BRAIN-1

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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.