Graeme F. Mason - US grants

technology /technique development; computers; nuclear magnetic resonance spectroscopy; biomedical resource;

Maintenance of the phosphorylation state of the brain, which is essential for cellular viability and normal brain function, requires an unimpeded supply of glucose and oxygen. Under conditions of oxygen insufficiency, changes in certain modulators (such as ATP, G-6-P, G-1, 6-DP) may enhance the rate of glucose utilization by their action on key enzymes in the respective metabolic pathways. In addition, with development in early life, the proportion of glucose oxidized to carbon dioxide and water in the citric acid cycle to that metabolized to pyruvate and lactate in the glycolytic and hexosemonophosphate pathways changes dramatically. The primary objective of this project is to investigate how hypoxia impacts upon the mechanisms which regulate brain glucose metabolism during development. This project will center on the enzymatic control of the respective fluxes through hexokinase, glucose-6-phosphate dehydrogenase, and alpha-ketoglutarate dehydrogenase complex in the neocortex of immature and mature rats exposed to acute and chronic periods of hypoxia. [unreadable] Our general hypothesis is that differences in the regulation of specific metabolic pathways contributing to the maintenance of the phosphorylation state are the result of changes in key enzymatic activities or their modulators. Using in-vivo and in-vitro NMR spectroscopy, MRI and enzymatic assays n-vitro, we will examine not only the maturation of glucose flux, the mechanisms involved and the effect of hypoxia on these but also the importance and the role that such fluxes play in nerve cell function and survival potential in the immature and mature subject.

Magnetic resonance spectroscopy (MRS) and imaging (MRI) allow studies of brain amino acid neurotransmitters in vivo. Recent developments of MR permit not only measurements of neurotransmitter levels, but of synthesis and release, also. The technique is enhanced by the candidate's use of mathematics to interpret measurements quantitatively. The candidate applied an unusual background in MRS, MRI, math, and neurochemistry to alcoholism and (2) improve his education in pharmacology, diagnosis, and treatment of alcoholism. The program will combine MR with other measurements to study neurotransmission and energetics, improve mathematical models of neurotransmission and metabolism, and apply them to pharmacology of alcoholism. His other long term goal is dissemination of MR methods and mathematics for study design and interpretation. The leaders of Psychiatry at Yale have deliberately fostered integration of basic and clinical research, and they have invested heavily in MR research. For education and collaboration with the candidate, there are NIAAA and VA Alcohol Research Centers and laboratories and faculty who are well-established in the field of alcoholism. The grant will free the candidate from other commitments so that he can focus on learning about and researching alcoholism. The development plan has three parts: (1) Education in diagnosis, treatment, neurochemistry, and pathophysiology of alcoholism, (2) Research for more detailed knowledge of pathways of neurotransmitter metabolism, clinical correlates of the basic data, and validation of models of neurotransmission. Two projects are proposed. (1) Measure rates of brain glutamate neurotransmission and glucose utilization in alcohol-dependent patients during detoxification, testing for correlations of those parameters with each other and with measures of cognitive function and electrophysiology, which is funded through the NIAAA Center for Translational Neuroscience in Alcoholism (CTNA) at Yale. (2) Validate and further develop the mathematical models of glutamate and GABA neurotransmission, combining measurements of isotopic labeling in rats with microdialysis measurements of ECF levels of brain glutamate, glutamine, and GABA.

drug /alcohol abstinence; genetic susceptibility; neurotransmitter metabolism; glutamine; brain metabolism; alcoholism /alcohol abuse; glutamates; neural transmission; detoxification; family genetics; cognition disorders; patient oriented research; stable isotope; nuclear magnetic resonance spectroscopy; carbon; human subject; clinical research;

