Targeting the NLRP3 inflammasome to Reduce Diet-induced Metabolic Abnormalities in Mice

Abstract

Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3^−/− littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3^−/− mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1 β and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-κB nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.

Introduction

Metabolically driven, chronic, low-grade inflammation has a crucial role in the pathogenesis of obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM) (1). Enhanced serum concentrations of proinflammatory cytokines play a key role in the development of metabolic derangements, and new antiinflammatory therapeutic approaches have recently been proposed for the treatment of these conditions (2,3). However, the identity of the specific inflammation-related signaling pathways that are responsible for these metabolic abnormalities are still unknown. Cytokines of the interleukin (IL)-1 family, particularly IL-1β, but also IL-18, are among the most critical proinflammatory cytokines that reduce insulin formation by pancreatic β-cells, thus promoting both pathogenesis and progression of diabetes (4). IL-1β and IL-18 are produced via cleavage of pro-IL-1β and pro-IL-18 by caspase-1, which in turn is activated by a multiprotein complex called the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Inflammasomes are newly identified multiprotein platforms responsible for the activation of innate inflammatory processes and instigation of inflammatory responses during a variety of chronic degenerative diseases (5). Among the inflammasomes, the most studied in the area of metabolic diseases is the NLRP3 inflammasome, which comprises (a) NLRP3, (b) an apoptosis-associated specklike protein containing a caspase activation recruitment domain (ASC) and (c) caspase-1. The NLRP3 inflammasome plays a substantial role in sensing obesity-associated inducers of caspase-1 activation and, therefore, regulates the magnitude of the inflammatory response and hence its downstream effects on insulin signaling in different organs, including liver and kidney (6). The fatty acid palmitate, cholesterol crystals, low-density lipoprotein and ceramide (generated from fatty acids), which are all increased in abundance during nutritional excess, can each activate NLRP3, resulting in increased IL-1β production (7–9). We and others have recently demonstrated that either a high-fat diet or a high-sugar diet trigger both NLRP3 inflammasome formation and activation in target organs of metabolic inflammation (10–12). In addition, mice genetically deficient of NLRP3 are protected against high-fat diet-induced insulin resistance (9,13). Although these data suggest that the NLRP3 inflammasome is a central player in the induction of insulin resistance, its potential role as a pharmacological target for therapeutic intervention in T2DM is ill defined, and no selective NLRP3 inhibitors have been tested in preclinical models of metabolic disease. No studies have compared the effects of pharmacological inhibition or gene silencing of the NLRP3 inflammasome in mice chronically fed a diet enriched in both sugars and saturated fats (high-fat, high-fructose diet [HD]), which are the two major components of the unhealthy diet that promotes obesity and insulin resistance. Hence, the present study was designed (a) to investigate the effects of NLRP3 inflammasome gene ablation on the metabolic alterations caused by chronic exposure to refined fat and fructose, the main ingredients of most processed foods, and (b) to determine the potential therapeutic value of the pharmacological modulation of NLRP3 inflammasome by the selective inhibitor BAY 11-7082.

Materials and Methods

Animals and Experimental Procedures

This study was carried out in 4-wk-old male NLRP3^−/− knockout (KO) mice on C57BL/6 background (14) and NLRP3^+/+ littermate control wild-type (WT) mice, housed in the same animal unit under conventional housing conditions at 25 ± 2°C. Both the WT and KO mice were randomly allocated into the following dietary regimens: normal diet (ND WT, n = 10, and ND KO, n = 4) and a high-fat, high-fructose diet (HD WT, n = 20, and HD KO, n = 6) for 12 wks. They were provided with diet and water ad libitum. HD WT mice were treated with vehicle (n = 10) or BAY 11-7082 (HD WT+BAY, 3 mg/kg IP 5 d/wk, n = 10) for the last 7 wks of dietary manipulation. The BAY 11-7082 dose used in this study was previously found to improve functional and biochemical deficits associated with experimental diabetic neuropathy (15). NLRP3 silencing was checked in all KO mice before starting dietary manipulation (Supplementary Figure S1). The HD diet contained 45% kcal fat (soybean oil and lard), 20% protein (casein) and 35% carbohydrate (fructose) (D03012907 diet, Research Diets). The in vivo procedures here described were approved by the local ethical committee (DGSAF 0021573-P-12/11/2013) and are in keeping with the European Directive 2010/63/EU as well as the 2011 Guide for the Care and Use of Laboratory Animals.

