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 NLRP3Inflammasome-MediatedAdipose Insulin ResistanceDiabetes 2014;63:3742–3747 | DOI: 10.2337/db13-1398

Statins reduce lipid levels and are widely prescribed.Statins have been associated with an increased in-cidence of type 2 diabetes, but the mechanisms areunclear. Activation of the NOD-like receptor family, pyrindomain containing 3 (NLRP3)/caspase-1 inflammasome,promotes insulin resistance, a precursor of type 2 di-abetes. We showed that four different statins increasedinterleukin-1b (IL-1b) secretion frommacrophages, whichis characteristic of NLRP3 inflammasome activation. Thiseffect was dose dependent, absent in NLRP32/2 mice,and prevented by caspase-1 inhibition or the diabetesdrug glyburide. Long-term fluvastatin treatment ofobese mice impaired insulin-stimulated glucose uptakein adipose tissue. Fluvastatin-induced activation of theNLRP3/caspase-1 pathway was required for the devel-opment of insulin resistance in adipose tissue explants,an effect also prevented by glyburide. Fluvastatin im-paired insulin signaling in lipopolysaccharide-primed3T3-L1 adipocytes, an effect associated with increasedcaspase-1 activity, but not IL-1b secretion. Our resultsdefine an NLRP3/caspase-1–mediated mechanism ofstatin-induced insulin resistance in adipose tissue andadipocytes, which may be a contributing factor to statin-induced development of type 2 diabetes. These resultswarrant scrutiny of insulin sensitivity during statin useand suggest that combination therapies with glyburide,or other inhibitors of the NLRP3 inflammasome, may beeffective in preventing the adverse effects of statins.

Therapy with statins inhibits hydroxymethylglutaryl-CoAreductase, a rate-limiting enzyme in cholesterol biosynthe-sis, and reduce LDL cholesterol levels. Statins have actions

beyond lipid-lowering effects, which include modulation ofimmune function (1). Statins decrease intermediates in themevalonate pathway that lie upstream of cholesterol for-mation, reducing protein prenylation, a post-translationallipid modification that occurs on many proteins, includingthose involved in immune responses (1). Immune proteinssuch as pattern recognition receptors (PRRs) have emergedas integrators of nutrient- and pathogen-sensing systems,and the inflammation that occurs during obesity (i.e., meta-flammation) has been characterized in terms of excessnutrients and energy (2). Drug-mediated changes in inflam-mation via engagement of PRRs should also be considered,particularly for therapeutic agents such as statins that areused to treat aspects of metabolic disease.

Statin-mediated decreases in protein prenylation aregenerally associated with anti-inflammatory responsesand can reduce levels of tumor necrosis factor andinterleukin (IL)-6 in lipopolysaccharide (LPS)-treatedperipheral blood (3). In contrast, statins have been asso-ciated with increased secretion of the proinflammatorycytokine IL-1b; an effect that requires caspase-1 activityand priming with another immunogenic agent such asLPS (4). These features are indicative of regulation bythe inflammasome containing the PRR, NOD-like receptorfamily, pyrin domain containing 3 (NLRP3; also referredto as NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 or cryopyrin) (5,6). The NLRP3 inflam-masome is causally linked to the development of insulinresistance in rodents (7) and has recently been shown tobe activated in macrophages of patients with newly di-agnosed insulin-resistant type 2 diabetes (8). Statin ther-apy has been associated with increased incidence of type 2

1Department of Biochemistry and Biomedical Sciences, McMaster University,Hamilton, Ontario, Canada2Department of Medicine, McMaster University, Hamilton, Ontario, Canada3Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada

Corresponding author: Jonathan D. Schertzer, schertze@mcmaster.ca.

Received 9 September 2013 and accepted 31 May 2014.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 3569.

3742 Diabetes Volume 63, November 2014

METABOLISM

diabetes, as high as 48% in certain populations (9,10).Glyburide, a drug that is widely prescribed for the treat-ment of diabetes, inhibits the NLRP3 inflammasome in-dependently of cyclohexylurea-mediated insulin secretion(11). We hypothesized that statin-mediated activation ofthe NLRP3 inflammasome promotes insulin resistance,which could be attenuated by glyburide.

