Simultaneous measurement of neuronal and glial metabolism in rat brain in vivo using co-infusion of [1,6-13C2]glucose and [1,2-13C2]acetate

Abstract

In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6-(13)C(2)]glucose and [1,2-(13)C(2)]acetate was investigated. Time courses of (13)C spectra were measured in vivo while infusing both (13)C-labeled substrates simultaneously. Individual (13)C isotopomers (singlets and multiplets observed in (13)C spectra) were quantified automatically using LCModel. The distinct (13)C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these (13)C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole (13)C-labeled substrate.

Fig. 1

Incorporation of ¹³C labels into the neuronal and astrocytic compartments following simultaneous infusion of [1,6-¹³C₂]glucose and [1,2-¹³C₂]acetate in the brain. (For simplicity, the uptake of [1,6-¹³C₂]glucose in the astrocytes is not shown.) In both cell types, [1,6-¹³C₂]glucose is converted to [3-¹³C]pyruvate which enters the neuronal or astrocytic TCA cycle via pyruvate dehydrogenase in the form of acetyl-CoA and leads to the formation of single-labeled [4-¹³C]glutamate and [4-¹³C]glutamine. In astrocytes, [3-¹³C]pyruvate can also enter the TCA cycle via pyruvate carboxylase to yield [2-¹³C]glutamate. At the same time, after conversion to [1,2-¹³C₂]acetyl-CoA in the astrocytes, acetate enters the TCA cycle to produce [4,5-¹³C₂]glutamate and [4,5-¹³C₂]glutamine during the first turn of the cycle. Glutamate released by neurons is taken up by astrocytes and converted into glutamine. Glutamine is then released and sent back to neurons where it is converted to glutamate. This cycling of neurotransmitter between astrocytes and neurons is referred to as the glutamate-glutamine cycle (V_NT).

Fig. 2

¹³C isotopic enrichment (left) and total concentration (right) of glucose, acetate and lactate in blood plasma. Error bars represent standard deviation (n = 5).

Fig. 3

LCModel fit of an in vivo ¹³C NMR spectrum obtained during simultaneous infusion of [1,6-¹³C₂]glucose and [1,2-¹³C₂]acetate (sum spectrum of 5 rats, voxel size of 400 μL, 4096 scans per rat measured during 3 hours infusion of the ¹³C-labeled substrates). From top to bottom: in vivo spectrum, LCModel fit and residuals. Ace: acetate; Ala: alanine; Asp: aspartate; Cr: creatine; GABA: gamma-aminobutyric acid; Glc: glucose; Glu: glutamate; Gln: glutamine; Lac: lactate; and NAA: N-acetyl aspartate.

Fig. 4

Decomposition of the individual isotopomers contributing to glutamate and glutamine C4 resonances from in vivo spectrum shown in Fig. 3. C4D45 doublets from [4,5-¹³C₂] glutamate and glutamine are synthesized from [1,2-¹³C₂]acetate while the C4S singlets and C4D43 doublets originate from [1,6-¹³C₂]glucose. Note the large signal from the C4D45 doublet in glutamine indicates that a large fraction of glutamine is synthesized from [1,2-¹³C₂]acetate metabolism in the glial TCA cycle.

Fig. 5

In vivo time courses of ¹³C concentration (μmol/g) of (a) glutamate C4 isotopomers (b) glutamine C4 isotopomers (c) total glutamate and total glutamine labeled at C2 and C3 positions and (b) acetate-D12 and lactate-C3 in the rat brain during co-infusion of [1,6-¹³C₂]glucose and [1,2-¹³C₂]acetate with a time resolution of 11 min (256 scans for each time point with a TR of 2.5s). Error bars represent mean Cramér-Rao Lower Bound (n = 5).