Figure 1 Effects of chronic exposure to a normal diet (ND) or a high-fat high-sugar diet (HD) on serum insulin (A), blood glucose (B) and oral glucose tolerance (C), evaluated in NLRP3−/− KO and WT mice treated or not with BAY 11-7082 (BAY, 3 mg/kg IP). Results are expressed as mean ± SEM of four to five animals per group.

Oral Glucose Tolerance Test

The oral glucose tolerance test (OGTT) was performed by administration of glucose (2 g/kg) by oral gavage after a fasting period of 6 h. The concentrations of fasting serum glucose were measured with a conventional glucometer (GlucoMen LX kit, Menarini Diagnostics).

Biochemical Analysis

After 12 wks of dietary manipulation, the mice were anesthetized by using isoflurane and killed by cardiac puncture and exsanguination. The plasma lipid profile was determined by measuring the content of triglycerides (TGs), total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) by standard enzymatic procedures using reagent kits (Hospitex Diagnostics). Plasma insulin was measured by using an enzyme-linked immunosorbent assay kit (ELISA) (R&D Systems). The albumin-to-creatinine ratio (ACR) was used to evaluate the urinary excretion of albumin. Urine samples were collected at 18 h in metabolic cages, urine creatinine concentrations were determined by using a creatinine kit (ArborAssays) and albumin concentrations were determined by using a mouse albumin ELISA quantification kit (Bethyl Labs).

Western Blot Analysis

Liver, kidney and gastrocnemius extracts were prepared as previously described (12). About 60 µg total proteins were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinyldenedifluoride membrane, which was then incubated with primary and secondary antibodies. To ascertain that blots were loaded with equal amounts of proteins, they were also incubated with antibody against β-actin or tubulin protein. The relative expression of the protein bands was quantified by densitometric scanning using Gel Pro®Analyzer 4.5 (Media Cybernetics) and standardized for densitometric analysis to β-actin levels.

Real-Time Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from liver and kidney samples using the All-Prep® DNA/RNA/Protein Kit (Qiagen) according to the manufacturer’s instructions. The total RNA concentration µg/mL) was determined by the fluorometer Qubit and the Quant-iT™ RNA Assay Kit (Invitrogen). A total of 500 ng total RNA was reversetranscribed by using the QuantiTect Reverse Transcription Kit (Qiagen), and the synthesized cDNA was used for real-time polymerase chain reaction (PCR). The cDNA was amplified by real-time PCR using SsoFast™ EvaGreen (Bio-Rad) and primers (Qiagen) specific for cytokines IL-1β (Mm_Il1b_2_SG, cat. no. QT01048355) and IL-18 (Mm_Il18_1_SG, cat. no. QT00171129). The PCR protocol conditions were as follows: HotStarTaq DNA polymerase activation step at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 55°C for 10 s. All samples were run in duplicate. At least two nontemplate controls were included in all PCR runs. The transcript of the reference gene ribosomal RNA 18S (Mm_Rn18s_3_SG, cat. no. QT02448075) was used to normalize mRNA data, and the quantification data analyses were performed by using the Bio-Rad CFX Manager Software, version 1.6 (Bio-Rad) according to the manufacturer’s instructions. These analyses were performed following the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) (16).

Histological Examination of the Kidney and Oil Red Staining of the Liver

Dewaxed 5-µm sections of kidney were stained with hematoxylin and eosin and examined as previously described (17). Neutral lipids were assessed on sections of frozen liver embedded in OCT (10 µm in thickness) by oil red O staining. The degree of fatty change was scored as following: mild when lipid droplets were observed in no more of 30% of the hepatocytes; moderate when it compromised between 31 and 60% of the parenchymal cells and severe when steatosis was observed in >60% of them.

Liver TG Levels

A sensitive assay kit was used to measure hepatic TGs following the provided protocol (Triglyceride Quantification Kit; Abnova Corporation).

Histochemistry Analysis of Collagen Deposition in Kidney and Liver Samples

Sirius red stain was performed in 4% formaldehyde-buffered solution fixed sections from kidney and liver to evaluate the degree of fibrosis. In the liver, portal tracts >100 µm in size were not considered, since they contain a large amount of collagen and therefore prevent evaluation of collagen-associated fibrosis.

