Douglas L. Rothman - US grants

The GABAergic system may play a significant role in the pathogenesis as well as in the pharmacological control of epilepsy and other brain diseases. Concentrations and rates of synthesis of GABA may reflect the activity of the GABAergic system. The goal of the proposed research is to develop quantitative single volume and multivolume 1H NMR methods for measuring brain gamma amino butyric acid (GABA) concentration and rates of synthesis in human brain. The methods will be applied in studies of human subjects to determine the precision of the measurement, the accuracy of the resonance assignment, and to measure the relaxation properties needed for GABA absolute quantitation. An animal model will be used to validate the NMR method by comparison with conventional chemical assay. The development of 1H NMR methods for measuring GABA will provide an important new tool for studying the human GABAergic system.

The relationships between brain cell metabolism and functional activity, studies of amino-acid metabolism and the effects of pharmacological agents basic studies of glucose metabolism and mechanisms of glucose homeostasis. New NMR-based functional brain imaging methods for assessing neuronal activation in response to specific cognitive and sensory stimuli. Susceptibility contrast effects from alterations in blood oxygenation in the microvasculature that can be used to detect regional brain activation. This application is unique in that it involves the combined efforts of scientists who are leaders in spectroscopic studies of metabolism and physiology, in imaging of brain functional activity and other aspects of MR imaging science, as well as leading neuroscientists. A high degree of synergy will occur between the spectroscopic and imaging studies, and the resource will be the primary system for advanced NMR applications serving an experienced community of users.

We are requesting funds to purchase a Bruker Avarice DBX NMR spectroscopy/imaging console and shielded gradient set with rf coil to be placed on a 7.OT 20 cm bore magnet located in the Yale Magnetic Resonance Center. The 7.OT system has been in the forefront in the development and application of MRS and MRI methods to understand the regulation of metabolism and how metabolism is altered in pathology. The studies which are described in the project section are addressing cutting edge issues in basic biochemistry/physiology and clinical science including the energy requirements of brain functional activity, the interaction between anti epileptic compounds and GABA metabolism and function, metabolic adaptations of the neonatal cortex to hypoxia, the role of altered muscle and liver carbohydrate metabolism in the pathogenesis of diabetes, and the role of metabolism in the regulation of neurotransmitter glutamate and GABA pools. The insights into metabolic regulation obtained from these studies has guided us in the application of similar methods in the study of pathology in human patients on our 2.1T whole body system which is presently equipped with an Avance console. Presently our ability to further improve the MR methodology is hampered by an obsolete console and gradient set. The requested equipment will allow us to continue to develop and apply state of the art NMR methodology to the research problems described in this proposal. In many experiments the new equipment will lead to a 2-4 fold improvement in sensitivity and spatial resolution improvement. The improvements in MRS and MRI provided by requested equipment will allow Yale researchers to stay in the forefront of basic and clinical studies of metabolism and physiology.

DESCRIPTION (provided by applicant): The last decade has seen a renaissance of interest in how astroglial metabolism supports neuronal function. Magnetic Resonance Spectroscopy (MRS) in combination with stable 13C labeled precursors is the only method which may be used for studying neuronal/glial metabolic relationships non invasively in humans. In our previous funding cycle we used 13C MRS to show that the neuronal/glial glutamate/glutamine cycle is highly active in human cerebral cortex, and is the major pathway of both neuronal glutamate repletion and astroglial glutamine synthesis. In the course of this research we developed a novel labeling strategy for human studies using 2-13C acetate. Acetate is unique as a brain fuel because it is almost exclusively metabolized in astroglial cells, a property which makes it ideal for studying neuronal/glial neurotransmitter cycles. The primary goals of this grant are to further develop the 2-13C acetate/MRS method at 4T, and then use it to answer several pivotal questions on the role of neuronal/glial neurotransmitter cycles in normal brain function. Our specific aims are in human cerebral cortex to (1) characterize acetate transport and metabolism to allow absolute rates to be determined from acetate tracer studies, (2) study the role of glia in supporting excitatory function by measuring with much higher spatial resolution the neuronal/glial glutamate/glutamine cycle and its coupling to neuronal and glial glucose oxidation (3) study the role of glia in supporting inhibitory function by measuring the GABA/glutamine cycle and GABAergic neuron glucose oxidation and (4) assess the importance of astroglial glutamate oxidation for maintaining proper neuronal function. In addition we will validate these measurements both through comparison with results obtained using a 1-13C glucose tracer, and by assessing test-retest stability of metabolic rates. Although no patient studies are proposed, our goal is for these studies to provide validated methodology and normative values to be used in future studies of neuronal-glial neurotransmitter cycles in development, aging, and disease.

