Fig 2: ACE2 pathway induced white adipose tissue browning in the Leprdb/db obese mice.(A) Representative H&E staining from subcutaneous white adipose tissue (scWAT) sections of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 6–7/each group). (B) Relative mRNA levels of mitochondrial related genes, fatty acid oxidation related genes and transcription factors in scWAT of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 3–5/each group). (C) Representative H&E staining from scWAT sections of Leprdb/db+ Ang-(1-7) and Leprdb/db + Vehicle mice exposure at 4 °C (n = 6–7/each group). (D) Relative mRNA levels of mitochondrial related genes, fatty acid oxidation related genes and transcription factors in scWAT of Leprdb/db+ Ang-(1-7) and Leprdb/db+ Vehicle mice exposure at 4 °C (n = 4/each group). (E) Representative H&E staining from epididymal white adipose tissue (eWAT) sections of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 6–7/each group). (F) Relative mRNA levels of mitochondrial related genes, fatty acid oxidation related genes, and transcription factors in eWAT of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 4/each group). Data represent mean ± SEM. *p < 0.05, **p < 0.01 vs GFP/Vehicle group by Student’s t-test. Figure 6—source data 1.Numerical quantification data for Figure 6.

Fig 3: Ace2 deficiency impairs thermogenesis, BAT activity, and energy metabolism.Eight-week-old male Ace2-/y mice and their wild-type (WT) mice (controls) had a high-fat diet (HFD) for 8 weeks. (A) Body weight of Ace2-/y and WT mice fed a HFD for 8 weeks (n = 4–5/each group). (B) Serum levels of Ang-(1-7), as determined by ELISA (n = 4–6/each group). (C–E) Energy expenditure was evaluated by measurement of oxygen consumption (VO2) (C), carbon dioxide release (VCO2) (D) and energy expenditure (EE) (E) over a 24 hours period (n = 5/each group). (F) Core body temperature at 30 °C, 22°C and 4°C for 8 hr in Ace2-/y and WT mice (n = 5/each group). (G) Infrared thermal images at 22 °C in Ace2-/y and WT mice. (H) Representative tomography–computed tomography (PET-CT) image and standard uptake values (SUVs) (n = 4/each group). (I) Representative haematoxylin and eosin (H&E) staining and uncoupling protein-1 (UCP1) immunostaining from BAT sections of Ace2-/y and WT mice exposure at 4 °C (n = 5/each group). (J) Representative western blots showing the changes of key proteins of energy expenditure and thermogenesis in BAT of Ace2-/y and WT mice exposure at 4 °C (n = 3/each group). (K) Representative western blots showing the key protein changes in primary brown adipocytes from Ace2-/y and WT mice (n = 3/each group). (L) Representative immunofluorescent images of in vitro differentiated primary brown adipocytes of Ace2-/y and WT mice, primary brown adipocytes show staining for UCP1 (red), boron-dipyrromethene (BODIPY) (green; neutral lipid dye), and DAPI (blue; nuclei). (M) Continuous measurement of oxygen consumption rate (OCR) in primary brown adipocytes from Ace2-/y mice and WT littermates. Oxygen consumption was performed under basal conditions, following the addition of oligomycin (1 μM), the pharmacological uncoupler FCCP (1 μM) or the Complex III and I inhibitor antimycin A and rotenone (0.5 μM) (n = 4–5/each group). Data represent mean ± SEM. *p < 0.05, **p < 0.01 vs WT group by Student’s t-test, or ANCOVA. Figure 2—source data 1.Numerical quantification data for Figure 2.

Fig 4: ACE2 pathway is activated by cold exposure.Eight-week-old male C57BL/6 J mice were housed at room temperature (RT) for 2 weeks before cold exposure at 4 °C for various time periods as indicated. (A) Levels of ACE2 and Mas1 protein from interscapular brown adipose tissue (BAT), subcutaneous and epididymal white adipose tissue (scWAT and eWAT) of C57BL/6 mice at room temperature (RT), as determined by Western blotting (n = 3/each group). (B) Levels of Ace2 and Mas1 mRNA from BAT, scWAT and eWAT of C57BL/6 mice at RT, as determined by qPCR (n = 3/each group). (C) Levels of ACE2 and Mas1 protein from interscapular BAT of C57BL/6 mice at RT or exposed to 4 °C for 6 hr, as determined by western blotting (n = 3/each group). (D, E) Levels of Ace2 and Mas1 mRNA from BAT, scWAT, eWAT and liver of C57BL/6 mice exposed to 4 °C for 24 hr, as determined by qPCR (n = 6/each group). (F, G) Serum levels of ACE2 (F) and Ang-(1-7) (G), as determined by ELISA (n = 4–6/each group). Data represent mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 vs Control group by Student’s t-test, or one-way ANOVA. Figure 1—source data 1.Numerical quantification data for Figure 1.

Fig 5: ACE2 enhances thermogenesis, BAT activity, and energy metabolism in Leprdb/db obese mice.Ace2 over-expression adenovirus (Ad-Ace2) and Ad-GFP (control) were introduced into the Leprdb/db obese mice by tail vein injection. The ad-Ace2 and Ad-GFP treated Leprdb/db mice were used at the 6th day post-virus injection. (A) Serum levels of Ang-(1-7), as determined by ELISA (n = 6–7/each group). (B) Body weight of ad-Ace2 and Ad-GFP treated Leprdb/db mice at the 6th day post-virus injection (n = 4–6/each group). (C–E) Energy expenditure was evaluated by measurement of oxygen consumption (VO2) (C), carbon dioxide release (VCO2) (D) and energy expenditure (EE) (E) over a 24-hr period (n = 4–6/each group). (F) Core body temperature at 30 °C, 22°C and 4°C for 8 hr (n = 5/each group). (G) Infrared thermal images at 22 °C in Leprdb/db+ Ace2 and Leprdb/db+ GFP mice (n = 4/each group). (H) Representative tomography–computed tomography (PET-CT) image and standard uptake values (SUVs) (n = 4/each group). (I) Representative H&E staining and UCP1 immunostaining from BAT sections of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 5/each group). (J) Representative western blots showing the changes of key proteins of energy expenditure and thermogenesis in BAT of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 3/each group). (K) Relative mRNA levels of mitochondrial related genes, fatty acid oxidation related genes and transcription factors in BAT of Leprdb/db+ Ace2 and Leprdb/db+ GFP mice exposure at 4 °C (n = 5–6/each group). Data represent mean ± SEM. *p < 0.05, **p < 0.01 vs Ad-GFP group by Student’s t-test, or ANCOVA. Figure 3—source data 1.Numerical quantification data for Figure 3.