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. 2024 Oct:88:102018.
doi: 10.1016/j.molmet.2024.102018. Epub 2024 Aug 24.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

Motahareh Solina Safari  1 , Priska Woerl  1 , Carolin Garmsiri  1 , Dido Weber  1 , Marcel Kwiatkowski  2 , Madlen Hotze  2 , Louisa Kuenkel  1 , Luisa Lang  1 , Matthias Erlacher  3 , Ellen Gelpi  4 , Johannes A Hainfellner  4 , Gottfried Baier  5 , Gabriele Baier-Bitterlich  1 , Stephanie Zur Nedden  6
Affiliations

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

Motahareh Solina Safari et al. Mol Metab. 2024 Oct.

Abstract

Objective: Glucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain.

Methods: The effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured.

Results: We found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels.

Conclusions: We provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

Keywords: Energy metabolism; Glucose-1,6-bisphosphate; Ischemia; Mitochondrial pyruvate carrier; Phosphoglucomutase 2 like 1; Protein kinase N1.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
PGM2L1 is important for post-ischemic neuronal survival. (A) G-1,6-BP levels were measured in organs of adult mice (one way ANOVA with Holm-Šídák's multiple comparisons test, (∗∗∗) P < 0.001, (∗∗∗∗) P < 0.0001)). (B) Expression of PGM2L1 in adult mouse brain. Image credit: Allen Institute for Brain Science (Allen Brain atlas Pgm2l1 - RP_040825_02_D01 – sagittal, http://mouse.brain-map.org/experiment/show/545100). (C) Human prefrontal cortical sections were stained for PGM2L1 (see also Supplemental Fig. 1), which was mainly seen in pyramidal neurons, identified by their triangular shape (arrowheads). (D) Hippocampal mouse brain slices were exposed to control conditions, 7 min OGD or 7 min OGD and 2 h Rep and G-1,6-BP was measured (one way ANOVA with Holm-Šídák's multiple comparisons test, (∗∗) P < 0.01, (∗∗∗∗) P < 0.0001). (E) Cortical mouse neurons were transfected with non-targeting siRNA (Co siRNA) or PGM2L1 siRNA and knockdown was confirmed after 48 h by western blotting (unpaired t-test (∗∗∗) P < 0.001). (F) Transfected neurons were exposed to 3 h OGD and 1 h Rep and stained for Hoechst, cleaved caspase-3 (casp-3, green) and the neuronal marker TAU (magenta). Image is representative of 3 separate experiments. (G) The percentage of cleaved casp-3 positive cells was calculated (two way ANOVA: Treatment (∗∗∗) P < 0.001, siRNA (∗) P < 0.05, Interaction (∗) P < 0.05, Holm-Šídák's multiple comparisons test (∗) P < 0.05, (∗∗∗) P < 0.001, (ns) not significant). (H) The percentage of TAU-positive cells was calculated (two way ANOVA: Treatment P > 0.05, siRNA (∗∗) P < 0.01, Interaction (∗) P < 0.05, Holm-Šídák's multiple comparisons test (∗∗) P < 0.01, (ns) not significant). All scale bars refer to 50 μm. All data are presented as scatter blot with mean ± S.E.M.
Figure 2
Figure 2
Reduction in G-1,6-BP levels is accompanied by reduced incorporation of13C6-glucose into M2 citrate. (A) Mouse hippocampal/neocortical brain slices were exposed to control conditions or 7 min OGD and 15 min Rep. Thereafter slices were incubated in aCSF containing 10 mM 13C6-glucose (M6) for 10 min. Metabolites were analyzed for the fractional enrichment of 13C into glycolytic and TCA metabolites (Supplemental Table 1). M0 refers to “normal” 12C-metabolites. Image was prepared in BioRender. (B) The abundance (corrected peak area/mg wwt) of glucose-6-phosphate (G-6-P), pyruvate, lactate or citrate was not affected by OGD/Rep. There was a significant reduction in G-1,6-BP. Data is presented as % downregulation compared to control conditions (one sample t-test compared to hypothetical value of 100%, (∗∗) P < 0.01, (ns) not significant). There was no difference (not significant (ns)) in 13C incorporation into (C) G-6-P, (D) G-1,6-BP, (E) pyruvate or (F) lactate. (G) There was a significant reduction in fractional enrichment of M2 citrate (multiple t-test with a false discovery rate (Q) of 1% employing the two-stage set-up, (∗∗∗∗) P < 0.0001). (H) The reduction in M2 citrate was not due to upstream differences in glycolysis as analyzed by the M2 citrate/M3 pyruvate ratio (unpaired t-test, (∗∗) P < 0.01). All data are presented as scatter blot with mean ± S.E.M.
Figure 3
Figure 3
