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

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.

from Dr. Hornung (hereafter referred to as iJ774 macrophages) ( 19 ). iJ774 macrophages were cultured in DMEM supplemented with L -glutamine, sodium pyruvate, and 10% (v/v) FBS (Gibco). To determine Gaussia luciferase (GLuc) activity, the BioLux GLuc assay kit (NEB Inc.) was used and read with a Synergy H1 multimode reader (BioTek). The cleavage product of the pro-IL-1 ␤ -iGLuc fusion protein was detected in the media by Western blot analysis using anti-GLuc antibody (NEB Inc.).

Glucose and insulin tolerance tests
A glucose tolerance test (GTT) was performed on fasted (overnight) db/db mice by intraperitoneal injection of 10% D -glucose solution [0.5 g/kg body weight (BW)]. Blood glucose levels (mg/dl) were measured at 0, 15, 30, 60, and 120 min after injection using a glucometer (Bayer, Contuor). Plasma insulin levels at basal and 30 min after glucose intraperitoneal injection were determined by ELISA (Crystal Chem). For insulin tolerance test (ITT), fasted (4 h) db/db mice were administered 1 U/kg BW of insulin (Novolin R); blood glucose levels were measured at 0, 15, 30, and 60 min after injection.

Quantitative real-time PCR
Gene expression analysis was performed as described previously ( 18 ). Relative gene expression was determined based on the 2 Ϫ ⌬ ⌬ CT method with normalization of the raw data to either 36b4 or Gapdh (see primer sequences in supplementary Table 1). For PCR microarray analysis, RT 2 profi ler PCR array for mouse infl ammasome (PAMM-097ZD) was used according to the manufacturer's instruction (Qiagen). For each treatment, equal amounts of total mRNA obtained from four mice were pooled. The results were analyzed using software provided by Qiagen. is regulated by two discrete signaling cues: the priming and assembly of the infl ammasome. The priming of NLRP3 infl ammasome consists of the transcription of infl ammasome components and requires NF B-mediated transcription of pro-Il-1 ␤ , pro-Il-18 , and Nlrp3 ( 13 ). Subsequently, the infl ammasome assembles in response to the engagement of pattern recognition receptors by pathogenassociated molecular pattern (PAMP) or damage-associated molecular pattern (DAMP) endogenous signals (sterile infl ammation); the latter include FFA, free cholesterol, uric acid, and ATP. One of the key mechanisms by which infl ammasome assembly can be repressed is autophagic degradation of these deleterious molecular factors (reviewed in Ref. 14 ).
Our previous work as well as others has shown that ␥ T3 can block NF B activation ( 5-7 ) and stimulate AMPactivated protein kinase (AMPK) and autophagy in a variety of cell types (15)(16)(17)(18). In the present study, we ask whether ␥ T3 represses the NLRP3 infl ammasome and associated infl ammatory processes in type 2 diabetes by modulating NF B, AMPK, and autophagy signaling pathways. Using murine macrophages and leptin receptor knockout ( db/db ) mice, we clearly show that ␥ T3 blocks NLRP3 infl ammasome activation and ameliorates type 2 diabetes.

Chemicals and reagents
␥ T3 (with 90% purity) was kindly provided by Carotech. ␥ T3 was prepared as previously described ( 18 ). Compound C was purchased from Calbiochem. All other chemicals and reagents were purchased from Sigma Chemical Co., unless otherwise stated.

Animals
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln. Male BKS.Cg-Dock 7 m+/+ Lepr db /J mice (referred to as db/db mice) were obtained from Jackson Laboratory. Animals were housed in a specifi c pathogen-free facility and given free access to food and water. Mice (6 weeks old) were fed a standard AIN93G diet (control) or the AIN93G containing 0.1% (w/w) ␥ T3 for 8 weeks. Mice were given fresh rations daily. Food and water consumption were measured every day for 3 days during the last week of feeding before euthanasia. Individual body weights were measured weekly.

Preparation of bone marrow-derived macrophages and stimulation for infl ammasome
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, then 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.