Determination of Tumor Necrosis Factor (TNF)-α, IL-1 β, IL-6 and IL-18 in Plasma and Tissues

Commercially available ELISA kits (R&D Systems) were used to measure concentrations of TNF-α, IL-1β, IL-6 and IL-18 in either plasma and tissue homogenates, according to the manufacturer’s instructions.

Materials

All compounds were from the Sigma-Aldrich. Mouse anti-IRS-1, rabbit anti-phospho-IRS1 (Ser³⁰⁷), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser⁴⁷³), rabbit anti-glycogen synthase kinase (GSK)-3β, rabbit anti-phospho-GSK-3β (Ser⁹), rabbit anti-AS160, rabbit anti-phospho-AS160 (Thr⁶⁴²), rabbit anti-NF-κB p65, rabbit anti-Smad2/3, rabbit anti-phospho-Smad2 (Ser^465/467)/Smad3 (Ser^423/425) and rabbit anti-β-actin were from Cell Signaling Technology. Rabbit anti-glucose transporter (GLUT)-4, rabbit anti-transforming growth factor (TGF)-β and rabbit anti-NALP3 were from Abcam. Rabbit anti-caspase-1 p-10 was from Santa Cruz.

Statistical Analysis

All values were expressed as mean ± standard error of the mean (SEM) for “n” observations. The results were analyzed by one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. P <0.05 was considered significant.

All supplementary materials are available online at https://doi.org/www.molmed.org .

Results

Chronic BAY 11-7082 Administration and NLRP3 Inflammasome Knockdown Normalized Diet-Induced Impairment of Metabolic Parameters

Wild-type mice fed with the experimental high-fat, high-sugar diet for 12 wks (HD WT) had greater body weights than control diet-fed littermates (ND WT) because of an increased caloric intake (Table 1). The body weight gain was slightly, but not significantly, reduced by BAY 11-7082. When compared with NLRP3^−/− mice fed a normal diet (ND KO), NLRP3^−/− mice exposed to the HD (HD KO) showed an increased body weight gain. The levels of fasting blood glucose and fasting serum insulin in both the ND KO and HD KO group were similar to those recorded in the ND WT group and were significantly lower than that in the HD WT group (P < 0.01; Figures 1A, B). The concentrations of fasting serum glucose and insulin in WT mice treated with BAY 11-7082 (HD+BAY WT) were significantly (P < 0.05) reduced when compared with that in the HD WT group, although insulin levels remained significantly higher than that in the ND WT group. The OGTT revealed that glycemia in the HD WT group after glucose challenge was higher than that in the ND WT group (Figure 1C). In contrast, the dynamic changes in the levels of blood glucose in either the HD+BAY WT and HD KO groups of mice were similar to those in the related control groups (ND WT and ND KO, respectively).

	ND WT	HD WT	HD+BAY WT	ND KO	HD KO
n	10	10	10	4	6
Body weight gain (g)	8.0 ± 0.4	14.9 ± 0.7*	12.1 ± 0.9*	7.8 ± 1.2	15.9 ± 0.8#
Food intake (g/day)	2.48 ± 0.48	2.34 ± 0.06	2.19 ± 0.10	2.72 ± 0.12	2.41 ± 0.21
Caloric intake (kcal/day)	9.23 ± 0.24	11.32 ± 0.24*	10.39 ± 0.45	9.52 ± 0.13	11.48 ± 0.19#
Serum triglyceride (mmol/L)	0.39 ± 0.04	0.60 ± 0.06*	0.40 ± 0.03§	0.30 ± 0.04	0.37 ± 0.04
Serum total cholesterol (mmol/L)	2.08 ± 0.09	2.52 ± 0.07*	2.20 ± 0.08§	2.02 ± 0.06	2.20 ± 0.04
Serum LDL (mmol/L)	0.76 ± 0.08	1.21 ± 0.09*	0.87 ± 0.14	0.084 ± 0.04	0.96 ± 0.05
Serum HDL (mmol/L)	1.05 ± 0.05	1.03 ± 0.07	1.01 ± 0.07	1.03 ± 0.06	1.04 ± 0.07

1. Data are means ± SEM. *P < 0.05 versus ND WT; ^§P < 0.05 versus HD WT; ^#P < 0.05 versus ND KO.

Effects of BAY 11-7082 Administration and NLRP3 Inflammasome Deficiency on Insulin Signaling Pathway in the Liver and Skeletal Muscle

