Fluvastatin Causes NLRP3 Inflammasome-Mediated ... - Diabetes

2 downloads 16 Views 1MB Size Report
Statins reduce lipid levels and are widely prescribed. Statins have been associated with an increased in- cidence of type 2 diabetes, but the mechanisms are.
3742

Diabetes Volume 63, November 2014

Brandyn D. Henriksbo,1 Trevor C. Lau,1 Joseph F. Cavallari,1 Emmanuel Denou,1 Wendy Chi,1 James S. Lally,2 Justin D. Crane,2 Brittany M. Duggan,1 Kevin P. Foley,1 Morgan D. Fullerton,1,2 Mark A. Tarnopolsky,2,3 Gregory R. Steinberg,1,2 and Jonathan D. Schertzer1,3

Fluvastatin Causes NLRP3 Inflammasome-Mediated Adipose Insulin Resistance

METABOLISM

Diabetes 2014;63:3742–3747 | DOI: 10.2337/db13-1398

Statins reduce lipid levels and are widely prescribed. Statins have been associated with an increased incidence of type 2 diabetes, but the mechanisms are unclear. Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3)/caspase-1 inflammasome, promotes insulin resistance, a precursor of type 2 diabetes. We showed that four different statins increased interleukin-1b (IL-1b) secretion from macrophages, which is characteristic of NLRP3 inflammasome activation. This effect was dose dependent, absent in NLRP32/2 mice, and prevented by caspase-1 inhibition or the diabetes drug glyburide. Long-term fluvastatin treatment of obese mice impaired insulin-stimulated glucose uptake in adipose tissue. Fluvastatin-induced activation of the NLRP3/caspase-1 pathway was required for the development of insulin resistance in adipose tissue explants, an effect also prevented by glyburide. Fluvastatin impaired insulin signaling in lipopolysaccharide-primed 3T3-L1 adipocytes, an effect associated with increased caspase-1 activity, but not IL-1b secretion. Our results define an NLRP3/caspase-1–mediated mechanism of statin-induced insulin resistance in adipose tissue and adipocytes, which may be a contributing factor to statininduced development of type 2 diabetes. These results warrant scrutiny of insulin sensitivity during statin use and suggest that combination therapies with glyburide, or other inhibitors of the NLRP3 inflammasome, may be effective in preventing the adverse effects of statins.

Therapy with statins inhibits hydroxymethylglutaryl-CoA reductase, a rate-limiting enzyme in cholesterol biosynthesis, and reduce LDL cholesterol levels. Statins have actions

1Department

of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada 2Department of Medicine, McMaster University, Hamilton, Ontario, Canada 3Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada Corresponding author: Jonathan D. Schertzer, [email protected]. Received 9 September 2013 and accepted 31 May 2014.

beyond lipid-lowering effects, which include modulation of immune function (1). Statins decrease intermediates in the mevalonate pathway that lie upstream of cholesterol formation, reducing protein prenylation, a post-translational lipid modification that occurs on many proteins, including those involved in immune responses (1). Immune proteins such as pattern recognition receptors (PRRs) have emerged as integrators of nutrient- and pathogen-sensing systems, and the inflammation that occurs during obesity (i.e., metaflammation) has been characterized in terms of excess nutrients and energy (2). Drug-mediated changes in inflammation via engagement of PRRs should also be considered, particularly for therapeutic agents such as statins that are used to treat aspects of metabolic disease. Statin-mediated decreases in protein prenylation are generally associated with anti-inflammatory responses and can reduce levels of tumor necrosis factor and interleukin (IL)-6 in lipopolysaccharide (LPS)-treated peripheral blood (3). In contrast, statins have been associated with increased secretion of the proinflammatory cytokine IL-1b; an effect that requires caspase-1 activity and priming with another immunogenic agent such as LPS (4). These features are indicative of regulation by the inflammasome containing the PRR, NOD-like receptor family, pyrin domain containing 3 (NLRP3; also referred to as NACHT, leucine-rich repeat, and pyrin domainscontaining protein 3 or cryopyrin) (5,6). The NLRP3 inflammasome is causally linked to the development of insulin resistance in rodents (7) and has recently been shown to be activated in macrophages of patients with newly diagnosed insulin-resistant type 2 diabetes (8). Statin therapy has been associated with increased incidence of type 2

© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See accompanying article, p. 3569.

