Evaluation of Cerebral Acetate Transport and Metabolic Rates in the Rat Brain in vivo Using ¹H-[¹³C]-NMR

Abstract

Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from 13C-acetate has been limited mainly to estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state 13C enrichments. In this study we used 1H-[13C]-NMR spectroscopy with intravenous infusion of [2-13C]acetate-Na+ in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the 13C turnover curves of glutamate-C4 and glutamine-C4 from [2-13C]acetate-Na+, brain acetate levels, and the dependence of steady state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4–5 mM. Analysis_assuming reversible, symmetric Michaelis-Menten kinetics for transport yielded 27±2 mM and 1.3±0.3 µmol/g/min for Kt and Tmax, respectively, and for utilization, 0.17±0.24 mM and 0.14±0.02 µmol/g/min for KM_util and Vmax_util, respectively. The distribution space for acetate was only 0.32±0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal TCA cycle fluxes were 0.37±0.03 µmol/g/min and 1.41±0.11 µmol/g, respectively; astrocytes thus comprised ~21±3% of total oxidative metabolism.

Characterization of Cerebral Glutamine Uptake from Blood in the Mouse Brain: Implications for Metabolic Modeling of13C NMR Data

“…Forty-five minutes after induction of anesthesia, [U- 13 C 5 ]glutamine (250 mmol/L dissolved in deionized water, pH B7.0) was administered intravenously for 3, 15, 30, or 60 minutes, using a bolus-variable rate infusion (1,220 mmol/kg per minute (0 to 15 seconds), 244 mmol/kg per minute (15 seconds to 4 minutes), 122 mmol/kg per minute (4 to 8 minutes), and 49 mmol/kg per minute (48 minutes). This protocol, modified from a previously described acetate infusion protocol, 19 elevated the blood glutamine level to B4.1 mmol/L within 3 minutes from a baseline value of B0.6 mmol/L and maintained it at B1.1 mmol/L thereafter. Four mice were used for each infusion time point.…”

“…A significant amount of free acetate is present in mouse blood (B0.2 to 0.9 mmol/L), 32 which can be readily oxidized in brain, mainly by astrocytes. 33 In the cerebral cortex of anesthetized rats, blood acetate is oxidized at a rate of 0.04 mmol/g per minute 19 for physiologic blood acetate level (0.94 mmol/L), contributing B13% of the rate of glutamine synthesis. The 13 C labeling of glutamine-C4 from [2-13 C]acetate, an astroglial specific substrate, has been shown to be higher than glutamate-C4.…”

“…The 13 C labeling of glutamine-C4 from [2-13 C]acetate, an astroglial specific substrate, has been shown to be higher than glutamate-C4. 19,34 Hence the dilution of astroglial acetyl-CoA from unlabeled acetate will result in additional dilution of glutamine-C4 when compared with that at glutamate-C4. The relevant dilution in glutamine-C4 relates to the dilution of astroglial glutamate-C4 (precursor of glutamine-C4) from astroglial sources relative to the much larger pool of neuronal glutamate which directly supports 70% to 80% of glutamine synthesis (and C4 labeling from C1-glucose).…”

“…We note that mass balance can also be achieved without the need to expel glutamine if the glutamate produced by anaplerosis replaces glutamate which is lost by oxidation, 17,35 effectively reducing the rate of glutamine efflux to the same degree. Although considerable in vitro evidence exists for glutamate oxidation in astrocytes, 36 studies conducted in vivo in rodents under euglycemic conditions did not show a significant glutamate oxidation, 19 emphasizing the need for new approaches to resolve this issue. Alternatively, considerable numbers of glutamine transporters may be bound at the luminal side of epithelium of the blood-brain barrier, which will reduce the available capacity for glutamine inflow, although the extracellular glutamine concentration of B0.39 mmol/L 37 is similar to plasma levels under physiologic conditions and therefore not saturating.…”

Progress towards in vivo brain 13C-MRS in mice: Metabolic flux analysis in small tissue volumes

“…3A) for the first time by integrating ex vivo and in vivo data measurements with direct detection in a 360 mL voxel [101]. Similar experiments under infusion of [2e 13 C] acetate could distinguish between C3 peaks of glutamate and glutamine in a 196 mL volume at 7 Tesla [132]. Again, labelling curves determined in vivo were supplemented with ex vivo data on C3 peaks to allow the use of a two-compartment model.…”