NISTP: NEUROIMAGING SCIENCES TRAINING PROGRAM This institutional grant requests funds for five postdoctoral positions for interdisciplinary training and research in neuroimaging. Two general classes of neuroimaging will be emphasized: radiotracer and nuclear magnetic resonance. The specific imaging methods are described by the following acronyms: PET (positron emission tomography, MRI (magnetic resonance imaging), including fMRI (functional magnetic resonance imaging) and DTI (diffusion tensor imaging), and MRS (magnetic resonance spectroscopy). These imaging methodologies offer real promise for the expanded clinical utility of neuroimaging in the diagnosis, treatment, and understanding of the pathophysiology of substance abuse and dependence. However, these new methodologies typically derive from complex, multidisciplinary sciences, which thereby create a challenging information barrier to their further development and application in medical practice. The purposes of the proposed program are the following: - Provide formal course training to postdoctoral fellows, with an emphasis on imaging and clinical research methodology, and instruction in their underlying multidisciplinary sciences. - Provide mentored training for fellows to apply these new methods to the interdisciplinary advance of our understanding of the function of the brain in health, substance abuse, and other psychiatric diseases. - Provide an integrated research experience for the fellows both in imaging applications and in methodology. All postdoctoral fellows will be expected to complete at least one research project with an applications mentor and one with a methodology mentor during their training period. The integration of training in methodology and imaging applications will be enhanced by the organization of this program as two interactive tracks of approximately equal size: Methodology and Application. We expect that the majority of postdoctoral fellows in the Methodology track will have Ph.D. degrees, and those in the Application track, primarily M.D. degrees or Ph.D. in neurosciences or behavioral sciences. Each fellow will have a primary and secondary faculty mentor to represent the two tracks.

[unreadable] DESCRIPTION (provided by applicant): This workshop will be focused primarily on psychiatric disorders, including mood disorders, schizophrenia, and abuse of substances such as alcohol and nicotine. The workshop has three primary goals: (1) Exchange of ideas and information between investigators involved primarily in technical development and those focused on neuropsychiatric research. (2) Technical education for psychiatric applications researchers. (3) Psychopharmacologic and etiologic education for technically oriented researchers. Attendees will include established researchers from technical and applications areas, but a special emphasis will be made to encourage attendance and participation by young investigators. METHODS: The program will include an international roster of speakers in a two and a half-day symposium. The audience is expected to include 150-250 researchers from around the world. The proposed meeting dates are October 13-15. The proposed agenda will include 2 main topics: Neurotransmitter function in neuropsychiatric disorders: pathophysiology and role in treatment, and Methods of Magnetic Resonance Spectroscopy (MRS). A general outline for the two and a half day meeting is: Introductory Lecture night before conference: Very basics of MRS: What is spin? What is shimming? How do we get a signal? Plenary Presentations What do we need to know about psychiatric disorders? How can imaging in general and spectroscopy in particular, help us learn more about key problem area? Review of neurocircuitry and neurotransmitter function related to psychiatric disorders. Plenary Sessions on each of the MRS methods and psychiatric applications: Teaching talks (30" each) followed by proffered, reviewed papers on current research (10 min each), concluding with open discussion. New Directions and Applications to Treatment: Talks on clinical applications, transition from basic research to pharmaceutical industry. COMMENTS: Topics will include predictors of diagnosis and treatment success; markers for treatment; neurocircuitry; intracellular targets; gene expression, etc. A call for abstracts will bring forward names and ideas from other groups that might be added to the main program. Young Investigator travel awards form an important part of this program. The editor of NMR in Biomedicine, Chris Boesch, has agreed to consider publishing peer-reviewed papers on cutting-edge work presented in the sessions in the conference. Magnetic resonance spectroscopy (MRS) is an imaging technology whose cutting edge applications can pose an intimidating technological barrier to clinical researchers, and our knowledge of neurotransmitter function and regulation has reached a point at which technically trained MRS researchers may lack knowledge of clinical applications and associate issues to apply their advanced techniques. This workshop will promote the cross-education of investigators from both areas, which is necessary to maximize the opportunities for and quality of translational research on neurotransmitter function in psychiatric disorders. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nicotine has been shown to affect GABAergic function in the brain. In rats, nicotine increases GABA release acutely, and human and animal studies have implicated the GABAergic system in the acquisition and maintenance of nicotine addiction. Transient increases in levels of human cortical GABA levels have been observed by magnetic resonance spectroscopy (MRS) with nicotine administration in preliminary studies in humans. Furthermore, nicotine has been seen to elevate brain GABA synthesis 2-4 fold in human brain. Knowing the effects of acute nicotine administration on brain GABA may provide insight into the interaction of tobacco smoking with prevalent disorders that are known to involve the GABAergic system, such as depression and alcoholism. This project may also provide further information about why it is so hard for some people to stop smoking. It has been shown that menstrual cycle related changes in brain GABA levels are abolished by smoking, and that men and women have some different responses to nicotine, so this project will also explore gender-based differences in GABAergic responses in the human brain. Several questions will be answered: 1) Does acute nicotine administration increase brain GABA? 2) How long does it take brain GABA to return to pre-nicotine levels? 3) Does nicotine increase GABA synthesis? 4) Does gender affect nicotine-induced changes in brain GABA levels and rates of synthesis? The concentration and rates of synthesis of brain GABA will be measured in 40 healthy smokers (20 men, 20 women) after overnight abstinence from smoking. The measurements will be conducted twice, once with a nicotine inhaler, and once with a placebo inhaler. Exclusion criteria will include current neuropsychiatric disorders, or a recent history of such. PUBLIC HEALTH RELEVANCE: This work will establish what changes in GABAergic neurons in the cerebral cortex are associated with nicotine and some of the subjective feelings that people experience with nicotine. The outcomes may help to explain why some of the newer drug-based smoking cessation approaches are effective and guide the development of new approaches. The results may also contribute to our understanding of why depressed people and patients with alcohol dependence have a greater likelihood of addiction to tobacco.