diabetes.diabetesjournals.org

diabetes, as high as 48% in certain populations (9,10). Glyburide, a drug that is widely prescribed for the treatment of diabetes, inhibits the NLRP3 inflammasome independently of cyclohexylurea-mediated insulin secretion (11). We hypothesized that statin-mediated activation of the NLRP3 inflammasome promotes insulin resistance, which could be attenuated by glyburide. RESEARCH DESIGN AND METHODS Mice and Materials

The McMaster University Animal Ethics Review Board approved all procedures. Male wild-type (WT) C57BL/6 (catalog #000664) and leptin-deficient ob/ob (catalog #000632) JAX mice were from The Jackson Laboratory. NLRP32/2 mice (.10 generations backcrossed to C57BL/6) were from Professor Nicolas Fasel (Université de Lausanne, Lausanne, Switzerland) and were provided by Dr. Dana Philpott (University of Toronto, Toronto, ON, Canada). To determine the effect of long-term statin treatment on insulin-stimulated tissue glucose uptake, ob/ob mice were orally administered 40–50 mg/kg fluvastatin or vehicle 5 days a week for 6 weeks, a dose of fluvastatin that has been used in other mouse models (12). Twenty-four hours after the last dose, mice were injected with 2 mCi of 3 H-2-deoxy-D-glucose (2DG) via tail vein, immediately followed by the administration of insulin (4 units/kg i.p.). Blood samples were taken at baseline, 5, 10, 15, and 20 min, and were analyzed for 2DG radioactivity. Mice were killed by cervical dislocation, and tissues were snap frozen in liquid nitrogen. Brown adipose tissue (BAT) and gonadal white adipose tissue (WAT) were analyzed for 2DG radioactivity with and without deproteinization (0.3 mol/L BaOH and 0.3 mol/L ZnSO4) to calculate the rates of tissue-specific glucose uptake. Statins were from Cayman Chemical (Ann Arbor, MI). InvivoGen (San Diego, CA) supplied ultra-pure LPS (Escherichia coli 0111:B4). z-WHED-FMK and caspase-1/3 kits were from R&D Systems (Denver, CO). The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) kit and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Macrophages

Bone marrow–derived macrophages (BMDMs) were cultured for 7–10 days in DMEM containing 10% FBS and 15% L929 conditioned media. BMDMs were washed in serum-free media and exposed to statin (1 mmol/L fluvastatin, unless otherwise stated) for 18 h in serumfree DMEM and LPS (200 ng/mL) was added during the final 4 h. GGPP (10 mmol/L), z-WHED-FMK (10 mmol/L), and glyburide (200 mmol/L) were used during the statin treatment period. IL-1b and IL-6 were quantified by ELISA. Transcript levels were analyzed by quantitative PCR, as described previously (13,14). Adipose Explants and Adipocytes

Mice were killed by cervical dislocation, and PBS-rinsed gonadal adipose tissue was minced into ;5-mg pieces in DMEM containing 10% FBS. After 2 h of equilibration,

Henriksbo and Associates

3743

25 mg explants were placed in serum-free DMEM and exposed to 10 mmol/L fluvastatin (18 h) and 2 mg/mL LPS (4 h), and were stimulated with 0.3 nmol/L insulin for 10 min. Adipose tissue lysates were used for determination of caspase-1/3 activity (over 4 h), immunoblotting, or ELISA determination of cytokines, as described previously (14). 3T3-L1 preadipocytes (ATCC, Rockville, MD) were differentiated (14), and fluvastatin/LPS treatment was similar to explants. 3T3-L1 media were used for ELISAs, and lysates were used to measure caspase-1 enzymatic activity fluorometrically or for immunoblotting after insulin stimulation at 0.3 or 100 nmol/L for 10 min. Statistical Analysis

Significance was determined by unpaired, two-tailed t tests or ANOVA, as appropriate. A Bonferroni or Tukey post hoc test was used when appropriate (Prism 4–6; GraphPad Software). RESULTS Statins Activate the NLRP3 Inflammasome