The levels of total expression of IRS-1, Akt and GSK-3β, a downstream target of Akt, were similar in all groups of mice (Figure 2) when measured in both liver and skeletal muscle, suggesting that BAY 11-7082 treatment and NLRP3^−/− downregulation did not alter the expression of the key components of the insulin-signaling pathway. In contrast, significant changes in the degree of protein phosphorylation were recorded in the liver and skeletal muscle of WT mice exposed to HD. These alterations in protein phosphorylation and, hence, activation status of the respective proteins indicate that HD led to an impairment in insulin signaling. In contrast, treatment of WT mice exposed to HD with BAY 11-7082 abolished all of the alterations in insulin signaling caused by HD in vehicle-treated animals. Moreover, the degree of phosphorylation of IRS-1, Akt and GSK-3β was similar between the ND KO and HD KO groups of mice. In the gastrocnemius of WT mice, the impairment of insulin sensitivity due to dietary manipulation correlated with a slight reduction in GLUT-4 expression, which was increased after BAY 11-7082 administration in WT mice and not affected by diet in KO mice (Figure 3). The Thr⁶⁴² phosphorylation of the Akt substrate 160 (AS160), which is needed for GLUT-4 translocation to the cell membrane, was reduced by HD in the gastrocnemius of WT mice, and it was restored to basal levels by BAY 11-7082, thus indicating an improvement in GLUT-4 translocation to the plasma membrane when the drug was administered to WT mice. In contrast, AS160 phosphorylation was not affected by dietary manipulation in NLRP3 KO mice.

Figure 2 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on insulin signal transduction in liver and skeletal muscle. Western blot analysis of total IRS-1 and Ser307 phosphorylation (A), total Akt and Ser473 phosphorylation (B) and total GSK-3β and Ser9 phosphorylation (C) were performed in the liver and gastrocnemius of NLRP3−/− KO and WT mice treated or not with BAY 11-7082 (BAY, 3 mg/kg IP). The relative expression of the protein bands was expressed as relative optical density (O.D.) and standardized to the corresponding β-actin contents. The data are means ± SEM of pooled data from three separate experiments (n = 4−6 mice per group). *P [less than] 0.05 versus ND WT.

Figure 3 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on GLUT-4 expression in the skeletal muscle. Western blot analysis of GLUT-4 (A) and AS160 total protein expression and Thr642 phosphorylation (B) were performed in the liver and gastrocnemius of NLRP3−/− KO and WT mice treated or not with BAY 11-7082 (BAY, 3 mg/kg IP). The relative expression of the protein bands was expressed as relative optical density (O.D.) and standardized to the corresponding β-actin contents. The data are means ± SEM of pooled data from three separate experiments (n = 4−6 mice per group). *P [less than] 0.05 versus ND WT.

Chronic BAY 11-7082 Administration or Ablation of NLRP3 Inflammasome Reduced Diet-Induced Dyslipidemia and Hepatic Lipid Accumulation

Concurrent with the development of insulin resistance, HD-fed WT animals displayed a two-fold increase in serum TG levels as well as a robust increase in total cholesterol and LDL concentrations in comparison with ND WT mice (Table 1). The diet-induced changes in serum lipid profile were suppressed by the pharmacological inhibition of NLRP3 inflammasome, whereas the same experimental diet did not evoke any significant change in the serum lipid profile of NLRP3^−/− mice. As shown by oil red O staining (Figure 4A), the HD diet resulted in a significant lipid deposition in the liver. Microscopically, the intensity of the steatosis was severe in almost all hepatocytes containing cytoplasmic micro- or macrovacuolar lipid droplets. Chronic administration of BAY 11-7082 attenuated neutral fat accumulation, and no difference in lipid accumulation was recorded between ND KO and HD KO groups of mice. These effects were confirmed by measurement of TG content (Figure 4B). Liver oil red O staining as well as assessment of hepatic TG levels showed no effects of the HD on lipid accumulation in the liver of KO mice. As shown in Figure 4C, the diet-induced lipid accumulation in the liver of WT mice was associated with significant local increase in TNF-α levels, which was counteracted by either pharmacological inhibition or genetic knockdown of the NLRP3 inflammasome.

Figure 4 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on liver lipid accumulation and inflammation. (A) Representative photomicrographs (40× magnification) of oil red O staining on liver sections from either NLRP3−/− KO or WT mice treated or not with BAY 11-7082 (BAY 3 mg/kg IP) and maintained on a normal diet (ND) or a high-fat high-sugar diet (HD). (B) Triglyceride (TG) content in mouse liver. (C) TNF-α concentrations in mouse liver. Data are mean ± SEM of four to six animals per group. *P [less than] 0.05 versus ND WT.