DESCRIPTION (provided by applicant): Alcohol abuse and dependence are global health concerns associated with numerous comorbidities. Hypoglycemia is a comorbidity particularly associated with binge-drinking. Under normal conditions glucose is the primary fuel for brain energy metabolism, so in hypoglycemia the brain relies increasingly on blood lactate, ketone bodies, and acetate, all of which cross the blood-brain barrier by the same monocarboxylic acid transporter. When drinking, the body converts alcohol to acetate, and rats are able to utilize the acetate, partially replacing glucose consumption. Studies of hypoglycemia in diabetes and in starvation show that the transport and utilization of monocarboxylic acids are enhanced by hypoglycemia and by elevations in monocarboxlyic acids. Therefore, we hypothesize that through repeated exposure to elevated acetate, ketones, lactate, and hypoglycemia, heavy drinkers who experience repeated episodes of hypoglycemia are more able to consume the acetate derived from ethanol than are light drinkers and non-drinkers. If the hypotheses of this project are supported, the fuel-generation aspect of alcohol may provide a novel award mechanism that promotes the continuation of heavy drinking and helps to prolong episodes of binge-drinking. The central question to be answered is this: Can heavy drinkers readily transport and utilize acetate for brain energy metabolism? A secondary question will be answered: For brain metabolism, do heavy drinkers more readily transport and utilize acetate than light/non- drinkers? These questions will be examined in three components during infusions of [2-13C]acetate. Measurements will be (1) brain acetate concentrations, (2) oxidize acetate more rapidly, and (3) blood-brain transport capacity of heavy drinkers relative to light/non-drinkers. The concentrations and rates of utilization acetate will be measured in 10 heavy drinkers and 10 light/non-drinkers during infusions of [2-13C]acetate. The measurements will utilize 13C MRS at 4 Tesla to detect the time courses of 13C-labeled acetate, glutamate, and glutamine in the brain. PUBLIC HEALTH RELEVANCE: Under most circumstances, people's brains derive nearly all their energy needs from the sugar glucose. Sometimes when people drink large quantities of alcohol, their blood sugar drops, particularly if they are not eating properly, and to survive, the brain must find alternatives to glucose. One alternative chemical that the brain can consume is acetate that the body forms from alcohol. In this study, we will determine if heavy drinkers are more able to use acetate as fuel for the brain. If they are, the possibility exists that heavy drinkers continue drinking not only for the known drug-effects of alcohol, but to provide sustenance for the brain when they do not eat properly, supporting an idea that nutrition is a key player in the ability to reduce heavy drinking or stop drinking alcohol.