All statins (10 mmol/L, 18 h) increased the secretion of IL-1b from WT BMDMs compared with LPS alone (Fig. 1A). Fluvastatin increased IL-1b secretion in a dose-dependent manner only with LPS priming (Fig. 1B), but LPS alone increased IL-6 secretion in BMDMs (Fig. 1C). Fluvastatin up to 100 mmol/L did not lower BMDM viability detected using the MTT assay (data not shown). The isoprenyl intermediate GGPP prevented fluvastatin-induced IL-1b secretion in LPS-primed BMDMs (Fig. 1D), suggesting that decreased prenylation drives statin-mediated inflammasome activation. Inhibition with z-WHED or glyburide prevented statin-induced IL-1b secretion in LPS-primed BMDMs (Fig. 1E). LPS treatment, but not fluvastatin treatment alone, increased transcript levels of NLRP3, IL-1b, and IL-6 (Fig. 1F and G). Therefore, statins alone did not alter inflammasome-priming events such as increased NLRP3 transcript levels (15). The combination of fluvastatin and LPS synergistically increased both IL1b and IL-6 transcript levels (Fig. 1F). Fluvastatin did not increase IL-1b secretion in LPS-primed BMDMs from NLRP32/2 mice (Fig. 1H). LPS increased IL-6 secretion BMDMs from NLRP32/2 mice (Fig. 1I). Fluvastatin Impairs Adipose Tissue Insulin Signaling Via the NLRP3 Inflammasome

We first established that long-term oral administration of fluvastatin impaired insulin-simulated glucose disposal into adipose tissue using an in vivo mouse model of obesity. 2DG uptake was .50% lower in WAT, but not BAT, of fluvastatin-treated ob/ob mice (Fig. 2A). We then used WAT explants to determine the mechanisms of statin-induced insulin resistance. Fluvastatin increased caspase-1 activity in LPS-primed adipose tissue from WT mice, but not from NLRP32/2 mice (Fig. 2B). Glyburide prevented this increased caspase-1 activity (Fig. 2B). Fluvastatin increased caspase-3 activity in LPS-primed adipose tissue from WT and NLRP32/2 mice and independently

3744

Statins Cause Inflammasome Insulin Resistance

Diabetes Volume 63, November 2014

Figure 1—Statins activate the NLRP3 inflammasome in macrophages. A: BMDMs from WT mice were treated with various statins (10 mmol/L, 18 h) and/or LPS (200 ng/mL, 4 h), and IL-1b in the media was quantified. BMDMs were treated with various doses of fluvastatin and/or LPS, and IL-1b (B) and IL-6 (C) in the media were quantified. D: BMDMs were treated with LPS, and 1 mmol/L fluvastatin combined with vehicle or 10 mmol/L GGPP and IL-1b in the media was quantified. E: BMDMs were treated with LPS, and 1 mmol/L fluvastatin combined with vehicle, or 10 mmol/L z-WHED-FMK, or 200 mmol/L glyburide and IL-1b in the media was quantified. Transcript levels of cytokines (F) and PRRs (G) in BMDMs after 1 mmol/L fluvastatin (18 h) and 200 ng/mL LPS (4 h) treatment. BMDMs from NLRP32/2 mice were treated with various doses of fluvastatin and/or 200 ng/mL LPS, and IL-1b (H) and IL-6 (I) in the media were quantified. *Significantly different from LPS alone or as indicated by connecting bars; fsignificantly different from conditions without LPS; #significantly different from fluvastatin plus LPS; ^significantly different from control or statin alone. Statin, fluvastatin. Values are shown as the mean 6 SEM. n > 3 for all conditions.

of glyburide treatment (Fig. 2C). Therefore, fluvastatin activated an NLRP3-dependent, glyburide-sensitive, caspase-1 inflammasome in adipose tissue. Surprisingly, LPS alone increased IL-1b in adipose explants from both WT and NLRP32/2 mice (Fig. 2D). Fluvastatin plus LPS further increased IL-1b levels compared with LPS in adipose explants from WT mice, but not NLRP32/2 mice (Fig. 2D). LPS alone did not change the ability of insulin to phosphorylate Akt at serine 473 in adipose tissue explants (Fig. 2E). Fluvastatin alone impaired insulin-mediated phosphorylated Akt (pAkt) in adipose tissue from WT mice, but not NLRP32/2 mice (Fig. 2E). The combination of LPS and fluvastatin prevented the ability of

insulin to increase the levels of pAkt in adipose tissue explants from WT mice, but not NLRP32/2 mice (Fig. 2E). Glyburide reversed fluvastatin-induced suppression of insulin-mediated pAkt in LPS-primed adipose explants, but glyburide did not increase pAkt levels on its own (Fig. 2F). Interestingly, changes in caspase-1 activity, but not Il-1b secretion mirrored statin-induced insulin action in adipose explants. There are many nonadipocyte cell types and potential sources of IL-1b processing in adipose tissue (16), so we next tested the adipocyte cell-autonomous response. Treatment with fluvastatin plus LPS increased caspase-1 activity in 3T3-L1 adipocytes, but did not increase IL-1b or IL-6 secretion (Fig. 3A–D). However, treatment with

