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, 38 (30), 6665-6681

Human ApoE Isoforms Differentially Modulate Brain Glucose and Ketone Body Metabolism: Implications for Alzheimer's Disease Risk Reduction and Early Intervention

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Human ApoE Isoforms Differentially Modulate Brain Glucose and Ketone Body Metabolism: Implications for Alzheimer's Disease Risk Reduction and Early Intervention

Long Wu et al. J Neurosci.

Abstract

Humans possess three genetic isoforms of apolipoprotein E (ApoE)-ApoE2, ApoE3, and ApoE4-that confer differential risk for Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. This study sought to investigate the impact of human ApoE isoforms on brain energy metabolism, an area significantly perturbed in preclinical AD. A TaqMan custom array was performed to examine the expression of a total of 43 genes involved in glucose and ketone body transport and metabolism, focusing on pathways leading to the generation of acetyl-CoA, in human ApoE gene-targeted replacement female mice. Consistent with our previous findings, brains expressing ApoE2 exhibited the most robust profile, whereas brains expressing ApoE4 displayed the most deficient profile on the uptake and metabolism of glucose, the primary fuel for the brain. Specifically, the three ApoE brains differed significantly in facilitated glucose transporters, which mediate the entry of glucose into neurons, and hexokinases, which act as the "gateway enzyme" in glucose metabolism. Interestingly, on the uptake and metabolism of ketone bodies, the secondary energy source for the brain, ApoE2 and ApoE4 brains showed a similar level of robustness, whereas ApoE3 brains presented a relatively deficient profile. Further, ingenuity pathway analysis indicated that the PPAR-γ/PGC-1α signaling pathway could be activated in the ApoE2 brain and inhibited in the ApoE4 brain. Notably, PGC-1α overexpression ameliorated ApoE4-induced deficits in glycolysis and mitochondrial respiration. Overall, our data provide additional evidence that human ApoE isoforms differentially modulate brain bioenergetic metabolism, which could serve as a potential mechanism contributing to their discrete risk impact in AD.SIGNIFICANCE STATEMENT We uncovered hexokinase as a key cytosolic point in the glucose metabolism that is differentially modulated by the three ApoE genotypes. The differences in hexokinase expression and activity exhibited in the three ApoE brains may underlie their distinct impact on brain glucose utilization and further susceptibility to AD. Therefore, a therapeutic approach that could circumvent the deficiencies in the cytosolic metabolism of glucose by providing glucose metabolizing intermediates, e.g., pyruvate, may hold benefits for ApoE4 carriers, who are at high risk for AD. The bioenergetic robustness may translate into enhanced synaptic activity and, ultimately, reduces the risk of developing AD and/or delays the onset of clinical manifestation.

Keywords: Alzheimer's disease; apolipoprotein E; energy metabolism; glucose; glycolysis; ketone body.