DESCRIPTION (provided by applicant): Alcohol abuse and dependence are global health concerns associated with numerous comorbidities. Hypoglycemia is a comorbidity particularly associated with binge-drinking. Under normal conditions glucose is the primary fuel for brain energy metabolism, but during hypoglycemia the brain may rely increasingly on blood lactate, ketone bodies, and acetate, all of which cross the blood-brain barrier by the same monocarboxylic acid transporter. When drinking, the body converts alcohol to acetate, raising blood acetate levels, and possibly providing an alternate brain fuel partially replacing glucose consumption. Studies of hypoglycemia in diabetes and in starvation show that the transport and utilization of monocarboxylic acids are enhanced by blood elevations in monocarboxlyic acids. We hypothesize that repeated exposure to elevated acetate, ketones, and lactate will increase consumption of acetate in rats chronically exposed to ethanol. We also hypothesize that some ethanol is oxidized within the brain. We propose two aims to measure the relative contributions of systemically generated acetate and intracerebral ethanol to brain metabolism in 12 brain regions and estimate the extent of potential glial and neuronal contributions to intracerebral ethanol consumption. If the hypotheses of this project are supported, the fuel-generation aspect of alcohol may provide a novel award that promotes continued heavy drinking and prolongs episodes of binging. The consumption of intracerebral ethanol may provide a nutritive reward, an acetaldehyde reward, which could lead to oxidative damage. The central questions to be answered are these: To what extent do systemically generated acetate and intracerebral ethanol provide substrates for brain energy metabolism, and can ethanol consumption increase those contributions? A secondary assessment is the relative glial and neuronal fractions of intracerebral ethanol oxidation. The concentrations and rates of utilization of acetate and intracerebral ethanol will be measured in rats during infusions of [2-13C]acetate and [2-13C]ethanol. The measurements will utilize 13C MRS of brain extracts to detect time courses of 13C-glutamate and glutamine in the brain. PUBLIC HEALTH RELEVANCE: People's brains usually derive nearly all their energy needs from the sugar glucose, but sometimes when people drink large quantities of alcohol, their blood sugar drops, particularly if they are not eating properly. To survive, the brain may turn to alternatives such as acetate that the liver makes from alcohol, or the alcohol itself, which leads to the formation of other chemicals that may lead people want to drink more, while damaging brain cells. In this study, we will determine if heavy drinkers are more able to use acetate and alcohol as fuels for the brain, and if they are, the possibility exists that heavy drinkers continue drinking not only for the known drug-effects of alcohol and its products, but to provide sustenance for the brain when not eating properly, supporting an idea that nutrition is a key player in the ability to reduce heavy drinking or stop drinking alcohol.

DESCRIPTION (provided by applicant): Alcohol abuse and dependence are global health concerns associated with numerous comorbidities. Hypoglycemia is a comorbidity particularly associated with binge-drinking. Under normal conditions glucose is the primary fuel for brain energy metabolism, so in hypoglycemia the brain relies increasingly on blood lactate, ketone bodies, and acetate, all of which cross the blood-brain barrier by the same monocarboxylic acid transporter. When drinking, the body converts alcohol to acetate, and the brain is able to utilize the acetate, partially replacing glucose consumption. Studies of hypoglycemia in diabetes and in starvation show that the transport and utilization of monocarboxylic acids are enhanced by hypoglycemia and by elevations in monocarboxlyic acids. Therefore, we hypothesize that through repeated exposure to elevated acetate and acute alcohol-induced hypoglycemia when not eating, heavy drinkers have a greater capacity to consume the acetate derived from ethanol than are light drinkers and non-drinkers. Our preliminary data support this hypothesis, and in this proposal we plan to test whether the condition is a state or a trait, by assessing if acetate consumption normalizes in alcohol-dependent people who have been sober for more than six months. We hypothesize that the heavy drinkers will consume more acetate than the light drinkers and long-term sober individuals. If the hypotheses of this project are supported, the fuel-generation aspect of alcohol may provide a novel reward mechanism that promotes the continuation of heavy drinking and helps to prolong episodes of binge-drinking. Another chemical derived from oxidation of alcohol is acetaldehyde, which is rewarding in the brain but aversive in the rest of the body. If the brain can derive energy not only from acetate, but also from the oxidation of ethanol within the brain, then the brain can generate acetaldehyde, creating another trigger to drink alcohol. We hypothesize that the brain does oxidize ethanol and that heavy drinkers oxidize more than light drinkers. If the human brain oxidizes ethanol, it provides a novel reward mechanism in humans, a mechanism that can be investigated with many approaches such as genetics and family history. These questions will be answered with 13C MRS during infusions of 13C-labeled acetate or ethanol.