␥ T3 ameliorated the progression of type 2 diabetes in db/db mice
To determine the effectiveness of ␥ T3 supplementation in preventing the progression of type 2 diabetes, db/db mice were fed either an AIN93G diet devoid of ␥ T3 (control) or an AIN93 diet containing 0.1% ␥ T3 (1 g/kg diet) for 8 weeks. Feeding ␥ T3 to db/db mice was associated with the improvement of diabetic hyperphagia ( Fig. 3A ) and diabetic thirst (polydipsia) ( Fig. 3B ), and a slight but significant increase in body weight ( Fig. 3C ). Importantly, results show a signifi cant decrease in fasting glucose levels ( Fig.  3D ), but an increase of adiponectin levels ( Fig. 3E ). ␥ T3 also improved glucose disposal during the GTT ( Fig. 3F ). There was no signifi cant change in fasting insulin levels between the two groups. However, 30 min within the GTT, plasma insulin failed to rise in control mice, whereas in ␥ T3-fed mice plasma insulin levels were increased ‫ف‬ 2-fold ( Fig. 3G ). During the ITT, the insulin injection (1 U/kg BW) markedly improved glucose clearance to the same extent in both groups of mice ( Fig. 3H ). H&E staining of pancreatic islet cells revealed that the average islet size was signifi cantly larger in ␥ T3-fed mice than in controls ( Fig. 3I ), and the islet size distribution shifted toward larger sizes in ␥ T3-fed mice ( Fig. 3J ). Moreover, the percentage of insulin-positive area per islet was signifi cantly higher in ␥ T3fed mice than controls (5.9% vs. 2.4%; Fig. 3K ). The immunostaining of the islets also showed that islet integrity was better preserved in ␥ T3-fed mice with a low degree of immune cell infi ltration ( Fig. 3L ). Collectively, these data indicate that ␥ T3 attenuates the loss of pancreatic ␤ -cells and delays the progression of type 2 diabetes in db/db mice.

Isolation of stromal vascular cells and fl ow cytometric analysis
Epididymal fat pads were collected at the time of necropsy and used for stromal vascular (SV) cell isolation by following the protocol of Orr et al. ( 20 ). For cell surface staining, cells were stained for 30 min at room temperature in FCS-PBS buffer (1%) with the following antibodies: FITC-CD4, PE-CD8, and APC/Cy7 CD45 and relative isotypes (BioLegend). SV cells were gated using forward and side scatter characteristics for lymphocytes on the basis of at least 10,000 events using a FACSCalibur TM fl ow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Treestar).

Hematoxylin and eosin staining, islet size determination, and insulin staining
Paraffi n-embedded epididymal adipose tissues from mice were sectioned (10 m) for hematoxylin and eosin (H&E) staining. Paraffi n-embedded pancreases were serially sectioned (5 m) at 50 m intervals and used for H&E staining and insulin immunostaining. For determination of average islet size, ‫ف‬ 150-200 islets were counted per mouse pancreas from the six serial sections of H and E-stained paraffi n block (using Image J software). For insulin staining, pancreas sections were incubated with a primary insulin antibody (dilution 1:50, Dako), followed by incubation with EnVision-AP TM (Dako) and visualized using a SIGMA-FAST TM Fast Red system with hematoxylin counterstaining.

siRNA transfection
siRNAs (On-Target smart pool) targeting mouse A20 ( Tn-faip3 ) or nontargeting siRNA control were transfected into iJ774 macrophages at a fi nal concentration of 100 nM using Dharma-FECT4 transfection reagent (GE Dharmacon). Forty-eight hours after transfection, cells were washed and stimulated as indicated in the fi gure legends.