We are submitting a request for $500,000 towards the purchase of a Magnex 11.74T/21 cm horizontal bore magnet for the development of an advanced magnetic resonance (MR) spectroscopy (MRS) and imaging (MRI) system for studying transgenic mice and rat models. The major aims of the research planned on the 11.74T system are to develop in vivo MRS and MRI measurements of metabolism and physiology, and apply these methods to understand the function of key gene products in transgenic mouse and rat models. The MR group at Yale, directed by Dr. Douglas L. Rothman who is the PI o this proposal, has been a leader in the development of MRS/MRI methods for studying metabolism in small animals and humans. The primary user group of the SIG consists of 8 NIH funded Yale investigators who will use the 11.74T to address fundamental questions relevant to human disease including the molecular regulation of muscle and liver carbohydrate and lipid metabolism in transgenic models of diabetes; the regulation of neurotransmitter glutamate and gamma-amino butyric acid release and recycling; the molecular mechanisms coupling glutamate neurotransmitter cycling to neuroenergetics; the neurophysiological basis of fMRI, and the functional neuroanatomy of mammalian sensory systems (e.g., whisker barrel cortex and olfactory bulb). The presence of an active human MR research program at the Magnetic Resonance Center (MRC) at Yale has facilitated the translation of these findings to clinical research studies in diabetes, epilepsy, neonatal hypoxia, obesity, and psychiatric disorders. These studies all have depended upon the development of quantitative high resolution MRS and MRI methods at the Yale MRC, made possible by state-of-the-art magnet technology. The importance of the further development of rodent MRS/MRI technology, for which the 11.74T system is critical, is emphasized by the recent approval of an award to Yale from the NIH to develop a transgenic Mouse Metabolic Phenotyping Center. This Center is a national resource which makes available to NIH funded investigators throughout the country with transgenic mouse models of diabetes the advanced MRS and MRI methods developed at Yale. This effort will make extensive use of the 11.74T system. We believe the long-standing tradition at Yale of innovation in MRS/MRI measurements and close interdisciplinary collaborations provide an ideal site for the development of an 11.74T in vivo MRS/MRI system and the application to transgenic mouse and other small animal models for understanding the molecular control of metabolism and physiology in health and disease.

Since its establishment in 1985 the Yale Magnetic Resonance Research Center (MRRC) has been a leader in the development and application of MRS technology and 13C isotopic labeling strategies for studying carbohydrate and lipid metabolism in insulin resistance. Much of the initial support for the development of the MRRC core research program came from NIDDK. Recently the MRRC has moved to a new 30,000 square foot building and has added a new 4.0T human MRS/MRI system dedicated to studying metabolism. The primary aim of the MRS/MRI Core will be to provide comprehensive MRS and MRI support on this system for the projects clinical core. Core services provided are i) MRS and MRI methodology at 4.0T for studying brain liver and muscle mitochondrial function ii) metabolic modeling to obtain metabolic fluxes from MRS measurements, iii) MRS/MR system operation and data analysis, and iv) regular quality control measures to ensure the reliability of the MRS and MRI. In addition to being critical for the success of the program project, the MRS and MRI methodology and quality control procedures implemented by the core will benefit all NIDDK investigators (both within and outside Yale) using the facility.

neurotransmitter metabolism; hepatic coma /encephalopathy; glutamates; brain metabolism; aminoacid metabolism; hyperglycemia; glutamine; gamma aminobutyrate; patient oriented research; carbon; nuclear magnetic resonance spectroscopy; human subject; stable isotope; clinical research;

