Triheptanoin alters [U-13C6]-glucose incorporation into glycolytic intermediates and increases TCA cycling by normalizing the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in a chronic epilepsy mouse model

Abstract

Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-¹³C₆]-glucose (i.p.), enrichments of ¹³C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of ¹³C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of ¹³C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of ¹³C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or - as an additional astrocytic fuel - it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of ¹³C was reduced in the interictal state. Triheptanoin restored the correlation between ¹³C enrichments of pyruvate relative to most of the TCA cycle intermediates in "epileptic" mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

Keywords: Glucose metabolism; anaplerosis; medium chain fatty acid; pilocarpine; temporal lobe epilepsy.

Figure 1.

Schematic of [U-¹³C₆]-glucose metabolism in the brain. Simplified schematic of ¹³C-labelling patterns following the metabolism of [U-¹³C₆]-glucose via glycolysis, pentose phosphate pathway (PPP) and the TCA cycle. Empty circles represent ¹²C and black-filled circles represent ¹³C. Grey coloured intermediates represent the metabolites that were not measured in this study. Glucose (GLC), glucose 1-phosphate (G1P); glucose 6-phosphate (G6P); fructose 6-phosphate (F6P); fructose 1,6-bisphosphate (F16BP); glyceraldehyde 3-phosphate (GA3P); dihydroxyacetone phosphate (DHAP); 1,3-bisphosphoglycerate (13BPG); 3-phosphoglycerate (3PG); 2-phosphoglycerate (2PG); phosphoenolpyruvate (PEP); pyruvate (PYR); Acetyl CoA (Ac-CoA); citrate (CIT); aconitate (ACO); 2-oxoglutarate (2OG); succinate (SUC); fumarate (FUM); malate (MAL); oxaloacetate (OAA), ribulose 5-phosphate (RL5P). Please note that G6P can be produced from glycogen and lactate can leave the brain.

Figure 2.

Similar % [U-¹³C₆]-glucose enrichment in plasma from no SE and SE mice in the chronic stage of the pilocarpine model. Plasma enrichment of [U-¹³C₆]-glucose as percentage of total glucose levels after i.p. injection of [U-¹³C₆]-glucose was compared between (“epileptic”) SE and (“non-epileptic') No SE mice, either untreated (Con) or treated with 35E% triheptanoin (trihep). There were no statistically significant differences between the groups (two-way ANOVA; p = 0.47 interaction, p = 0.14 treatment, p = 0.10 SE vs. No SE, n = 6–8 mice per group).

Figure 3.

The effect of triheptanoin on the interictal metabolism of [U-¹³C₆]-glucose via glycolysis in SE mice in the chronic stage of the pilocarpine model. (a) Hippocampal ¹³C enrichment of glycolytic metabolites after i.p. injection of [U-¹³C₆]-glucose was compared between (“epileptic”) SE and (“non-epileptic') No SE mice, either untreated or treated with 35E% triheptanoin. The top row indicates outcomes of the two-way ANOVAs regarding effects of treatment or SE, if found significant. The horizontal lines with stars indicate significances found in post hoc Fisher's LSD comparisons between specific groups. *p = 0.05 and **p < 0.01. Having undergone SE and thus being in the chronic epileptic stage affected the % ¹³C enrichment of glucose 6-phosphate (G6P, p = 0.016), fructose 6-phosphate (F6P, p = 0.002), fructose 1,6-bisphosphate (F16BP, p = 0.044), dihydroxyacetone phosphate (DHAP, p = 0.028), phosphoenolpyruvate (PEP, p = 0.040), pyruvate (PYR, p = 0.040) and ribulose 5-phosphate (RL5P, p = 0.024 all two-way ANOVAs). Triheptanoin treatment affected the ¹³C enrichment of G6P (p = 0.004), DHAP (p = 0.042), 3- and 2-phosphoglycerate (3PG+2PG, p = 0.018), PEP (p = 0.027) and RL5P (p = 0.028). A Fisher's LSD post-test showed a significant increase in the % ¹³C incorporation in glucose 6-phosphate in triheptanoin-treated mice compared to controls (1.2-fold higher, p = 0.044, No SE mice; 1.3-fold higher, p = 0.032, SE mice; p < 0.05). A significant loss in the % ¹³C incorporation in fructose 6-phosphate was observed in SE mice compared to No SE mice in both the untreated (21%, p = 0.039) and triheptanoin treated (23%, p = 0.011) groups. There was a 1.3-fold increase in the % ¹³C incorporation of 3PG+2PG in triheptanoin-treated No SE mice compared to untreated No SE mice (p = 0.031). Finally, there was a 34% decrease in the ¹³C enrichment of RL5P between the SE and No SE mice fed a standard diet. No significant differences were seen when the SE triheptanoin-treated mice were compared to the No SE untreated group. n = 9–13 mice per group. (b–g) Comparisons between % ¹³C enrichments of G6P vs. its downstream metabolites with solid lines using the same colours as in panel A showing linear correlations calculated by GraphPad Prism. Significant correlations were observed across all four treatment groups (r = 0.89–0.91, p = 0.003–0.0001) when correlated with the % enrichment of (b) F6P, (c) F16BP, (d) DHAP, (f) PEP and (g) PYR, whereas, (e) 2 + 3PG was only significantly correlated in the No SE group that was not treated (r = 0.76, p = 0.01).

Figure 4.

The effect of triheptanoin on the interictal metabolism of [U-¹³C₆]-glucose in the TCA cycle in SE mice in the chronic stage of pilocarpine model. (a) Percent ¹³C enrichments in the TCA cycle metabolites from the first turn of the TCA cycle were compared between SE and No SE mice treated with or without 35E% triheptanoin. The top row shows outcomes of the Two-way ANOVAs regarding effects of treatment or SE when found significant. The horizontal lines with stars indicate significances found in post hoc Fisher's LSD comparisons between specific groups. *p = 0.05 and **p < 0.01. Being in the chronic epileptic stage (SE mice) affected the % ¹³C enrichment of citrate (CIT, 30.4%, p < 0.001), aconitate (ACO, 26.2%, p < 0.001), 2-oxoglutarate (2OG, 17%, p = 0.08), succinate (SUC, 19.4%, p = 0.003), fumarate (FUM, 24.3%, p = 0.001) and malate (MAL, 21.2%, p = 0.003). However, triheptanoin treatment only affected the % ¹³C enrichment of CIT (7.1%, p = 0.038). A Fisher's LSD post-test showed that ¹³C enrichment was reduced in CIT (17%, p = 0.003), ACO (16%, p = 0.019), SUC (34%, p = 0.002), FUM (23%, p = 0.004), and MAL (17%, p = 0.021) in the untreated SE mice compared to No SE mice. The percent ¹³C enrichments of CIT (15%, p = 0.003), ACO (18%, p = 0.005), 2OG (18%, p = 0.043) and MAL (14%, p = 0.010) were also reduced between the triheptanoin-treated SE and No SE mouse groups. In the SE mice treated with triheptanoin compared to the untreated SE mice, the % ¹³C enrichment of malate was reduced (15%, p=0.030). Compared to the untreated SE group, the SE mice treated with triheptanoin had a 30% increase in the % ¹³C enrichment of succinate (p = 0.048) n = 9–13 per group. (b) Comparisons between the ¹³C enrichment of pyruvate and citrate with solid lines using the same colours as in panel A to depict linear correlations for each of the four treatment groups. Strong positive correlations were found in the No SE untreated, and both No SE and SE groups treated with triheptanoin (r = 0.8–0.86, p = 0.05–0.001), but not in the untreated SE group (yellow, r = 0.34; p = 0.33).