Statistics
The two-tailed Student's t -test was used for statistical analyses of two-group comparisons. Multigroup comparisons were performed by a one-way ANOVA followed by Tukey's multiple comparison test. All statistical analyses were performed using GraphPad Prism 6 (version 6.02).
We also investigated the role of ␥ T3 in autophagy in peritoneal macrophages of db/db mice. p-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 ). Because p62 is degraded in autolysosome, p62 levels are inversely correlated with autophagic fl ux. To confi rm that the autophagic fl ux was stimulated by ␥ T3, BMDMs were treated with chloroquine (CQ), a lysosomal ␥ T3 suppressed NLRP3 infl ammasome activation in peritoneal macrophages of db/db mice We asked whether the observed improvement of type 2 diabetes sequelae by ␥ T3 was due to the suppression of NLRP3 infl ammasome activation and associated infl ammatory processes. We found that plasma IL-18 was decreased by ‫ف‬ 2-fold in ␥ T3-fed versus control mice ( Fig. 4A ), whereas IL-1 ␤ levels were below detection limit in both groups. Next, we isolated the peritoneal macrophages from db/db mice fed the experimental diets for 8 weeks to carry out further analysis. Consistent with the results in Fig. 1 , the mRNA levels of NF B target genes, which included Il-6 , Tnf ␣ , Il-1 ␤ , Il-18 , and Nlrp3 , were signifi cantly downregulated in the macrophages of ␥ T3-fed mice ( Fig.  4B ). There was a ‫ف‬ 40-fold increase in A20 transcript levels, a negative feedback regulator of NF B ( Fig. 4B ). The infl ammasome 84-gene array revealed that no gene was A20 in WAT ( Fig. 5D ). Flow cytometry data revealed that the total leukocytes (CD45 + ) and cytotoxic T-cell populations (CD45 + /CD8 + ) were signifi cantly decreased in the WAT of ␥ T3-fed mice compared with control WAT ( Fig. 5E ).
To assess whether ␥ T3 could preserve insulin sensitivity of adipocytes from the deleterious effects of IL-1 ␤ , primary mouse adipocytes differentiated from mesenchymal stem cells were fi rst cultured with or without ␥ T3 (1 M). Then adipocyte cultures were exposed to the IL-1 ␤containing CM obtained from LPS/Ng-treated iJ774 ( Fig.  5H , left). The insulin-stimulated Akt phosphorylation was signifi cantly reduced following exposure to the CM ( Fig. 5H , degradation inhibitor. ␥ T3 (but not control) led to LC3II accumulation and p62 degradation in CQ-treated BMDMs ( Fig. 4F ) implying that ␥ T3 coregulates autophagosome formation and infl ammasome activation. Taken together, these data revealed that ␥ T3 decreased NLRP3 infl ammasome activation in macrophages of db/db mice.

␥ T3 decreased immune cell infi ltration into white adipose tissue and preserved insulin sensitivity
The impact of ␥ T3-mediated suppression of NLRP3 infl ammasome activation on adipose tissue infl ammation was further investigated in epididymal white adipose tissue (WAT) of db/db mice. H&E staining revealed that ␥ T3 supplementation signifi cantly reduced immune cell infi ltration into WAT ( Fig. 5A ). Flow cytometric analysis of the SV fractions showed that total macrophage infi ltration (F4/80 + ) was decreased by ‫ف‬ 30% in ␥ T3-fed versus control mice (12.3% vs. 8.3%; Fig. 5B ). In addition, Cd11c gene expression was substantially decreased in the WAT of ␥ T3-fed mice ( Fig. 5C ). In accordance with the anti-infl ammatory role of ␥ T3 in peritoneal macrophages ( Fig. 4C ), feeding ␥ T3 to db/db mice decreased the mRNA levels of Il-1 ␤ , Il-18 , and Nlrp3 but raised those of phosphorylates I B, which undergoes proteosomal degradation thereby allowing NF B to dimerize into its active form ( 21 ). To test these hypotheses, BMDMs were pretreated with or without ␥ T3 (1 M) and then stimulated with LPS, and mRNAs and proteins were collected over a 3 h time course. We found that Il-1 ␤ mRNA levels were inversely associated with those of A20 ( Fig. 6A ). The elevation of A20 protein abundance induced by ␥ T3 coincided with: 1 ) the decrease of p-I B kinase (IKK) ␣ / ␤ , p-ERK, and p-I B ␣ ; 2 ) the reappearance of I B ␣ ; and 3 ) the disappearance of TRAF6, suggesting a negative feedback of A20 on TRAF6/IKK/NF B signaling ( Fig. 6B ).
It has been shown that A20 can transfer TRAF6-bound Ub from residue K63 to K48 thereby causing the proteasomal degradation of TRAF6 ( 22 ). Thus, we performed immunoprecipitation to determine K63-versus K48-Ub status of TRAF6. As opposed to the study by Muroi and lane 5), unless the adipocytes had received ␥ T3 ( Fig. 5H ,  lane 6). Collectively, these data demonstrate that ␥ T3 reduced NF B and NLRP3 infl ammasome activation in WAT resulting in attenuation of adipose tissue infl ammation and preservation of insulin sensitivity.