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In intensively treated subjects with T1DM during hypoglycemia there is often a loss of both the counterregulatory response and the mild cognitive symptoms prior to severe cognitive dysfunction. Both of these adaptations are believed to contribute significantly to hypoglycemic unawareness, which increases the risk of severe hypoglycemia. Using a novel method combining 13C MRS and [2-13C] acetate infusion we found that cortical monocarboxylic acid transport (MCT) and metabolism is up regulated 2-fold in patients with intensively treated T1DM. In Aim 1 we will assess whether increased usage of lactate, the monocarboxylic acid (MCA) with the highest concentration in blood during hypoglycemia, plays a significant role in preserving neuronal energy metabolism in subjects with intensively treated T1DM. In Aim 2 we will assess whether MCT upregulation is specifically caused by recent exposure to extended hypoglycemia. The detailed understanding provided by these studies of cortical metabolic adaptations to hypoglycemia should aid in the development of strategies for counteracting them, which may help restore awareness of hypoglycemia in such patients. In Aim 3 we will use a novel 1H MRS method for measuring brain glucose transport to assess upregulation of glucose transport in intensively treated TIDM. In Aim 4 we will use an animal model to validate our interpretation that the increase in acetate transport and metabolism due to repeated hypoglycemia is due to upregulation of blood brain barrier MCT activity. Finally in Aim 5 we will further assess in the animal model whether the upregulation of MCT activity can potentially protect against hypoglycemic energy failure. If MCAs or alternatively medium chain fatty acids can protect against hypoglycemic energy failure they may potentially be administered as a protective therapy for nocturnal hypoglycemic episodes. [unreadable] [unreadable]

The central hypothesis of this program project is that altered mitochondrial function is a key determinant of metabolic syndrome. However while metabolic syndrome has been extensively characterized in muscle, liver, kidney and pancreas, little is known about how it impacts brain function. The potential importance of understanding the impact of metabolic syndrome on brain function is highlighted by two converging lines of evidence. Firstly, recent studies have strongly implicated impaired brain mitochondrial function in a host of neurodegenerative diseases, including Alzheimer's and temporal lobe epilepsy. Secondly, epidemiological studies have found a several fold enhancement in the total incidence of neurodegenerative diseases in patients with type 2 Diabetes Metlitus. Reduced brain mitochondria in people with severe insulin resistance may be a factor in this increased susceptibility. In project 3 we will take advantage of our recent developments of MRS methods for non-invasively imaging brain mitochondrial metabolic function to assess whether it is altered in three groups of subjects with metabolic syndrome. These groups are 1) insulin resistant offspring of parents with Type II diabetes, 2) elderly subjects and 3) patients with an identified mutation in a gene coding for mitochondrial tRNA. Recent MRS studies by the Shulman laboratory have shown that all three groups have altered muscle mitochondrial metabolism. Our general hypothesis in this project is that there are similar alterations in brain mitochondrial metabolism in the brain of these subjects as in the muscle and liver. This project will )rovide the first comprehensive study of metabolic syndrome to brain, and may provide new insight nto why subjects with metabolic syndrome are at increased risk of neurodegenerative disease.

technology /technique development

[unreadable] DESCRIPTION (provided by applicant): The development of MR technology has allowed biomedical researchers to make major advances in understanding and treating human disease among the major areas of strength in our MR program are applications to diabetes, epilepsy and image guided surgery, psychiatric disease, and learning disorders. Our progress in these areas has gone hand in hand with the development of MR technology focused on obtaining quantitative images of metabolic fluxes and metabolite levels, and quantitative fMRI measures of blood flow, blood volume and metabolism. All of these measurements put extreme demands on MR sensitivity and spatial resolution. At present the highest resolution and sensitivity achievable in humans is from 7T systems. Based on published studies a 3 fold sensitivity in MRSI and a similar enhancement for quantitative fMRI measures can be achieved. However the application of 7T MR to clinical research has been restricted due to the inability, primarily as a consequence of the increased Bo inhomogeneity, to obtain whole brain distortion free MRSI and fMRI. For example applications of MR to epilepsy, neurodegenerative diseases, and image guided neurosurgical planning requires high quality MRSI and fMRI in regions near the base of the brain such as the hippocampus, where the worst Bo inhomogeneity is present. At present none of the existing 7T systems have presented adequate quality MRS or fMRI in these regions for research purposes much less surgical applications. We feel strongly, based upon our extensive experience developing and applying MR technology to important questions in clinical research, that the several fold higher sensitivity made possible by 7T magnet technology has the potential of greatly advancing the field. Therefore the major goals of this project are to 1) purchase and install a Varian/Magnex 7T 68 system with gradients and shims optimized for whole brain dynamic shimming, 2) develop quantitative whole brain and limb ultra high resolution MRSI and fMRI using dynamic shim updating, Bo and B1 robust pulse sequences, and parallel imaging approaches and 3) apply this technology to allow the programs at Yale in diabetes, epilepsy and image guided neurosurgery, psychiatry, and learning disorders to continue to make important strides in the understanding and treatment of human disease. [unreadable] [unreadable] [unreadable]