Chronic BAY 11-7082 Administration or Ablation of NLRP3 Inflammasome Limited Diet-Induced Renal Damage

Histological analysis of kidneys from the HD WT group revealed a marked degree of vacuolar degeneration at level of the S1-S2 segments of the proximal convoluted tubules. Others tubular structures of the nephrons were histologically preserved. Neither glomerular changes nor interstitial inflammation were detected (Figure 5). Kidneys of the BAY 11-7082-treated mice, as well as those from the NLRP3^−/− mice, appeared protected against the deleterious effects of the dietary manipulation. Although vacuolar degeneration was detectable, it was limited to <20% of the proximal convoluted tubules examined. The remaining parenchyma did not show any significant alterations. When compared with ND WT mice, WT mice subjected to HD demonstrated a significant increase in the albumin-to-creatinine ratio (ACR), which is an indicator of albuminuria (401.23 ± 33.16 versus 98.06 ± 13.16 µg/mg, P < 0.05; Figure 5F). Administration of BAY 11-7082 significantly attenuated the diet-induced rise in ACR (271.36 ± 5.63 versus 401.23 ± 33.16 µg/mg, P < 0.05). An impaired ACR was also recorded in the kidney of NLRP3^−/− mice fed the experimental diet in comparison to control NLRP3^−/− mice (263.95 ± 48.18 versus 114.03 ± 41.94 µg/mg, P < 0.05). However, the diet-induced increase in ACR in HD KO was significantly lower than that recorded in HD WT mice (P < 0.05).

Figure 5 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on kidney structure and function. (A–E) Histological appearance of kidneys from either NLRP3−/− KO or WT mice treated or not with BAY 11-7082 (BAY, 3 mg/kg IP) and maintained on a normal diet (ND) or a high-fat high-sugar diet (HD). (F) Urinary albumin-to-creatinine (ACR) levels in the absence or presence of diet manipulation and BAY 11-7082 treatment. Values are mean ± SEM of four to six animals per group. *P [less than] 0.05 versus ND WT; •P [less than] 0.05 versus HD WT; ◆P [less than] 0.05 versus HD KO.

Chronic BAY 11-7082 Administration or Ablation of NLRP3 Inflammasome Reduced Diet-Induced Overexpression of Profibrotic Markers in Liver and Kidney

As shown in Figure 6, Western blot analysis of the expression levels of TGF-β in the liver and kidney of WT mice demonstrated a significant increase in this profibrotic marker after dietary manipulation, which led to downstream phosphorylation of Smad2, suggestive of activation. However, these effects did not lead to a marked fibrotic process in the liver, at least when determined at 12 wks, since no difference among the groups was shown in the amount of collagen detected in the portal spaces and around the terminal hepatic venules; and the acinar structure of the liver was preserved. In contrast, kidneys of WT mice exposed to HD showed a moderate peritubular fibrosis, with Sirius red collagen accumulation observed mainly around the S1-S2 portion of the proximal convoluted tubule. Interestingly, chronic BAY 11-7082 administration as well as NLRP3 inflammasome deficiency blunted the diet-induced activation of the TGF-β/Smad2 profibrotic signaling cascade and the renal collagen accumulation.

Figure 6 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on hepatic and renal fibrotic response. Western blot analysis of TGF-β (A, D) and Smad2 (B, E) proteins in the liver and kidney of NLRP3−/− KO or WT mice exposed or not to diet manipulation in the absence or presence of BAY 11-7082 (BAY, 3 mg/kg IP). Representative Sirus red staining of liver and kidney sections from WT or KO mice fed a control diet, HD diet in the absence or presence of BAY 11-7082 (BAY, 3 mg/kg IP) are reported in (C) and (F), respectively. The autoradiograms reported here are representative of three independent experiments. The relative expression of the protein bands was expressed as relative optical density (O.D.) and standardized to the corresponding β-actin contents. The data are means ± SEM of pooled data from three separate experiments (n = 4−6 mice per group). *P [less than] 0.05 versus ND WT.

