Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis

Abstract

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

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Figures

Figure 1.

Effect on the PGC1-family. (A) PGC1α mRNA was higher in the 2 g/kg/day and combined OAA groups. (B) The PGC1α nucleus:cytoplasm ratio was higher in the 1 g/kg/day, 2 g/kg/day, and combined OAA groups. (C) PRC mRNA was higher in the 2 g/kg/day OAA group. (D) On post hoc analysis, the PRC nucleus:cytoplasm ratio was higher in the 2 g/kg/day OAA group. (E) PGC1β mRNA levels were comparable. (F) PGC1β protein levels were comparable. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005; #ANOVA comparison was not significant, but the post hoc LSD test between the specified treatment group and the control group was significant at P < 0.05.

Figure 2.

Effect on other mitochondrial biogenesis-related parameters. (A) NRF1 mRNA was higher in the combined OAA group. (B) On post hoc analysis, TFAM mRNA was higher in the 2 g/kg/day group. (C) COX4I1 mRNA was higher in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. (C) Cytoplasmic COX4I1 protein was higher in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005; #ANOVA comparison was not significant, but the post hoc LSD test between the specified treatment group and the control group was significant at P < 0.05.

Figure 3.

Effect on proteins that regulate PGC1α. (A) Normalized to total AMPK, AMPK was more phosphorylated at Thr172 in the 1 g/kg/day and combined OAA groups. (B) Normalized to GAPDH, AMPK was more phosphorylated at Thr172 in the 1 g/kg/day and combined OAA groups. (C) Normalized to total p38 protein, levels of p38 phosphorylation at Thr180/Tyr182 were comparable across groups. (D) Normalized to GAPDH protein, p38 phosphorylation was greater at Thr180/Tyr182 in the 1 g/kg/day OAA group. (E) Normalized to total CREB, CREB was more phosphorylated at Ser133 in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. (F) When normalized to GAPDH, CREB was more phosphorylated at Ser133 in the combined OAA group. Although the ANOVA was not significant, on post hoc analysis CREB phosphorylation was higher in the 2 g/kg/day OAA group than in the control group. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005; #ANOVA comparison was not significant, but the post hoc LSD test between the specified treatment group and the control group was significant at P < 0.05.

Figure 4.

Effect on proteins involved in insulin signaling. (A) Normalized to total Akt, Akt was more phosphorylated at Ser473 in the 2 g/kg/day and combined OAA groups. (B) Normalized to GAPDH, Akt was more phosphorylated at Ser473 in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. (C) Normalized to total mTOR protein, mTOR was more phosphorylated at Ser2448 in the 2 g/kg/day and combined OAA groups. (D) Normalized to GAPDH, mTOR was more phosphorylated at Ser2448 in the 2 g/kg/day and combined OAA groups. (E) Normalized to total P70S6K, P70S6K was more phosphorylated at Thr389 in the 1 g/kg/day and combined OAA groups. (F) Normalized to GAPDH, levels of P70S6K phosphorylation at Thr389 were comparable between the groups. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005.

Figure 5.

Effect on inflammation. (A) TNFα mRNA levels were comparable. (B) CCL11 mRNA was lower in the 2 g/kg/day OAA group. (C) Nuclear NFκB protein was lower in the combined OAA group. Although the ANOVA was not significant, on post hoc analysis nuclear NFκB protein was lower in the 1 g/kg/day OAA group. (D) The NFκB nucleus:cytoplasm ratio was lower in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005; #ANOVA comparison was not significant, but the post hoc LSD test between the specified treatment group and the control group was significant at P < 0.05.

Figure 6.

Effect on growth factors and neurogenesis. (A) BDNF mRNA was comparable. (B) VEGF mRNA was higher in the combined OAA group. (C) DCX mRNA was higher in the 1 g/kg/day, 2 g/kg/day and combined OAA groups. (D) Representative confocal microscopy pictures of the hippocampal dentate gyrus. The top image used the ×10 objective, the bottom image represents an enlarged view of the boxed area from the 10× picture. (E) Dentate gyrus DCX pixel density was higher in the 2 g/kg/day OAA group. (F) Mice in the 1 g/kg/day, 2 g/kg/day and combined OAA groups had more intensely DCX-positive neurons. (G) Mice in the 1 g/kg/day, 2 g/kg/day and combined OAA groups had longer DCX-positive neurites. Values shown are relative group means ± SEM. *P < 0.05; **P < 0.005.

Figure 7.

Pre- and post-OAA brain MRS. (A) Lactate was higher post-treatment. (B) GABA was higher post-treatment. (C) Glutathione was higher post-treatment. (D) Glutamate did not change. (E) Glutamine did not change. (F) Alanine did not change.

Figure 8.

OAA effects and inter-effect relationships. OAA, a bioenergetic intermediate, affects bioenergetic flux. This produces a number of molecular changes. CREB phosphorylation and CREB activity increase, which in turn promotes the expression of PGC1 family member genes. AMPK and p38 MAP phosphorylation increase, and these activated kinases enhance PGC1α co-activator function. PGC1-induced co-activation of the NRF1 transcription factor stimulates COX4I1 production, while PGC1-induced co-activation of the ERRα transcription factor stimulates VEGF gene expression (61). OAA-induced flux changes also stimulate the pro-growth insulin signaling pathway and reduce inflammation. The pro-growth effects of increased insulin pathway signaling and increased VEGF, in conjunction with a more favorable bioenergetic status and less inflammation, cooperatively stimulate hippocampal neurogenesis.