Exogenous L-lactate administration in rat hippocampus increases expression of key regulators of mitochondrial biogenesis and antioxidant defense

Abstract

L-lactate plays a critical role in learning and memory. Studies in rats showed that administration of exogenous L-lactate into the anterior cingulate cortex and hippocampus (HPC) improved decision-making and enhanced long-term memory formation, respectively. Although the molecular mechanisms by which L-lactate confers its beneficial effect are an active area of investigations, one recent study found that L-lactate supplementation results in a mild reactive oxygen species burst and induction of pro-survival pathways. To further investigate the molecular changes induced by L-lactate, we injected rats with either L-lactate or artificial CSF bilaterally into the dorsal HPC and collected the HPC after 60 minutes for mass spectrometry. We identified increased levels of several proteins that include SIRT3, KIF5B, OXR1, PYGM, and ATG7 in the HPC of the L-lactate treated rats. SIRT3 (Sirtuin 3) is a key regulator of mitochondrial functions and homeostasis and protects cells against oxidative stress. Further experiments identified increased expression of the key regulator of mitochondrial biogenesis (PGC-1α) and mitochondrial proteins (ATPB, Cyt-c) as well as increased mitochondrial DNA (mtDNA) copy number in the HPC of L-lactate treated rats. OXR1 (Oxidation resistance protein 1) is known to maintain mitochondrial stability. It mitigates the deleterious effects of oxidative damage in neurons by inducing a resistance response against oxidative stress. Together, our study suggests that L-lactate can induce expression of key regulators of mitochondrial biogenesis and antioxidant defense. These findings create new research avenues to explore their contribution to the L-lactate's beneficial effect in cognitive functions as these cellular responses might enable neurons to generate more ATP to meet energy demand of neuronal activity and synaptic plasticity as well as attenuate the associated oxidative stress.

Keywords: PGC-1 alpha; SIRT3; hippocampus; lactate; mitochondrial biogenesis; oxidative stress; proteomics.

Figure 1

Volcano plot with all identified proteins from proteomic analysis of HPC of L-lactate treated rats and ACSF treated rats. Thirty four upregulated and four downregulated proteins were found in lactate group compared to control.

Figure 2

L-lactate causes increased SIRT3 expression in neurons of HPC. (A,B) Representative confocal micrograph of SIRT3 (red) co-labelled with NeuN (green) in the CA1 (A) and CA3 (B) areas of HPC of control and lactate groups. L-lactate infusion increased SIRT3 expression in both areas. Scale bars: 20 μm. (C) Representative confocal micrograph of SIRT3 (red) co-labelled with GFAP (green) or NeuN (green) in the HPC of lactate group. Scale bars: 5 μm. (D,E) Fluorescence intensity of SIRT3 stained sections in the CA1 (D) and CA3 (E) areas of HPC of lactate group was assessed and normalized to control group of rats. Data is shown as mean ± SD (n = 5 rats per group). p*** < 0.001, unpaired Student’s t-test.

Figure 3

L-lactate increases CREB, pCREB, and pCREB/CREB in HPC. (A,C) Representative confocal micrograph of pCREB (red) co-labelled with NeuN (green) in the CA1 (A) and CA3 (C) areas of HPC of control and lactate groups. L-lactate infusion increased pCREB in both areas of HPC. Scale bars: 20 μm. (B,D) Number of pCREB+ cells in CA1 (B) and CA3 (D) areas of HPC of control and lactate groups. Data are shown as mean ± SD (n = 5 rats per group). p*** < 0.001, unpaired Student’s t-test. (E–H). Representative WB images of pCREB and CREB in the HPC extracts from control and lactate groups. Intensity of pCREB and CREB was quantified and normalized with β-actin and was found to be significantly increased in lactate group compared to control (F,G). pCREB/CREB ratio was also increased (H). Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, p** < 0.01, unpaired Student’s t-test.

Figure 4

L-lactate increases PGC-1α expression in neurons of HPC. (A,C) Representative confocal micrograph of PGC-1α (red) co-labelled with NeuN (green) in the CA1 (A) and CA3 (C) areas of HPC of control and lactate groups. L-lactate infusion increased PGC-1α expression in both areas of HPC. Scale bars: 20 μm. (B,D) Fluorescence intensity of PGC-1α stained sections in the CA1 (B) and CA3 (D) areas of HPC of lactate group was assessed and normalized to control group. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test. (E) Representative confocal micrograph of PGC-1α (red) co-labelled with GFAP (green) or NeuN (green) in the HPC of lactate group. Scale bars: 10 μm. (F) Representative WB images of PGC-1α in the HPC extracts from control and lactate groups. Intensity of PGC-1α was quantified and normalized with β-actin. PGC-1α was found to be significantly increased in lactate group compared to control. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test.

Figure 5

L-lactate increases ATPB expression in neurons of HPC. (A,C) Representative confocal micrograph of ATPB (red) co-labelled with NeuN (green) in the CA1 (A) and CA3 (C) areas of HPC of control and lactate groups. L-lactate infusion increased ATPB expression in both areas of HPC. Scale bars: 20 μm. (B,D) Fluorescence intensity of ATPB stained sections in the CA1 (B) and CA3 (D) areas of HPC of lactate group was assessed and normalized to control group. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test. (E) Representative confocal micrograph of ATPB (red) co-labelled with GFAP (green) or NeuN (green) in the HPC of lactate group. Scale bars: 10 μm. (F) Representative WB images of ATPB in the HPC extracts from control and lactate groups. Intensity of ATPB was quantified and normalized with GAPDH. ATPB was found to be significantly increased lactate group compared to control. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test.

Figure 6

L-lactate increases Cyt-c expression in neuron of HPC and increases mtDNA copy number. (A,C) Representative confocal micrograph of Cyt-c (red) co-labelled with NeuN (green) in the CA1 (A) and CA3 (C) areas of HPC of control and lactate groups. L-lactate infusion increased Cyt-c expression in both areas of HPC. Scale bars: 20 μm. (B,D) Fluorescence intensity of Cyt-c-stained sections in the CA1 (B) and CA3 (D) areas of HPC of lactate group was assessed and normalized to control group. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test. (E) Representative confocal micrograph of Cyt-c (red) co-labelled with GFAP (green) or NeuN (green) in the HPC of lactate group. Scale bars: 10 μm. (F) Representative WB images of Cyt-c in the HPC extracts from control and lactate groups. Intensity of Cyt-c was quantified and normalized with β-actin. Cyt-c was found to be significantly increased in lactate group compared to control. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test. (G) mtDNA copy number abundance in the HPC of control and lactate groups relative to nDNA. Relative mtDNA copy number was significantly increased in lactate group compared to control. Data are shown as mean ± SD (n = 4 rats per group). p*** < 0.001, unpaired Student’s t-test.