Elimination of the NLRP3-ASC inflammasome protects against chronic obesity-induced pancreatic damage

Abstract

Clinical evidence that the blockade of IL-1β in type-2 diabetic patients improves glycemia is indicative of an autoinflammatory mechanism that may trigger adiposity-driven pancreatic damage. IL-1β is a key contributor to the obesity-induced inflammation and subsequent insulin resistance, pancreatic β-cell dysfunction, and the onset of type 2 diabetes. Our previous studies demonstrated that the ceramides activate the Nod-like receptor family, pyrin domain containing 3 (Nlrp3) inflammasome to cause the generation of mature IL-1β and ablation of the Nlrp3 inflammasome in diet-induced obesity improves insulin signaling. However, it remains unclear whether the posttranslational processing of active IL-1β in pancreas is regulated by the NLRP3 inflammasome or whether the alternate mechanisms play a dominant role in chronic obesity-induced pancreatic β-cell exhaustion. Here we show that loss of ASC, a critical adaptor required for the assembly of the NLRP3 and absent in melanoma 2 inflammasome substantially improves the insulin action. Surprisingly, despite lower insulin resistance in the chronically obese NLRP3 and ASC knockout mice, the insulin levels were substantially higher when the inflammasome pathway was eliminated. The obesity-induced increase in maturation of pancreatic IL-1β and pancreatic islet fibrosis was dependent on the NLRP3 inflammasome activation. Furthermore, elimination of NLRP3 inflammasome protected the pancreatic β-cells from cell death caused by long-term high-fat feeding during obesity with significant increase in the size of the islets of Langerhans. Collectively, this study provides direct in vivo evidence that activation of the NLRP3 inflammasome in diet-induced obesity is a critical trigger in causing pancreatic damage and is an important mechanism of progression toward type 2 diabetes.

Fig. 1.

Ablation of ASC lowers caspase-1 activation and improves insulin action in DIO. A, Western blot analysis of caspase-1 in epididymal fat (eFat) of C57/B6 mice. The activated p20 isoform of caspase-1 increases during obesity and ablation of ASC, a critical adaptor for NLRP3 inflammasome prevents autocatalytic activation of caspase-1. B, The percent change in glucose levels after the ITT in WT and ASC-deficient mice fed 60% HFD for 6 wk. The data are presented as mean (sem) with n = 6. The drop in glucose levels after insulin injection was significant at each time point (*, P < 0.01). C, The GTT in WT and ASC-deficient mice fed 60% HFD for 6 wk. The ASC mice displayed significant reduction in glucose values after the ip glucose injection (n = 6).

Fig. 2.

Ablation of Nlrp3 inflammasome improves pancreatic insulin production in chronic obesity. A, The kinetics of fasting serum insulin levels in male C57/B6 mice fed the normal chow diet and 60% HFD. The data are presented as mean (sem) with n = 5–8 per age group. B, The kinetics of fasting serum insulin levels in WT, NLRP3^−/−, and ASC^−/− mice fed normal chow diet and 60% HFD up to 12 months. The data are presented as mean (sem) with n = 5–8 (*, P < 0.01). C and D, Fasting glucose levels (C) and kinetics (D) of fasting serum leptin levels in in WT, NLRP3^−/−, and ASC^−/− mice fed normal chow diet and 60% HFD up to 12 months. The data are presented as mean (sem) with n = 5–8.

Fig. 3.

Elimination of Nlrp3 inflammasome protects pancreas from obesity-induced IL-1β damage and fibrosis. A, Immunoblotting for active (p17) IL-1β in pancreas tissue lysates of WT and Nlrp3^−/− mice maintained on chow and HFD for 9 months. B, Fasting serum insulin levels in a different cohort of WT and Nlrp3^−/− mice fed chow and HFD for 6 and 9 months. All data are presented as mean ± sem; n = 6–10 mice. *, P < 0.05. C, The trichrome staining of pancreatic cryosections from 9-month-old WT and Nalp3^−/− mice fed 60% HFD. The deposition of collagen and fibrosis can be identified by light blue regions within the pancreatic islets. D, The pancreatic cryosections from 12-month-old WT and Nlrp3^−/− mice fed 60% HFD were stained with antiinsulin (red) and anti-IL-1β antibodies. The extreme right panel shows representative image of pancreatic cryosections of 12-month-old DIO mice stained with macrophage marker F4/80 (with membrane staining, in red) and IL-1β (cytoplasmic, in green). The nuclei are counterstained blue with 4′,6-diamidino-2-phenylindole. The confocal images are representative of at least four mice and were repeated thrice.

Fig. 4.

Ablation of Nlrp3 inflammasome protects β-cell death and increases islet size in chronic mouse model of middle-age obesity. A, The confocal immunofluorescence microcopy of pancreatic islets stained for terminal transferase dUTP nick end labeling (TUNEL) (in red) in 12-month-old chow-fed WT and 60% HFD-fed WT and NLRP3^−/− mice. TUNEL-positive cells are marked by arrowheads. The negative control of pancreatic cryosections was prepared by omitting terminal transferase, and positive control includes permeabilization of cryosections with micrococcal nuclease or deoxyribonuclease 1 to induce DNA strand breaks. B, The pancreatic islet morphometry revealed that compared with 12-month-old WT obese mice, in age-matched NLRP3^−/− mice, the pancreatic islet size was significantly larger. C, A total of 250–286 islets from four to six mice of each strain were counted, and the area of each islet was determined by ImageJ software. Six nonserial sections from each mouse were used to count the islets. The ablation of NLRP3 causes significant increase in islet size (*, P < 0.01).