Regional differences in brain glucose metabolism determined by imaging mass spectrometry

Abstract

Objective: Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS).

Methods: To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 μm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism.

Results: In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate.

Conclusion: These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution.

Keywords: ATP; Brain imaging; Glucose metabolism; Glycolysis; Mass spectrometry; Pentose phosphate pathway.

Figure 1

Differences in glucose metabolism between cortex and amygdala are not apparent despite different distributions of cells types between these regions. (A) Regions dissected for analysis are indicated by black outlines. (B) Immunohistochemistry for glucose-6-phosphate dehydrogenase across brain regions. (C) mRNA taken from amygdala (Amy), thalamus (Thal) and motor cortex (Cor) were compared by qPCR for expression of markers of neurons (NeuN), microglia (Iba1), astrocytes (GFAP) and oligodendrocytes (MBP), as well as (D) expression of enzymes involved in glycolysis and the PPP. N = 8. (E) Hexokinase activity N = 6, Glucose-6-phosphate dehydrogenase (G6PD) activity N = 3, and Phosphofructokinase type M (PFK M) activity N = 6 were performed on separate cohorts of mice. (F) Expression of glucose transporters across brain regions as measured by qPCR. N = 8.

Figure 2

Glycolytic intermediate hexose bisphosphate differs by brain region. Using imaging mass spectrometry (IMS) at 100 μm resolution, (A) the distributions of hexose phosphate (top) and hexose bisphosphate (middle) were determined in coronal brain sections. Nissl stain was performed on a serial brain section (bottom). (B) To determine the identity of the hexose bisphosphate MS³ was performed, demonstrating that the majority of the hexose bisphosphate measured was the glycolytic intermediate fructose-1,6-bisphosphate. N = 4. (C) Overlay image of hexose monophosphate and hexose bisphosphate with brain region map showing identifiable brain regions on IMS. (D) Relative abundance of hexose phosphate, hexose bisphosphate and a ratio of the two for amygdala, thalamus, and cortex and (E) 8 additional brain regions. N = 3.

Figure 3

ATP levels vary dramatically across the brain. (A) IMS images of ATP (top), ADP (middle), and the two overlaid (bottom) from the same brains used in Figure 2. (B) Brain map overlaying IMS images highlights much higher ATP in white matter tracts and low ATP in the amygdala and thalamus. (C) Relative abundance of ATP, ADP, and the ratio of the two for amygdala, thalamus, and cortex and (D) 8 additional brain regions. N = 3.

Figure 4

Fasting causes regional increases in brain lactate. (A) Fasting did not induce changes in hexose phosphate, hexose bisphosphate, ATP, or ADP compared to random fed mice. (B) Fasting induced increases in lactate in the cortex and hippocampus without changing levels in other brain regions. N = 5. (C) After glucose enters a cell it can be metabolized through glycolysis (left) or the pentose phosphate pathway (right). Enzymes assessed are in green ovals. Metabolites measured are in bold. G6PD = glucose-6-phosphate dehydrogenase, PGI = phosphoglucoisomerase, PFK = phosphofructokinase.