The Ketogenic Diet and Neuroinflammation: The Action of Beta-Hydroxybutyrate in a Microglial Cell Line

Abstract

The ketogenic diet (KD), a diet high in fat and protein but low in carbohydrates, is gaining much interest due to its positive effects, especially in neurodegenerative diseases. Beta-hydroxybutyrate (BHB), the major ketone body produced during the carbohydrate deprivation that occurs in KD, is assumed to have neuroprotective effects, although the molecular mechanisms responsible for these effects are still unclear. Microglial cell activation plays a key role in the development of neurodegenerative diseases, resulting in the production of several proinflammatory secondary metabolites. The following study aimed to investigate the mechanisms by which BHB determines the activation processes of BV2 microglial cells, such as polarization, cell migration and expression of pro- and anti-inflammatory cytokines, in the absence or in the presence of lipopolysaccharide (LPS) as a proinflammatory stimulus. The results showed that BHB has a neuroprotective effect in BV2 cells, inducing both microglial polarization towards an M2 anti-inflammatory phenotype and reducing migratory capacity following LPS stimulation. Furthermore, BHB significantly reduced expression levels of the proinflammatory cytokine IL-17 and increased levels of the anti-inflammatory cytokine IL-10. From this study, it can be concluded that BHB, and consequently the KD, has a fundamental role in neuroprotection and prevention in neurodegenerative diseases, presenting new therapeutic targets.

Keywords: beta-hydroxybutyrate (BHB); ketogenic diet; microglial cell line (BV2); neuroinflammation; neuroprotection.

Figure 1

Cell viability analysis of BHB. Cell viability evaluated by MTT test, initially with BV2 cells, with dose–response curves for concentrations from 5 mM to 100 mM (A). The 5 mM concentration of BHB was used in the next experimental phase to treat cells in the absence or in the presence of LPS (1 µg/mL) (B). Results are presented as means ± SDs of three independent experiments performed in triplicate of percentages compared to control values. * p < 0.05 compared to the same time points for the CTR. # p < 0.05 compared to the LPS time points.

Figure 2

Morphological assay after administration of BHB with or without LPS. Morphological assay of BV2 cells in different conditions: control (A), 1 µg/mL LPS (B), 5 mM BHB (C) and BHB with 1 µg/mL LPS (D). Bar: 75 µm (20× objective). The arrows indicate the cells that underwent morphological change. Cell areas expressed in µm² were quantified using ImageJ software (Panel E). Data are expressed as the means of three cell areas calculated from three independent experiments performed in triplicate ± SDs from three independent experiments. * p < 0.05 compared to CTR. # p < 0.05 compared to LPS.

Figure 3

BV2 cell wound-closure assay results following administration of BHB with or without LPS. The cuts on the BV2 cell monolayer were evaluated 24 h after treatment in the different conditions: BV2 cells at time 0 (A), BV2 control (B), LPS (C), 5 mM BHB (D) and BHB + LPS (1 µg/mL) (E). The images are representative of three independent replicates for each experiment. The results are expressed as means ± SDs of the percentages of wound closures compared to the 0-time condition (F), using ImageJ software. * p < 0.05 compared to CTR. # p < 0.05 compared to LPS.

Figure 4

Effects of BHB on cytokine expression. Analysis of cytokine expression, performed by ELISA assay, of IL-17 (A) and IL-10 (B) with stimulation of 5 mM of BHB in the presence or absence of LPS (1 µg/mL). Experimental data are expressed as the means (pg/mL) ± SDs of three independent experiments. * p < 0.05 compared to the CTR. # p < 0.05 with respect to the LPS condition.

Figure 5

The main mechanism of ketogenic diet action on microglia through BHB production.