Robert G. Shulman - US grants

The aim is to develop NMR spectroscopy of animal models, in vivo, using 13C, 31P and 1H to follow metabolites, to monitor pH and the energy state, so as to prepare future clinical applications to humans. The core project plans to install and NMR spectrometer with a 20 cm diameter bore capable of measuring animals as large as cats. The magnetic field of 1.89T allows obersvation of 13C NMR at 20 MHz, 31P at 32 MHz and 1H at 80 MHz. By the use of surface coils and a focussed magnetic field it will be possible to obtain signals from localized regions as small as 1.5 cm diameter. We will install proton decoupling for 13C observe and will minimize the heating effects so as not to be harmful. Various instrumental improvements will be attempted so as to obtain well resolved NMR spectra, capable of following metabolic flows in real time. The specific projects include studies of brain, liver and tumors in living animals. The brain studies will determine normal glucose pathways to amino acids and other products by 13C NMR generally starting from 13C labeled glucose. It is expected that we should be able to detect glutamate, glutamine, Gamma-amino butyric acid, aspartate and lactate and in some cases to determine biosynthetic pathways and compartmentation. The effects upon these pathways of hypoxia, restraints and seizures will be measured in order to improve and extend our biochemical understanding of these states. The liver studies will follow the gluconeogenic pathway, following fluxes and hormonal effects. In the tumor studies attempts will be made with 31P and 13C NMR to determine the hypoxic fraction of tumors, in vivo, and to monitor its response to different radiation treatments in order to increase their effectiveness. In these studies we shall be preparing for related investigations of disease states in humans.

nuclear magnetic resonance spectroscopy;

Glucose is the normal fuel of the mammalian brain. However the molecular regulation of brain glucose metabolism is poorly understood. We propose using 1H, 13C and 31P NMR in conjunction with in vitro enzyme reconstitution to study the control of glycolytic flux in the rat brain. First we are going to measure the rates of glucose metabolism down the glycolytic pathway (Vgly) and the subsequent flows through the TCA cycle (Vtca). This will be done by proton observe carbon edit (POCE) methods that we have previously developed for brain studies. In these measurements 1-13C glucose is infused and the rates of 13C turnover of cerebral lactate and glutamate pools are measured by POCE. These turnover rates are used to determine Vgly and Vtca respectively by the use of a computer model that we have developed. The particular conditions of hypocapnia and seizures have been selected because they modify the glycolytic rate and also have been shown to induce high enough steady state levels of lactate (several millimolar) to allow the time course of its turnover by 13C to be measured in the POCE experiment. Second under the same conditions we will measure the concentrations of cerebral glucose over a range of blood glucose concentrations. These measurements, which take advantage of the ability of 13C NMR to measure brain glucose concentrations in vivo, will be used to determine the kinetic parameters of glucose transport into the brain. In accordance with previous studies, showing a saturable glucose transport system, we will at first analyze the data in terms of a Michaelis-Menten model. In order to combine the glucose transporter kinetics, so determined, with the control of glycolytic flux it will be necessary to make the third kind of experiment which is to study the enzymatic control of hexokinase (HK) and phosphofructokinase (PFK) both in vivo and in vitro. This will be done for PFK by measuring the concentrations of all of its allosteric effectors and substrates in vivo and measuring the enzymatic flux after establishing these concentrations in an in vitro assay system. When the in vitro flux is the same as the in vivo flux the sensitivity of the flux to the different effectors will be evaluated, giving a dependence that will have significance in vivo. The in vitro kinetics will be used to understand in vivo glycolysis by application of control theory. Spectroscopic improvements in signal to noise and spectral localization will be developed for the rat brain on a 7.OT Biospec Spectrometer.

We are requesting funds to purchase a Bruker Biospec 7 Tesla, 15- cm horizontal bore NMR spectrometer. The instrument will be used to extend and expand our current research efforts into amino acid chemistry and pathology in the brain, interactions between ion transport, carbohydrate and amino acid metabolism in the kidney, and carbohydrate metabolism in the liver. These studies will make use of new and verified multipulse lH and 13C spectroscopic techniques developed in our laboratory for this purpose. The application of these methods to the metabolic research problems contained in this proposal will lead to further improvements in pulse sequence design and quantitative accuracy. The new 7 T magnet/system has full imaging capability and will provide both a high field strength and a large homogeneous volume in a horizontal orientation--permitting us to expand our current work to investigations of regional metabolism in larger animals in vivo. The use of these techniques to study complex metabolic problems on carefully controlled and unstressed animal models, under condition of optimal sensitivity and resolution, will define the potential scope for human investigations.