Abstract Mitochondrial dysfunction has been proposed as a major factor in insulin resistance, aging, and metabolic diseases. 13C NMR in vivo has been the main method to assess mitochondrial fluxes like the TCA cycle and anaplerosis. NMR measures 13C flow from labeled substrates like [3-13C]lactate into the amino acids aspartate and glutamate, with the rationale that via 13C exchange with the TCA intermediate ?-ketoglutarate, glutamate is a ?trap? for 13C mixing with TCA cycle intermediates. Because NMR in vivo requires major technical expertise, methods exist to measure plasma glucose labeling from precursors that enter hepatic metabolism and, from steady-state C labeling, estimate VTCA, particularly using C-propionate. Although in principle these methods 13 13 should agree with tissue measurements, large discrepancies have been observed in several rates, including VTCA. The divergence is the subject of several recent commentaries, letters, and symposia but lacks a clear resolution. Resolving the controversy is key to understand the role of mitochondria in the pathogenesis and treatment of hepatic insulin resistance, nonalcoholic steatohepatitis, and type 2 diabetes. A solution to the controversy is to measure 13C positional labeling of TCA cycle intermediates. NMR in vivo and steady-state plasma glucose methods yield indirect measures of mitochondrial metabolism and depend on some incompletely tested assumptions about relationships with cytosolic glutamate and aspartate. We recently published the Mass Isotopomeric Multi Ordinate Spectral Analysis (MIMOSA) platform for comprehensive, stepwise, integrated analysis of intracellular metabolism (see Alves et al., Cell Metabolism, 2015). The ?mass isotopomer? aspect of MIMOSA uses MS/MS-based ion fragmentation analysis of stable- isotope-labeled metabolites to identify carbon-specific label positions. The ?multi-ordinate? aspect is a major innovation that allows direct assessment of label flow along intersecting pathways, including mitochondrial intermediates that are inaccessible by positional NMR due to sensitivity limitations. We used MIMOSA in a cell model and found that previous measures of anaplerosis by steady-state glutamate labeling were up to 3x too high due to mitochondrial dilution pathways that could not otherwise be measured. We propose to apply MIMOSA in an animal model in vivo to establish the ground truth for hepatic VTCA and other key fluxes (Aim 1). We will use the information to test the accuracy of present methods used in vivo for human and rodent studies (Aim2) and develop improved measurement methods. Aim 3 will assess plasma labeling patterns resulting from tissue-specific metabolism that can impact the interpretation of tissue data. Our preliminary data identify a lactate-glycerol shunt in adipose that may have pathologic effects in addition to confounding flux measurements in vivo. Consequently, targeting this pathway may be a novel treatment for diabetes or other metabolic diseases. A major translational goal is to develop a cross-validated in vivo analytic platform using either MS or NMR either humans or rodents.

Abstract When people drink, the ethanol can provide energy for the brain, and that supply increases with chronic, heavy exposure in rats and humans. The energy derives partly from acetate that is generated hepatically and released into the blood, which is then carried to the brain. However, ethanol can also be oxidized inside the brain. This process contributes to behavioral effects of ethanol, including the locomotor reduction seen with ethanol administration in rats, and there may be relationships between vulnerability to alcohol abuse and how brain and systemic ethanol metabolism occurs, even though both processes are toxic. We propose to quantify brain ethanol oxidation, including a way to assess rates of flow through catalase relative to total brain ethanol oxidation. We will examine how brain ethanol metabolism interacts with gender and chronic ethanol exposure to affect behavior following acute and chronic ethanol exposure. Catalase is believed to dominate brain ethanol oxidation. We propose to obtain measures of locomotor activity and metabolism with and without catalase inhibition to quantify the portion of brain ethanol oxidation that flows through catalase. There is strong reason to believe that male and female rats will differ with respect to enzyme activity, metabolism, and behavioral responses to ethanol, including following chronic ethanol exposure, so we will study male and female rats and use vapor chambers to achieve daily exposure. Other enzymes, cytochrome P450, specifically the Cyp2e1 isoform, and possibly to a limited extent brain alcohol dehydrogenase, may also contribute to brain ethanol oxidation. However, we are focusing the resources of this R21 project on (1) establishing the enzyme procedure in the context of the metabolic rate measurements as related to behavior, (2) determining how much of the exposure-induced increase in brain ethanol oxidation is due to catalase versus other enzymes, and (3) assessing how metabolism and exposure interact to affect behavior differences by sex. The project relates to human health because men and women are known to be affected differently by ethanol for multiple reasons, and this project may illuminate metabolic mechanisms that can contribute to these sex differences. The results will ultimately provide information about enzyme targets of potential importance to prevent the neurotoxic effects of ethanol that may be related to vulnerability to alcohol abuse and dependence.