Short Term Isocaloric Ketogenic Diet Modulates NLRP3 Inflammasome Via B-hydroxybutyrate and Fibroblast Growth Factor 21

Abstract

A ketogenic diet (KD) is known to have beneficial health effects. Various types of KD interventions have been applied to manage metabolic syndrome based on modification of diet parameters such as duration of intervention, macronutrient components, and total calories. Nevertheless, the beneficial health impact of isocaloric KD is largely unknown, especially in healthy subjects. The present study investigated the acute effects of a 3-day isocaloric KD. In this non-randomized intervention study, we recruited 15 healthy volunteers aged 24-38 years (7 men and 8 women) and placed them on an isocaloric KD restricting intake of carbohydrates but not energy (75% fat, 20% protein, 5% carbohydrate) for 3 days. Biochemical profiles and laboratory measurements were performed. Peripheral blood monocular cells were cultured, and measured cell stimulated cytokines. After short-term isocaloric KD, subjects lost body weight and serum free fatty acid levels were increased. These results accompanied elevated serum β-hydroxybutyrate (BHB) concentration and fibroblast growth factor 21 (FGF21) levels and improved insulin sensitivity. Regarding the direct effect of BHB on inflammasome activation, interleukin-1β (IL-1β) and tumor necrosis factor-α secretion in response to adenosine triphosphate or palmitate stimulation in human macrophages decreased significantly after isocaloric KD. In ex-vivo experiments with macrophages, both FGF21 and BHB further reduced IL-1β secretion compared to either BHB or FGF21 alone. The inhibitory effect of FGF21 on IL-1β secretion was blunted with bafilomycin treatment, which blocked autophagy flux. In conclusion, isocaloric KD for 3 days is a promising approach to improve metabolic and inflammatory status.

Clinical trial registration: clinicaltrials.gov (NCT02964572).

Keywords: FGF21; IL-1β (interleukin 1β); Ketogenic diet (KD); NLRP3 inflammasome; isocaloric; β-hydroxybutyrate.

Figure 1

Schematic of the isocaloric ketogenic diet protocol. Healthy volunteers were recruited, and blood samples were collected on day 0 and day 3 of isocaloric ketogenic diet. PBMC, peripheral blood mononuclear cell.

Figure 2

Effects of isocaloric ketogenic diet on metabolic parameters. Fasting serum levels of BHB (A), insulin (B), glucose (C), QUICK (D), HOMA-IR (E), FFA (F), total cholesterol (G) from baseline to day 3 of isocaloric KD (H); Body weight changes from baseline to day 3 of isocaloric KD. Two-sided paired t-test or Wilcoxon signed rank test; *P <0.05, **P <0.01, ***P <0.001, and ****P <0.0001 vs. baseline. BHB, β-hydroxybutyrate; FFA, free fatty acid; KD, ketogenic diet; QUICKI, quantitative insulin sensitivity check index; HOMA-IR, homeostatic model assessment of insulin resistance; N.S., non-significant.

Figure 3

Effects of isocaloric ketogenic diet on secretion of IL-1β and TNF-α from macrophages. Secretion of IL-1β: ELISA assay measurement of IL-1β secretion from macrophages exposed to 2 mM ATP (A) or 0.2 mM palmitate (B) with 0.1 µg/ml LPS priming; changes in serum IL-1β levels (C); TNF-α secretion from macrophages exposed to ATP (D) or palmitate (E). Experiments with macrophage were repeated two or three times per sample; graphs are drawn using mean values of those results per sample, whereas statistical significance is derived from raw data. Two-sided paired t-test or Wilcoxon signed rank test; *P <0.05, **P <0.01, and ***P <0.001 vs. baseline. PA, palmitate; ATP, adenosine triphosphate; LPS, lipopolysaccharide; IL-1β, interleukin-1β; KD, ketogenic diet; TNF-α, tumor necrosis factor-α.

Figure 4

Effects of FGF21 as a mediator of ketogenic diet on NLRP3 inflammasome signaling activation in human macrophages. Changes in serum FGF21 levels from baseline to day 3 of isocaloric KD (A); ELISA assay measurement of IL-1β secretion from macrophages primed with 0.1 µg/ml LPS and stimulated with 2 mM ATP in the presence of various dose of FGF21 (15, 30, 100 nM) for 24h (B), BHB (1 mM), FGF21 (100 nM), or both (C), bafilomycin (10 nM), FGF21 (30 nM), or both (D). Data are represented as mean ± SEM. One-way analysis of variance (ANOVA) and two-sided paired t-test or Wilcoxon signed rank test; *P <0.05, **P <0.01, ***P <0.001, and ****P <0.0001. ATP, adenosine triphosphate; LPS, lipopolysaccharide; FGF21, fibroblast growth factor 21; N.S., non-significant; KD, ketogenic diet; IL-1β, interleukin-1β; BHB, β-hydroxybutyrate.

Figure 5

The summary of effects of the isocaloric ketogenic diet.