The Flux from Glucose to Glutamate in the Rat Brain in vivo as Determined by ¹-Observed, ¹³C-Edited NMR Spectroscopy

Abstract

Summary:The rate of incorporation of carbon from [1-13C]glucose into the and [3-CH21 of cerebral glutamate was measured in the rat brain in vivo by IH_ observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 6O-min infusion of [l-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved resonances of glu tamate and glutamine increased exponentially during the infusion and attained a steady state in -20 min with a first-order rate constant of 0. 130 ± 0.010 min -I (t1/2 = 5. 3 ± 0. 5 min). The appearance of the [3-13CH21 resonance in the POCE difference spectrum lagged behind that of the [4-13CH21 resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26. 6 ± 4. 1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the Cerebral glutamate functions as a neurotransmit ter, is necessary for cerebral ammonia detoxifica tion, and is directly related to cerebral energy me tabolism (see Cooper and Plum, 1987, and ref. therein). Glutamate metabolism occurs in at least two morphologically distinct cellular compartments (Hertz, 1979). A small, rapidly turning over pool of glutamate, thought to be the precursor of brain glu- 170 total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intrac ellular pH as determined by 31p NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate a-ketoglutarate, the rate of glutamate label ing provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be = 1.4 fLmol g -1 min -I. These experiments demon strate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1 H observed, 13C-decoupled NMR spectroscopy. Key Words: Brain metabolism-[1-13C]Glucose-Glutamate Glycolysis-Nuclear magnetic resonance-Tricarboxylic acid cycle-Two-dimensional heteronuclear spectros copy.tamine, is located within the astrocytes. The pool of glutamate localized to the neurons is larger (at least 80% of total brain glutamate) and has a slower turn over rate than the astrocytic glutamate pool. Each glutamate pool is in equilibrium with a distinct tri carboxylic acid (TCA) cycle (Van den Berg et aI., 1969; Berl et aI., 1970). The flow of carbon from glucose to glutamate and the turnover time of the glutamate pool depend both on the rate of glycolysis and on the rate of entry of glucose carbon into the TCA cycle. Although turnover rates for glutamate have been estimated by radioactive tracer and sta ble isotope studies (Berl et aI., 1962(Berl et aI., , 1970 Cooper et aI., 1988) there has been no acceptable way to measure these rates in vivo.The turnover of cerebral glutamate and other me tabolites can be determined in vivo by monitoring the site-specific incorporation of l 3C using l 3C_ en...

Glucose is Necessary to Maintain Neurotransmitter Homeostasis during Synaptic Activity in Cultured Glutamatergic Neurons

“…This, in turn, is dependent on TCA cycling. Tricarboxylic acid cycling may in any given microenvironment be monitored using labeling of glutamate since aspartate aminotransferase activity is several fold higher than that of the TCA cycling (Drejer et al, 1985;Fitzpatrick et al, 1990;Mason et al, 1992). The enzymatic pathways involved in the conversion of glucose/lactate to glutamate including glycolysis, lactate dehydrogenase, and TCA cycle reactions are most likely compartmentalized and heterogeneous (Eloqayli et al, 2002;Waagepetersen et al, 2003, and references therein).…”

Caloric Restriction Impedes Age-Related Decline of Mitochondrial Function and Neuronal Activity

“…After surgery anesthesia was maintained with a-chloralose (initial 80 mg/kg, plus 40 mg/kg/hour) and D-tubocurarine-Cl (0.3 mg/kg) was administered for immobilization. The left femoral artery and vein were catheterized for continuous monitoring of arterial blood pressure and blood sampling, and for infusion of [1,[6][7][8][9][10][11][12][13] C]-D-glucose, respectively (Cambridge Isotopes, Andover, MA, USA). Blood gases (pCO 2 , pO 2 , and pH) were measured periodically in arterial blood samples (ABL5 blood gas system, Radiometer America, Westlake, OH, USA).…”

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons

“…[3-13 C]pyruvate produced from [1-13 C] glucose by glycolysis enters the TCA cycle as [2-13 C]acetyl-CoA, generating [4-13 C]α-ketoglutarate, which undergoes transport/exchange transamination with the larger cytosolic pool of glutamate producing [4-13 C]glutamate (Fitzpatrick et al, 1990). Subsequent metabolism in the TCA cycle leads to labeling of [3-13 C], [2-13 C], and [1-13 C]glutamate and is eventually lost as CO 2 with continued turns of the cycle.…”

“…The concentrations of metabolites were determined both in neurons and neuronal medium relative to a known concentration of [2-13 C]glycine, added during tissue extraction as an internal standard. The 13 C isotopic enrichments of [4-13 C]glutamate and [2-13 C]GABA in neuronal extracts were calculated from the ratio of the areas of their resonances in the 1 H-{ 13 C}-NMR difference spectrum (2 × 13 C only) and the non-edited spectrum ( 12 C+ 13 C) as described previously (Fitzpatrick et al, 1990). The concentration and percent 13 C enrichment of [3-13 C]lactate and [3-13 C]alanine in the neuronal medium was determined from the 1 H-{ 13 C}-NMR spectrum using a known amount of TSP.…”