G-1,6-BP regulates mitochondrial pyruvate uptake. (A) Protein from mouse brain mitochondria was isolated and probed for mitochondrial markers (ATPB and Cox IV) and PGM2L1, which was localized to both, the cytosolic (Cyto) and mitochondrial fraction (Mito). Blot is representative of 3 separate experiments. (B) The mitochondrial G-1,6-BP content remained stable over a 15 min incubation period (unpaired t-test, P > 0.05). (C) Addition of G-1,6-BP (100 μM) to pyruvate dehydrogenase (PDH) did not alter its activity (paired t-test, P > 0.05). (D) A DARTS assay was performed with mouse hippocampal protein extracts incubated with low (-PI) or high protease inhibitor cocktail (+PI) after addition of vehicle (double distilled H2O, 0 mM) or 0.5 mM or 1 mM G-1,6-BP. G-1,6-BP (0.5 mM and 1 mM) protected (E) MPC2 from degradation by endogenous proteases (one way ANOVA with Holm-Šídák's multiple comparisons test: (∗∗) P < 0.01), while (F) MPC1 was only protected by 1 mM G-1,6-BP (one way ANOVA with Holm-Šídák's multiple comparisons test: (∗∗∗) P < 0.001, (ns) not significant). The protective effects were lost upon incubation with high PI (+PI) cocktail. (G) Control plasmids (empty vector, Co Plasmid) or human (h) PGM2L1 were overexpressed in human HEK cells expressing MPC1-mVenus (V)/MPC2-RLuc8 (R). Blot is representative of 3 separate experiments. (H) Transfected HEK MPC1V/MPC2R were treated with pyruvate (5 mM, arrowhead) or PBS and the BRET ratio was monitored. Values are expressed as fold of baseline with the PBS values subtracted (see Supplemental Figs. 3E and F for raw values). Areas marked in grey were used for analysis (N = 3). (I) The mean of 2 values (baseline and 15 min after addition of pyruvate) and the first value after addition of pyruvate of the areas marked in grey in H were compared (two way ANOVA Plasmid (∗∗∗∗) P < 0.0001, treatment (∗∗∗) P < 0.001, interaction P > 0.05, Holm-Šídák's multiple comparisons test (∗) P < 0.05). (J) Brain mitochondria were isolated and 14C3-pyruvate uptake was measured in the presence of vehicle (double distilled H2O, control, N = 8), 100 μM G-1,6-BP (N = 6) or 10 μM UK-5099 (N = 4). All values were related to each respective time point 0 (0 min, which was immediately stopped by the addition of 10 μM UK-5099). Pyruvate uptake with 0 and 100 μM G-1,6-BP was compared (two way ANOVA, Time (∗∗∗) P < 0.001, Treatment (∗∗) P < 0.01, Interaction P > 0.05, Holm-Šídák's multiple comparisons test: (∗) P < 0.5). (K) Mouse brain mitochondria were isolated and analyzed for their oxygen consumption rates (OCR). Mitochondria were stimulated with pyruvate (5 mM), ADP (4 mM) and malate (1 mM) and the effect of 100 μM G-1,6-BP on pyruvate-mediated OCR was analyzed. Raw relative fluorescence units (RFU) values are shown. (L) The slopes of each curve (from 1 to 45 min, shown in K) of the response was analyzed (unpaired t-test, (∗) P < 0.05). All data are presented as scatter blot with mean ± S.E.M., except for H, J and K were only mean ± S.E.M is shown.
Figure 4
Figure 4
Regulation of PGM2L1 activity. (A) Human PGM2L1 protein structure was predicted with AlphaFold Monomer v2.0 pipeline. The amino acids T173-H176 are shown in green in the predicted structure and encompass the predicted phosphorylation site S175. (B) Human SH-SY5Y cells were transfected with empty vectors (EV for the PKN1 and PGM2L1 plasmids; Control), HA-tagged PKN1+EV (for PGM2L1 plasmid), untagged PGM2L1+EV (for PKN1 plasmid) and PKN1+PGM2L1 and G-1,6-BP levels were analyzed (one way ANOVA with Holm-Šídák's multiple comparisons test ((∗) P < 0.05, (∗∗∗) P < 0.001). Blot is representative of 3 separate experiments. (C) The top 50 (rank) kinases predicted to phosphorylate human PGM2L1 S175 were analyzed with WebGestalt, using Network-Topology based analysis and PPI Biogrid. A pie chart summarizing the enrichment ratio (% of total) of the top 10 predicted networks are shown. (D) Scheme summarizing the metabolic processes involved in PGM2L1 regulation. Glycolysis, shown in green, has a stimulatory- and metabolic stress an inhibitory effect on G-1,6-BP levels, due to degradation by PMM1. Additionally PGM2L1 is inhibited by citrate, encompassing a potential feedback inhibition. The phosphorylation of S175 is further predicted to inhibit PGM2L1 activity. All data are presented as scatter blot with mean ± S.E.M. A and D were prepared in BioRender.
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