RESEARCH DESIGN AND METHODS

Mice and MaterialsThe McMaster University Animal Ethics Review Boardapproved 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. DanaPhilpott (University of Toronto, Toronto, ON, Canada).To determine the effect of long-term statin treatmenton insulin-stimulated tissue glucose uptake, ob/ob micewere orally administered 40–50 mg/kg fluvastatin or ve-hicle 5 days a week for 6 weeks, a dose of fluvastatin thathas been used in other mouse models (12). Twenty-fourhours after the last dose, mice were injected with 2 mCi of3H-2-deoxy-D-glucose (2DG) via tail vein, immediately fol-lowed by the administration of insulin (4 units/kg i.p.).Blood samples were taken at baseline, 5, 10, 15, and20 min, and were analyzed for 2DG radioactivity. Micewere killed by cervical dislocation, and tissues were snapfrozen in liquid nitrogen. Brown adipose tissue (BAT) andgonadal white adipose tissue (WAT) were analyzed for2DG radioactivity with and without deproteinization(0.3 mol/L BaOH and 0.3 mol/L ZnSO4) to calculate therates of tissue-specific glucose uptake. Statins were fromCayman 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 Sys-tems (Denver, CO). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit and all otherchemicals were from Sigma-Aldrich (St. Louis, MO).

MacrophagesBone marrow–derived macrophages (BMDMs) were cul-tured for 7–10 days in DMEM containing 10% FBS and15% L929 conditioned media. BMDMs were washed inserum-free media and exposed to statin (1 mmol/Lfluvastatin, unless otherwise stated) for 18 h in serum-free DMEM and LPS (200 ng/mL) was added during thefinal 4 h. GGPP (10 mmol/L), z-WHED-FMK (10 mmol/L),and glyburide (200 mmol/L) were used during the statintreatment period. IL-1b and IL-6 were quantified byELISA. Transcript levels were analyzed by quantitativePCR, as described previously (13,14).

Adipose Explants and AdipocytesMice were killed by cervical dislocation, and PBS-rinsedgonadal adipose tissue was minced into ;5-mg pieces inDMEM containing 10% FBS. After 2 h of equilibration,

25 mg explants were placed in serum-free DMEM andexposed to 10 mmol/L fluvastatin (18 h) and 2 mg/mLLPS (4 h), and were stimulated with 0.3 nmol/L insulinfor 10 min. Adipose tissue lysates were used for determi-nation of caspase-1/3 activity (over 4 h), immunoblotting,or ELISA determination of cytokines, as described previ-ously (14). 3T3-L1 preadipocytes (ATCC, Rockville, MD)were differentiated (14), and fluvastatin/LPS treatmentwas similar to explants. 3T3-L1 media were used for ELISAs,and lysates were used to measure caspase-1 enzymaticactivity fluorometrically or for immunoblotting after in-sulin stimulation at 0.3 or 100 nmol/L for 10 min.

Statistical AnalysisSignificance was determined by unpaired, two-tailed t testsor ANOVA, as appropriate. A Bonferroni or Tukey post hoctest was used when appropriate (Prism 4–6; GraphPadSoftware).

RESULTS

Statins Activate the NLRP3 InflammasomeAll statins (10 mmol/L, 18 h) increased the secretion ofIL-1b fromWT BMDMs compared with LPS alone (Fig. 1A).Fluvastatin increased IL-1b secretion in a dose-dependentmanner only with LPS priming (Fig. 1B), but LPS aloneincreased IL-6 secretion in BMDMs (Fig. 1C). Fluvastatinup to 100 mmol/L did not lower BMDM viability detectedusing the MTT assay (data not shown). The isoprenyl in-termediate GGPP prevented fluvastatin-induced IL-1b se-cretion in LPS-primed BMDMs (Fig. 1D), suggesting thatdecreased prenylation drives statin-mediated inflamma-some activation. Inhibition with z-WHED or glyburideprevented statin-induced IL-1b secretion in LPS-primedBMDMs (Fig. 1E). LPS treatment, but not fluvastatintreatment alone, increased transcript levels of NLRP3,IL-1b, and IL-6 (Fig. 1F and G). Therefore, statins alonedid not alter inflammasome-priming events such as in-creased NLRP3 transcript levels (15). The combinationof fluvastatin and LPS synergistically increased both IL-1b and IL-6 transcript levels (Fig. 1F). Fluvastatin did notincrease IL-1b secretion in LPS-primed BMDMs fromNLRP32/2 mice (Fig. 1H). LPS increased IL-6 secretionBMDMs from NLRP32/2 mice (Fig. 1I).

Fluvastatin Impairs Adipose Tissue Insulin SignalingVia the NLRP3 InflammasomeWe first established that long-term oral administration offluvastatin impaired insulin-simulated glucose disposalinto adipose tissue using an in vivo mouse model ofobesity. 2DG uptake was .50% lower in WAT, but notBAT, of fluvastatin-treated ob/ob mice (Fig. 2A). We thenused WAT explants to determine the mechanisms ofstatin-induced insulin resistance. Fluvastatin increasedcaspase-1 activity in LPS-primed adipose tissue from WTmice, but not from NLRP32/2 mice (Fig. 2B). Glyburideprevented this increased caspase-1 activity (Fig. 2B).Fluvastatin increased caspase-3 activity in LPS-primed adi-pose tissue fromWT and NLRP32/2mice and independently

diabetes.diabetesjournals.org Henriksbo and Associates 3743

of glyburide treatment (Fig. 2C). Therefore, fluvastatinactivated an NLRP3-dependent, glyburide-sensitive, caspase-1inflammasome in adipose tissue.

