Heptanoate as a neural fuel: energetic and neurotransmitter precursors in normal and glucose transporter I-deficient (G1D) brain

Abstract

It has been postulated that triheptanoin can ameliorate seizures by supplying the tricarboxylic acid cycle with both acetyl-CoA for energy production and propionyl-CoA to replenish cycle intermediates. These potential effects may also be important in other disorders associated with impaired glucose metabolism because glucose supplies, in addition to acetyl-CoA, pyruvate, which fulfills biosynthetic demands via carboxylation. In patients with glucose transporter type I deficiency (G1D), ketogenic diet fat (a source only of acetyl-CoA) reduces seizures, but other symptoms persist, providing the motivation for studying heptanoate metabolism. In this work, metabolism of infused [5,6,7-(13)C(3)]heptanoate was examined in the normal mouse brain and in G1D by (13)C-nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). In both groups, plasma glucose was enriched in (13)C, confirming gluconeogenesis from heptanoate. Acetyl-CoA and glutamine levels became significantly higher in the brain of G1D mice relative to normal mice. In addition, brain glutamine concentration and (13)C enrichment were also greater when compared with glutamate in both animal groups, suggesting that heptanoate and/or C5 ketones are primarily metabolized by glia. These results enlighten the mechanism of heptanoate metabolism in the normal and glucose-deficient brain and encourage further studies to elucidate its potential antiepileptic effects in disorders of energy metabolism.

Figures

Figure 1

Schematic diagram of [5,6,7-13C3]heptanoate and [3,4,5-13C]C5-ketone metabolism in the liver and brain. Standard metabolic model depicting that dietary [5,6,7-13C3]heptanoate can be metabolized in the liver to produce [3,4,5-13C3]C5-ketones, which are subsequently released to the bloodstream. C5-ketones cross the blood–brain barrier through monocarboxylate transporters and are metabolized in the brain. On the other hand, heptanoate can directly cross the blood–brain barrier by passive diffusion and be oxidized in the brain as well. In the brain, [5,6,7-13C3]heptanoate and [3,4,5-13C3]C5-ketones produce unlabeled acetyl-CoA (2 molecules from heptanoate, one from C5-ketones) and one molecule of [1,2,3-13C3]propionyl-CoA. Acetyl-CoA is further oxidized in the tricarboxylic acid (TCA) cycle and [1,2,3-13C3]propionyl-CoA can be converted to [1,2,3-13C3]methyl-malonyl-CoA and this can be converted into [1,2,3-13C3]succinyl-CoA or [2,3,4-13C3]succinyl-CoA, since this molecule is structurally symmetrical. As a consequence of the first cycle through the TCA cycle, glutamate and glutamine will be labeled in carbons 2 and 3 or in carbons 1,2 and 3. Note that carbons 4 and 5 of glutamate are derived directly from acetyl-CoA and carbons 1, 2 and 3 originate from oxaloacetate. Potential hepatic or glial 4-carbon ketone body formation and subsequent metabolism by the brain are not represented. Hpt, heptanoate; C5-KB, C5 ketone bodies; Ac-CoA, acetyl-CoA; CIT, citrate; α-KG, α-ketoglutarate; GLU, glutamate; GLN, glutamine; Prop-CoA, propionyl-CoA; SucCoA, succinyl-CoA. C#: carbon labeled in position #. Open circle: carbon 12, closed circle: carbon 13.

Figure 2

Mass isotopomers of 13C-glucose in plasma. Plasma 13C-glucose was enriched from [5,6,7-13C3]heptanoate in both normal and mutant (glucose transporter type I deficiency, G1D) animal groups. Data were corrected for the natural abundance of 13C. Glucose was mostly enriched in M+2 (Control: 4.63%±1.5% G1D: 5.28%±1.22%) and M+3 (Control: 3.65%±0.86% G1D: 3.45%±0.69%). No statistical differences were detected between both animal groups.

