The essential function of CARD9 in diet-induced inflammation and metabolic disorders in mice

Abstract

Inflammation and metabolic disorder are common pathophysiological conditions, which play a vital role in the development of obesity and type 2 diabetes. The purpose of this study was to explore the effects of caspase recruitment domain (CARD) 9 in the high fat diet (HFD)-treated mice and attempt to find a molecular therapeutic target for obesity development and treatment. Sixteen male CARD9^-/- and corresponding male WT mice were fed with normal diet or high fat diet, respectively, for 12 weeks. Glucose tolerance, insulin resistance, oxygen consumption and heat production of the mice were detected. The CARD9/MAPK pathway-related gene and protein were determined in insulin-responsive organs using Western blotting and quantitative PCR. The results showed that HFD-induced insulin resistance and impairment of glucose tolerance were more severe in WT mice than that in the CARD9^-/- mice. CARD9 absence significantly modified O₂ consumption, CO₂ production and heat production. CARD9^-/- mice displayed the lower expression of p38 MAPK, JNK and ERK when compared to the WT mice in both HFD- and ND-treated groups. HFD induced the increase of p38 MAPK, JNK and ERK in WT mice but not in the CARD9^-/- mice. The results indicated that CARD9 absence could be a vital protective factor in diet-induced obesity via the CARD9/MAPK pathway, which may provide new insights into the development of gene knockout to improving diet-induced obesity and metabolism disorder.

Keywords: CARD9; energy metabolism obesity; inflammation; insulin resistance.

Figure 1

Changes in body weight (b.w.), food intake and the ratio of organ/b.w in the CARD9^−/− and WT mice after ND and HFD feeding. Weekly changes of b.w. (A), b.w. at the end of 12 weeks feeding (B), Weekly food intake (C),Average food intake during the entire experiment (D), The ratios of organ/b.w. in liver, WAT, BAT and spleen (E). *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 2

Glucose homeostasis and insulin sensitivity in the CARD9^−/− and WT mice at week 8 and 12. IPGTT at week 8 (A) and week 12 (B), IPITT at week 8 (C) and week 12 (D), Fasting blood glucose level (E), Fasting blood insulin level (F), HOMA‐IR index (G). *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 3

Energy homeostasis in the CARD9^−/− and WT mice after ND and HFD feeding. O₂ consumption (A), CO ₂ consumption (B), Heat production (C), Respiratory exchange ratio (D). Left panel: the trends of above four indicators with time, Right panel: the bar graph of above four indicators in the four groups, respectively. *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 4

Systemic inflammation in the CARD9^−/− and WT mice after ND and HFD feeding. IL‐6 (A), TNF‐α (B). *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 5

Representative histological images of liver, white adipose fat (WAT) and brown adipose fat (BAT) stained with haematoxylin and eosin (H&E). BAT (A), original magnification, ×400, scale bar = 100 μm; WAT (B), original magnification, ×200, scale bar = 100 μm; Liver (C), original magnification, ×400, scale bar = 100 μm. Adipocyte area was calculated from 100 adipocytes of BAT (D) and WAT (E) in each mouse.*P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 6

mRNA expressions of IL‐6, TNF‐α, p38, ERK1/2, JNK and NF‐κB in the CARD9^−/− and WT mice after ND and HFD feeding. The expression in liver (A), WAT (B). *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.

Figure 7

Protein expression of inflammation and MAPK signalling pathways in the liver of the CARD9^−/− and WT mice after ND and HFD feeding, along with the UCP‐1 expression in BAT. Western blotting for phosphorylated NF‐κB (P‐NF‐κB)/total NF‐κB (A). Western blotting of signalling molecules involved in MAPK pathway [P‐p38/p38, (B); P‐JNK/JNK, (C); P‐ERK/ERK, (D)]. Western blotting of UCP‐1 (E). *P < 0.05, **P < 0.01, HFD vs. ND; ^# P < 0.05, ^## P < 0.01, CARD9^−/− vs. WT.