Gasdermin D Deficiency Does Not Protect Mice from High-Fat Diet-Induced Glucose Intolerance and Adipose Tissue Inflammation

Abstract

The adipose tissue NLRP3 inflammasome has recently emerged as a contributor to obesity-related metabolic inflammation. Recent studies have demonstrated that the activation of the NLRP3 inflammasome cleaves gasdermin D (GSDMD) and induces pyroptosis, a proinflammatory programmed cell death. However, whether GSDMD is involved in the regulation of adipose tissue function and the development of obesity-induced metabolic disease remains unknown. The aim of the present study was to investigate the role of GSDMD in adipose tissue inflammation as well as whole-body metabolism using GSDMD-deficient mice fed a high-fat diet (HFD) for 30 weeks. The effects of GSDMD deficiency on adipose tissue, liver, and isolated macrophages from wild-type (WT) and GSDMD knockout (KO) mice were examined. In addition, 3T3-L1 cells were used to examine the expression of GSDMD during adipogenesis. The results demonstrate that although HFD-induced inflammation was partly ameliorated in isolated macrophages and liver, adipose tissue remained unaffected by GSDMD deficiency. Compared with the WT HFD mice, GSDMD KO HFD mice exhibited a mild increase in HFD-induced glucose intolerance with increased systemic and adipose tissue IL-1β levels. Interestingly, GSDMD deficiency caused accumulation of fat mass when challenged with HFD, partly by suppressing the expression of peroxisome proliferator-activated receptor gamma (PPARγ). The expression of GSDMD mRNA and protein was dramatically suppressed during adipocyte differentiation and was inversely correlated with PPARγ expression. Together, these findings indicate that GSDMD is not a prerequisite for HFD-induced adipose tissue inflammation and suggest a noncanonical function of GSDMD in regulation of fat mass through PPARγ.

Figure 1

Body weight and food intake in wild-type (WT) and gasdermin D knockout (GSDMD KO) mice fed a high-fat diet (HFD). (a and b) Measurements of changes in body weight during a HFD or normal chow diet (NCD) in WT and GSDMD KO mice. (c and d) Measurements of food intake in WT and GSDMD KO mice fed a NCD or HFD. (e–g) Tissue weight and ratio of tissue weight per body weight in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and liver. ^∗p < 0.05 vs. same genotype mice fed a NCD and ^#p < 0.05 vs. WT HFD group.

Figure 2

Glucose and insulin tolerance in wild-type (WT) and gasdermin D knockout (GSDMD KO) mice fed a high-fat diet (HFD). (a) Oral glucose tolerance test and (b) intraperitoneal insulin tolerance test performed at 15 weeks on a HFD in WT and GSDMD KO mice. Blood glucose levels were measured and the area under the curve (AUC) was determined. (c) Oral glucose tolerance test and (d) intraperitoneal insulin tolerance test performed at 30 weeks on a HFD in WT and GSDMD KO mice. Plasma IL-1β (e) and IL-18 (f) measured by ELISA. ^∗p < 0.05 vs. same genotype mice fed a NCD, ^†p < 0.05 vs. WT NCD group, and ^#p < 0.05 vs. WT HFD group.

Figure 3

Liver inflammation and metabolism in wild-type (WT) and gasdermin D knockout (GSDMD KO) mice fed a high-fat diet (HFD). (a) Representative western blots and quantitative analysis of GSDMD in liver. (b) IL-1β protein expression in liver was measured by ELISA assay. (c) Representative western blots and quantitative analysis of pro-caspase1 and caspase1 in liver. The expression level of protein was normalized using β-tubulin. (d–h) Real-time PCR analysis of genes in the liver. The expression level of each target was normalized using 18S rRNA. (i) GSH concentration in liver was evaluated by GSH Detection Assay kit. ^∗p < 0.05 vs. same genotype mice fed a NCD, ^†p < 0.05 vs. WT NCD group, and ^#p < 0.05 vs. WT HFD group.

Figure 4

Adipose tissue inflammation and metabolism in wild-type (WT) and gasdermin D knockout (GSDMD KO) mice fed a high-fat diet (HFD). (a, c, d, g, h, and i) Real-time PCR analysis of genes in visceral adipose tissue (VAT). The expression level of each target was normalized using 18S rRNA. (b) IL-1β protein expression in VAT was measured by ELISA assay. (e, f, and j) Representative western blots and quantitative analysis of protein expression in VAT. The protein expression level was normalized using β-actin. (k) GSH concentration in VAT was evaluated by GSH Detection Assay kit. ^∗p < 0.05 vs. same genotype mice fed a NCD, ^†p < 0.05 vs. WT NCD group, and ^#p < 0.05 vs. WT HFD group.

Figure 5

PPARγ and gasdermin D (GSDMD) expression in 3T3-L1 cells during adipogenesis. (a) Real-time PCR analysis of PPARγ genes in adipose tissue of wild-type (WT) and GSDMD KO mice fed a normal chow diet (NCD) or high-fat diet (HFD). (b) Representative western blot image and quantitative analysis of PPARγ in adipose tissue of WT and GSDMD KO mice. (c and d) Real-time PCR analysis of PPARγ and GSDMD genes during adipogenesis. (e) Representative western blot image and quantitative analysis of GSDMD protein during adipogenesis. Samples were taken before and 2, 4, and 6 days after treatment of 3T3-L1 preadipocytes with differentiation media. (f) Correlation analysis between GSDMD and PPARγ genes during adipogenesis. Protein expression levels were normalized to β-actin. Gene expression levels were normalized to 18S rRNA. ^∗p < 0.05 vs. same genotype mice fed a NCD, ^†p < 0.05 vs. WT NCD group, ^#p < 0.05 vs. WT HFD group, and ^ǂp < 0.05 vs. D+0.

Figure 6

Macrophage activation in wild-type (WT) and gasdermin D knockout (GSDMD KO) mice fed a high-fat diet (HFD). (a and c) Bone marrow-derived macrophages were isolated from WT and GSDMD KO mice fed a normal chow diet (NCD) or HFD. Bone marrow-derived macrophages (BMDMs) were treated with LPS, and gene expression of GSDMD and IL-1β was measured by real-time PCR. Gene expression levels were normalized to 18S rRNA. (b) Representative western blot image and quantification of NLRP3 after LPS and ATP treatment. Protein expression levels were normalized to β-tubulin. (d) Secretion of IL-1β protein from BMDMs was measured by ELISA after treatment with LPS and ATP. ^∗p < 0.05 vs. control, ^†p < 0.05 vs. LPS, ^‡p < 0.05 vs. NCD LPS+ATP, and ^ɤp < 0.05 vs. HFD LPS+ATP.