Figures

Figure 1.
Figure 1.
Human ApoE isoforms differentially modulate brain energy metabolism. A, Schematic representation of brain energy substrate uptake and generation of acetyl-CoA. Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate, a “branch-point” metabolite that has alternative metabolic fates. Acetyl-CoA is a critical point of convergence of glucose and ketone body metabolism. Brain glucose and ketone body uptake are strictly controlled by their respective transporters, GLUTs, and MCTs. The figure shows the key enzymes involved in the glucose and ketone body metabolic pathways. B, Gene expression profiles on energy substrate uptake and metabolism in the cortices of 6-month-old hApoE-TR female mice. The group heat map shows the expression of genes involved in glucose and ketone body transport and metabolism for all three ApoE genotypes. n = 4 per ApoE genotype. A complete list of the 48 genes grouped according to the functional class can be found in Figure 1-1. The relative expression (FC) with the corresponding p value are indicated in Figure 1-2.
Figure 2.
Figure 2.
ApoE2-expressing brains exhibit the most robust profile on glucose uptake and glycolysis. A, The group heat map shows the expression of genes involved in glucose transport and glycolysis for all three ApoE genotypes. B, C, The volcano plots illustrate FCs (x-axis) and p values (y-axis) between (B) ApoE2 brains versus ApoE3 brains and (C) ApoE4 brains versus ApoE3 brains; significantly altered expression: Hk1/2, Slc2a3 (Glut3), Slc2a4 (Glut4). p < 0.05 was considered statistically significant with Student's t test. D, The heat map shows the expression of key genes involved in pyruvate metabolism, glycogen utilization, and pentose phosphate pathways for all three ApoE genotypes. E, F, The volcano plots show FCs (x-axis) and p values (y-axis) between (E) ApoE2 brains versus ApoE3 brains and (F) ApoE4 brains versus ApoE3 brains; significantly altered expression: Dlat and Pgd. p < 0.05 by Student's t test. n = 4 per group. GI, Gene and protein expression levels of Hk1 were significantly lower in ApoE4 brains than in ApoE2 and ApoE3 brains. HJ, ApoE2 brains exhibited significantly higher levels of mRNA and protein expression of Hk2. Results were compared using one-way ANOVA with Bonferroni post hoc test. All of the analyses were performed in the cortical tissues of the same 6-month-old hApoE-TR mice. *p < 0.05, **p < 0.01. n = 4–5 per group.
Figure 3.
Figure 3.
Characterization of N2a cells expressing human ApoE isoforms. A, Gene expression levels of ApoE were determined in N2a-ApoE cells by real-time PCR. Amplification plots of three ApoE genotypes are shown. NC, Negative control. Four replicates each group. B, Genomic DNA extracted from N2a cells transfected with human ApoE cDNA or empty vector were amplified with specific primers for ApoE2, ApoE3, and ApoE4 genotyping. Three replicates per group. The difference in Ct values between positive and negative reactions across all genotypes was at least 10 cycles, which indicated the high specificity of the primers. C, ApoE protein levels were comparable among the three ApoE-transfected groups. D, E, Hexokinase expression was assessed in N2a-ApoE cells. Protein levels of Hk1 and Hk2 were significantly lower in ApoE4-transfected group than in ApoE2- and ApoE3-transfected groups. F, Hexokinase activities were determined in the cell lysates of N2a-ApoE cells. Hexokinase activity was the highest in N2a-ApoE2 cells, but the lowest in N2a-ApoE4 cells. Results were normalized to N2a-ApoE3 group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA. n = 4–5 per group.
Figure 4.
Figure 4.
ApoE2-expressing cells are most robust in glycolytic function and mitochondrial respiration. A, B, Glycolytic stress tests were performed using the Seahorse XF96 Extracellular Flux Analyzer to measure the basal glycolysis, glycolytic capacity, and glycolytic reserve in N2a-ApoE cells. Results were compared using one-way ANOVA with Bonferroni post hoc test. n = 18–20 per group. *p < 0.05, **p < 0.01 vs N2a-ApoE3 group. ###p < 0.001, ####p < 0.0001 vs N2a-ApoE2 group by one-way ANOVA with Bonferroni post hoc test. CE, Mitochondrial stress tests were performed using the Seahorse XF96 Extracellular Flux Analyzer to measure mitochondrial basal respiration, maximal respiration capacity and ATP-linked respiration in N2a-ApoE cells. n = 18–20 per group. F, ATP levels were determined in cell lysates of N2a-ApoE cells. n = 5 per group. Results were compared using one-way ANOVA with Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs N2a-ApoE3 group. ###p < 0.001, ####p < 0.0001 vs N2a-ApoE2 group.
Figure 5.
Figure 5.
