Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes

Yongeun Kim,Wei Wang,Meshail Okla,Inhae Kang,Regis Moreau,Soonkyu Chung

doi:10.1194/jlr.m062828

Abstract

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1β secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice with γT3 attenuated immune cell infiltration into adipose tissue, decreased circulating IL-18 levels, preserved pancreatic β-cells, and improved insulin sensitivity. Mechanistically, γT3 regulated the NLRP3 inflammasome via a two-pronged mechanism: 1) the induction of A20/TNF-α interacting protein 3 leading to the inhibition of the TNF receptor-associated factor 6/nuclear factor κB pathway and 2) the activation of AMP-activated protein kinase/autophagy axis leading to the attenuation of caspase-1 cleavage. Collectively, we demonstrated, for the first time, that γT3 inhibits the NLRP3 inflammasome thereby delaying the progression of type 2 diabetes. This study also provides an insight into the novel therapeutic values of γT3 for treating NLRP3 inflammasome-associated chronic diseases.

Highlights

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals
We investigated the role of ␥T3 in autophagy in peritoneal macrophages of db/db mice. p-AMPactivated protein kinase (AMPK)␣ (Thr 172) as well as markers of autophagic activation, including Beclin-1, autophagy protein 5 (ATG5), and LC3II, were markedly increased by ␥T3, whereas p62 was decreased by ␥T3 (Fig. 4E)
The activation of the NOD-like receptor protein 3 (NLRP3) inflammasome is positively associated with the prevalence of type 2 diabetes

Summary

RESEARCH DESIGN AND METHODS

Chemicals and reagents ␥T3 (with 90% purity) was kindly provided by Carotech. ␥T3 was prepared as previously described [18]. Primary bone marrow cells were isolated from the femurs of 6- to 10-week-old C57BL/6 mice and stimulated to differentiate for 7–10 days in L-cell conditioned medium (CM) as we described previously [5]. The resulting differentiated bone marrow-derived macrophages (BMDMs) were pretreated with ␥T3 or vehicle (DMSO) for 24 h, primed with lipopolysaccharide (LPS) (100 ng/ml) for 1 h, and stimulated either with nigericin (Ng; 6.5 ␮M, a K+/H+ ionophore) for 1 h or palmitate (PA; 400 ␮M complexed with BSA) for 12 h. Pro-IL-1␤-Gaussia luciferase reporter assay The J774 macrophages stably expressing pro-IL-1␤-Gaussia luciferase (iGLuc) fusion construct (Fig. 1A) were a generous gift. ␥T3 suppressed NLRP3 inflammasome procaspase reporter activity in iJ774 macrophages. A: Structure of the iGLuc (NLRP3 inflammasome and caspase activity reporter constructs). The cleavage product of the pro-IL1␤-iGLuc fusion protein was detected in the media by Western blot analysis using anti-GLuc antibody (NEB Inc.)

Glucose and insulin tolerance tests

Isolation of stromal vascular cells and flow cytometric analysis

RESULTS

Findings

DISCUSSION