NMI Regulates Adipose Adaptive Thermogenesis Through TLR4/IRF3 Signaling to Promote Obesity

Figures

• 

  FIGURE 1. Nmi deficiency attenuates HFD‐induced adiposity and hepatic steatosis. (A) mRNA expression of damage‐associated molecular pattern (DAMP) genes in normal and overweight individuals (n = 770). (B) Pearson correlation between NMI expression and homeostatic model assessment of insulin resistance (HOMA‐IR) in humans (n = 770). (C) Changes in NMI expression in adipose tissue after dietary intervention‐induced weight loss and during the maintenance period in a weight‐loss cohort (n = 46). (D) Nmi mRNA expression in adipose tissues from lean and obese mice (n = 6). (E) Body weight (BW) of WT (Nmi+/+ ) and Nmi‐deficient (Nmi−/− ) mice fed normal chow diet (NCD) (n = 9–12). (F, G) BW of Nmi+/+ and Nmi−/− mice under high‐fat diet (HFD) for 12 weeks. Representative images (F) and BW growth curve (G, n = 6–9). (H) Fat mass and fat mass percentage measured by magnetic resonance imaging (MRI) in Nmi+/+ and Nmi−/− mice after 12 weeks of HFD (n = 6–9). (I, J) Fat depot weights in Nmi+/+ and Nmi−/− mice after 12 weeks of HFD. Representative images (I) and weights of total white adipose tissue (total WAT), subcutaneous WAT (scWAT), epididymal WAT (eWAT), and perirenal WAT (pWAT) (J, n = 6–9). (K)…

• 

  FIGURE 2. Nmi deficiency enhances energy expenditure. (A, B) Food consumption (A) and cumulative energy intake (B) in Nmi+/+ and Nmi−/− mice (n = 6–8). (C‐E) Oxygen consumption (VO2, C), carbon dioxide production (VCO2, D) and total energy expenditure (E) in Nmi+/+ and Nmi−/− mice (n = 6–8). (F) Analysis of covariance (ANCOVA) of energy expenditure with BW as a covariate (n = 6–8). Data are represented as mean ± SEM. Statistical analyses: two‐way ANOVA followed by Bonferroni's multiple comparisons test (A), two‐tailed unpaired Student's t‐test (B–E), ANCOVA with BW as covariate (F). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

• 

  FIGURE 3. Nmi deficiency promotes BAT adaptive thermogenesis and scWAT browning in HFD‐fed mice. (A, B) UCP1 protein levels in adipose tissue of HFD‐fed Nmi+/+ and Nmi−/− mice, assessed by Western blotting (A, n = 8) and immunohistochemistry (B, n = 6). (C) Time‐course changes in core body temperature over 24 h under thermoneutral conditions (30°C) in Nmi+/+ and Nmi−/− mice (n = 12–14). (D) Thermogenic protein levels in BAT under thermoneutral conditions (30°C) in Nmi+/+ and Nmi−/− mice, determined by Western blotting (n = 6). (E) Representative UCP1 immunohistochemistry images in scWAT and BAT under thermoneutral conditions (30°C). (F, G) Core body temperature (F, n = 5) and surface temperature (G, n = 7) in 8‐week‐old Nmi+/+ and Nmi−/− mice under cold exposure (4°C), with representative infrared thermal images of the lumbar back region and interscapular BAT. (H) Representative UCP1 immunohistochemistry images in scWAT and BAT under cold exposure (4°C). (I) Thermogenic protein levels in BAT under cold exposure (4°C), determined by Western blotting (n = 6). (J, K) Mitochondrial biogenesis‐related gene expression in BAT (J) and scWAT (K) of Nmi+/+ and Nmi−/− mice under HFD feeding (n = 8).…

• 

  FIGURE 4. NMI is enriched in mature adipocytes. (A, B) Nmi mRNA expression (A) and NMI protein levels (B) across adipose depots in 8‐week‐old male mice (n = 4). (C) Nmi mRNA expression in stromal vascular fraction (SVF) and mature adipocytes from 8‐ to 10‐week‐old chow‐fed mice (n = 3–6). (D) NMI protein levels in BAT from lean and diet‐induced obesity (DIO) mice, determined by Western blotting (n = 3). (E, F) Nmi mRNA (E, n = 3) and NMI protein (F, n = 6) during 3T3‐L1 differentiation into beige adipocytes (Day 0 and Day 8). (G) Schematic of fluorescence‐activated cell sorting (FACS) strategy for separating adipocyte precursor cells (CD45−) and immune cells (CD45+) from the SVF. (H) Nmi mRNA levels in SVF, immune cells, and mature adipocytes from lean mice and DIO mice (n = 3). Data are represented as mean ± SEM. Statistical analyses: one‐way ANOVA followed by Bonferroni's multiple comparisons test (A, B), two‐tailed unpaired Student's t‐test (C‐F, H). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