ApoE2 and ApoE4 brains present similar level of robustness on ketone body uptake and metabolism. A, The heat map shows the expression of genes involved in brain ketone body transport and metabolism in the cortices of 6-month-old hApoE-TR mice. B, C, The volcano plots illustrate fold-changes and p values between (B) ApoE2 brains versus ApoE3 brains and (C) ApoE4 brains versus ApoE3 brains; Highlighted significantly altered expression: Bdh1 and Slc16a1. p < 0.05 was considered statistically significant with Student's t test. DG, The impact of ApoE isoforms on ketone body utilization was evaluated in N2a cells expressing human ApoE isoforms. N2a-ApoE cells received sequential injections of substrate (BHB or vehicle) and modulating compounds, and OCR was measured by Seahorse XF96 Extracellular Flux Analyzer. The ketone body-metabolizing capacity of cells was represented by the maximum OCR measurement after FCCP injection. FCCP-induced OCR of cells treated with BHB was normalized to the vehicle-treated group. Results were compared using one-way ANOVA with Bonferroni post hoc test. n = 8 per group. *p < 0.05, **p < 0.01 vs N2a-ApoE3 group.
Figure 6.
Figure 6.
PGC-1α overexpression ameliorates ApoE4-induced deficiencies in glycolysis and mitochondrial respiration. A, qRT-PCR was performed to examine the gene expression level of PGC-1α in the cortices of 6-month-old hApoE-TR mice. B, C, Protein expression of PGC-1α was determined in the cortices of the same hApoE-TR mice (B) and N2a-ApoE cells (C). Results were normalized to ApoE3 group. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA, n = 4–5 per group. D, E, Protein levels of Hk1 (D) and Hk2 (E) were measured in ApoE4-expressing cells transfected with vectors expressing mouse PGC-1α or mock control. PGC-1α overexpression increased Hk1 but decreased Hk2 in cells expressing ApoE4. F, Hexokinase activity was determined in the whole-cell lysates for the indicated groups. PGC-1α overexpression improved hexokinase activity in ApoE4-expresssing cells. *p < 0.05, ****p < 0.0001 by one-way ANOVA, n = 4–5 per group. G, ECAR was measured following sequential injections of glucose, oligomycin, and 2-DG in ApoE4-expressing cells transfected with mouse PGC-1α. H, Individual parameters for glycolysis, glycolytic capacity, and glycolytic reserve were calculated for the indicated groups. I, ATP levels were measured in ApoE4-expressing cells transfected with PGC-1α or empty vectors. Results were normalized to ApoE2 group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Bonferroni post hoc test. n = 6–7 per group. J, Mitochondrial OCR was measured in ApoE4-expressing cells transfected with mouse PGC-1α after sequential additions of oligomycin, FCCP, and antimycin A and rotenone as indicated. K, L, Quantification of individual parameters for basal respiration, maximal respiration, spare respiratory capacity, and ATP-linked respiration. Results were compared using one-way ANOVA with Bonferroni post hoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs ApoE2 group. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs ApoE4 group. n = 18–20 per group.
Figure 7.
Figure 7.
Differentiated N2a cells expressing ApoE2 displayed the most potent hexokinase expression and glycolytic activity. A, B, N2a cell morphology was evaluated by phase contrast imaging. The formation of neuronal processes were observed after RA incubation for 96 h. Scale bar = 40 μm. CE, The differentiation of N2a cells was also confirmed by immunoblotting for markers of mature neurons. Expressions of NeuN, synaptophysin, and PSD95 were significantly increased in the differentiated N2a cells. n = 3–8 per group. F, ApoE expression levels were comparable in differentiated N2a cells transfected with ApoE isoforms. n = 6 per group. G, H, Protein expressions of Hk1 1 and Hk2 were significantly higher in neuron-like differentiated N2a cells expressing ApoE2. n = 7–13 per group. I, J, Differentiated N2a cells expressing ApoE2 exhibited the most potent hexokinase activity and glycolytic function. Results were normalized to ApoE3 group. n = 3–6 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Bonferroni post hoc test.
Figure 8.
Figure 8.
Our working hypotheses and conclusions. The ApoE2 brain exhibits the most metabolically robust profile, whereas the ApoE4 brain presents the most deficient profile on the uptake and metabolism of glucose, the primary energy source for the brain. Particularly, these two ApoE genotypes are significantly different in the expression of GLUTs and hexokinases. On the uptake and metabolism of ketone bodies, the secondary fuel for the brain, both ApoE2 and ApoE4 brains show more robust profiles than the ApoE3 brain. PGC-1α may serve as an upstream master regulator of the bioenergetic robustness of ApoE2 whereas an inhibition of PPARγ signaling pathway may underlie the energy deficiency associated with ApoE4. In support of this notion, PGC-1α overexpression attenuated the bioenergetic deficits induced by ApoE4. A therapeutic approach that can bypass the deficiency in glucose uptake and glycolysis by providing glucose metabolizing intermediates, e.g., pyruvate, may hold the promise to reduce AD risk in ApoE4 carriers; however, a ketogenic strategy may provide a more meaningful benefit in ApoE3 than in ApoE4 carriers.

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