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.
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