作的传播是通过关键的皮层下结构传播的。这些皮层下分支点可能对控制癫痫的扩散具有潜在的重要作用。因此，酮饮食对临床改善的另一个可能贡献可能是能量改善的神经解剖学分布扭曲的结果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The importance of ketosis stems not only from its critical role in the ketogenic diet, but also because of its normally frequent occurrence. It develops quickly during a mild-to-moderate fast and with vigorous exercise. For example, a 36hour fast will typically result in a 7 fold elevation of plasma BHB from 0.2mM to 1.5mM in adults, rising further to 2.5mM after another 12-18hours. It has also been reported that in adults, plasma ketone concentrations can climb to 8 to 11mM while glucose is either normal or low- normal at 3-5mM. Thus it is clear that in ketosis, ketones not only serve to supplement glucose but can also become a major cerebral fuel. In spite of this, relatively little is known about the transport and utilization of ketones in the human brain. The difficulty in studying brain metabolism in ketosis is related to the absence of a stable radioactive ketone body tracer analogous to FDG for glucose. Blomqvist et al, using C-BHB PET has reported that the regional uptake of ketones in human brain can be described by a one compartment model, suggesting that the cerebral pool of ketones is negligible. However, the measurement itself is subject to scatter, given the calculated cerebral metabolic rate of ketones (CMRkb) values, at 0.49 - 10.31 nM/ml-min (one compartment). In spite of the difficulties in measuring the transport parameters for ketones, several studies have shown that ketone body oxidation contributes significantly to total brain energy metabolism. With an acute infusion of BHB in non-fasted volunteers, Hasselbalch et al found that global CMRkb increased from 0.011uM/g-min to 0.081uM/g-min, offsetting a global decrease in CMRglc, from 0.26uM/g-min to 0.17uM/g-min. This suggests that the extent to which ketones can replace glucose can be quite large. Owens et al studied 3-week fasted obese patients with global AV difference measurements, and found a drop of 50% in brain glucose use, with 45% of the remaining glucose glycolyzed and released as lactate. In these patients, ketones accounted for approximately 65% of total fuel utilized. However the degree to which ketones may replace glucose necessarily depends on the roles both have in brain metabolism and function. The hypothesis of Magistretti and colleagues suggests that glucose is an obligate oxidative fuel for neurotransmission. This contrasts with the hypothesis of Hertz et al, which has no specific prediction for the interdependence of glucose and ketone oxidation. In considering these possibilities, the degree to which ketones are utilized would be most clearly assessed by evaluating brain oxidation rates and lactate levels. (Oxidation is the ultimate energy producing path for both glucose and ketones.) A.2 Low carbohydrate diets and epilepsy It has long been known that the ketogenic diet (KD, a diet where more than 80% of calories are derived from fats) can be successful in seizure control in intractable epilepsy. Although numerous hypotheses have been proposed as explanations for seizure control by the ketogenic diet, the evidence for a metabolic contribution has been the strongest. As demonstrated in two rat models of chronic ketosis, the clearest effect of chronic ketosis is an increase in brain bioenergetic reserve. Devivo et al reported that a sum of total energetic storage (glycogen, glucose, phosphocreatine) increased by 12% with chronic ketosis while Nakazawa et al reported a direct increase in ATP concentrations. Although human data have been difficult to obtain, studies of Schwartz et al reported that patients placed on the KD had better seizure control; this was correlated with the achievement of ketosis, although no correlation could be found between plasma concentrations of ketones and seizure control. More recently, the report of Kossoff et al has revived consideration of ketosis, as they found that even the low carbohydrate diet (LCD) could improve seizure control. In implementing this diet, Kossoff et al maintained subjects in the induction phase of the diet, in which approximately 10 to 20grams of carbohydrates/day are used. Although no systematic study has been undertaken to compare the KD and LCD in epilepsy patients, regulating the availability of glucose through ketone replacement is a common aspect of these diets. Given the models of glucose utilization that interlace fuel consumption with brain functional activity, we anticipate that a better understanding of how ketones affect cerebral metabolism will provide information as to how such diets may be clinically efficacious. SPECIFIC AIMS It is well known that