Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet (13)C NMR of the adult mouse brain

Abstract

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the (13)C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-(13)C(2)]glucose (oxidized by both neurons and glia) and [1,2-(13)C(2)]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by (13)C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of (13)C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The (13)C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-(13)C]acetate relative to [1,6-(13)C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron-glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.

Figure 1

Expected glutamate C4 spectra from simulation studies under physiological (A) and PDC-deficient conditions (B). The simulations were enclosed in a single cell compartment assuming that the enrichments of glucose-derived [2-¹³C]acetyl-CoA and acetate-derived [1,2-¹³C₂]acetyl-CoA were 50% and 100%, respectively. PDC activity was considered normal when its flux was 80% of the total TCA cycle flux and deficient (in 25%) when its rate was 60% of the total TCA cycle flux. S: singlet, Dxx: doublet, Q: quartet.

Figure 2

Schematic metabolic diagram depicting the ¹³C-label distribution during the first cycle (“turn”) of the TCA cycle in brain cells when [1,2-¹³C₂]acetate and [1,6-¹³C₂]glucose are simultaneously administered. [1,2-¹³C₂]acetate is exclusively metabolized in glial cells, giving rise to glutamine and, to a lesser extent, to glutamate labeled in carbon positions 4 and 5, whereas glucose can be metabolized in both cell types (neurons and glia) leading to the synthesis of both glutamate (which is primarily produced in neurons) and glutamine labeled in carbon 4. Pyruvate carboxylase (not illustrated) is exclusively present in glia. In red: glucose-derived ¹³C; in blue: acetate-derived ¹³C; in purple when red and blue converge. Glc-6-P: glucose 6-phosphate, HK: hexokinase, PYR: pyruvate, PDC: pyruvate dehydrogenase complex, Ac-CoA: acetyl-CoA, CIT: citrate, α-KG: α-ketoglutarate.

Figure 3

A segment of a representative cortical ¹³C spectrum from a PDC-deficient mouse co-infused with [1,2-¹³C₂]acetate and [1,6-¹³C₂]glucose exhibiting the key metabolites of interest. The insets display the labeling patterns of glutamate and glutamine in carbons 2, 3 and 4. 1: Lactate C3, 2: N-acetylaspartate C6, 3: GABA C3, 4: GABA C2, 5: Taurine C2, 6: Aspartate C2, 7: Creatine C2, 8: GABA C4, 9: N-acetylaspartate C3, 10: taurine C1, 11: Aspartate C2, 12: N-acetylaspartate C2. C#: carbon labeled in position #. Sx: singlet, Dxx: doublet, T: triplet, Q: quartet.

Figure 4

Detail of a cortical ¹³C spectrum obtained from a normal mouse co-infused with [1,2-¹³C₂]acetate and [1,6-¹³C₂]glucose. The insets display the labeling patterns of glutamate and glutamine in carbons 2, 3 and 4. 1: Lactate C3, 2: N-acetylaspartate C6, 3: GABA C3, 4: GABA C2, 5: Taurine C2, 6: Aspartate C2, 7: Creatine C2, 8: GABA C4, 9: N-acetylaspartate C3, 10: taurine C1, 11: Aspartate C2, 12: N-acetylaspartate C2. C#: carbon labeled in position #. Sx: singlet, Dxx: doublet, T: triplet, Q: quartet.

Figure 5

Labeling pattern of glutamate and glutamine C4 in the cortex of PDC-deficient (A) and normal (B) mice infused with [1,6-¹³C₂]glucose and [1,2-¹³C₂]acetate, a representative ¹³C NMR spectra (separately obtained for comparative purposes) from the cortex of a normal mouse infused with [1,6-¹³C₂]glucose (C) and from the forebrain of a normal mouse infused with [1,2-¹³C₂]acetate (D). The labeling pattern of both C4 isotopomers is very similar when only ¹³C-glucose is administered, probably demonstrating that glucose is metabolized both by neurons and glia without indication of compartmentation. As ¹³C-acetate is infused alone or together with ¹³C-glucose, the labeling pattern of glutamate C4 and glutamine C4 differs significantly supporting the notion that acetate and glucose are oxidized differently in each cell compartment, and that there is a pool of these metabolites that is not rapidly exchangeable across both major cell types. C#: carbon labeled in position #. Sx: singlet, Dxx: doublet, T: triplet, Q: quartet.

Figure 6

Diagram of [1,2-¹³C₂]acetate and [1,6-¹³C₂]glucose metabolism in glia and neurons as decteted by ¹³C-NMR spectroscopy from cortex extracts. At least two potential pools of brain glutamate are observed when both ¹³C-labeled substrates are infused: 1) an exchangeable glutamate pool, mostly originating from glial, acetate-derived glutamine, and 2) a slowly exchangeable pool of neuronal glutamate, principally produced by glucose oxidation in the neuronal TCA cycle. The GABA labeling pattern matches that of glutamate, suggesting that it directly communicates with the exchangeable and slowly-exchangeable pools of glutamate. The metabolism of [1,2-¹³C₂]acetate also allowed for detection of alternative pathways that may contribute to the different labeling pattern of glutamate and glutamine, such as the recycling of pyruvate which, under these experimental conditions, originates primarily from glial glutamine oxidized in the neuronal compartment. These metabolic aspects are preserved in PDC-deficient mice. In red: glucose-derived ¹³C; in blue: acetate-derived ¹³C; in purple when red and blue converge. Glc-6-P: glucose 6-phosphate, HK: hexokinase, PYR: pyruvate, PDC: pyruvate dehydrogenase complex, Ac-CoA: acetyl-CoA, CIT: citrate, α-KG: α-ketoglutarate., OAA: oxaloacetate, MAL: malate.