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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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Biophysical Chemistry Study Section (BBCB)
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Yale University
Schools of Medicine
New Haven
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Shulman, Robert G; Rothman, Douglas L (2017) The Glycogen Shunt Maintains Glycolytic Homeostasis and the Warburg Effect in Cancer. Trends Cancer 3:761-767
Patel, Anant B; Lai, James C K; Chowdhury, Golam I M et al. (2017) Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-13C2]Glucose Metabolism in Anesthetized Rats. Neurochem Res 42:173-190
Shulman, Robert G; Rothman, Douglas L (2015) Homeostasis and the glycogen shunt explains aerobic ethanol production in yeast. Proc Natl Acad Sci U S A 112:10902-7
Patel, Anant B; de Graaf, Robin A; Rothman, Douglas L et al. (2015) Effects of ?-Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and ?-Aminobutyric acid metabolism in the anesthetized rat In vivo. J Neurosci Res 93:1101-8
Chowdhury, Golam M I; Jiang, Lihong; Rothman, Douglas L et al. (2014) The contribution of ketone bodies to basal and activity-dependent neuronal oxidation in vivo. J Cereb Blood Flow Metab 34:1233-42
Patel, Anant B; Lai, James C K; Chowdhury, Golam M I et al. (2014) Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle. Proc Natl Acad Sci U S A 111:5385-90
Herzog, Raimund I; Jiang, Lihong; Herman, Peter et al. (2013) Lactate preserves neuronal metabolism and function following antecedent recurrent hypoglycemia. J Clin Invest 123:1988-98
de Graaf, Robin A; Chowdhury, Golam M I; Behar, Kevin L (2011) Quantification of high-resolution (1)H NMR spectra from rat brain extracts. Anal Chem 83:216-24
de Graaf, Robin A; Rothman, Douglas L; Behar, Kevin L (2011) State of the art direct 13C and indirect 1H-[13C] NMR spectroscopy in vivo. A practical guide. NMR Biomed 24:958-72
Jiang, Lihong; Mason, Graeme F; Rothman, Douglas L et al. (2011) Cortical substrate oxidation during hyperketonemia in the fasted anesthetized rat in vivo. J Cereb Blood Flow Metab 31:2313-23

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