␥ T3 blocked NF B-mediated priming of the infl ammasome
NF B activation is a critical step for the priming of the infl ammasomes. Based on multiple observations that ␥ T3 increased A20 expression ( Figs. 4, 5 ), we hypothesized that ␥ T3 inhibits NF B signaling pathway via activation of A20, an ubiquitination-editing enzyme. We further hypothesized that A20 activation hinders the ubiquitination of TRAF6. The formation of a polyubiquitin chain (K63-Ub) on TRAF6 is critical for TRAF6-mediated phosphorylation and activation of IKK. Following activation, IKK  Fig. 1 for quantifi cation). F: BMDMs were stimulated for infl ammasome formation by PA/LPS for 12 h. CQ (10 M, an autophagy inhibitor) was added 3 h before harvest. Protein levels of LC3I/II, p62, and ␤ -actin were determined by Western blot (n = 3). Data in A and B are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. production of IL-1 ␤ , and 3 ) ␥ T3 can inhibit IL-1 ␤ production independently of A20.

DISCUSSION
The activation of the NLRP3 infl ammasome is positively associated with the prevalence of type 2 diabetes. Consequently, targeting the inhibition of NLRP3 infl ammasome is an attractive strategy against type 2 diabetes. In this study, we addressed the question of whether ␥ T3, an unsaturated form of vitamin E, is effective in suppressing the NLRP3 infl ammasome and mitigating the pathophysiological consequences of infl ammation in a mouse model of type 2 diabetes. Our results showed that ␥ T3 inhibits the NLRP3 infl ammasome activity by two-pronged mechanism: 1 ) the blockage of infl ammasome priming and 2 ) the impediment of infl ammasome activation. To our knowledge, this is the fi rst study to characterize the role of ␥ T3 in inhibiting the NLRP3 infl ammasome and show that, by acting through the infl ammasome, ␥ T3 dietary supplementation can improve insulin resistance in mice.
Intense research is currently being devoted to the discovery of chemical inhibitors of the NLRP3 infl ammasome ( 23 ), blocking agents of the IL-1 ␤ signaling such as IL-1R (IL-1 receptor) antagonist ( 24 ), and neutralizing antibodies ( 25,26 ). Although, there is growing evidence that nutrition is a critical modulator of the infl ammasome, few attempts have been made to inhibit the NLRP3 infl ammasome through diet. For instance, elevated levels of circulating FFAs were shown to activate the NLRP3 infl ammasome and weaken insulin sensitivity ( 27,28 ). In contrast, -3 FA ( 29 ) and MUFAs ( 30 ) have been shown to curtail NLRP3 Tanamoto ( 22 ), inhibition of proteasomal degradation by MG132 failed to rescue the ␥ T3-mediated decrease of TRAF6 or to increase K48-Ub of TRAF6 ( Fig. 6C ). Similarly, ␥ T3 also lowered K63-Ub of TRAF6 compared with controls ( Fig. 6D ). Taken together, we posit that A20 induction by ␥ T3 inhibited the ubiquitination of both the K48 and K63 residues of TRAF6 and promoted TRAF6 nonproteasomal degradation, leading to early blockage of the NF B signaling pathway and repression of infl ammasome priming.