Diet-Induced NLRP3 Activation Was Suppressed by BAY 11-7082 Administration or NLRP3 Gene Silencing

HD caused a marked increase in expression of NLRP3 and activated caspase-1 in WT mice (Figure 7), which was paralleled by a robust increase in expression of IL-1β and IL-18 mRNA (Supplementary Figure S2) associated with upregulated local and systemic concentrations of mature IL-1β and IL-18 (Figure 8). Chronic treatment with BAY 11-7082 reduced the expression of the components of the NLRP3 inflammasome complex as well as the mRNA levels and active forms of the cytokines IL-1β and IL-18. In the kidney (but not in the liver), the levels of IL-1β and IL-18 in the HD+BAY group remained higher than those in the ND WT group. As expected, the NLRP3 inflammasome complex was not activated in KO mice and, hence, the HD diet did not trigger the production of the active forms of IL-1β and IL-18 in these mice. The significant reduction in systemic and local levels of NLRP3 inflammasome-dependent cytokines, but not IL-6 (Supplementary Figure S3), by BAY 11-7082 administration or NLRP3^−/− gene silencing confirm that the prevention/reduction of the HD-induced metabolic alterations were secondary to the lack of activation of the inflammasome.

Figure 7 Western blot analysis of NLRP3, procaspase-1 and activated caspase-1 proteins in the liver and kidney of NLRP3−/− KO or WT mice exposed or not to diet manipulation in the absence or presence of BAY 11-7082 (BAY, 3 mg/kg IP). The autoradiograms here reported are representative of three independent experiments.

Figure 8 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on local and systemic levels of IL-1 β and IL-18. IL-1 β and IL-18 concentrations were analyzed by ELISA in liver (A, B), kidney (C, D) and serum (E, F) of NLRP3−/− KO or WT mice maintained on a normal diet (ND) or a high-fat high-sugar diet (HD), in the absence or presence of BAY 11-7082 (BAY, 3 mg/kg IP). Data are means ± SEM of four to six animals per group. *P [less than] 0.05 versus ND WT.

BAY 11-7082 Administration, but Not NLRP3 Silencing, Inhibited Diet-Induced NF-κ B Activation

When compared with WT mice exposed to control diet, mice that underwent the experimental diet showed an increased phosphorylation of IκBα on Ser^32/36 (Figures 9A, C), which was associated with an increased translocation of the p65 NF-κB subunit from the cytosol to the nucleus (Figures 9B, D), in both liver and kidney. In contrast, chronic administration of BAY 11-7082 to HD WT mice diminished both IκBα phosphorylation and p65 nuclear translocation (Figure 9B). The experimental diet induced a robust induction of both IκBα phosphorylation and NF-κB activation in the livers and kidneys of NLRP3^−/− mice, similar to that recorded in the HD WT group, thus indicating that NLRP3 silencing does not affect NF-κB nuclear translocation.

Figure 9 Effects of diet manipulation and NLRP3 inflammasome inhibition or silencing on NF-κB signaling pathway. Signaling events were assessed in liver (A, B) and kidney (C, D) tissues from NLRP3−/− KO or WT mice maintained on a normal diet (ND) or a high-fat high-sugar diet (HD), in the absence or presence of BAY 11-7082 (BAY, 3 mg/kg IP). The relative expression of the protein bands was expressed as relative optical density (O.D.) and standardized to the corresponding β-actin contents. NF-κB p65 subunit translocation was evaluated expressing p65 subunit levels as a nucleus:cytosol ratio corrected for the β-actin contents and normalized using the ND WT band. The data are means ± SEM of pooled data from three separate experiments (n = 4−6 mice per group). *P [less than] 0.05 versus ND WT; •P [less than] 0.05 versus HD WT; ◆P [less than] 0.05 versus ND KO.

