METTL3 is essential for postnatal development of brown adipose tissue and energy expenditure in mice

Abstract

Brown adipose tissue (BAT) undergoes rapid postnatal development and then protects against cold and obesity into adulthood. However, the molecular mechanism that determines postnatal development and maturation of BAT is largely unknown. Here we show that METTL3 (a key RNA methyltransferase) expression increases significantly in interscapular brown adipose tissue (iBAT) after birth and plays an essential role in the postnatal development and maturation of iBAT. BAT-specific deletion of Mettl3 severely impairs maturation of BAT in vivo by decreasing m⁶A modification and expression of Prdm16, Pparg, and Ucp1 transcripts, which leads to a marked reduction in BAT-mediated adaptive thermogenesis and promotes high-fat diet (HFD)-induced obesity and systemic insulin resistance. These data demonstrate that METTL3 is an essential regulator that controls iBAT postnatal development and energy homeostasis.

Fig. 1. METTL3 is enriched in iBAT and associated with postnatal development of iBAT.

a METTL3 and UCP1 protein levels in iBAT, iWAT, and eWAT of C57BL/6 male mice at 8 weeks of age. b METTL3 and UCP1 protein levels in iBAT at 0, 1, 5, 10, 20, 30, and 60 days after birth. These experiments were repeated three times independently with similar results. Source data are provided as a Source Data file.

Fig. 2. METTL3 is required for postnatal development of iBAT.

a Gross appearance of iBATs in Mettl3^flox/flox and BKO mice at 0, 1, 5, 10, 20, 30, and 60 days after birth. The scale bar represents 1 cm. b Gross appearance of iBATs in Mettl3^flox/flox and BKO mice at 8 weeks of age. c Hematoxylin and eosin (H&E) staining of iBATs in Mettl3^flox/flox and BKO mice at 0, 1, 5, 10, 20, 30, and 60 days after birth. Scale bars represent 100 μm. Three mice for each group were used for H&E staining with similar results. d, e iBAT and body weight of METTL3 ^flox/flox and BKO mice at 0, 1, 5, 10, 20, 30, and 60 days after birth (n = 5–8 for each group). f METTL3 and UCP1 protein levels in iBAT of Mettl3^flox/flox and BKO mice at 0, 1, 5, 10, 20, 30, and 60 days after birth. This immunoblotting experiment was repeated three times independently with similar results. Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.

Fig. 3. BAT-specific deletion of Mettl3 dramatically decreases expression of BAT-selective genes RNA-seq analysis was performed in the iBATs of Mettl3^flox/flox and BKO mice at 8 weeks old.

a The differentially expressed genes (DEGs) (BKO VS flox/flox) including 530 downregulated genes and 1552 upregulated genes were illustrated in a volcanoplot (fold change > 2 and q value < 0.005). b Top GO biological process terms enriched in downregulated and upregulated genes. c Relative mRNA levels (n = 5–8 for each group). d UCP1, PGC-1α, PPARγ, PRDM16, and GAPDH protein levels in iBATs of 8-week-old Mettl3^flox/flox and BKO mice were determined by Western blot (n = 3 for each group). e Relative mitochondria number in iBATs of 8-week-old Mettl3^flox/flox and BKO mice (Mettl3^flox/flox, n = 8; BKO, n = 6). f Mitochondrial complex protein levels in iBATs of 8-week-old Mettl3^flox/flox and BKO mice were determined by western blot (n = 3 for each group). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.

Fig. 4. METTL3 is essential for mRNA m⁶A modification in iBAT.

The m⁶A RNA immunoprecipitation sequencing (m⁶ARIP-seq) analysis of iBATs were performed in 8-week-old Mettl3^flox/flox and BKO mice. a The enrichment of m⁶ARIP-seq peaks in iBAT of 8-week-old Mettl3^flox/flox and BKO mice. b Consensus motif of m⁶A sites in iBAT of 8-week-old Mettl3^flox/flox and BKO mice. c Heatmap of FoldEnrich in m⁶A peaks (BKO VS flox/flox).

Fig. 5. METTL3 is essential for m⁶A modification of Prdm16, Pparg, and Ucp1 transcripts.

a–c The read density from m⁶A-RIP-seq experiments on BKO–flox/flox pairs showing the m⁶A peaks identified in the Prdm16, Pparg, and Ucp1 transcripts. d–f The m⁶A modification in Prdm16, Pparg, and Ucp1 transcripts in iBAT of 8-week-old Mettl3^flox/flox and BKO mice were measured by m⁶ARIP-RT-qPCR (n = 3 for each group). g–j Primary brown preadipocytes seeded in 24-well plates were co-transfected with pMIR-REPORT Luciferase vectors (Prdm16, Pparg, Ucp1), siRNAs (Scramble siRNA, siMettl3-1, siMettl3-2) and β-galactosidase (β-Gal) reporter plasmid by X-tremeGENE siRNA Transfection Reagent for 24 h. Cells were then induced to differentiate for 48 h. METTL3 and GAPDH protein levels were measured by immunoblotting g. Relative Prdm16, Pparg, and Ucp1 luciferase activity were measured and normalized to β-Gal levels h–j (Scr in h, n = 4; others, n = 5 biologically independent cell samples for each group). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.