the brain can switch from its primary fuel of glucose to ketone bodies under states of fasting, vigorous exercise, low carbohydrate diets as well as a number of clinical disorders. However, the way in which the human brain uses ketones is not well defined. Furthermore, how the brain accommodates under conditions of ketotic diets is of particular interest given the numerous models of brain function that give specifically glucose a critical role in activation. The distinctive roles that glucose and ketones have in fueling cerebral functional activity may be specifically contributing to the long held fact that the ketogenic diet can significantly improve seizure frequency in profound epilepsy. Recent data further has strongly suggested that even milder forms of the ketogenic diet (a low carbohydrate diet such as the Atkins diet) may be similarly effective. Thus the goals of this project are to better understand the metabolic physiology of human brain ketone body metabolism. We will do this using methods of MR spectroscopy and imaging, a non-invasive and non-radioactive means to biochemically and physiologically evaluate the living brain. To better define the physiology of ketone body use in human brain, we will first examine the transport of ketones, testing the hypothesis that induction of the monocarboxylic acid transporter occurs with fasting. Using MR spectroscopic methods of editing to detect hydroxybutyrate and lactate, we will test the hypothesis that in the 3 day fasted subjects significant elevations of brain BHB occur and that brain BHB levels increase with increasing plasma ketone levels. We will also test the hypothesis that fasting induces the brain ketone body transporter, resulting in higher brain BHB levels in fasted subjects as compared to non-fasted volunteers for equivalent plasma ketone levels. By better understanding the transporter characteristics, we will improve our understanding as to how brain ketone depends on plasma levels. If the cerebral level of ketones is a key variable in terms of gauging the utility of ketosis in patient populations, such measurements may be important for determining the neurological efficacy of ketosis and ketotic diets in patients. Aim 1: To better understand the metabolic compartment (neuronal vs. astrocytic) in which ketones are oxidized, we can use C labeled (MR detectable) ketones. For example, the model of brain activity as described by Pellerin and Magistretti describes neuronal fuel as largely being lactate, glycolytically produced from astrocytic glucose consumption. Depending on the metabolic processing of ketones (astrocytic vs. neuronal), the distribution of C label will demonstrate a characteristic pattern that can be evaluated by in vivo C spectroscopy. Thus we will test the hypothesis that the fractional contribution of ketones to oxidative metabolism is small, and is preferentially used by neurons to a greater extent than astrocytes. Aim 2: As an alternate fuel, our hypothesis is that ketones may provide the neuron with a fuel source that does not require astrocytic processing. This path that potentially functionally circumvents the astrocyte may be special interest, as astrocytic dysfunction has been suggested to be a potentially important influence on hyperexcitability in epilepsy. In the first two aims of the project we have evaluated the metabolic processing of ketones in human brain. However, at a fundamental level, an important difference between glucose and ketones is the process and efficiency with which ATP is generated. Fuel shifts may thus be expected to induce energetic changes. Aim 3: We will induce chronic ketosis by using low carbohydrate diets in overweight volunteers to establish the neuro-energetic consequences. In preliminary data, we have studied 5 overweight volunteers on a low carbohydrate diet, and have found increases in ATP concentrations primarily in gray matter, subcortical nuclei including the thalamus and hypothalamus. In Aim 3 we will complete testing the hypothesis that A low carbohydrate diet results in an increase in cerebral energetics that is tissue specific, with gray matter and subcortical structures showing the greatest effect. This effect of cerebral ketosis may be again especially pertinent to epilepsy, where substantial human and animal model work in epilepsy have demonstrated a network process, i.e., spread of seizures is propagated through key subcortical structures. These subcortical branch points may be viewed as potentially important for controlling seizure spread. Thus another possible contribution to the clinical improvement seen with ketotic diets may be a consequence of a skewed neuroanatomical distribution of energetic improvement.