Article Figures & Data
Figures
- Fig. 1.
Schematic of [U-13C]glucose in the brain. Simplified schematic of 13C-labeling patterns after the metabolism of [U-13C]glucose via glycolysis and the TCA cycle. Empty circles, 12C; black filled circle, 13C; gray filled circles, 13C derived from 13C-labeled oxaloacetate that enters the second turn of the TCA cycle (gray dotted lines). *Metabolites that were not measured in this study. GA3P, glyceraldehyde 3-phosphate; 13BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; Ac-CoA, acetyl CoA; OAA, oxaloacetate.
- Fig. 2.
Metabolism of [U-13C]glucose via glycolysis in SE mice in the chronic stage of pilocarpine model. A, Hippocampal 13C enrichment of glycolytic metabolites after i.p. injection of [U-13C]glucose was compared between SE and No SE mice. Reduced 13C enrichment in SE mice was found in G6P (22% reduction, p = 0.030), F6P (21%, p = 0.038), DHAP (17%, p = 0.05), and PEP (20%, p = 0.023). No significant differences were found in F16BP, 2 + 3PG, and PYR. Two-way ANOVA, SE status p < 0.001, n = 9–11 mice. B, The activities of all cytosolic enzymes, namely HK, PGI, PFK, PK, LDH, and G6PDH, were unaltered between control No SE mice and mice after SE within the chronic stage of the model (p > 0.05 for all); n = 7–9. C, Correlation analysis between percentage 13C enrichment of G6P and 13C enrichment in downstream metabolites in No SE mice. A significant correlation was observed with each metabolite, specifically: F6P, r = 0.89, p < 0.001; F16BP, r = 0.97, p < 0.001; DHAP r = 0.96, p < 0.001; 2 + 3PG, r = 0.76, p < 0.01; PEP, r = 0.96, p < 0.001; and PYR, r = 0.95, p < 0.001. No significant correlation was found between body weight (g) and percentage 13C enrichment of G6P, r = –0.28, p > 0.05. D, Correlation analysis between percentage 13C enrichment of G6P and 13C enrichment in downstream metabolites in SE mice. Similar to the No SE group, a strong correlation was observed with each downstream metabolite apart from 2 + 3PG. F6P, r = 0.91, p < 0.001; F16BP, r = 086, p < 0.001; DHAP, r = 0.90, p < 0.001; 2 + 3PG, r = 0.10, p > 0.05; PEP, r = 0.72, p < 0.05; and PYR, r = 0.64, p < 0.05. No significant correlation was found between body weight (g) and percentage 13C enrichment of G6P, r = –0.21, p > 0.05.
- Fig. 3.
Metabolism of [U-13C]glucose via the TCA cycle is impaired in SE mice in the chronic stage of pilocarpine model. A, Percentage 13C enrichment in the TCA cycle metabolites from the first turn of the TCA cycle were compared between SE and No SE mice. Reduced 13C enrichment was found in CIT (17% reduction, p < 0.006), ACO (17%, p = 0.0001), SUC (35%, p = 0.005), and FUM (23%, p = 0.001) in the hippocampal formation of mice in the chronic epileptic state. No changes were found in the 13C enrichment of 2OG (p > 0.05) or MAL (p > 0.05). Two-way ANOVA, SE status p < 0.001, n = 9–11 mice. B, Percentage 13C enrichment of TCA cycle metabolites when labeled oxaloacetate condenses with [1,2-13C]acetyl CoA. A reduction in 13C enrichment was observed in the intermediates 2OG (47%, p = 0.03), SUC (55%, p = 0.037), FUM (25%, p = 0.044), and MAL (29%, p = 0.003). Two-way ANOVA, seizure status p < 0.001, n = 9–11 mice. C, Maximal activities of mitochondrial enzymes were compared between SE and No SE mice. SE mice had lower activity of both PDH (33%, p = 0.045) and 2-OGDH (55%, p = 0.027), two key enzymes involved in the entry and rate of TCA cycling, compared with No SE controls. No changes were found in the enzymes PCX, GDH, GPT, and GOT (all p > 0.05); n = 7–9 mice for all enzymes. D, Correlation analysis between percentage 13C enrichment in PYR to all first-turn TCA cycle intermediates in No SE mice. A significant correlation exists for all metabolites compared with PYR in this group. CIT, r = 0.86, p < 0.001; ACO, r = 0.80, p < 0.01; 2OG, r = 0.78, p < 0.01; SUC, r = 0.86, p < 0.01; FUM, r = 0.70, p < 0.05; and MAL, r = 0.91, p < 0.001. E, Correlation analysis of percentage 13C enrichment in SE mice between PYR and first-turn TCA cycle intermediates. No significant correlation was found between PYR and the TCA cycle metabolites. CIT, r = 0.34, p > 0.05; ACO, r = 0.54, p > 0.05; 2OG, r = 0.40, p > 0.05; SUC, r = 0.48, p > 0.05; FUM, r = 0.37, p > 0.05; and MAL, r = 0.46, p > 0.05. F, Correlation between percentage enrichment of 13C from the first turn of the TCA cycle between 2OG and SUC. A strong correlation was observed in 13C enrichment between the two metabolites in No SE mice (r = 0.95, p < 0.001), whereas no correlation was found in 13C enrichment of 2OG and SUC in SE mice (r = 0.42, p > 0.05).
