SGLT2 inhibition modulates NLRP3 inflammasome activity via ketones and insulin in diabetes with cardiovascular disease

Abstract

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in humans with type 2 diabetes (T2D); however, the underlying mechanism remains unclear. Activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and subsequent interleukin (IL)-1β release induces atherosclerosis and heart failure. Here we show the effect of SGLT2 inhibitor empagliflozin on NLRP3 inflammasome activity. Patients with T2D and high cardiovascular risk receive SGLT2 inhibitor or sulfonylurea for 30 days, with NLRP3 inflammasome activation analyzed in macrophages. While the SGLT2 inhibitor's glucose-lowering capacity is similar to sulfonylurea, it shows a greater reduction in IL-1β secretion compared to sulfonylurea accompanied by increased serum β-hydroxybutyrate (BHB) and decreased serum insulin. Ex vivo experiments with macrophages verify the inhibitory effects of high BHB and low insulin levels on NLRP3 inflammasome activation. In conclusion, SGLT2 inhibitor attenuates NLRP3 inflammasome activation, which might help to explain its cardioprotective effects.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1. Participant flow diagram.

Numbers of participants who were initially screened, underwent random allocation, withdrew, and were included in the final analysis. SGLT2 sodium–glucose cotransporter 2.

Fig. 2. Effects of SGLT2 inhibitor and sulfonylurea on metabolic parameters.

a–i Changes in metabolic parameters from baseline to end of treatment (sulfonylurea group: n = 32, SGLT2 inhibitor group: n = 29). †Statistical significance for the time × group interaction evaluated by repeat-measures analysis of variance (ANOVA) (Non-normally distributed variables were log transformed for analysis and back transformed for presentation). Data are represented as mean ± SEM or median (interquartile range). Two-sided paired t test or Wilcoxon signed rank test; *P < 0.05, **P < 0.01, and ***P < 0.001 versus baseline. BHB β-hydroxybutyrate, FFA free fatty acid, HOMA-IR homeostatic model assessment of insulin resistance, QUICKI quantitative insulin sensitivity check index, SEM standard error of the mean, SGLT2 sodium–glucose cotransporter 2. Source data are provided as a Source Data file.

Fig. 3. Effects of SGLT2 inhibitor and sulfonylurea on secretion of IL-1β and TNF-α from macrophages.

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 (sulfonylurea group: n = 32, SGLT2 inhibitor group: n = 29). TNF-α secretion from macrophages exposed to ATP (c) or palmitate (d). Experiments were repeated twice or three times per sample; bar graphs are drawn using mean values of those results per sample, whereas the statistical significances are derived from raw data. Data are represented as mean ± SEM. Two-sided paired t test; *P < 0.05, **P < 0.01, and ***P < 0.001 versus baseline. mRNA levels encoding IL1B (e), TNFA (f), and NLRP3 (g) (n = 6 per group). Two-sided paired t test or Wilcoxon signed rank test. †Statistical significance for the time × group interaction evaluated by repeat-measures analysis of variance (ANOVA) (Non-normally distributed variables were log transformed for analysis and back transformed for presentation). h Representative protein levels of molecules regarding NLRP3 inflammasome activation with or without LPS and ATP stimulation. IL-1β interleukin-1β, NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells, NLRP3 NLR family, pyrin domain-containing 3, PA palmitate, SEM standard error of the mean, SGLT2 sodium–glucose cotransporter 2, TNF-α tumor necrosis factor-α. Source data are provided as a Source Data file.

Fig. 4. Effects of BHB, glucose, and insulin on NLRP3 inflammasome activation in human macrophages by ELISA assay.

IL-1β secretion when exposed to vehicle or 2 mM ATP with 0.1 μg/mL LPS priming and increased BHB (1, 10, 20, and 30 mM) or 100 nM MCC950, a small-molecule inhibitor of NLRP3 inflammasome (administered for the last 5 hours as a positive control) (a); increased 2-DG (b); increased glucose (11.5 mM vs. 25 mM) (c); 11.5 mM glucose with/without 10 nM insulin (d); increased BHB (10 mM vs. 20 mM) with/without 10 nM insulin (e). 5–64 biologically independent samples per treatment; experiments on each treatment were repeated up to three times per sample. Symbols are data points from independent experiments: n = 137, 90, 58, 64, 28, 24, 5 (a, left to right), 66, 44, 12, 12 (b, left to right), 39, 27, 28, 11 (c, left to right), 48, 33, 21 (d, left to right), 39, 26, 18, 14, 14, 8 (e, left to right). Data are represented as mean ± SEM. One-way analysis of variance (ANOVA) using Tukey’s test or a two-tailed Student’s t test with the Bonferroni method for adjusting P values; *P < 0.05, **P < 0.01, and ***P < 0.001. BHB β-hydroxybutyrate, IL-1β interleukin-1β, NLRP3 NLR family, pyrin domain-containing 3, SEM standard error of the mean, 2-DG 2-deoxyglucose. Source data are provided as a Source Data file.

Fig. 5. Scheme representing the proposed effects of SGLT2 inhibitor on NLRP3 inflammasome activation.

BHB β-hydroxybutyrate, FFA free fatty acid, IL-1β interleukin-1β, NLRP3 NLR family, pyrin domain-containing 3, SGLT2 sodium–glucose cotransporter 2.