Surprisingly, LPS alone increased IL-1b in adiposeexplants from both WT and NLRP32/2 mice (Fig. 2D).Fluvastatin plus LPS further increased IL-1b levels com-pared with LPS in adipose explants from WT mice, butnot NLRP32/2 mice (Fig. 2D). LPS alone did not changethe ability of insulin to phosphorylate Akt at serine 473 inadipose tissue explants (Fig. 2E). Fluvastatin alone impairedinsulin-mediated phosphorylated Akt (pAkt) in adipose tis-sue from WT mice, but not NLRP32/2 mice (Fig. 2E). Thecombination of LPS and fluvastatin prevented the ability of

insulin to increase the levels of pAkt in adipose tissueexplants from WT mice, but not NLRP32/2 mice (Fig. 2E).Glyburide reversed fluvastatin-induced suppression ofinsulin-mediated pAkt in LPS-primed adipose explants, butglyburide did not increase pAkt levels on its own (Fig. 2F).

Interestingly, changes in caspase-1 activity, but not Il-1bsecretion mirrored statin-induced insulin action in adiposeexplants. There are many nonadipocyte cell types and po-tential sources of IL-1b processing in adipose tissue (16), sowe next tested the adipocyte cell-autonomous response.Treatment with fluvastatin plus LPS increased caspase-1activity in 3T3-L1 adipocytes, but did not increase IL-1bor IL-6 secretion (Fig. 3A–D). However, treatment with

Figure 1—Statins activate the NLRP3 inflammasome in macrophages. A: BMDMs from WT mice were treated with various statins (10mmol/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 fluvastatinand/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 combinedwith vehicle or 10 mmol/L GGPP and IL-1b in the media was quantified. E: BMDMs were treated with LPS, and 1 mmol/L fluvastatincombined with vehicle, or 10 mmol/L z-WHED-FMK, or 200 mmol/L glyburide and IL-1b in the media was quantified. Transcript levels ofcytokines (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 micewere 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. *Significantlydifferent from LPS alone or as indicated by connecting bars; fsignificantly different from conditions without LPS; #significantly differentfrom fluvastatin plus LPS; ^significantly different from control or statin alone. Statin, fluvastatin. Values are shown as the mean6 SEM. n > 3for all conditions.

3744 Statins Cause Inflammasome Insulin Resistance Diabetes Volume 63, November 2014

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

DISCUSSION

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

over reassessing the benefits and risks of statin use (18).Understanding how statins promote adverse effects such asthe progression to diabetes may promote improvements inthis drug class. We show that statins activate the NLRP3inflammasome in various immune and metabolic cells ofadipose tissue. Fluvastatin-induced impairments in insulinsignaling were dependent upon the NLRP3 inflammasome.The commonly used diabetes drug glyburide inhibited

Figure 2—Fluvastatin activates the NLRP3 inflammasome and impairs insulin signaling in adipose tissue. A: In vivo insulin-stimulated2DG uptake in BAT and WAT from ob/ob mice orally treated with vehicle (Control) or fluvastatin (Statin) for 6 weeks (n $ 3 mice pergroup). 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 andNLRP32/2 mice. F: Representative immunoblots (left) and quantification (right) of basal and insulin-mediated phosphorylation of Aktafter treatment with fluvastatin and LPS with various doses of glyburide in adipose tissue explants from WT mice. fSignificantlydifferent from control or basal; «significantly different from L+S; ^significantly different from LPS alone in WT; #significantly differentfrom vehicle control (with insulin). Statin, fluvastatin. Values are shown as the mean 6 SEM. n $ 8 explants per group. AU, arbitraryunits.

diabetes.diabetesjournals.org Henriksbo and Associates 3745

statin-induced inflammasome activation and preventeddefects in adipose tissue insulin action.