diabetes.diabetesjournals.org

Henriksbo and Associates

3745

Figure 2—Fluvastatin activates the NLRP3 inflammasome and impairs insulin signaling in adipose tissue. A: In vivo insulin-stimulated 2DG uptake in BAT and WAT from ob/ob mice orally treated with vehicle (Control) or fluvastatin (Statin) for 6 weeks (n $ 3 mice per group). Caspase-1 (B) and caspase-3 (C ) activity in adipose tissue explants from WT mice and NLRP32/2 mice, where explants (n > 7) were treated with vehicle (control), LPS (2 mg/mL) plus 10 mmol/L fluvastatin (L+S) or L+S plus 10 mmol/L glyburide (L+S+Glyb). D: Quantification of IL-1b in adipose tissue lysates from WT and NLRP32/2 mice after treatment of explants with vehicle (control), LPS, fluvastatin, or LPS plus fluvastatin. E: Representative immunoblots (left) and quantification (right) of basal (Bas; i.e., no insulin) and insulin-mediated pAkt (serine 473) after treatment with fluvastatin and/or LPS in adipose tissue explants (n > 6) from WT and NLRP32/2 mice. F: Representative immunoblots (left) and quantification (right) of basal and insulin-mediated phosphorylation of Akt after treatment with fluvastatin and LPS with various doses of glyburide in adipose tissue explants from WT mice. fSignificantly different from control or basal; «significantly different from L+S; ^significantly different from LPS alone in WT; #significantly different from vehicle control (with insulin). Statin, fluvastatin. Values are shown as the mean 6 SEM. n $ 8 explants per group. AU, arbitrary units.

fluvastatin plus LPS significantly lowered insulin-stimulated pAkt in 3T3-L1 adipocytes (Fig. 3E). DISCUSSION

Treatment with statins lowers blood lipid levels and reduces the number of cardiovascular disease–related events (17). Paradoxically, statins have been associated with an increased incidence of diabetes. This has sparked debate

over reassessing the benefits and risks of statin use (18). Understanding how statins promote adverse effects such as the progression to diabetes may promote improvements in this drug class. We show that statins activate the NLRP3 inflammasome in various immune and metabolic cells of adipose tissue. Fluvastatin-induced impairments in insulin signaling were dependent upon the NLRP3 inflammasome. The commonly used diabetes drug glyburide inhibited

3746

Statins Cause Inflammasome Insulin Resistance

Diabetes Volume 63, November 2014

Figure 3—Cell-autonomous actions of fluvastatin in adipocytes. Time course (A) and quantification (B) of relative caspase-1 activity in 3T3-L1 adipocytes after treatment with vehicle (Control) or LPS plus fluvastatin (L+S) (n $ 7/group). Quantification of IL-1b (C ) and IL-6 (D) secreted in the media after control or L+S (n $ 8/group). E: Representative immunoblots (left) and quantification (right) of 0.3 and 100 nmol/L insulin-stimulated phosphorylation of Akt (serine 473) in 3T3-L1 adipocytes after treatment with control or L+S (n = 4/group). *Significantly different from control (no insulin); #significantly different from control at the same dose of insulin. AU, arbitrary units; Con, control; nd, not detected; Statin, fluvastatin. Values are shown as the mean 6 SEM.

statin-induced inflammasome activation and prevented defects in adipose tissue insulin action. Endogenous and exogenous stimuli activate the NLRP3 inflammasome, which prompted the theory that this PRR is a metabolic danger sensor (19). Production of bioactive IL-1b (or IL-18) by the NLRP3 inflammasome requires priming and stimuli-promoting assembly of a caspase-1 protein complex. We confirm that statins increased IL-1b secretion in adequately primed macrophages (5,6,20), and we demonstrated the requirement of the NLRP3 inflammasome. Glyburide, an existing diabetes drug, inhibited statin-induced increases in IL-1b in macrophages, which is consistent with its inhibitory effect on other inflammasome activators (11). All statins tested activated NLRP3-mediated increases in IL-1b, which is similar to the class effect of these hydroxymethylglutaryl-CoA reductase inhibitors increasing the risk of diabetes, independently of potency or lipophilic properties (18). A standard dose of fluvastatin can equate to micromolar serum levels in humans (21), and other statins can reach serum levels .10 mmol/L (22), which corresponds with the effective dose range of our in vitro models. The dose response of fluvastatin-induced IL-1b secretion that we report is consistent with higher doses of statins increasing the risk of diabetes to a greater extent (18). This is important because of the diminishing returns of lipid lowering as the dose of statins is increased, the high dose of statins required to achieve adequate lowering in many patients, and the incidence of statin intolerance in clinical practice (23).