We will develop and improve the spectroscopic methods needed to study tissue metabolism by (1)H Nuclear Magnetic Resonance. We will also develop computer models of metabolic pathways and design (13)C tracer experiments to test and improve these models. Finally we will perform initial experiments designed to interpret the flow of (13)C label from 1-13C glucose into lactate and glutamate in tenus of metabolic fluxes. The specific aims are: 1. To develop 1H and 13C NMR methods to measure kinetics and steady state 13C labeling patters of lactate and amino acids in vivo. Specific areas for improvement are: i) Water Suppression (1H). ii)Sensitivity (1H, 13C). iii)Localization (1H, 13C). iv)Editing (1H). v)Quantification (1H, 13C). 2. To develop kinetic models to interpret the patterns and rates of 13C labeling of lactate and amino acids from 13C-glucose in rabbit brain. Two general areas of research are proposed: 1) To interpret the observed 13C flows into lactate and glutamate pools in terms of a computer model which only includes metabolites on the direct or linear pathway. 2) To evaluate the assumptions made in the linear model about equilibrium and contributions from other pathways.

The goal is to study ethanol metabolism in the rat liver using in vivo NMR methods. These noninvasive studies will first require NMR developments in order to answer current questions about ethanol metabolism in both acute and chronic rats. The first specific aim is to develop NMR localization methods for following, non invasively, hepatic metabolism of 13C labeled precursors i.e. ethanol, alanine and acetate. This will be done by direct 13C NMR measurements and by heteronuclear edited 1H NMR spectra in which the high sensitivity of 1H NMR will be used to detect the 13C label flow into neighboring, coupled carbons. To quantitate these fluxes, it will be necessary to determine the fractional enrichment which will be done by NMR, and to specify the local volume giving rise to the signal. Simultaneously, the energy status of the liver will be monitored by 31P NMR. The second aim is to try to develop in vivo NMR methods for following the redox states. This will follow the indirectly responding ratios of lactate/pyruvate for cytosolic potential and of beta- hydroxybutyrate/acetoacetate for the mitochondrial. Direct 13C NMR and heteronuclear 1H-13C detection will be tried. The third aim is to follow the labeled precursors into TCA cycle pools of glutamate and, when possible, of glutamine and aspartate. Additional studies of the flows into pools of the ketone bodies and gluconeogenic products will be made. These experiments will utilize the methods developed in the first two specific aims to study both the acute response to ethanol and the response of rats administered two different chronic ethanol diets. The goal is to specify the differences in these pathways between the chronic and acute metabolism of ethanol. The fourth specific aim is to study the "empty calorie" effect in which chronic ethanol infusion reduces the energetic efficiency of ethanol metabolism. The pathways will be studied in conjunction with measurement, by 2H NMR, of the futile cycling between alcohol dehydrogenase and the MEOS enzyme that has been proposed to waste ethanol reducing equivalents in both rats and humans.

The metabolic basis of brain activation remains an unsettled question because of recent results suggesting that anaerobic glycolysis provides the additional energy required for activation The rat brain will be studied by in vivo NMR at 7.OT in order to understand the metabolic basis of brain activity. The somatosensory cortex will be activated by a forepaw stimulation protocol which has already been established. The fMRI of the activation will be measured at shorter times after the stimulation and by gradient echo and T1 weighted imaging so as to understand its mechanism. Instrumental improvements of transmitter/receiver coils, gradients and pulse sequences will be implemented so as to improve sensitivity and spatial resolution. 1H NMR spectroscopy will be used to measure localized glucose and lactate pools and their changes during forepaw stimulation. During 1-13C glucose infusion the turnover of glucose, lactate and glutamate will be measured by our well established Proton Observe Carbon Edit sequence. These measurements will be interpreted by a quantitative model to give deltaCMRO(glucose), deltaCMRO2 and changes of lactate during stimulation so as to quantify the contribution of anaerobic glycolysis to the energy. Brain extracts of metabolites and of hexokinase in the somatosensory cortex will be made comparing resting conditions with forepaw stimulation. Mitochondrial and cytosolic isoforms of the enzyme will be tested separately in order to complete our understanding of their control by glucose-6-phosphate and glucose-l,6-bisphosphate. Control of the flux by hexokinase will be calculated from these values using control theory approach. Finally 13C NMR measurements of larger volumes of brain will evaluate changes in the TCA cycle activity and the flux from glutamate to glutamine at different levels of alpha-chloralose anesthesia. The hypotheses that glutamine synthesis is regulated by glutamate release from neurons to glia as a neurotransmitter will be tested by measuring the relative rates of glutamate and glutamine labeling by a label that originates in the glia and by inhibiting glutamate release by carbetapentane. A coherent study of brain activation, glucose and oxygen consumption and flux control by hexokinase is proposed that can be accomplished in the rat and will answer questions about the energetics of rat and human cerebral metabolism.