Statins Promote Interleukin-1β–Dependent Adipocyte Insulin Resistance Through Lower Prenylation, Not Cholesterol

Statins lower blood cholesterol and reduce the risk of adverse cardiovascular events, but these drugs can increase blood glucose and risk of diabetes (1). Statins inhibit HMG-CoA reductase, lowering cholesterol biosynthesis and promoting hepatic cholesterol uptake. Statins have cholesterol-independent actions that depend on HMG-CoA inhibition but are often termed pleiotropic effects. Statin-mediated lowering of mevalonate pathway intermediates can alter immunity, independent of cholesterol (2). Inhibition of mevalonate synthesis reduces the isoprenoid production required for protein prenylation, a posttranslational modification that occurs on hundreds of cellular proteins (3). Statin lowering of specific isoprenoids can limit farnesylation or geranylgeranylation (3).

Statin-mediated lowering of prenylation is generally associated with reduced inflammation, including lower levels of circulating interleukin-6 (IL-6) and tumor-necrosis factor-α (4). Statins paradoxically increase IL-1β and IL-18, and reduced geranylgeranylation is sufficient to increase IL-1β and IL-18 in monocytes (5). It is also known that statins increase caspase-1 activity in immune cells (6). Therefore, statins activate a caspase-1 inflammasome and IL-1β/IL-18 responses despite widespread anti-inflammatory actions of this drug class. We previously characterized how an inflammasome contributes to the balance of these immune effects and endocrine control of metabolism. We found that the nucleotide-binding oligomerization (NOD)-like receptor family, pyrin-domain containing 3 (NLRP3) contributes to statin-induced insulin resistance in adipose tissue (7). It was not known which metabolites in the mevalonate pathway promote statin-induced insulin resistance. Statin-mediated inhibition of the cholesterol biosynthesis pathway can influence immunity by altering intermediates such as 25-hydroxycholesterol (25-HC), which has been directly linked to NLRP3 inflammasome activation in macrophages (8). Statins can actually prevent inflammasome assembly and attenuate caspase-1–mediated IL-1β secretion by acutely depleting endoplasmic reticulum–resident cholesterol in macrophages (9). Statins can also attenuate NLRP3 activation and IL-1β release when lipopolysaccharide (LPS)-primed monocytes are activated with cholesterol crystals, but the same data show that statins alone increase active caspase-1 and IL-1β secretion (10). Here, we tested whether prenylation or cholesterol underpinned statin-induced adipose insulin resistance.

It is unknown which inflammasome effector propagates adipose insulin resistance as a result of statins. NLRP3/caspase-1 inflammasome regulation of IL-1β promotes insulin resistance in adipocytes and dysglycemia in rodents (11). Markers of NLRP3 inflammasome activation are higher in patients with type 2 diabetes, and IL-1 receptor antagonism can improve glycemia (12,13). IL-1β is a good candidate to test for statin-mediated insulin resistance, but caspase-1 targets beyond IL-1β or IL-18 can link NLRP3 inflammasome responses to metabolic defects in insulin-responsive tissues (14,15).

We show that statin-induced adipocyte insulin resistance occurs through IL-1β and the lowering of isoprenoids required for prenylation, independent of changes in cellular cholesterol or LDL-mediated pathways to insulin resistance. Statins cause cell-autonomous impairment in insulin-stimulated adipocyte lipogenesis rather than glucose uptake or inhibition of lipolysis.

The McMaster University animal ethics review board approved all procedures. Male C57BL/6J mice were from The Jackson Laboratory (#000664). IL-1β^−/− mice were from Yoichiro Iwakura (University of Tokyo, Tokyo, Japan) and bred in-house. IL-18^−/− mice were from A.A.A. Explants from mouse gonadal adipose depots were exposed to statins, zoledronate, cholesterol derivatives, geranylgeranyl pyrophosphate (GGPP) or z-YVAD (18 h), LPS (final 4 h), or insulin (final 10 min). Lysates were immunoblotted (16), and IL-1β was quantified by ELISA (7). Adipocytes were separated from the stromal vascular fraction (SVF) (17). 3T3-L1 adipocytes and bone marrow–derived macrophages (BMDMs) were immunoblotted or analyzed by quantitative PCR (18).

3T3-L1 adipocyte lipolysis was determined as previously described (18). Lipogenesis was measured in lipids from adipose explants using [¹⁴C]U-glucose (2 μCi/mL) and 3T3-L1 adipocytes (1 μCi/mL) ± insulin (0.3 nmol/L) for the final 2 h (explants) or 1 h (3T3-L1). Glucose uptake was measured using [³H]-2-deoxyglucose (16).

Each experimental replicate represents a single adipose explant or 3T3-L1 culture well. Statistical significance was determined by ANOVA with Tukey post hoc test. Unpaired t tests were used to compare two conditions.

