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. 2020 Jan 31;21(3):934.
doi: 10.3390/ijms21030934.

Ketone Bodies Promote Amyloid-β 1-40 Clearance in a Human in Vitro Blood-Brain Barrier Model

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Free PMC article

Ketone Bodies Promote Amyloid-β 1-40 Clearance in a Human in Vitro Blood-Brain Barrier Model

Romain Versele et al. Int J Mol Sci. .
Free PMC article

Abstract

Alzheimer's disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood-brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.

Keywords: Alzheimer’s disease; acetoacetate; amyloid-β peptide; blood–brain barrier; ketone bodies; β-hydroxybutyrate.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The effect of KBs on BLEC permeability. (A) Schematic representation of the human in vitro BBB model used for the KB study. This model is composed of an apical compartment in the filter containing endothelial cells and a basolateral compartment in the well with brain pericytes. After 5 days of co-culture, BLECs were incubated with various concentrations of KBs, AcAc, or βHB alone (5–20 mM) or in in combination (20 mM AcAc/20 mM βHB) for 48 h. Following KB treatment, the BLECs on the insert were transferred to a new plate for experiments. (B) The effects of KBs on cell viability were analyzed by MTT assay. (C) BLEC monolayer integrity was determined by measuring the endothelial lucifer yellow permeability (PeLY). Each bar represents the mean ± SEM relative to the control conditions (PeLY = 0.92 ± 0.03 × 10−3 cm∙min−1). The results are representative of three independent experiments performed in triplicate (*** p < 0.001). (D) Associated tight junction protein ZO-1 (green) and tight junction protein claudin-5 (red) staining were stained using immunofluorescence. Interruptions in the staining are indicated by white arrows. Nuclei were stained with Hoechst reagent and appear in blue. Scale bar: 50 µm.
Figure 2
Figure 2
Effect of KBs on the human in vitro BBB model. (A) BLECs were incubated with various concentrations of KBs for 48 h: 20 mM AcAc, 20 mM βHB, or ratio (20 mM AcAc/20 mM βHB). The glucose concentration was measured in the apical compartment every 8 h. (B) The βHB concentration was measured in the apical and basolateral compartments and in BLECs. The data are represented as mean ± SEM obtained from three independent experiments. (C) The presence of MCT1 (green) and GLUT1 (red) in the BLECs was performed using immunostaining. Nuclei were stained with Hoechst reagent, and appear in blue. Scale bar: 50 µm. (D) The effects of KBs on MCT1 and GLUT1 protein levels were analyzed by Western blot. The protein level was normalized using β-actin. The protein data represent the mean ± SEM obtained from at least three independent experiments relative to control conditions (* p < 0.05, ** p < 0.01). (E) The images are representative of at least three independent experiments.
Figure 3
Figure 3
Effects of KBs on the protein expression of actors involved in Aβ peptide transport in BLECs. The protein levels of (A) P-gp, (B) BCRP, (C) LRP1, (D) RAGE, and (E) PICALM were quantified using Western blot. The data were normalized using β-actin. The results represent the mean ± SEM obtained from at least three independent experiments relative to the control conditions (* p < 0.05, ** p < 0.01, *** p < 0.001). (F) The images are representative of at least three independent experiments.
Figure 4
Figure 4
Effect of KBs on Aβ transport across BBB. (A) Schematic representation of the human in vitro BBB model used for (A) apical-to-basolateral (red arrow) and (C) basolateral-to-apical (green arrow) Aβ transport. After 48 h of treatment with KBs, Aβ1–40Cy5 or [3H]inulin was added to the (B) apical compartment or (D) basolateral compartment, incubated for 30 min, and permeability was then assessed. The data represent the mean ± SEM obtained from at least three independent experiments relative to the control conditions, each of which was performed in triplicate (*** p < 0.001).
Figure 5
Figure 5
Involvement of LRP1 and P-gp in basolateral-to-apical transport of Aβ1–40Cy5 peptide after ratio KB treatment. (A) Schematic representation of the human in vitro BBB model used for basolateral to-apical Aβ transport (green arrow) in the presence of (A) LRP1 or (B) P-gp inhibitors, respectively RAP (red ban symbol) or elacridar (orange ban symbol). (C) Aβ1–40Cy5 or [3H]inulin basolateral-to-apical transport was performed with RAP or elacridar. The data represent the mean ± SEM obtained from at least three independent experiments relative to the control conditions, each of which was performed in triplicate (*** p < 0.001).

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