- Fig. 4.
Mitochondrial functional parameters of isolated hippocampal mitochondria from SE and No SE mice measured with the extracellular flux analyzer. A, Representation of the stages of the coupling assay to measure mitochondrial functions based on OCR. B, An example of the stages of the electron flow assay to measure electron flow through the electron transport chain base on the OCR. No differences were found in any of the parameters measured using the coupling assay state 2 respiration (C), state 3 respiration after the addition of ADP (D), state 3 uncoupled respiration (E), and respiration associated with ATP synthesis (F). Similarly, no significant differences were observed in the parameters measured using the electron flow assay including complex I–driven respiration (G) and complex II–driven respiration (H) between No SE and SE mice (n = 6–8 mice).
Tables
- Table 1.
Analyte-dependent parameters for the transitions used in scheduled multiple reaction monitoring data acquisition
Analyte Q1 (Da) 12C analyte Q3 (Da) 12C analyte RT (min) DP (volts) CE (volts) CXP (volts) Glucose 6-phosphate 258.89 96.7 8.1 –20 –30 –15 Fructose 6-bisphosphate 259.02 96.8 9.6 –20 –30 –15 Fructose 1,6-bisphosphate 339.08 96.9 21.9 –20 –30 –15 Dihydroxyacetone phosphate 168.84 97 11.9 –50 –14 –5 2- and 3-Phosphoglycerate 184.91 97 21.5 –50 –20 –5 Phosphoenolpyruvate 166.83 79 22.3 –40 –18 –5 Pyruvate 87.02 43 11.9 –45 –12 –1 Citrate 190.96 110.9 22.6 –50 –18 –7 Aconitate 172.94 84.9 22.6 –30 –18 –5 2-Oxoglutarate 144.95 100.8 20.5 –40 –12 –5 Succinate 117 73 18.5 –45 –16 –3 Fumarate 115.01 70.9 21.1 –45 –12 –1 Malate 133 70.8 19.7 –40 –22 –3 Metabolite (nmol/g tissue) No SE (n = 6–9) SE (n = 6–7) Glucose 6-phosphate 20.1 ± 1.7 24.2 ± 3.4 Fructose 6-phosphate 33.0 ± 2.2 36.2 ± 5.9 Fructose 1,6-bisphosphate 16.5 ± 1.0 17.8 ± 1.4 Dihydroxyacetone phosphate 0.70 ± 0.08 0.67 ± 0.08 2- and 3-Phosphoglycerate 11.2 ± 1.0 10.9 ± 1.2 Phosphoenolpyruvate 8.93 ± 1.42 7.50 ± 1.33 Pyruvate 38.2 ± 2.7 34.2 ± 6.0 Citrate 109 ± 5 110 ± 17 Aconitate 1.84 ± 0.12 2.24 ± 0.28 2-Oxoglutarate 90.8 ± 6.5 83.9 ± 17.4 Succinate 10.1 ± 0.8 8.1 ± 1.6 Fumarate 12.5 ± 0.9 13.2 ± 2.7 Malate 45.9 ± 3.9 46.1 ± 6.8
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