Discussion

In the present work, we demonstrated that NLRP3^−/− deficiency exerts protective effects against the metabolic alterations evoked by exposure to a high-fat diet combined with an overconsumption of the simple sugar fructose, for which use as a sweetener in food processing has dramatically increased over the last decade. Prior observations indicated that activation of the NLRP3 inflammasome plays a role in T2DM pathogenesis, possibly by driving inflammation, obesity and insulin resistance (7,9,10,18,19). Nevertheless, its role as specific pharmacological target for drug therapy of insulin resistance and related metabolic diseases has been poorly investigated. There are limited experimental data showing that pharmacological tools may ameliorate diabetic injury by regulating NLRP3 inflammasome activity (20–23). However, none of the proposed pharmacological strategies is based on the use of selective and specific NLRP3 inflammasome inhibitors. Thus, the described effects could be due to interferences up- or downstream of inflammasome activation. Only the evaluation of small molecules that are able to selectively inhibit the NLRP3 inflammasome may allow the future design of novel and effective therapeutics for diseases caused by excessive activation of the NLRP3 inflammasome. However, efficacious NLRP3 inflammasome inhibitors are still at an early stage of development. BAY 11-7082 is one of the few compounds that has been demonstrated to directly target the NLRP3 inflammasome and selectively inhibits the ATPase activity of NLRP3 required for its activation (24). Our study provides the first evidence that the chronic administration of BAY 11-7082 protects against the diet-induced metabolic alterations and that the qualitative and quantitative effects of BAY 11-7082 (with very minor exceptions discussed below) are similar to those recorded in NLRP3^−/− mice. The improved glucose tolerance here documented was, at least in part, due to an improvement in the signaling pathway of insulin in HD-fed mice. The IRS-1/Akt/GSK-3β cascade is a key regulator of glucose transportation, glycogen synthesis and glycolysis (25). Here we demonstrated that the defects in the insulin signaling observed in both the livers and skeletal muscles of HD-fed mice could be restored by pharmacological inhibition of NLRP3 activity. Accordingly, the dietary manipulation did not evoke any significant impairment in phosphorylation of IRS-1, Akt and GSK-3β, a substrate of Akt, in NLRP3^−/− mice, thus confirming that NLRP3 suppression potentiates Akt activity. NLRP3 suppression was associated with a significant improvement in expression and membrane translocation of GLUT-4, the most abundant glucose transporter isoform in skeletal muscle (26), thus facilitating glucose transport. As previous findings convincingly showed that Akt regulates translocation, targeting and fusion of GLUT-4-containing vesicles in mouse skeletal myocytes (27,28), we speculate that GLUT-4 translocation and subsequent glucose uptake in skeletal muscle after NLRP3 modulation is mainly due to activation of the IRS-1/Akt/GSK-3β pathway. Overall, our data demonstrate for the first time that activation of the NLRP3 inflammasome directly modulates the Akt pathway, thus affecting a crucial pathogenic mechanism responsible for the development of insulin resistance. Preservation of insulin sensitivity may also account for the improved lipid profile detected in both HD+BAY WT and HD KO groups. As previously documented (29), the hyperinsulinemic state due to chronic exposure to HD may induce greater lipid accumulation, thus enhancing lipotoxicity. Mice exposed to HD showed obvious lipid accumulation, and hepatic steatosis was associated with local activation of NLRP3 inflammasome. Interestingly, NLRP3 gene silencing as well as BAY 11-7082 treatment effectively prevented lipid accumulation as well as local and systemic inflammation by suppressing the release of TNF-α, IL-1β and IL-18. Similarly, inhibition of NLRP3 inflammasome activity within the mouse kidney attenuated both renal injury (histology) and dysfunction (albuminuria) caused by HD. Consistent with our results, a recent study showed that a high-fat diet increases NLRP3 inflammasome activity in glomeruli, resulting in glomerular inflammation and consequent glomerular injury (30). Moreover, we previously observed a similar activation of this inflammatory machinery in the kidney of mice exposed to an excessive intake of fructose (12). Inflammatory microenvironments have been previously demonstrated to promote TGF-β signaling, which in turn can stimulate Smad2/3 phosphorylation, thus increasing profibrogenic responses in the liver and kidney (31,32). In this study, collagen deposition in liver was not detectable and was minimal in kidneys of HD WT mice, despite significant sustained inflammation. However, we could detect a robust increase in TGF-β levels in both liver and kidney of WT mice after HD exposure, and this effect was associated with enhanced phosphorylation of Smad2, which has an essential role in the development and progression of obesity-related liver and kidney diseases. Recently, the TGF-β/Smad2 signaling has also been shown to be involved in regulating insulin gene transcription and energy homeostasis (33). Thus, on the basis of our data, it appears that HD diet induces a local inflammatory response, resulting in activation of the TGF-β/Smad2 signaling, which may potentially contribute to development of insulin