Inhibition of A20 and AMPK activation altered IL-1 ␤ secretion and caspase-1 cleavage by ␥ T3
To determine whether AMPK mediates ␥ T3's inhibition of the infl ammasome, we treated iJ774 macrophages with Compound C (10 M), a chemical inhibitor of AMPK. Results showed that Compound C partially reversed ␥ T3-dependent decreases in IL-1 ␤ secretion ( Fig. 7A ), caspase-1 cleavage ( Fig. 7B , top), and iGLuc activity ( Fig.  7B , bottom). To ascertain the contribution of A20 induction by ␥ T3 in the lowering of IL-1 ␤ secretion and caspase-1 activation, A20 was knocked down in iJ774 macrophages using siRNAs . As expected, the silencing of A20 effectively decreased ␥ T3-mediated A20 induction but did not reverse ␥ T3-mediated blockage of caspase-1 cleavage ( Fig. 7C ). Although total IL-1 ␤ levels were significantly increased in siA20-transfected cells versus siControl cells, the extent to which ␥ T3 attenuated IL-1 ␤ secretion was not signifi cantly different between siA20 and siControl cells ( ⌬ of columns 1 and 2 vs. ⌬ of columns 3 and 4; Fig. 7D ). Taken together, our results suggest that 1 ) AMPK mediates the blockage of caspase-1 cleavage afforded by ␥ T3, 2 ) A20 downregulates NF B-mediated the composition of NLRP3 infl ammasomes, the extent of immune cell infi ltration, and insulin sensitivity. We found that the lowering of NLRP3 infl ammasome in the WAT of ␥ T3-fed mice ( Fig. 5F ) was associated with the lowering of cytotoxic CD8 + T cells and macrophages in WAT ( Fig. 5A,  B ), and with the amelioration of insulin resistance ( Figs. 3,  5H ).
The destruction of pancreatic ␤ -cells and impairment of insulin production are hallmarks of late-stage type 2 diabetes ( 32,33 ). Because of a lack of specimens, we were unable to determine whether the protection of ␤ cells afforded by ␥ T3 ( Fig. 3I-L ) was due to the blockage of infl ammasome formation within the islets or the insensitivity of ␤ cells to the deleterious effects of IL-1 ␤ paracrine signaling. We are currently investigating whether ␥ T3 inhibits the infl ammasome of macrophages residing in islets or the infl ammasome of ␤ cells themselves, or both.
Mechanistically, we had anticipated that ␥ T3 would inhibit the priming of NLRP3 infl ammasome based on ␥ T3's intrinsic ability to suppress NF B ( 5 ). However, we did not anticipate that ␥ T3 would augment A20 induction, an ubiquitin-editing enzyme ( 34 ). Single nucleotide polymorphisms in the Tnfaip3/A20 are associated with increased susceptibility to chronic infl ammatory diseases ( 35,36 ). Deletion of A20 has been shown to cause hypersensitivity to TNF-␣ due to the constitutive activation of NF B, leading to premature death ( 37 ). In addition, an inverse correlation between diabetes incidence and A20 mRNA levels was found in patients with both type 1 and type 2 diabetes ( 38 ). In our study, multiple lines of evidence demonstrated that A20 is augmented by ␥ T3 to mitigate the priming of the NLRP3 infl ammasome ( Figs. 4, 6, and 7 ). Recently, Wang et al. ( 39 ) have reported that ␥ T3 causes A20 induction via regulation of sphingolipid metabolism. It is presently unknown whether alterations to sphingolipid metabolism by ␥ T3 affected NLRP3 infl ammasome activity in our study. Nonetheless, the work by Wang et al. corroborates our claim that ␥ T3 protects against abnormal stimulation of the innate immune system in part through the upregulation of A20 and feedback inhibition of priming of the NLRP3 infl ammasome. Indeed, A20 silencing with siRNA did not completely reverse ␥ T3's inhibition of IL-1 ␤ secretion ( Fig. 7C, D ), suggesting that A20 activation is important but not suffi cient, and full inhibition of NLRP3 infl ammasome necessitates the coordinated involvement of additional factors. One of these factors may be autophagy because Matsuzawa et al. ( 40 ) reported that A20 activation and autophagy cooperate in T-cell survival.