Endogenous and exogenous stimuli activate the NLRP3inflammasome, which prompted the theory that this PRRis a metabolic danger sensor (19). Production of bioactiveIL-1b (or IL-18) by the NLRP3 inflammasome requirespriming and stimuli-promoting assembly of a caspase-1protein complex. We confirm that statins increased IL-1bsecretion in adequately primed macrophages (5,6,20), andwe demonstrated the requirement of the NLRP3 inflam-masome. Glyburide, an existing diabetes drug, inhibitedstatin-induced increases in IL-1b in macrophages,which is consistent with its inhibitory effect on otherinflammasome activators (11). All statins tested acti-vated NLRP3-mediated increases in IL-1b, which is similarto the class effect of these hydroxymethylglutaryl-CoAreductase inhibitors increasing the risk of diabetes, inde-pendently of potency or lipophilic properties (18). A stan-dard dose of fluvastatin can equate to micromolar serumlevels in humans (21), and other statins can reach serumlevels .10 mmol/L (22), which corresponds with the ef-fective dose range of our in vitro models. The dose re-sponse of fluvastatin-induced IL-1b secretion that wereport is consistent with higher doses of statins increas-ing the risk of diabetes to a greater extent (18). This isimportant because of the diminishing returns of lipidlowering as the dose of statins is increased, the highdose of statins required to achieve adequate loweringin many patients, and the incidence of statin intolerancein clinical practice (23).

Adipose tissue is a key site of inflammation duringinsulin resistance, and the NLRP3/caspase-1 inflamma-some regulates adipose tissue inflammation and function(24). We first showed that 6 weeks of oral fluvastatintreatment in ob/ob mice impaired insulin-stimulated glu-cose uptake in WAT, a depot where insulin normallyincreases glucose disposal from the blood. Statin feedingin 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 impairedadipose tissue insulin action via the NLRP3 inflamma-some, which was also prevented with glyburide ex vivo.The combination of LPS and fluvastatin was most effec-tive in preventing insulin-mediated signals in adipose tis-sue explants. However, fluvastatin did not require LPS tocause impaired insulin action, suggesting that adipose tis-sue contains endogenous NLRP3 inflammasome-primingsignals. This is consistent with the NLRP3 inflammasomemediating obesity-associated insulin resistance in re-sponse to saturated lipids (7). Intriguingly, NLRP3 wasnot necessarily required for IL-1b secretion from adiposetissue explants. Since the regulation of IL-1b did not mir-ror changes in insulin action, our results suggest thatcaspase-1 rather than IL-1b provides the link to adiposetissue insulin resistance. Further, a cell-autonomous pro-gram that increases caspase-1 activity can be engaged byfluvastatin in LPS-primed clonal adipocytes. This responsein 3T3-L1 adipocytes did not increase IL-1b secretion,but impaired insulin action. Therefore, our results sug-gest that fluvastatin acts through the NLRP3/caspase-1

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

3746 Statins Cause Inflammasome Insulin Resistance Diabetes Volume 63, November 2014

inflammasome in multiple cells within adipose tissue andculminates in insulin resistance that does not necessarilyrequire Il-1b. Our results concerning insulin resistancehave focused on fluvastatin, but the type of statin andpleiotropic actions on inflammation (which are often con-flicting) in immune cells, liver, muscle, adipose tissue, andpancreas should be considered in obese and prediabeticmice and patients (25,26). Little is known about the con-tributing factors to the statin-diabetes relationship. Wepropose a role for inflammation and that inflammasome-mediated insulin resistance is positioned as a contributorto the development of diabetes. It is enticing to speculatethat metabolic endotoxemia or other priming agents forthe inflammasome may play a role (27).

We propose that the inhibition of NLRP3/caspase-1inflammasome may attenuate statin-induced insulin resis-tance. This is particularly relevant in mitigating any contri-bution of statins to insulin resistance leading to diabetes inobese hyperlipidemic patients who commonly use this classof drugs for lipid lowering, but are often at risk for thedevelopment of diabetes. The next generation of statins maybe driven by combination therapy or statin derivatives thatmaintain or enhance lipid-lowering properties, but allayadverse effects by evading the NLRP3 inflammasome.

Acknowledgments. The authors thank the Université de Lausanne (Lau-sanne, 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 CanadianInstitutes of Health Research (CIHR) (grants PNI123793 and MOP130432), theCanadian Diabetes Association (CDA), Natural Sciences and Engineering Re-search Council (grant 435474-2013), and a CIHR grant to G.R.S. J.F.C. wassupported by a Canada Graduate Scholarship (CIHR). J.D.C. was supported byMAC-Obesity research funds (McMaster University). M.D.F. was supported byBanting and CIHR postdoctoral fellowships. G.R.S. holds a Tier II Canada Re-search Chair. J.D.S. is supported by a CDA Scholar award.Duality of Interest. G.R.S. is on the scientific advisory board and receivedhonoraria from Esperion Therapeutics. No other potential conflicts of interestrelevant to this article were reported.Author Contributions. B.D.H. researched the data, contributed to thediscussion, 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 tothe discussion and edited the manuscript. J.D.S. researched the data, derived thehypothesis, 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 forthe integrity of the data and the accuracy of the data analysis.

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