Applications of magnetic resonance (MR) techniques to research in the life sciences are growing rapidly. State-of-the-art heteronuclear MR spectroscopy (MRS) and multi-modal MR imaging (MRI) methodologies cultivated at the Magnetic Resonance Research Center (MRRC) at Yale have moved in vivo animal research into central roles in experimental neuroscience, addressing fundamental issues with far reaching implications for brain function. Since the formation of the Yale MRRC in the early in 1980s, the number of . horizontal-bore magnets for in vivo studies have multiplied three-fold in 2004. The present number of ' magnets for in vivo studies - three each for animals and humans - were needed to match the growing number of investigators across many disciplines - a majority of whom are supported by NINDS. The strength of the Yale MRRC has been, and still is, the dynamic interaction between rodent and human research. Active interplay between heteronuclear MRS and multi-modal MRI methods in rodents and humans have furthermore rapidly progressed. Because MR technology requires unwavering infrastructural support for state-of-the-art exploits to be successfully applied, long-term stability of Yale MRRC is contingent on sustained support. A program in Quantitative Neuroscience with Magnetic Resonance (QNMR) at Yale will support shared resources and facilities used by NINDS-funded investigators at Yale, and thereby generate greater productivity than would be possible via independent efforts. QNMR will consist of three research Cores - each dedicated to improving effectiveness of ongoing research based upon multimodal MRI, heteronuclear MRS, neurophysiology - and one service Core - designed for rapid data analysis, access, sharing, and backup using high-performance cluster of workstations. We expect that QNMR will promote a more cooperative and interactive research environment for neuroscientists who are utilizing MR technology at Yale, and will nurture new cross-disciplinary approaches in medicine, physiology, and neuroscience.

DESCRIPTION (provided by applicant): Mitochondrial dysfunction has been implicated in age-related neurodegenerative diseases and may play a role in the decline of cognitive and sensory function with healthy aging. However there is no direct in vivo evidence for altered brain mitochondrial function. Over the last decade we have developed non invasive 13C Magnetic Resonance Spectroscopy (MRS) methods, in conjunction with stable 13C isotope labeled substrates, to study brain metabolism in humans. MRS has the unique ability to non-invasively measure the rates of the neuronal and glial TCA cycles - a direct measure of in vivo mitochondrial oxidative energy production. Using 13C MRS we found profound alterations in energy metabolism in the occipital lobe of healthy elderly subjects including a 28% reduction in the neuronal TCA cycle, a parallel 24% reduction in the glutamate/glutamine cycle, and a 30% increase in the glial TCA cycle. Our general hypothesis is that in healthy aging there is a loss of capacity of neuronal mitochondria to support brain functional energetic requirements. We will address three questions in the proposed research - 1) Are these metabolic changes present in the prefrontal cortex, which has been implicated in the loss of cognitive function with aging? 2) Does the severity of impairment of the neuronal TCA cycle correlate with performance on an established test of executive function and 3) are the alterations in glial mitochondrial metabolism due to enhanced glutamate oxidation and anaplerosis, which may reflect impaired synaptic glutamate clearance. Answering these questions will have immediate significance in understanding the role of mitochondrial function in the cognitive declines associated with normal aging, potentially provide novel, non-invasive biomarkers of this process, and also will provide critical baseline information for the application of these methods to study the role of mitochondrial dysfunction in the development of Alzheimer's and other neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: The proposed studies will use 13C MRS to study in human brain whether mitochondrial oxidative metabolism and glutamate neurotransmitter cycling are altered in the prefrontal cortex of healthy elderly subjects. They will test directly whether altered mitochondrial metabolism is associated with loss of cognitive function with aging. If an association is found it will provide further evidence for a key role of mitochondrial dysfunction in the etiology of the cognitive declines associated with the aging process. Although there are no patient studies proposed, these findings will form the basis for characterizing abnormal alterations in mitochondrial metabolism that may play a role in the etiology of Alzheimer's and other neurological diseases.

