GRIM19 Impedes Obesity by Regulating Inflammatory White Fat Browning and Promoting Th17/Treg Balance

Abstract

Obesity, a condition characterized by excessive accumulation of body fat, is a metabolic disorder related to an increased risk of chronic inflammation. Obesity is mediated by signal transducer and activator of transcription (STAT) 3, which is regulated by genes associated with retinoid-interferon-induced mortality (GRIM) 19, a protein ubiquitously expressed in various human tissues. In this study, we investigated the role of GRIM19 in diet-induced obese C57BL/6 mice via intravenous or intramuscular administration of a plasmid encoding GRIM19. Splenocytes from wild-type and GRIM19-overexpressing mice were compared using enzyme-linked immunoassay, real-time polymerase chain reaction, Western blotting, flow cytometry, and histological analyses. GRIM19 attenuated the progression of obesity by regulating STAT3 activity and enhancing brown adipose tissue (BAT) differentiation. GRIM19 regulated the differentiation of mouse-derived 3T3-L1 preadipocytes into adipocytes, while modulating gene expression in white adipose tissue (WAT) and BAT. GRIM19 overexpression reduced diet-induced obesity and enhanced glucose and lipid metabolism in the liver. Moreover, GRIM19 overexpression reduced WAT differentiation and induced BAT differentiation in obese mice. GRIM19-transgenic mice exhibited reduced mitochondrial superoxide levels and a reciprocal balance between Th17 and Treg cells. These results suggest that GRIM19 attenuates the progression of obesity by controlling adipocyte differentiation.

Keywords: GRIM19; STAT3; Th17; obesity.

Figure 1

Genes associated with retinoid-interferon-induced mortality 19 (GRIM19) regulates the transcript levels of genes associated with white adipose tissue (WAT) and brown adipose tissue (BAT). Scale bar, 100 µm. (A) Representative images of Oil Red O-stained 3T3-L1 cells. Mock and GRIM19-overexpressing 3T3-L1 cells were stained with Oil Red O. (B,C) Transcript levels of genes associated with WAT (B) and BAT (C) in 3T3-L1 cells expressing control plasmids (MOCK) and plasmids encoding GRIM19 (Grim19). (D) Transcript levels of leptin and lipoprotein lipase (LPL) in 3T3-L1 cells expressing control plasmids (MOCK), plasmids encoding GRIM19 (Grim19), or GRIM19-depleted siRNA (siGrim19). (E) GRIM19 mRNA levels were determined using real-time polymerase chain reaction. GAPDH was used as the internal control. Data are presented as the means ± standard deviations (SDs) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns = not significant.

Figure 2

GRIM19 regulates BAT differentiation. (A) Representative images of Oil Red O-stained C2C12 cells. Mock, GRIM19-overexpressing, control siRNA (siRNA Con), or GRIM19 siRNA C2C12 cells were stained with Oil Red O. Scale bar, 100 µm. The bar graph shows averaged percentage of Oil Red O-stained area. (B) Transcript levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome C oxidase polypeptide 7A1 (Cox7a1), fatty acid elongase 3 (Elovl3), PGC1a, and UCP1 in mock-vector (MOCK) and GRIM19-overexpressing C2C12 cells. (C) Transcript levels of Cidea, Cox7a1, Elovl3, PGC1a, and UCP1 in control and GRIM19-depleted C2C12 cells. (D) Transcript levels of p53 and PRDM16 in mock-vector and GRIM19-overexpressing C2C12 cells. GAPDH was used as the internal control. Data are presented as the means ± SDs of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Figure 3

Overexpression of GRIM19 by two different strategies leads to the attenuation of high-fat diet (HFD)-induced obesity. (A) Kinetics of body weight changes (left) and rates of body weight gain (right) in HFD-fed control and GRIM19-administered mice over three independent experiments. (B) Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right) results for HFD-fed control and GRIM19-administered mice (n = 5). (C) Measurements of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and free fatty acid levels in HFD-fed control and GRIM19-administered mice (n = 5). (D) Splenocytes were obtained from wild-type (WT) and GRIM19-Tg mice. Cells were prepared to examine the expression of GRIM19. The bar graph shows relative intensity. (E) Kinetics of body weight changes in HFD-fed control and GRIM19-Tg mice. The bar graph shows the averaged body weights of HFD-fed WT and GRIM19-Tg mice at 10 weeks (n = 5–7). (F) Measurements of glucose, AST, and ALT levels in serum in HFD-fed control and GRIM19-Tg mice (n = 5–7). Data are presented as the means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Figure 4

GRIM19 protects against diet-induced obesity and hepatic steatosis as well as regulates the fat profile. (A) Representative hematoxylin and eosin (H&E)- and Oil Red O-stained images of liver tissues from HFD-fed WT and GRIM19-Tg mice. The bar graph shows the averaged hepatic levels of triglycerides. Scale bar, 200 µm. (B) Representative electron microscopy images of liver tissue from HFD-fed WT and GRIM19 mice. Scale bar, 2 µm. (C) Representative H&E-stained images of the epididymal fat pad from HFD-fed WT and GRIM19-Tg mice (×200). (D) Representative images of epididymal fat from normal-fat diet (NFD)- and HFD-fed WT mice and HFD-fed GRIM19-Tg mice. The bar graph shows the averaged epididymal fat weight of HFD-fed WT and GRIM19-Tg mice. (E) Representative electron microscopy images of interscapular brown fat from HFD-fed WT and GRIM19-Tg mice. Scale bar, 2 µm. Bar graphs show the transcript levels of UCP1, Cidea, PGC1a, and Cox7a1. The transcript levels were normalized to that of GAPDH. Data are presented as the means ± SDs. * p < 0.05 and ** p < 0.01.

Figure 5

Effect of GRIM19 on Th17/Treg cell regulation in obese mice. (A) Representative flow cytometry plots showing the frequencies of CD4⁺pSTAT3 (Tyr705)⁺ and CD4⁺pSTAT3 (Ser727)⁺ cells in spleens from HFD-fed WT and GRIM19-Tg mice. Bar graphs show frequency of each cell type. (B) Representative confocal microscopy images of CD4⁺IL-17⁺ cells and CD4⁺CD25⁺Foxp3⁺, as well as CD4⁺pSTAT3 (Tyr705)⁺, CD4⁺pSTAT3 (Ser727)⁺, and CD4⁺pSTAT5⁺ cells in spleens from HFD-fed WT and GRIM19-Tg mice. Bar graphs show the numbers of each cell type in four independent quadrants. Data are presented as the means ± SDs of three independent experiments. Scale bar, 20 µm * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

GRIM19-mediated anti-adipogenic and thermogenic effects in GRIM19-Tg and WT mice. (A) Representative images of interscapular white fat and interscapular brown fat from HFD-fed WT and GRIM19-Tg mice (top). Bar graphs show the averaged weights of interscapular white fat and interscapular brown fat, and ratios of interscapular white fat weight to that of interscapular brown fat in interscapular fat pads (bottom). (B) Transcript levels of UCP1, Elovl3, PGC1a, Cox7a1, cytochrome C1, fibroblast growth factor (FGF) 21, and PRDM16 in epididymal fat pads from HFD-fed WT and GRIM19-Tg mice. The transcript levels were normalized to that of GAPDH. Mice were housed at 4 °C for 1 day before the experiments. Data are presented as the means ± SDs of three independent experiments. * p < 0.05 and ** p < 0.01.