The dentate gyrus differentially metabolizes glucose and alternative fuels during rest and stimulation

Abstract

The metabolic demands of neuronal activity are both temporally and spatially dynamic, and neurons are particularly sensitive to disruptions in fuel and oxygen supply. Glucose is considered an obligate fuel for supporting brain metabolism. Although alternative fuels are often available, the extent of their contribution to central carbon metabolism remains debated. Differential fuel metabolism likely depends on cell type, location, and activity state, complicating its study. While biosensors provide excellent spatial and temporal information, they are limited to observations of only a few metabolites. On the other hand, mass spectrometry is rich in chemical information, but traditionally relies on cell culture or homogenized tissue samples. Here, we use mass spectrometry imaging (MALDI-MSI) to focus on the fuel metabolism of the dentate granule cell (DGC) layer in murine hippocampal slices. Using stable isotopes, we explore labeling dynamics at baseline, as well as in response to brief stimulation or fuel competition. We find that at rest, glucose is the predominant fuel metabolized through glycolysis, with little to no measurable contribution from glycerol or fructose. However, lactate/pyruvate, β-hydroxybutyrate (βHB), octanoate, and glutamine can contribute to TCA metabolism to varying degrees. In response to brief depolarization with 50 mM KCl, glucose metabolism was preferentially increased relative to the metabolism of alternative fuels. With an increased supply of alternative fuels, both lactate/pyruvate and βHB can outcompete glucose for TCA cycle entry. While lactate/pyruvate modestly reduced glucose contribution to glycolysis, βHB caused little change in glycolysis. This approach achieves broad metabolite coverage from a spatially defined region of physiological tissue, in which metabolic states are rapidly preserved following experimental manipulation. Using this powerful methodology, we investigated metabolism within the dentate gyrus not only at rest, but also in response to the energetic demand of activation, and in states of fuel competition.

Keywords: brain metabolism; glucose metabolism; ketone body metabolism; lactate metabolism; mass spectrometry imaging; stable isotope tracing.

Figure 1:. Glucose metabolism in the DGC layer at baseline.

a) Experiments are performed on acute hippocampal slices (450 μm thick) in a perfusion chamber, allowing for application of different substrates or stable isotope tracers. At the chosen experimental timepoint, the tissue is thermally denatured to preserve its metabolic state. b) Slices are cryosectioned to 10 μm thickness, thaw-mounted onto a conductive glass slide, and coated with matrix. Each laser location generates an individual mass spectrum. c) By repeating the process throughout the tissue, an image is generated, with one spectrum at each ‘pixel’ location. The shown ion images correspond to the m/z value indicated beneath each. Using these images, a region of interest outlining the dentate granule cell (DGC) layer is drawn (white dotted line). An average DGC layer spectrum is exported for further data analysis. d) Experimental design for isotopic fuel labeling time course. Slices are thermally preserved following 0, 3, 10, or 30 minutes of ¹³C₆-glucose perfusion. e) Time course of glucose metabolism into glycolytic and TCA cycle metabolites. Empty circles represent ¹²C molecules, filled circles represent ¹³C molecules, and position of carbon removal is indicated with orange line in (iso)citrate and αKG. ¹³C molecules from a second turn of the TCA cycle are shown in dashed boxes. Symmetry in succinate leads to two possible isotopomers in the second turn of the TCA cycle. N=6 biological replicates, defined as separate mice. *< 0.05; **<0.01; ***<0.001 by unpaired One-way ANOVA with Dunnett’s correction for multiple comparisons to the 0 timepoint condition. GAP/DHAP: glyceraldehyde-3-phosphate/dihydroxyacetone phosphate; bPG: bisphosphoglycerates; PG: phosphoglycerates; PEP: phosphoenolpyruvate; αKG: α-ketoglutarate.

Figure 2:. Alternative fuel metabolism in the DGC layer at baseline.

a) Schematic of metabolic incorporation of alternative fuels. Time course of (b) U-¹³C lactate and pyruvate, (c) U-¹³C β-hydroxybutyrate, (d) U-¹³C octanoate, or (e) U-¹³C glutamine metabolism into the TCA cycle. f) Total incorporation of all isotopologues from 10 mM ¹³C₆-glucose, or from 2 mM of each labeled species into the TCA cycle. Alternative fuels are provided in the continued presence of 10 mM unlabeled glucose. N=6 biological replicates. *< 0.05; **<0.01; ***<0.001 by unpaired One-way ANOVA with Dunnett’s correction for multiple comparisons to the 0 timepoint condition.

Figure 3:. Metabolic response to KCl stimulation.

a) Experimental design schematic. Slices were incubated for 5 minutes ± 30 seconds of 50 mM KCl stimulation immediately prior to thermal preservation. b) Representative ion images of acute hippocampal slices that were unstimulated (left) or stimulated (right). Top row shows ion intensities for phosphocreatine and bottom row shows ion intensities for AMP. The color bar represents the relative intensity of metabolites and is scaled separately for pCreatine and AMP. Total pool sizes are shown for metabolites in glycolysis (c), the TCA cycle (e), or energy supply (f), with or without KCl stimulation. For each metabolite, ion counts are normalized to the average of the unstimulated control condition. d) To evaluate glucose uptake and hexokinase activity, 2-Deoxy-D-glucose is used as a tracer to indicate changes in hexose phosphate pool size. g) Experimental design schematic to determine changes in metabolic flux following KCl stimulation. 30 seconds of KCl was applied either after 3 minutes of labeling (in the labeling dynamic phase), or after 30 minutes of labeling (nearing isotopic steady-state). h) Labeled fraction of glycolytic metabolites from U-¹³C glucose in each condition with or without KCl stimulation. (Iso)citrate labeled fraction in each condition, when supplied with (i) U-¹³C glucose, (j) U-¹³C lactate/pyruvate, (k) U-¹³C β-hydroxybutyrate, (l) U-¹³C octanoate, or (m) U-¹³C glutamine. In c, e, f, N = 19 biological replicates; In d, h-m, N=6 biological replicates. *< 0.05; **<0.01 by paired two-tailed Student’s t-tests. GAP/DHAP: glyceraldehyde-3-phosphate/dihydroxyacetone phosphate; bPG: bisphosphoglycerates; PG: phosphoglycerates; PEP: phosphoenolpyruvate.

Figure 4:. Metabolic preference during fuel competition.

a) Schematic of experimental design. All slices were perfused for 10 minutes with 10 mM U-¹³C glucose in the presence of 0, 2, 5, or 10 mM unlabeled lac/pyr or βHB. Where indicated, 50 mM KCl stimulation was performed for 30 seconds (dark bars). b) Schematic of possible effects of unlabeled fuels on labeling fraction from U-¹³C glucose. Labeled fraction could decrease due to unlabeled carbon incorporation, or due to feedback inhibition and decreased metabolism of U-¹³C glucose. Glycolytic metabolites and (iso)citrate labeling fractions from U-¹³C glucose throughout increasing concentrations of (c) unlabeled lac/pyr or (d) unlabeled βHB. N=6 biological replicates. *< 0.05; **<0.01; ***<0.001. Statistical comparisons across fuel concentration (without KCl) were performed by unpaired One-way ANOVA with Dunnett’s correction for multiple comparisons to the 0 timepoint condition. Effects of KCl stimulation are compared by unpaired Student’s t-test within each concentration group. GAP/DHAP: glyceraldehyde-3-phosphate/dihydroxyacetone phosphate; bPG: bisphosphoglycerates; PG: phosphoglycerates; PEP: phosphoenolpyruvate.