DESCRIPTION (provided by applicant): The Yale MRRC is a pioneer and leader in the in the study of metabolism by in vivo Magnetic Resonance Spectroscopy (MRS) by 1H and 13C MRS. The 4.0T 94 cm Bruker system has been the traditional workhorse for human metabolic studies and is the only system at the MRRC capable of 13C MRS studies of body and brain. At present the system supports the research of 15 faculty including 9 major users with R01 funding. The NIH support for the user group is 17 grants: 10 R01s, 4 Center grants (R24, P30, and two P50s), an R21, a K02, and an STTR. Most of the PIs using the system have over 10 years of experience using MRS in their research. However the continued development of novel experimental methodology and groundbreaking applications to disease, that have been the hallmark of the research program on system, are severely compromised by the limited capabilities and decreasing reliability of its 8 year old console and electronics. In order to sustain the productivity of the User group we propose to replace the failing Bruker Avance console with a state of the art Agilent console with multiple receiver capability. Our selection is based upon our extensive in house experience in operating and maintaining similar Agilent consoles and integrating them with our home built RF coils and gradient/shim hardware and the extensive MRS capabilities of the system. The new console will allow for years to come the reliable performance of a wide range of sophisticated metabolic experiments by the User group, and insure the continued development of advanced 13C and 1H MRS methods. PUBLIC HEALTH RELEVANCE: Magnetic Resonance Spectroscopy (MRS) is a unique technology that allows chemistry to be imaged throughout the human body. The requested new console for an existing MRS/MRI 4.0T scanner will greatly enhance the research of a highly productive group of NIH funded scientists studying aging, diabetes, neurological and psychiatric disease. Their work has direct relevance for finding new targets for disease treatment and using MRS to evaluate therapies.

DESCRIPTION (provided by applicant): Insulin resistance has been found to be a strong risk factor in the development of Alzheimer's disease and other age related neurological disorders. We have shown that reduced muscle mitochondrial oxidative metabolism plays a key role in the etiology of insulin resistance in aging, pre-diabetes, and diabetes. However it is not known whether there is a similar decrease in the brain. Mitochondrial dysfunction has been implicated in the cognitive decline with aging, Alzheimer's disease and other neurodegenerative disorders. In this proposal we will test whether reduced mitochondrial metabolism is a risk factor for age related neurological disorders in insulin resistance. We will combine 13C MRS to measure the rate of the neuronal TCA cycle with 31P MRS to measure energetic stress. 13C MRS has the unique capability to measure the rates of the neuronal and glial TCA cycles - a direct measure of in vivo mitochondrial oxidative energy production. We and others have used MRS to show that 80% of neuronal mitochondrial energy production supports neuronal signaling, with the implication that even small impairments in mitochondrial capacity could compromise brain function. Recently we applied these methods to study healthy elderly subjects. We found profound alterations in energy metabolism in the occipital lobe, including a 28% reduction in the neuronal TCA cycle. Our general hypothesis is that healthy non-diabetic, insulin-resistant subjects have a reduced capacity of neuronal mitochondria to support energetic requirements of brain function which may predispose them to age-related neurological disorders. We will test our hypothesis in otherwise healthy non-obese, insulin-resistant subjects who are the offspring of at least one parent with type 2 diabetes. In our metabolic studies we have extensively phenotyped a large cohort of these subjects because they allow the etiology and health impacts of chronic insulin resistance to be studied independent of the complications of diabetes and obesity.