We previously showed that NLRP3 was required for fluvastatin to impair insulin-stimulated Ser⁴⁷³ phosphorylation of Akt/phosphokinase B (PKB) in explanted mouse adipose tissue. Adipose tissue was primed with a dose of LPS (2 μg/mL, 4 h) that does not alter insulin signaling but allows investigation of inflammasome responses (7) (Fig. 1A). The bisphosphonate zoledronate (1 and 5 μmol/L), which inhibits the mevalonate pathway distal to HMG-CoA reductase, lowered insulin-stimulated Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed explanted adipose tissue (Fig. 1A). Multiple statins impaired insulin signaling in adipose tissue, indicating a drug class effect on insulin sensitivity. We found that 1 μmol/L atorvastatin or 1 μmol/L pravastatin (Fig. 1B and C) but 0.1 μmol/L cerivastatin decreased insulin-stimulated Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed adipose explants (Fig. 1D). Atorvastatin impaired insulin-stimulated lipogenesis in LPS-primed adipose explants (Fig. 1E). Compared with adipose explants from lean mice, explants from high-fat–fed obese mice had impaired insulin signaling where LPS, but not statin exposure, further decreased insulin action (Fig. 1F). These results show that inhibiting the mevalonate pathway at multiple steps or with multiple statins impaired insulin signaling wherein one functional outcome is lower insulin-stimulated adipose tissue lipogenesis as a result of statin exposure.

We next tested whether lowering cholesterol metabolites or isoprenoids underpinned statin-mediated insulin resistance. We found that supplementation with LDL cholesterol (0.01 and 1 mg/mL) or free cholesterol (1 and 20 μmol/L) did not cause a further reduction of (and did not restore) statin-mediated lowering of Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed adipose explants (Fig. 2A and B). LDL cholesterol treatment alone (1 mg/mL) lowered insulin signaling independently of LPS priming or statin treatment (Fig. 2A). Lower 25-HC can lead to activation of caspase-1 and increase IL-1β in macrophages (8). However, we found that supplementation of LPS-primed adipose explants with 25-HC (1 and 20 μmol/L) did not cause a further reduction of (and did not restore) impaired insulin signaling as a result of atorvastatin (Fig. 2C). In contrast to all experiments using cholesterol derivatives, supplementation of LPS-primed adipose explants with the isoprenoid GGPP at 50 μmol/L (but not 5 μmol/L) restored atorvastatin-induced suppression of insulin-stimulated phosphorylation of Akt/PKB at Ser⁴⁷³ and Thr³⁰⁸ (Fig. 2D and E). Importantly, GGPP restored insulin action as a result of statin exposure but not LDL cholesterol (1 mg/mL) (Fig. 2A). These data show that a statin-mediated reduction in a geranylgeranyl isoprenoid is required for impaired insulin signaling, which occurs independently of cholesterol or LDL-mediated effects on insulin action.

We next tested whether caspase-1 regulation of IL-1β or IL-18 was involved in impaired insulin action in adipose tissue (Fig. 3A). Inhibition of caspase-1 with 1 or 10 μmol/L z-YVAD restored atorvastatin-mediated lowering of Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed adipose explants stimulated with insulin (Fig. 3B). Atorvastatin, pravastatin, or cerivastatin did not lower Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed adipose explants derived from IL-1β^−/− mice (Fig. 3C and D). Similar to wild-type mice, atorvastatin lowered Ser⁴⁷³ phosphorylation of Akt/PKB in LPS-primed adipose explants derived from IL-18^−/− mice (Fig. 3E). Overall, these data show that statins require caspase-1 and IL-1β to impair insulin signaling in adipose tissue.

We next sought to determine the contributions of adipocytes versus the immune cell-enriched SVF. When adipose tissue explants were LPS primed and treated with atorvastatin, we found that both adipocytes and SVF had higher IL-1β (Fig. 4A). IL-1β was more abundant in the SVF compared with adipocytes (Fig. 4A). Previous research in BMDM reported that statin-mediated lowering of isoprenoids can also activate the pyrin inflammasome to promote IL-1β processing (19). The requirement for pyrin in macrophages contradicts our previous report wherein statins engaged the NLRP3 inflammasome in adipose tissue (7). We assessed whether cell type was the underlying factor for this discrepancy. We found that transcript levels of NLRP3 were increased with LPS treatment in the adipocyte and SVF, although no increase in transcripts of pyrin could be detected (Fig. 4B). We found that transcript levels of pyrin were detectable and augmented by LPS priming in BMDMs and adipose tissue (containing the SVF), but pyrin transcripts were not detectable in 3T3-L1 adipocytes (Fig. 4C). NLRP3 transcripts were present in BMDM, adipose tissue, and 3T3-L1 adipocytes, and NLRP3 transcript levels were augmented by LPS priming in all three cell/tissue types (Fig. 4C). This prompted us to determine whether a cell-autonomous response underpinned NLRP3 statin-induced adipocyte insulin resistance.