Fig. 6. METTL3 is essential for brown adipogenesis.

a The Mettl3 mRNA levels in mature primary brown adipocytes (10 days after differentiation) and preadipocytes (before differentiation) (n = 9 for each group). b Relative Mettl3 mRNA levels in primary brown adipocytes from Mettl3^flox/flox mice infected with Ad-Cre or Ad-βGal (Ad-Cre, n = 12; Ad-βGal, n = 11). c Oil red O staining of primary brown adipocytes from Mettl3^flox/flox mice infected with Ad-Cre or Ad-βGal. Scale bars represent 100 μm. d, e Real-time qPCR analysis of mRNA levels of genes involved in adipogenesis, lipolysis, lipogenesis, thermogenesis, fatty acid oxidation, thermogenesis and mitochondrial oxidative phosphorylation in primary brown adipocytes infected with Ad-Cre or Ad-βGal (n = 10–12 for each group). f Western blot analysis of protein levels of METTL3, UCP1, PGC-1α, PPARγ, PRDM16, and GAPDH in primary brown adipocytes described in d (n = 4 for each group). g Relative mitochondria number in primary brown adipocytes infected with Ad-Cre or Ad-βGal (n = 8 for each group). n was the number of biologically independent cell samples, and these cell culture experiments were repeated three times independently with similar results. Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.

Fig. 7. Mettl3 deficiency in BAT decreases energy expenditure and impairs cold tolerance.

a The Mettl3 mRNA levels in iBATs of WT mice after acute cold exposure for 6 h (22 °C, n = 8; 4 °C; n = 7). b, c The O₂ consumption rates in 8-week-old Mettl3^flox/flox and BKO mice at 22 °C (n = 5–6 for each group). d, e The CO₂ production rates in 8-week-old Mettl3^flox/flox and BKO mice at 22 °C (n = 5–6 for each group). f The rectal temperature of 8-week-old Mettl3^flox/flox and BKO mice during acute cold exposure (4 °C) (Mettl3^flox/flox, n = 10; BKO, n = 9). g Serum FFAs levels of 8-week-old Mettl3^flox/flox and BKO mice housed at room temperature or after cold exposure (4 °C) for 6 h (22 °C Mettl3^flox/flox, n = 9; 22 °C BKO, n = 8; 4 °C Mettl3^flox/flox, n = 8; 4 °C BKO, n = 8). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file.

Fig. 8. BAT-specific knockout of Mettl3 predisposes to HFD-induced obesity.

a–c The METTL3 protein and mRNA levels in iBATs of HFD-fed mice, ob/ob mice and their respective control mice (NC, n = 11; HFD, n = 10; WT, n = 7; ob/ob, n = 7). d The growth curve of METTL3 BKO and Mettl3^flox/flox mice fed HFD diet (5 weeks Mettl3^flox/flox and BKO, n = 10 for each group; 6 weeks Mettl3^flox/flox, n = 10; 6 weeks BKO, n = 12; 7–16 weeks Mettl3^flox/flox and BKO, n = 12 for each group). e The fat and lean mass of 20-week-old HFD-fed mice (n = 5 for each group). f The rectal temperature of HFD-fed Mettl3^flox/flox and BKO mice during acute cold exposure (4 °C) (Mettl3^flox/flox, n = 6; 0, 60, 120 min BKO, n = 5; 240 min BKO, n = 4). g The relative tissue weight of iBAT and liver in 20-week-old HFD-fed mice (Mettl3^flox/flox, n = 8; BKO, n = 7). h Representative images of iBAT and liver from 20-week-old HFD-fed mice. i Hematoxylin and eosin (H&E) staining of iBAT and liver from 20-week-old HFD-fed mice. Scale bars represent 100 μm. Three mice for each group were used for H&E staining with similar results. j The serum TG levels of 20-week-old HFD-fed mice (n = 5 for each group). Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis.*p < 0.05.**p < 0.01. Source data are provided as a Source Data file.

Fig. 9. BAT-specific knockout of Mettl3 predisposes to HFD-induced glucose intolerance and insulin resistance.

a GTTs of 16-week-old HFD-fed mice (Mettl3^flox/flox, n = 11; BKO, n = 10). b ITTs of 16-week-old HFD-fed mice (n = 10 for each group). c The serum insulin levels of 20-week-old HFD-fed mice (Mettl3^flox/flox, n = 9; BKO, n = 6). d p-AKT(S473) and AKT protein levels of 20-week-old HFD-fed mice. This experiment was repeated three times by using three different groups of mouse samples with similar results. Data represent the mean±SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. *p < 0.05. Source data are provided as a Source Data file.