? DESCRIPTION (provided by applicant): The Yale MRRC is a pioneer and leading site for using 1H, and 13C MRS and ultra-high resolution fMRI to study metabolism and function in small animal models. The 9.4T 21 cm animal system at the MRRC is the workhorse for these studies since installation in 2004. It presently supports the research of 22 NIH funded faculty at Yale and other institutions including 9 major users. The NIH support for the user group is 25 awards including 2 Center grants (P30, U24) a P01, 18 R01 awards, a DP1 award, an R21, and 2 K awards. It is a major resource for Yale's NINDS P30 MR Core Center and NIDDK Mouse Metabolic Phenotyping Center. However the continued development of novel MR methodology and groundbreaking applications to disease, that have been the hallmark of the research programs on the system, is severely compromised by limitations in its 7 year old Agilent console and 10 year old gradient hardware. Furthermore with the withdrawal of Agilent from the MRI market there is minimal support available for the console which is already leading to substantial system down time. In order to sustain and enhance the productivity of the User group we propose to replace the present console and gradient system with a state of the art Bruker console and gradient coil. Our selection is based upon the high performance capabilities of the Bruker console and gradient coil and our extensive in house experience in operating and maintaining Bruker systems The new console will insure for years to come the reliable performance of a wide range of state of the art MR methods by the User group, and the continued development of advanced MR methods.

? DESCRIPTION (provided by applicant): GABA is the major inhibitory neurotransmitter in the mammalian brain, and dysfunction of the GABAergic system underlies a number of neurological and neuropsychiatric disorders such as depression, schizophrenia and epilepsy. Currently 1H MRS is the only noninvasive method to measure brain GABA in humans. Altered GABA levels detected by MRS occur in a variety of clinical conditions such as depression and epilepsy, and recent reports indicate a strong relationship between GABA concentration and cortical excitability (as assessed by fMRI and gamma EEG) and cortical interconnectivity (as assessed by EEG and resting state fMRI). While the GABA MRS measurement has gained increasing importance as a biomarker in both clinical and basic neuroscience research, key questions about its meaning remain: ? Does the GABA MRS measurement provide an accurate assessment of total brain GABA? ? Does cytoplasmic GABA, which is most likely the primary component of the GABA MRS signal, determine the concentration of extracellular fluid (ECF) GABA, and therefore act as an important mechanism for tonic extrasynaptic GABA inhibition? ? Can the cytoplasmic and vesicular subcomponents of the GABA MRS signal be separately quantitated? ? What is the relation between physiological changes in cellular and extracellular GABA concentration and changes in cortical excitability? We propose to address these questions in a well-established rodent model that allows MRS, fMRI, microdialysis, and electrophysiology to be performed in parallel. In Aim 1 the in vivo MRS visibility of GABA will be determined, and whether the 1H MRS GABA signal can act as a biomarker of extracellular fluid GABA and tonic inhibition, through measurement of ECF GABA, cellular GABA, and cortical excitability. In Aim 2 a novel diffusion-weighted MRS method will be employed to characterize the cytoplasmic and vesicular components to the 1H MRS GABA signal to allow the two components of the GABA inhibition system to be studied in vivo. These aims will leverage our extensive experience and advances in 1H MRS GABA measurements with our strong collaborations with clinical neuroscientists who will provide expertise in microdialysis, electrica recordings, and pharmacological and functional manipulations of the GABAergic system. Successful completion will provide critical methodological validation of the MRS GABA measurement, develop a novel method to differentiate cytoplasmic and vesicular GABA pools, and gain new insight into how physiological and pharmacologically-induced shifts in GABA levels relates to GABA inhibitory function, and thus the meaning of [GABA]MRS as a translational biomarker.