Adipose tissue is a key site of inflammation during insulin resistance, and the NLRP3/caspase-1 inflammasome regulates adipose tissue inflammation and function (24). We first showed that 6 weeks of oral fluvastatin treatment in ob/ob mice impaired insulin-stimulated glucose uptake in WAT, a depot where insulin normally increases glucose disposal from the blood. Statin feeding in these mice had no effect on glucose uptake in BAT, highlighting the specificity of this statin-mediated effect. We then provided genetic evidence that statins impaired adipose tissue insulin action via the NLRP3 inflammasome, which was also prevented with glyburide ex vivo. The combination of LPS and fluvastatin was most effective in preventing insulin-mediated signals in adipose tissue explants. However, fluvastatin did not require LPS to cause impaired insulin action, suggesting that adipose tissue contains endogenous NLRP3 inflammasome-priming signals. This is consistent with the NLRP3 inflammasome mediating obesity-associated insulin resistance in response to saturated lipids (7). Intriguingly, NLRP3 was not necessarily required for IL-1b secretion from adipose tissue explants. Since the regulation of IL-1b did not mirror changes in insulin action, our results suggest that caspase-1 rather than IL-1b provides the link to adipose tissue insulin resistance. Further, a cell-autonomous program that increases caspase-1 activity can be engaged by fluvastatin in LPS-primed clonal adipocytes. This response in 3T3-L1 adipocytes did not increase IL-1b secretion, but impaired insulin action. Therefore, our results suggest that fluvastatin acts through the NLRP3/caspase-1

diabetes.diabetesjournals.org

inflammasome in multiple cells within adipose tissue and culminates in insulin resistance that does not necessarily require Il-1b. Our results concerning insulin resistance have focused on fluvastatin, but the type of statin and pleiotropic actions on inflammation (which are often conflicting) in immune cells, liver, muscle, adipose tissue, and pancreas should be considered in obese and prediabetic mice and patients (25,26). Little is known about the contributing factors to the statin-diabetes relationship. We propose a role for inflammation and that inflammasomemediated insulin resistance is positioned as a contributor to the development of diabetes. It is enticing to speculate that metabolic endotoxemia or other priming agents for the inflammasome may play a role (27). We propose that the inhibition of NLRP3/caspase-1 inflammasome may attenuate statin-induced insulin resistance. This is particularly relevant in mitigating any contribution of statins to insulin resistance leading to diabetes in obese hyperlipidemic patients who commonly use this class of drugs for lipid lowering, but are often at risk for the development of diabetes. The next generation of statins may be driven by combination therapy or statin derivatives that maintain or enhance lipid-lowering properties, but allay adverse effects by evading the NLRP3 inflammasome. Acknowledgments. The authors thank the Université de Lausanne (Lausanne, Switzerland) and the Institute of Arthritis Research for the NLRP32/2 mice. Funding. This work was supported by grants to J.D.S. from the Canadian Institutes of Health Research (CIHR) (grants PNI123793 and MOP130432), the Canadian Diabetes Association (CDA), Natural Sciences and Engineering Research Council (grant 435474-2013), and a CIHR grant to G.R.S. J.F.C. was supported by a Canada Graduate Scholarship (CIHR). J.D.C. was supported by MAC-Obesity research funds (McMaster University). M.D.F. was supported by Banting and CIHR postdoctoral fellowships. G.R.S. holds a Tier II Canada Research Chair. J.D.S. is supported by a CDA Scholar award. Duality of Interest. G.R.S. is on the scientific advisory board and received honoraria from Esperion Therapeutics. No other potential conflicts of interest relevant to this article were reported. Author Contributions. B.D.H. researched the data, contributed to the discussion, and edited the manuscript. T.C.L., J.F.C., E.D., W.C., J.S.L., J.D.C., K.P.F., B.M.D., and M.D.F. researched the data. M.A.T. and G.R.S. contributed to the discussion and edited the manuscript. J.D.S. researched the data, derived the hypothesis, and wrote the manuscript. J.D.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

