Exercise training differentially impacts brain microvascular endothelial cell function in individuals with the APOE ε3 versus ε4 genotype

Introduction:: Over 10% of Americans over the age of 65 have been diagnosed with Alzheimer’s disease (AD), making it the 7th leading cause of death in the United States [1]. The apolipoprotein (APOE) ε4 allele indicates a high risk for AD compared to the APOE ε3 allele, which presents a neutral risk [2]. Transgenic mice with the APOE ε4 allele demonstrate reduced blood-brain barrier function and glucose transport [3] indicating that the APOE ε4 allele has detrimental effects on brain microvascular endothelial cell (BMEC) function and metabolism.

Aerobic exercise training has been proposed as an effective method of combating AD development and progression. Aerobic exercise training increases expression of the dominant glucose transporter GLUT1 [4], which may enhance BMEC metabolism. Furthermore, exercise training elevates endothelial protein levels of Sirtuin 1 (SIRT1) [5], a protein deacetylase that promotes BMEC barrier function by decreasing permeability and attenuating reactive oxygen species (ROS) production [6]. APOE ε4 proteins bind to the SIRT1 promotor, effectively decreasing SIRT1 levels [7-8]. Exercise-induced SIRT1 may be another mechanism through which exercise training improves BMEC function and reduces risk for AD.

In this work, we examine how aerobic exercise training combats BMEC glycolytic metabolic dysfunction and low SIRT1 protein levels in APOE ε3 BMEC compared to APOE ε4 BMEC. We hypothesize that serum from exercise-trained older adults will elevate GLUT1, glycolysis, and SIRT1 in genotype matched APOE ε3 and APOE ε4 BMEC relative to their baseline sedentary serum.


Materials and Methods:: Serum was collected from cognitively healthy physically inactive adults (4 participants per APOE genotype) between the ages of 60-90 pre- and post- 6 months of moderate intensity aerobic exercise training as a part of clinical trial NCT03727360. CRISPR/Cas9 was used to edit the genotype of induced pluripotent stem cells (iPSC) to develop APOE ε3 and APOE ε4 lines [9]. iPSC were then derived into BMEC-like cells [10] and treated with 20% genotype-matched serum for 24 hours. We then analyzed differences from cells treated with serum from each individual participant pre- (Sedentary) and post- exercise training (Trained). Western blots were run on 4-12% Bis-Tris gels and transferred to nitrocellulose membranes before incubating in primary antibodies for GLUT1 (21829-1-AP; Thermo Fisher), HK2 (sc-374091; Santa Cruz Biotechnology), PFKFB3 (13123; Cell Signaling), and SIRT1 (9475S; Cell Signaling) overnight at 4°C and secondary antibodies for 2 hours at room temperature. Protein levels were normalized to β-actin (sc-47778; Santa Cruz Biotechnology). Concurrent with Western blot experiments, media samples were collected from cells at 0- and 24-hours after treating with 20% human serum. Glucose and lactate concentrations in the media were quantified using a YSI 2950, enabling glucose uptake and lactate production to be calculated over the 24-hour time-period. Data were analyzed using a Mann-Whitney test for statistical significance.


Results, Conclusions, and Discussions:: Exercise training tended to decrease glycolytic enzymes in APOE ε4 BMEC. GLUT1, responsible for selective glucose uptake from the serum, hexokinase 2 (HK2), responsible for initial phosphorylation of glucose, and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a rate limiting enzyme of glycolysis, all tended to decrease in APOE ε4 BMEC treated with exercise-trained serum compared to serum from sedentary individuals (Fig. 1A). In contrast, exercise-trained serum had varied effects on the glycolytic enzymes in APOE ε3 BMEC; tending to increase GLUT1 and PFKFB3 but decrease HK2 (Fig. 1A). These data suggest exercise training may impact glycolysis through different mechanisms in APOE ε3 versus ε4 BMEC.

Glucose uptake was slightly elevated in ε3 BMEC (Fig. 1B). APOE ε4 BMEC, however, tended to increase lactate production with the exercise-trained serum compared to the sedentary serum (Fig. 1B). This resulted in increased lactate:glucose ratio, generally indicative of a higher glycolytic rate (Fig 1B), which may indicate altered glucose use in other metabolic pathways such as the pentose phosphate pathway or the TCA cycle.

SIRT1 was elevated in APOE ε3 BMEC with exercise-trained compared to sedentary serum, while SIRT1 decreased in the APOE ε4 BMEC with exercise-trained compared to sedentary serum (Fig. 1C). This may indicate decreased barrier function and increased ROS production in APOE ε4 BMEC following exercise training.

Serum from exercise trained APOE ε4 individuals trended towards decreasing glycolytic enzymes in APOE ε4 BMEC, while serum from exercise trained APOE ε3 individuals trended towards increasing glycolytic enzymes and glucose uptake in APOE ε3 BMEC. SIRT1 levels followed a similar trend of increasing with exercise training in APOE ε3 but decreasing in APOE ε4. Future steps will focus on increasing participant numbers to validate the current results, running metabolomics and computational metabolic modeling to predict glucose flux through various metabolic pathways, and measuring barrier function and ROS production to determine how differential SIRT1 levels impact the BMEC.


Acknowledgements (Optional): : The authors gratefully acknowledge funding support from the Brain and Behavior Initiative at the University of Maryland through the BBI Seed Grant Program to AMC and JCS, R01HL140239-01 to AMC, R01AG057552 to JCS, NSF-GRFP (DGE 1840340) to CMW and the University of Maryland ASPIRE program to SMZ.


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