Resting-state fMRI has implicated hubs of high functional connectivity in the resting human brain, e.g., the default mode network (DMN), compared to other regions. In this proposal we will directly assess the role of metabolism in supporting high connectivity and assess whether metabolic dysfunction leads to connectivity reduction in DMN hubs seen in neurodegenrative (e.g., aging, Alzheimer's) and neuropsychiatric disorders. The complex nature of functional connectivity in healthy human brain has very high-energy demands, a need that is met by high ATP yielded from glucose oxidation (CMRglc(ox)). Using H[ C] MRS, we found that 1 13 neuronal CMRglc(ox) changes linearly with glutamatergic neurotransmission and that at rest most of the cerebral cortex's energy production is devoted to signaling. However there is a significant fraction of energy devoted to housekeeping needs such as synaptogenesis and maintaining membrane potentials. Given the tight link of energetics and signaling we found surprisingly, using quantitative PET imaging, that total CMRglc(ox) in the DMN is similar to regions with lower fMRI-derived connectivity. A potential explanation for this paradox is that the hubs in DMN vs. other cortical regions have a greater fraction of their total energy devoted to signaling than to nonsignaling, thus making the DMN hubs more vulnerable to functional energy failure. Alternatively it has been proposed that higher nonsignaling needs (e.g., synaptic remodeling) is present in DMN. To answer this novel question with large implications for interpreting resting-state fMRI data and to study how dysfunction of energy metabolism and tissue composition impacts function, there is a need for novel measurement and computational tools. To address this challenge we will develop a computational model to calculate signaling and nonsignaling energy costs. The model uses data from individual subjects on tissue composition obtained from high- resolution MRI. The model will be validated in both a rodent model and humans by comparison with 1H[13C] MRS, which can uniquely measure the signaling and nonsignaling components of neuroenergetics. The relative ratios of signaling to nonsignaling will be measured and calculated in high functional connectivity regions of the DMN and control low connectivity cortical regions in healthy young and elderly adults. We hypothesize that regions of high functional connectivity will have a greater fraction of energy production devoted to signaling and that this fraction will decline with age. Once the computational budget model is developed, and validated, it will provide a powerful noninvasive tool for studying alterations in cortical energetics and tissue composition that lead to loss of fMRI-derived connectivity as well as potentially as a novel clinical biomarker for assessing prognosis and treatment.

Project Summary/Abstract Hypoglycemia and its adverse effects on brain function remain the major factor limiting the use of intensified insulin therapy that has been shown to prevent or delay the long-term complications in type 1 diabetes (T1DM). Higher cognitive functions (e.g. working memory) that involve the prefrontal cortex are particularly sensitive to neuroglycopenia. This proposal seeks continued support of a long-term RO-1 grant with the long-term goal of documenting the health benefits of insulin delivery strategies that minimize the risk of frequent bouts of hypoglycemia in T1DM patients. The specific aims of the current project outlined below use functional magnetic resonance imaging (fMRI) and magnetic resonance spectroscopy (MRS) techniques to assess the changes in brain function and fuel metabolism caused by acute hypoglycemia and acute hyperglycemia in T1DM patients with hypoglycemia unawareness (versus hypoglycemia-aware T1DM patients and healthy controls) as well as the potential beneficial impact of employing closed-loop insulin delivery systems to improve brain function in hypoglycemia unaware T1DM individuals. The protocols rely heavily on human investigation involving non-diabetic as well as hypoglycemia aware and unaware T1DM subjects exposed to experimental mild and moderate hypoglycemia and acute hyperglycemia using the glucose clamp technique while undergoing brain imaging. However, we also take advantage of the power of rodent diabetic models to test specific mechanistic hypotheses. The primary hypothesis of this proposal is that hypoglycemia unaware T1DM patients not only have impaired hormonal and symptomatic responses, but also lack another key hypoglycemia defense mechanism, namely the capacity of the brain to activate motivation/reward circuits due to adaptive increases in brain glucose transport and metabolism as well as stimulation of the polyol pathway. The specific aims are to determine: 1) If T1DM patients with hypoglycemia unawareness (vs. T1DM and non-diabetic controls) lose the capacity to elicit brain responses to visual food cues as well as functional connectivity in striatal and a variety of other brain regions in response to food cues during mild and moderate hypoglycemia using the glucose clamp technique; 2) If patients with T1DM and hypoglycemia unawareness display adaptive changes causing excessive increases in brain glucose transport and metabolism in response to acute hyperglycemia that induce adverse neurocognitive effects within the pre-frontal cortex, a key brain region for cognitive function not previously examined in humans using magnetic resonance spectroscopy (MRS). In addition, mechanistic studies will be conducted in diabetic rats exposed to recurrent hypoglycemia to define the molecular mechanisms driving the changes in brain fuel metabolism induced by intensive insulin treatment; and 3) if reducing glycemic variability with a closed loop insulin delivery system in patients with hypoglycemia unawareness can reverse alterations in brain glucose transport/metabolism as well as functional connectivity, thereby reverse brain dysfunction induced by current intensive T1DM insulin treatment.