References 1. Greenwood J, Steinman L, Zamvil SS. Statin therapy and autoimmune disease: from protein prenylation to immunomodulation. Nat Rev Immunol 2006; 6:358–370 2. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol 2011;29:415–445 3. Rosenson RS, Tangney CC, Casey LC. Inhibition of proinflammatory cytokine production by pravastatin. Lancet 1999;353:983–984 4. Mandey SHL, Kuijk LM, Frenkel J, Waterham HR. A role for geranylgeranylation in interleukin-1b secretion. Arthritis Rheum 2006;54: 3690–3695

Henriksbo and Associates

3747

5. Liao Y-H, Lin Y-C, Tsao S-T, et al. HMG-CoA reductase inhibitors activate caspase-1 in human monocytes depending on ATP release and P2X7 activation. J Leukoc Biol 2013;93:289–299 6. Xu J-F, Washko GR, Nakahira K, et al.; COPDGene Investigators. Statins and pulmonary fibrosis: the potential role of NLRP3 inflammasome activation. Am J Respir Crit Care Med 2012;185:547–556 7. Wen H, Gris D, Lei Y, et al. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat Immunol 2011;12:408–415 8. Lee H-M, Kim J-J, Kim HJ, Shong M, Ku BJ, Jo E-K. Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes. Diabetes 2013;62: 194–204 9. Culver AL, Ockene IS, Balasubramanian R, et al. Statin use and risk of diabetes mellitus in postmenopausal women in the Women’s Health Initiative. Arch Intern Med 2012;172:144–152 10. Ridker PM, Pradhan A, MacFadyen JG, Libby P, Glynn RJ. Cardiovascular benefits and diabetes risks of statin therapy in primary prevention: an analysis from the JUPITER trial. Lancet 2012;380:565–571 11. Lamkanfi M, Mueller JL, Vitari AC, et al. Glyburide inhibits the Cryopyrin/ Nalp3 inflammasome. J Cell Biol 2009;187:61–70 12. Moriyama T, Kawada N, Nagatoya K, et al. Fluvastatin suppresses oxidative stress and fibrosis in the interstitium of mouse kidneys with unilateral ureteral obstruction. Kidney Int 2001;59:2095–2103 13. Jorgensen SB, O’Neill HM, Sylow L, et al. Deletion of skeletal muscle SOCS3 prevents insulin resistance in obesity. Diabetes 2013;62:56–64 14. Schertzer JD, Tamrakar AK, Magalhães JG, et al. NOD1 activators link innate immunity to insulin resistance. Diabetes 2011;60:2206–2215 15. Bauernfeind FG, Horvath G, Stutz A, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 2009;183:787–791 16. Stehlik C. Multiple interleukin-1b-converting enzymes contribute to inflammatory arthritis. Arthritis Rheum 2009;60:3524–3530 17. LaRosa JC, Grundy SM, Waters DD, et al.; Treating to New Targets (TNT) Investigators. Intensive lipid lowering with atorvastatin in patients with stable coronary disease. N Engl J Med 2005;352:1425–1435 18. Goldfine AB. Statins: is it really time to reassess benefits and risks? N Engl J Med 2012;366:1752–1755 19. Schroder K, Zhou R, Tschopp J. The NLRP3 inflammasome: a sensor for metabolic danger? Science 2010;327:296–300 20. Montero MT, Hernández O, Suárez Y, et al. Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells. Atherosclerosis 2000;153:303–313 21. Tse FLS, Jaffe JM, Troendle A. Pharmacokinetics of fluvastatin after single and multiple doses in normal volunteers. J Clin Pharmacol 1992;32:630–638 22. Holstein SA, Knapp HR, Clamon GH, Murry DJ, Hohl RJ. Pharmacodynamic effects of high dose lovastatin in subjects with advanced malignancies. Cancer Chemother Pharmacol 2006;57:155–164 23. Matteucci E, Giampietro O. Statin intolerance: why and what to do - with a focus on diabetic people. Curr Med Chem 2013;20:1397–1408 24. Stienstra R, Joosten LAB, Koenen T, et al. The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity. Cell Metab 2010;12:593–605 25. Kesh SB, Sikder K, Manna K, et al. Promising role of ferulic acid, atorvastatin and their combination in ameliorating high fat diet-induced stress in mice. Life Sci 2013;92:938–949 26. Satoh M, Tabuchi T, Itoh T, Nakamura M. NLRP3 inflammasome activation in coronary artery disease: results from prospective and randomized study of treatment with atorvastatin or rosuvastatin. Clin Sci (Lond) 2014;126: 233–241 27. Cani PD, Bibiloni R, Knauf C, et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 2008;57:1470–1481