• 

  FIGURE 5. Transcriptomic analysis reveals enhanced PPAR signaling and adaptive thermogenesis in BAT of Nmi−/− mice. (A) RNA sequencing (RNA‐seq) of BAT from HFD‐fed Nmi+/+ and Nmi−/− mice. Differentially expressed genes (DEGs, |log2 fold change|> 1, false discovery rate [FDR] < 0.05) were primarily enriched in adaptive thermogenesis and fatty acid oxidation pathways (n = 4). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of upregulated genes (|log2 fold change| > 1 and FDR < 0.05). (C) Gene set enrichment analysis (GSEA) showing major upregulated pathways, including PPAR signaling, adaptive thermogenesis, and oxidative phosphorylation (normalized enrichment score [NES] > 1.7 and FDR < 0.002). (D) Heatmap of pathway‐related genes (PPAR signaling, adaptive thermogenesis, and oxidative phosphorylation) from RNA‐seq (n = 4). (E, F) Validation of RNA‐seq results by quantitative PCR (E) and Western blotting (F) (n = 8). Data are represented as mean ± SEM. Statistical analyses for RNA‐seq: differential expression analysis was performed using DESeq2 (|log2 fold change| > 1, FDR < 0.05); KEGG and GSEA were performed using clusterProfiler. For validation experiments:…

• 

  FIGURE 6. NMI regulates adaptive thermogenesis through PPAR signaling. (A) Triacylglycerol (TAG) content in differentiated primary brown adipocytes from Nmi+/+ and Nmi−/− mice (n = 8). (B, C) Thermogenic gene expression in primary brown adipocytes treated with 10 µm GW6471 (PPARα antagonist), or 10 µm GW9662 (PPARγ antagonist) for 24 h (B) or 48 h (C) (n = 4–6). (D, E) Thermogenic protein levels in primary brown adipocytes treated as in (B, C) for 24 h (D) or 48 h (E) (n = 4–6). (F) Thermogenic gene expression in primary adipocytes transfected with scramble siRNA or Ppara siRNA (100 nm) for 7 days (n = 4). (G) Model of NMI‐mediated suppression of adaptive thermogenesis via PPAR signaling. NMI induces transrepression of PPARα and PPARγ transcriptional activity, leading to UCP1 downregulation (Created with BioRender.com). Data are represented as mean ± SEM. Statistical analyses: two‐tailed unpaired Student's t‐test (A), one‐way ANOVA followed by Bonferroni's multiple comparisons test (B–F). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

• 

  FIGURE 7. NMI suppresses adaptive thermogenesis through TLR4/IRF3 signaling. (A) Mature adipocytes were pretreated with TAK‐242 (1 µm, TLR4 inhibitor) for 2 h, followed by recombinant NMI (5 µg mL−1) treatment for 24 h. Thermogenic proteins were subsequently analyzed by Western blotting (n = 6). (B) Mature adipocytes were pretreated with Amlexanox (50 µm, IRF3 inhibitor) for 1 h, followed by Poly I:C (50 µg mL−1, 1 h) treatment to induce IRF3 phosphorylation. Cell lysates were collected for detection of the indicated proteins (n = 6). (C) Mature adipocytes were stimulated with IFN‐γ (20 ng mL−1) and LPS (100 ng mL−1) for 24 h. Culture supernatants were analyzed for NMI secretion (n = 3). (D) Conditioned media from stimulated Nmi+/+ and Nmi−/− adipocytes were transferred to WT primary adipocytes. Expression of the indicated proteins was subsequently examined by Western blotting (n = 6). (E) BW gain in DIO mice treated with monoclonal antibody‐mediated neutralization of NMI (mAb‐NMI, 10 mg kg−1) or PBS (n = 8). (F, G) GTT (F) and ITT (G) in DIO mice after 12 weeks of HFD and mAb‐NMI or PBS treatment, quantified by AUC (n = 8). (H) HFD‐induced adipose tissue inflammation in Nmi+/+ and Nmi−/−…