Cholesterol concentration in 3T3-L1 adipocytes did not change during treatment with atorvastatin (Fig. 4D). Atorvastatin lowered insulin-stimulated Ser⁴⁷³ phosphorylation of Akt/PKB in 3T3-L1 adipocytes, and supplementation with the isoprenoid geranylgeraniol (GGOH) (25 μmol/L) restored this aspect of insulin signaling (Fig. 4E). Supplementation with farnesol did not restore atorvastatin-mediated lowering of insulin-stimulated Ser⁴⁷³ phosphorylation of Akt/PKB in 3T3-L1 adipocytes (Fig. 4F). We next sought to determine the functional consequences of these adipocyte-autonomous statin-mediated responses. Atorvastatin did not alter lipolysis (with or without isoproterenol) in 3T3-L1 adipocytes (Fig. 4G). However, insulin-stimulated lipogenesis was decreased by atorvastatin, an effect that was not observed when 3T3-L1 adipocytes were supplemented with 25 μmol/L GGOH (Fig. 4H). Insulin-stimulated glucose uptake into 3T3-L1 adipocytes was not changed by atorvastatin or GGOH (Fig. 4I). Overall, these data show that atorvastatin lowers insulin-stimulated adipocyte-autonomous lipogenesis by lowering isoprenoids required for protein prenylation but not farnesylation.

Statins lower cholesterol, risk of cardiovascular disease, and all-cause mortality, but this drug class can increase blood glucose (1). The mechanisms linking statins and increased risk of diabetes should be clarified because warning labels now include an increased risk of blood glucose and diabetes (Health Canada, RA-16949). It is not known whether statin-induced changes in glycemia are related to cholesterol lowering. We sought to determine whether statins impaired insulin action by lowering cholesterol or another mevalonate pathway metabolite. We found that LDL cholesterol and 25-HC were dispensable for statin-mediated insulin resistance in adipose tissue. Cell type is a key determinant of how statins engage specific inflammasomes. Others have shown that statins can attenuate ligand-induced NLRP3 or pyrin inflammasome responses in macrophages or monocytes (9,10,19). We show that isoprenoids required for protein prenylation were sufficient to prevent statin-mediated defects in insulin signaling in adipocytes. This is important because coadministration of statin and isoprenoids may mitigate adipose tissue side effects but not interfere with lipid/cholesterol-lowering benefits of statins. Determination of the prenylated proteins that alter inflammasome activation and insulin resistance is warranted. Targeting specific prenylation events may be superior to widespread reversal of isoprenoid lowering that could mitigate longevity and cardiac benefits of statins shown in Drosophila (20).

We show that statins impaired insulin-stimulated lipogenesis in an adipocyte-autonomous manner, which was prevented by supplementing isoprenoids required for prenylation. This is important because impaired insulin signaling does not always correlate with impaired insulin action. Given that statins did not impair glucose uptake in adipocytes, our results are consistent with statins impairing selective and heterogeneous effects of insulin resistance that manifest in lipid metabolism (21).

We show that statin-induced insulin resistance is a drug class effect. Cerivastatin was removed from the market because of side effects. Our results show that cerivastatin was the most potent statin in promoting adipose insulin resistance (and the most potent activator of IL-1β release from BMDMs [data not shown]). IL-18 was dispensable for statin-induced adipose insulin resistance. IL-1β was the key mediator of statin-induced adipocyte insulin resistance, which ultimately impaired lipogenesis. IL-1β alone lowers insulin-stimulated lipogenesis in rodent and human adipocytes (22). IL-1 receptor antagonism can improve glycemia, but the design of clinical trials using IL-1β inhibition, such as the Canakinumab Anti-Inflammatory Thrombosis Outcome Study (CANTOS) trial, does not necessarily always reveal reduced diabetes incidence (13,23). Statins are pervasive in these clinical trials and should be considered as a confounding variable. It is not clear why patients with familial hypercholesteremia on lifelong statins can have a low prevalence of type 2 diabetes as opposed to patients who are obese or with metabolic disease in whom stains can increase the risk of type 2 diabetes (24). Our results suggest that impaired adipocyte lipogenesis underpins the relationship between lipids and glucose during statin treatment because higher blood triglycerides are a known risk factor predicting statin-induced diabetes (25). Our results appear relevant to statin intolerance and diminishing returns of increased statin dose on triglyceride/lipid lowering. Statin engagement of an NLRP3/caspase-1/IL-1β response may limit the effectiveness of statins to lower blood triglycerides independent of cholesterol lowering. Targeting NLRP3/caspase-1/IL-1β may allow enhanced blood lipid lowering at a given statin dose and/or mitigate side effects at a statin dose that achieves lipid-lowering goals.

Funding. This work was supported by grants to J.D.S. from the Canadian Institutes of Health Research (FDN-154295) and Hamilton Health Sciences New Investigator fund (NIF-16401). J.D.S. holds a Canada Research Chair in Metabolic Inflammation.

Duality of Interest. No 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. B.D.H. and J.D.S. derived the hypothesis and wrote the manuscript. A.K.T., J.X., B.M.D., J.F.C., and J.P. researched the data. M.R.S. and A.A.A. contributed to the discussion and provided mice. 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.