Figure 3

13C-NMR (nuclear magnetic resonance) spectra of brain hemispheres from normal and glucose transporter type I deficiency (G1D) mice. Proton-decoupled 13C-NMR spectra were acquired from extracts of brain tissue taken from animals infused with [5,6,7-13C3]heptanoate. Spectra from both animals demonstrate 13C labeling in brain glutamate and glutamine in carbons 2, 3 and 4 and lactate in carbons 2 and 3. In both animals, the glutamate and glutamine 13C labeling pattern differed from one another in all carbon positions, suggesting that heptanoate metabolism is compartmentalized in the brain. In carbons 2 and 3, the total signal from 13C-multiplets was more prominent in glutamine compared with glutamate. In carbon 4, the glutamate resonance was greater than that of glutamine. A doublet due to J45 (arising from coupling between carbons 4 and 5) was present in glutamate but not glutamine. Of note, the resonance of glutamine C2 doublet D2,3 (purple arrows) was statistically higher in G1D animals compared with controls. The lactate resonance was also prominent in the 13C spectrum of both animals. In lactate C2 and C3, the doublet D2,3 was the most abundant signal, indicating other 13C substrates (i.e., glucose) may be oxidized in the brain as a result of gluconeogenesis from [5,6,7-13C3]heptanoate. 1. N-acetyl-aspartate C2, 2. Aspartate C2, 3. Taurine C1, 4. N-acetyl-aspartate C3, 5. GABA C4, 6. Creatine C2, 7. Aspartate C3, 8. Taurine C2, 9. GABA C2, 10. GABA C3, 11. N-acetyl-aspartate C6. GLU, glutamate; GLN, glutamine; LAC, lactate. C#: carbon labeled in position #. Sx, singlet; Dxx, doublet; T, triplet; Q, quartet. The color reproduction of this figure is available on the Journal of Cerebral Blood Flow and Metabolism journal online.

Figure 4

Analysis of 13C spectra in normal and glucose transporter type I deficiency (G1D) mice. (A) Overall, the 13C abundance of glutamine in carbons 2 and 3 was greater than glutamate; this difference was more prominent in G1D mice although did not reach statistical significance. Carbon 4 was not illustrated since glutamine C4 did not contain multiplets. (B) The fractional amount of the glutamine C3 doublet was significantly higher than the glutamate C3 doublet in both mice with no difference between animal groups. (C) In carbon 2, the glutamine doublet D2,3 was statistically more abundant in G1D mice relative to normal and greater than the glutamate doublet D2,3 in both animals. No differences were observed in the fractional amount of the doublet D1,2 within and between animal groups. The quartet (Q) was more prominent in glutamine than glutamate, although it was statistically significant only in the G1D group. (D) No difference was detected between animals relative to the fractional amount of glutamate doublet D4,5. Values are expressed as mean±s.e.m. **P<0.01,***P<0.001.

Figure 5

Heptanoate stimulation of brain metabolism. The higher concentration, 13C resonance and 13C enrichment of glutamine relative to glutamate indicates that [5,6,7-13C3]heptanoate supplied [1,2,3-13C3]propionyl-CoA in the glial compartment, either directly or after conversion to C5 ketones in the liver. The presence of J45 in glutamate and not in glutamine, and the measured 13C enrichment in plasma glucose indicates that [5,6,7-13C3]heptanoate was metabolized to multiply enriched glucose followed by oxidation via pyruvate dehydrogenase primarily by neurons. This labeling pattern is also compatible with metabolism of labeled glucose to [1,2-13C2]acetyl-CoA by glia followed by dilution with unlabeled acetyl-CoA derived from heptanoate oxidation. Thus, (astrocytic) acetyl-CoA may be derived both from labeled plasma glucose and from unlabeled carbons of heptanoate. The different labeling pattern of glutamine relative to glutamate supports the notion that heptanoate and/or C5 ketone metabolism is compartmentalized in the brain. Additionally, the GABA doublet D3,4 was observed in the 13C spectra (Supplementary Figure 1), which indicates that part of the glial glutamine produced from heptanoate is transferred to neurons. Any potential contribution of hepatic or glial 4-carbon ketone body formation to the labeling patterns observed in the brain tissue is not represented. Hpt, heptanoate; C5-KB, C5 ketone bodies; Glc, glucose; PYR, pyruvate; Ac-CoA, acetyl-CoA; CIT, citrate; α-KG, α-ketoglutarate; GLU, glutamate; GLN, glutamine; Prop-CoA, propionyl-CoA; SucCoA, succinyl-CoA. C#: carbon labeled in position #. Open circle: carbon 12, closed circle: carbon 13.