Specific regions of the brain are capable of fructose metabolism

Abstract

High fructose consumption in the Western diet correlates with disease states such as obesity and metabolic syndrome complications, including type II diabetes, chronic kidney disease, and non-alcoholic fatty acid liver disease. Liver and kidneys are responsible for metabolism of 40-60% of ingested fructose, while the physiological fate of the remaining fructose remains poorly understood. The primary metabolic pathway for fructose includes the fructose-transporting solute-like carrier transport proteins 2a (SLC2a or GLUT), including GLUT5 and GLUT9, ketohexokinase (KHK), and aldolase. Bioinformatic analysis of gene expression encoding these proteins (glut5, glut9, khk, and aldoC, respectively) identifies other organs capable of this fructose metabolism. This analysis predicts brain, lymphoreticular tissue, placenta, and reproductive tissues as possible additional organs for fructose metabolism. While expression of these genes is highest in liver, the brain is predicted to have expression levels of these genes similar to kidney. RNA in situ hybridization of coronal slices of adult mouse brains validate the in silico expression of glut5, glut9, khk, and aldoC, and show expression across many regions of the brain, with the most notable expression in the cerebellum, hippocampus, cortex, and olfactory bulb. Dissected samples of these brain regions show KHK and aldolase enzyme activity 5-10 times the concentration of that in liver. Furthermore, rates of fructose oxidation in these brain regions are 15-150 times that of liver slices, confirming the bioinformatics prediction and in situ hybridization data. This suggests that previously unappreciated regions across the brain can use fructose, in addition to glucose, for energy production.

Keywords: Brain energy metabolism; Fructose toxicity; Fructose transporters.

Fig 1. Fru-1-P and Fru-6-P pathways for fructose catabolism

Arrows depict pathways from fructose to pyruvate. Plasma membrane is indicated by double line. Transporters are all capital bold and critical enzymes are bold. Intermediates Fru 6-P, Fru 1-P, Fru 1,6-P₂, DHAP, Glyceraldehyde-3-P, Pyruvate, and Glyceraldehyde are indicated.

Fig 2. Genes required in the Fru-1-P pathway are expressed in Purkinje cells

Coronal slices (14 μm) of adult mouse brain (n = 6) were probed with DIG-conjugated antisense mRNA complementary to the genes for ketohexokinase (khk), aldolase C (aldoC), GLUT5 (glut5), and GLUT9 (glut9) at the same concentration. Expression was visualized by color development from horseradish peroxidase-conjugated anti-DIG antibody. Scale bar represents 50 μm. An index micrograph from the Allen Brain Atlas (ABA) for the same layer of these coronal sections is shown to the left with a box indicating the field of observation for the micrographs. The cell layers are labeled in the upper left micrograph; PCL, Purkinje cell layer; MCL, molecular cell layer; GCL, granule cell layer. Arrow notes positive expression and arrowheads indicate negative expression.

Fig 3. Cells of the hippocampus express genes necessary for fructose metabolism

Antisense probes for the genes listed in the legend for Fig 2 and noted in the bottom left of each panel were used for ISH of coronal slices (14 μm) of adult mouse brain (n = 6) with ABA index micrograph and field of observation boxed scale bar represents 100 μm. The cell layers are labeled in the upper left micrograph; CA1-3, dorsal hippocampus (DH), and dentate gyrus (DG).

Fig 4. Select cells in the cortex express genes necessary for fructose metabolism

Antisense probes for the genes listed in the legend for Fig 2 and noted in the bottom left of each panel were used for in situ hybridization of coronal slices (14 μm) of adult mouse brain (n = 6) with ABA index micrograph and field of observation boxed. Scale bar represents 100 μm. The cell layers (I–VI) are labeled in the upper left micrograph.

Fig 5. Cells in the olfactory bulb express genes necessary for fructose metabolism

Antisense probes for the genes listed in the legend for Fig 2 and noted in the bottom left of each panel were used for ISH of coronal slices (14 μm) of adult mouse brain (n = 6) with ABA index micrograph and field of observation boxed. Scale bar represents 50 μm. The cell layers are labeled in the upper left micrograph; glomerular layer (GL), mitral cell layer (MCL), and granule cell layer (GCL).

Fig 6. Immunoblotting for detecting GLUT Expression

Membrane proteins were extracted from pooled samples of Fru-1-P positive brain regions. Protein (50 μg) extracted from membrane fractions was separated by SDS-PAGE and blotted with GLUT3, GLUT5, or GLUT9 antibodies as described in Experimental Procedures. The loading control was an antibody to the A1 subunit of the ubiquitous membrane protein Na⁺/K⁺ ATPase pump, used to probe the same blots. Representative blots of combined membrane preparations from the cerebellum (CB), hippocampus (HP), cortex (CX), and olfactory bulbs (OB) (the Fru-1-P-positive brain regions) or liver, from adult mice fed standard chow and water (−) or from mice fed standard chow and a 40% (w/v) fructose drinking solution (+) for 3 weeks. Arrows indicate the migration distance of GLUT3, 5, 9, and the loading control, which migrated as proteins of 50, 45, 54, and 100 kDa, respectively, based on known molecular weight marker proteins from the Kaleidoscope pre-stained markers (BioRad) as indicated to the left with dashed on the left of each blot.

Fig 7. Inhibiting fructose phosphorylation by both KHK and HK is required to reduce total fructose oxidation

Oxidation of radioactive fructose (1 mM)(U-¹⁴C at 0.065 – 0.073 μCi/mmole) was measured in Fru-1-P positive brain samples (n = 4–6) with fructose alone (Fru only), in the presence of 2.5 mM cold glucose (to compete for HK phosphorylation; Fru + Glc), 1 μM of a pyrimindinopyrimidine inhibitor of KHK (Fru + KHK inhibitor), or both cold glucose and the KHK inhibitor (Fru + Glc + KHK inhibitor), for 1 hr. Protein content, measured by dye-binding assays (Bradford, 1976), was used to normalize samples. The measured oxidation rates were the average of ≥3 assays and statistical significance among the groups analyzed by ANOVA. Error bars indicate 1σ SEM with an asterisk indicating p < 0.05 and the # indicating no significance (p>0.10).