resistance and to late liver and kidney fibrosis (not yet detectable at the time point here measured). Our observation is in concordance with previous reports indicating that local inflammation precedes fibrosis, when the latter is determined by measuring collagen production and accumulation (34,35). Our data showing reduced activation of the TGF-β/Smad3 signaling in KO mice exposed to the same diet manipulation raise the possibility of an involvement of the NLRP3 inflammasome pathway in modulation of the early markers of fibrosis. The present study did not aim to elucidate the exact mechanisms mediating the liver and kidney injury evoked by NALP3 inflammasome activation. However, we speculate that the diet-induced increased production of inflammatory cytokines, such as IL-1β and IL-18, resulting from activation of NALP3 inflammasomes, may act in an autocrine or paracrine fashion to change hepatic and renal cell function. Moreover, we cannot rule out a contribution of the “non-inflammatory effects” of NALP3 inflammasome activation such as pyroptosis, cytoskeleton changes and alteration of cell metabolism, which have also been reported to mediate the detrimental local action of inflammasome activation (36,37). Another unresolved question is whether the NLRP3 inflammasome activation occurs at the level of resident cells, since results so far obtained from preclinical models of obesity and insulin resistance are quite contrasting. There is good evidence that both hematopoietic and nonhematopoietic cells are involved in NLRP3 inflammasome activation in both renal and hepatic tissues (38–41). The above data provide evidence in support of NLRP3 inflammasome-dependent cellular and molecular events mediating the protective effects of BAY 11-7082 against diet-induced metabolic abnormalities. It has to be stressed, however, that BAY 11-7082 is not only an inhibitor of NLRP3 inflammasome activity, since this molecule may act also as an inhibitor of NF-κB activation and other inflammatory signaling pathways (42). For this reason, we also investigated the effects of BAY 11-7082 on NF-κB nuclear translocation, since BAY 11-7082 is known to block IκBα phosphorylation and the subsequent NF-κB activation, independently of its inhibitory effects on NLRP3 inflammasome formation and activation (24). Suppression of the NF-κB pathway by targeted KO mice or pharmacological inhibition of this pathway can reduce insulin resistance in mice exposed to dietary manipulation (43). Thus, both NF-κB nuclear translocation and activation of the NLRP3 inflammasome pathway coordinately contribute to insulin resistance in obesity. Here we demonstrated a diet-induced activation of the NF-κB pathway in both liver and kidney and we confirmed that the administration of BAY 11-7082 inhibited phosphorylation and subsequently the degradation of IκBs, which reduces nuclear translocation of NF-κB via its sequestration in an inactive state in the cytoplasm. Intriguingly, the inhibition of NF-κB activity was not seen in organs from NLRP3 KO mice. Thus, based on the considerable qualitative and quantitative similarities between the pharmacological effects elicited by BAY 11-7082 and the NLRP3 gene silencing in our experimental conditions, we speculate that NLRP3 inhibition alone is sufficient to evoke a marked improvement in insulin resistance and organ dysfunction, which is not further strengthened by the drug-induced NF-κB inhibition. However, we acknowledge that further studies with more selective NLRPL3 inflammasome inhibitors are required to demonstrate a phencopy of data obtained from the genetic absence of the NALP3-deficient mouse. In addition, we are aware that an important limitation of the present study is that WT and NLRP3 inflammasome KO mice were not littermates, thus involving potential differences at epigenetic and microbiome levels. Because recent findings clearly show that both NLRP3 inflammasome silencing and dietary manipulation evoke dysbiosis (44–46), it will be important to perform future studies aiming at directly determining possible microbiome-dependent differences in the role of NLRP3 inflammasome in modulating diet-induced metabolic abnormalities.

Conclusion

Overall, our results support the view that activation of the NLRP3 inflammasome drives the development of T2DM and the associated end-organ injury and, most notably, highlights the use of selective inhibitors of the NLRP3 inflammasome as novel and promising treatment options for T2DM. In fact, while NLRP3 inflammasome silencing demonstrates that the suppression of this pathway prevents the development of insulin resistance in response to HD feeding, the use of the selective inflammasome inhibitor BAY 11-7082, which was administered only for the last 7 wks of the 12-wk dietary manipulation, demonstrates that the deleterious effects of HD exposure may be reversed by the pharmacological inhibition of the NLRP3 inflammasome. Interestingly, selective inhibition of NLRP3 by a small molecule such as BAY 11-7082 might present certain advantages over the use of biological agents targeted at IL-1β and its receptor, including fewer immunosuppressive effects and better pharmacokinetics and cost-effectiveness. Further preclinical and clinical studies are needed to further explore this possibility and to investigate/ensure the safety of this innovative pharmacological approach.

Disclosure

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.

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