Lower NLRP3 inflammasome activity in NAG-1 transgenic mice is linked to a resistance to obesity and increased insulin sensitivity

Abstract

Objective: The NLRP3 inflammasome plays an important regulatory role in obesity-induced insulin resistance. NSAID activated gene-1 (NAG-1) is a divergent member of the TGF-β superfamily. NAG-1 Tg mice are resistant to dietary- and genetic-induced obesity and have improved insulin sensitivity. The objective was to examine whether NLRP3 inflammasome activity is associated with this observed phenotype in NAG-1 Tg mice.

Methods: Key components of the NLRP3 inflammasome were examined in NAG-1 Tg mice on both regular and high fat diet (HFD) conditions.

Results: The expression of caspase-1 and ASC, key components of the NLRP3 inflammasome, is significantly reduced at mRNA and protein levels in white adipose tissue (WAT) of NAG-1 Tg mice. HFD increases the expression of caspase-1 and ASC in WT mice, but their expression is reduced in NAG-1 Tg mice. Furthermore, there is reduced IL-18, IL-1β, and TNF-α expression in the WAT of NAG-1 Tg mice. NAG-1 Tg mice have significantly lower serum leptin and insulin levels and reduced expression of macrophage infiltration markers (F4/80, CD11b, and CD11c) in WAT.

Conclusions: The study suggests the lower NLRP3 inflammasome activity may play a role in the resistance of NAG-1 Tg mice to diet-induced obesity and improved insulin sensitivity.

Figure 1. NAG-1 Tg mice have reduced body/WAT weight and reduced IL-18 and IL-1β expression

(A) Body weight is reduced in 30 wk old male NAG-1 Tg mice (n=6 mice per group) fed on chow diet. (B) Total WAT is reduced in NAG-1 Tg mice (n=6) fed on chow diet. (C) Expression of IL-18 at mRNA level in WAT is reduced in NAG-1 Tg mice. (D) Expression of IL-β at mRNA level in WAT is reduced in NAG-1 Tg mice. The solid bars are the wild-type littermates and open bars are the NAG-1 mice (n=6 mice per group). Data are presented as mean ± SE. *p<0.05, **p<0.01 and ***p<0.001 as determined by Student’s t-test.

Figure 2. NAG-1 Tg mice have reduced caspase-1 activity determined by qRT-PCR and Western blot

(A) Real-time PCR analysis of mNLRP3, PYCARD and caspase-1 gene expressions at mRNA level in WAT of 15 wk old NAG-1 Tg and WT mice fed on chow diet (n=6 mice per group). (B) Western blot analyses of NLRP3 (NALP3), ASC (PYCARD), pro-caspase-1 (45kDa), and cleaved-caspase-1 (10kDa) basal expression levels in WAT fed on chow diet. GAPDH was used as a loading control. Expressions of PYCARD and both pro- and cleaved-caspase-1 are lower in NAG-1 Tg mice compared to WT mice (data represent 3 mice per group). Data is presented as ± SE. *p<0.05 as determined by Student’s t-test.

Figure 3. NAG-1 Tg mice on HFD have reduced body/WAT weight and reduced IL-18, IL-1β and TNF-α expression

(A) Body weight is reduced in 32 wk old NAG-1 Tg mice (n=6 mice per group) fed on HFD. (B) Total WAT is reduced in NAG-1 Tg mice (n=5–7) fed on HFD. Relative gene expression of IL-18 (C), IL-β (D), and TNF-α (E) at mRNA level in WAT is reduced in NAG-1 Tg mice both on LFD and HFD as determined by qRT-PCR. The solid bars are the wild-type littermates and open bars are the NAG-1 mice (n=5–7 mice per group). Data are presented as mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 as determined by Student’s t-test.

Figure 4. NAG-1 Tg mice on a HFD have lower inflammasome activity in WAT

(A) Relative gene expression of components of the NLRP3 inflammasome: PYCARD (ASC), NLRP3, and pro-caspase-1 in the WAT of WT and NAG-1 Tg mice fed on HFD determined by Real-time PCR analysis (n=5–7 mice per group). (B) Western blot analysis of pro-caspase-1 (45kDa), cleaved-caspase-1 (10kDa), NLRP3 (NALP3) and PYCARD (ASC) in WAT of WT and NAG-1Tg mice fed on HFD. GAPDH was used as a loading control. Expressions of PYCARD and both pro- and cleaved-caspase-1 are lower in NAG-1 Tg mice compared to WT mice. PYCARD expression is lower in the WAT of NAG-1 Tg mice on HFD compared to WT mice on HFD, while NLRP3 (NALP3) expression is unchanged (data represent 4 mice per group). Data are presented as ± SE. *p<0.05, **p<0.01, and ***p<0.001 as determined by Student’s t-test.

Figure 5. NAG-1 Tg mice have improved insulin sensitivity and reduced macrophage infiltration

(A) Serum levels of leptin, adiponectin and insulin in WT and NAG-1 Tg mice upon LFD or HFD treatment. (B). Adipocytes are smaller in NAG-1 Tg mice compared to WT littermates as determined by H&E staining (200x magnification). (C). Insulin tolerance test demonstrates NAG-1 Tg mice have improved insulin sensitivity (n=7–8/group). (D) Relative gene expression of macrophage infiltration markers F4/80, CD11b, and CD11c, and MCP-1, a contributor to macrophage infiltration, upon HFD diet are significantly inhibited in WAT of NAG-1 Tg mice compared to WT littermates (n=5–7 /group). Data are presented as ± SE. *p<0.05, **p<0.01, and ***p<0.001 as determined by Student’s t-test.