Autophagy regulates the fate of infl ammasomes by engulfi ng PAMP and DAMP signals ( 41,42 ). We clearly demonstrated that ␥ T3 activates AMPK and autophagy ( Figs. 2, 4, and 5 ). We further showed that the inhibition of AMPK with Compound C almost completely reversed ␥ T3's suppression of NLRP3 infl ammasome and caspase-1 cleavage, indicating that AMPK activation is critically important in the mechanism of ␥ T3 ( Fig. 7 ). Further research exploring the cross-talks between the A20 and AMPK/autophagy signaling pathways is needed to fully understand the infl ammasome activation. In this study, we report on the previously unrecognized function of ␥ T3 [at physiologically relevant concentration ( 1 )] as a potent negative regulator of the NLRP3 infl ammasome. Our data clearly show that ␥ T3 inhibited the NLRP3 infl ammasome in three macrophage models [i.e., iJ774 macrocytic cells ( Fig. 1 ), primary BMDMs ( Fig. 2 ), and peritoneal macrophages freshly isolated from db/db mice ( Fig. 4 )]. In addition, the suppression of NLPR3 infl ammasomes by ␥ T3 was evident upon stimulation of PAMP with Ng, and of DAMP with PA ( Fig. 2 ). We also found that ␥ T3 altered the NLR-subset infl ammasome, but not the AIM2-scaffold infl ammasome, the adaptor protein ASC, or the other caspases ( Fig. 4C ), suggesting that ␥ T3 has therapeutic potential against metabolic diseases where the NLRP3 infl ammasome is implicated, such as obesity, atherosclerosis, and type 2 diabetes ( 8 ).
In the obese, the chronic stimulation of Toll-like receptor 4 by FAs has been shown to activate the NLRP3 infl ammasome of macrophages residing in the adipose tissue and provoke the massive infl ux of immune cells into WAT ( 27,31 ). Here, we investigated the potential mitigating properties of dietary ␥ T3 on the infl ammatory and immunological consequences of obesity toward glycemia, insulinemia, and WAT remodeling. To that end, we determined Fig. 7. AMPK and A20 contributed to ␥ T3-mediated lowering of IL-1 ␤ secretion. iJ774 macrophages were treated with Compound C (10 M) to inhibit AMPK (A-C) or transfected with either A20targeting siRNA (siA20+) or nontargeting siRNA (siA20 Ϫ ) followed by LPS/PA stimulation for 12 h (C, D). A: Secreted IL-1 ␤ was measured by ELISA (n = 6). B: Relative GLuc activity of iGLuc (bottom) and caspase-1 cleavage (top). C: Western blot analysis of A20, cleaved caspase-1, and ␤ -actin (representative of triplicate samples). D: Secreted IL-1 ␤ was measured by ELISA (n = 6). The dotted line indicates the basal IL-1 ␤ level of siCont cells primed with LPS/PA. All data are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. ns, nonsignifi cant. precise mechanism by which ␥ T3 suppresses NLRP3 infl ammasome activation.
Most tocotrienol dietary supplements are provided as mixed formulations of ␣ -, ␥ -, and ␦ -tocotrienols, with the exception of tocotrienol supplmentation isolated from annatto seeds, which contain >90% of ␦ -tocotrienol. The bioavailability of ␥ T3 has been shown to vary in humans depending on the tocotrienol composition of the dietary source, the target population, and the study design (reviewed in Ref. 43 ). Tocotrienols circulate in blood bound to lipoproteins, in particular to HDL particles, suggesting that the plasma lipoprotein profi le affects tocotrienol metabolism. Due to the low binding affi nity of tocotrienols for the ␣ -tocopherol transfer protein, the transfer of tocotrienols to HDL particles must occur via alternative, yet to be identifi ed pathways ( 44,45 ). The tocotrienol-rich fraction supplementations are considered safe. They obtained the GRAS (Generally Recognized as Safe) designation from the Food and Drug Administration. Based on a recent review by Podszun et al. ( 46 ), there is no clear evidence that high doses of vitamin E (>300 mg/day), either as tocotrienols or tocopherols, cause adverse effects in either animals or humans. However, we note that a meta-analysis of human studies with vitamin E raised some concerns with regard to high-dose vitamin E supplementation, which may have adverse effects when consumed with certain drugs, such as aspirin ( 47 ). Despite some uncertainties regarding the metabolism of tocotrienols, multiple clinical studies support their benefi cial effects against metabolic diseases ( 48 ). By showing that ␥ T3 inhibits the NLRP3 infl ammasome and considering that the innate immune response plays a central role in metabolic disorders, our study may provide a possible explanation for some of the clinical positive outcomes of tocotrienol supplementation ( 8 ).
In summary, the critical role of the NLRP3 infl ammasome in the pathophysiology of insulin resistance was confi rmed in human studies ( 49,50 ). Hence, by revealing that ␥ T3 supplementation blunts NLRP3 infl ammasome activity and delays the progression of type 2 diabetes, our present work has clinical relevance. Although further investigations in human subjects are necessary to validate the therapeutic applicability of our work, we herein present seminal evidence that ␥ T3 is an anti-infl ammatory vitamin by inhibiting the NLRP3 infl ammasome in macrophages.