ASK1 inhibits browning of white adipose tissue in obesity

Fig. 1. LPS treatment blunts UCP1 expression in subcutaneous adipocytes ASK1-dependently. a, b Western blot and quantified protein levels of UCP1 in total lysates of inguinal white adipose tissue harvested from cold-exposed C57BL/6J mice chronically treated with or without LPS. n = 3 mice per group. **p = 0.004. c Ucp1 mRNA expression in inguinal fat explants harvested from C57BL/6J mice were treated with or without 100 ng/ml LPS and subsequently with 10 µM isoproterenol for 8 h. n = 3 biological replicates. *p = 0.036. d Ucp1 mRNA expression in subcutaneous adipocytes pre-treated with or without 100 ng/ml LPS for 24 h followed by stimulation with 0.1 µM isoproterenol for 6 h. n = 7 (LPS) or n = 8 (control) biological replicates. **p = 0.007. e, f Western blot and quantified protein levels of ASK1 in total lysates of inguinal white adipose tissue harvested from C57BL/6J mice fed a chow or HFD for 20 weeks. n = 8 (chow) and n = 7 (HFD) mice per group. *p = 0.037. g Ask1 mRNA expression in subcutaneous adipocytes treated with or without 100 ng/ml LPS treatment for 24 h. n = 7 biological replicates. *p = 0.046. h Ucp1 mRNA expression in subcutaneous adipocytes transfected with control shRNA…

Fig. 2. Reduced body weight and improved glucose tolerance in HFD-fed ASK1Δadipo mice. a Protein levels of ASK1 in lysate of isolated epididymal adipocytes harvested from ASK1F/F and ASK1Δadipo mice (n = 3 mice per group). b, c Body weight and body weight gain during 12 weeks of chow (ASK1F/F, n = 20 mice; ASK1∆adipo, n = 13 mice) or HFD feeding (ASK1F/F, n = 52 mice; ASK1∆adipo, n = 58 mice). **p = 0.02, ***p < 0.001. d, e Intraperitoneal glucose-tolerance test (chow-fed: ASK1F/F, n = 10 mice; ASK1∆adipo, n = 8 mice; HFD-fed: ASK1F/F, n = 13 mice; ASK1∆adipo, n = 14 mice) at 18 weeks of age. **p = 0.03, ***p < 0.001. f Glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamps in HFD-fed ASK1F/F (n = 6) and ASK1∆adipo (n = 8) mice. *p = 0.011. g Glucose uptake into respective tissues during hyperinsulinemic-euglycemic clamps in HFD-fed ASK1F/F (n = 5 (ing WAT, epiWAT and skeletal muscle) or n = 6 (BAT)) and ASK1∆adipo (n = 4 (BAT), n = 6 (ingWAT) or n = 7 (epiWAT and skeletal muscle)) mice. *p = 0.034, #p = 0.062. h Endogenous glucose production (EGP) during hyperinsulinemic-euglycemic clamps in HFD-fed ASK1F/F (n = 6) and ASK1∆adipo (n = 8) mice. *p = 0.040. i Liver…

Fig. 3. Increased heat production in HFD-fed ASK1Δadipo mice. a Light (day) and dark (night) oxygen consumption in HFD-fed ASK1F/F (n = 8) and ASK1Δadipo (n = 11) mice during a 24-h period at RT. **p = 0.020 (light), **p = 0.010 (dark). b Light (day) and dark (night) oxygen consumption in HFD-fed ASK1F/F (n = 7) and ASK1Δadipo (n = 6) mice during a 24-h period after 20 days housing at thermoneutrality. #p = 0.073, *p = 0.042. c Light (day) and dark (night) heat production in HFD-fed ASK1F/F (n = 8) and ASK1Δadipo (n = 11) mice during a 24-h period at RT. **p = 0.009. d Rectal temperature measured in HFD-fed ASK1F/F (n = 7) and ASK1Δadipo (n = 7) mice. *p = 0.015. Values are expressed as mean ± SEM. Statistical tests used: two-sided t-tests for (a–d). Source data are provided as a Source Data file.

Fig. 4. Increased adipose tissue browning in HFD-fed ASK1Δadipo mice. a, b Fat pad mass as well as total body fat mass (chow-fed: ASK1F/F, n = 13; ASK1∆adipo, n = 10; HFD-fed: ASK1F/F, n = 10; ASK1∆adipo, n = 16) in mice at 18 weeks of age. **p = 0.005; ***p < 0.001. c Representative image of inguinal adipose tissue of a HFD-fed ASK1F/F and ASK1∆adipo mouse (scale bar: 10 mm). d Representative immunohistochemistry for UCP1 in inguinal adipose tissue of a HFD-fed ASK1F/F (n = 3 mice) and ASK1∆adipo mouse (n = 3 mice) (scale bar: 50 µm). e mRNA expression of respective genes in inguinal adipose tissue of HFD-fed ASK1F/F (n = 8) and ASK1Δadipo (n = 9) mice. *p = 0.027 (Ucp1), *p = 0.028 (Cidea). f, g Representative western blot and quantification of UCP1 and PGC1α protein levels in inguinal adipose tissue harvested from HFD-fed ASK1F/F (n = 8) and ASK1Δadipo (n = 10) mice. *p = 0.031 (Ucp1), *p = 0.015 (Cidea). h, i Representative western blot and quantified protein levels of UCP1 in total lysates of inguinal white adipose tissue harvested from cold-exposed mice chronically treated with or without LPS (saline ASK1F/F, n = 6 mice; saline ASK1∆adipo, n = 5 mice; LPS ASK1F/F, n = 7 mice; LPS…

Fig. 5. Adipocyte-specific ASK1 overexpression decreases adipose tissue browning. a Protein levels of ASK1 in lysate of isolated adipocytes harvested from CAG-ASK1F/F and CAG-ASK1+adipo mice (n = 2 mice per group). b mRNA expression of respective genes in inguinal adipose tissue of cold-exposed CAG-ASK1F/F (n = 5) and CAG-ASK1+adipo (n = 6) mice. #p = 0.094, *p = 0.042, *p = 0.017. c, d Representative western blot and quantification of UCP1 protein levels in inguinal adipose tissue harvested from cold-exposed CAG-ASK1F/F (n = 7) and CAG-ASK1+adipo (n = 8) mice. *p = 0.037. Values are expressed as mean ± SEM. Statistical tests used: two-sided t-tests for (b) (Ucp1 and Pgc1α), d two-sided Mann–Whitney for (b) (Cidea). Source data are provided as a Source Data file.

Fig. 6. ASK1 phosphorylates IRF3. a, b Representative western blot and quantification of pIRF3 protein levels in isolated adipocytes of HFD-fed ASK1F/F and ASK1Δadipo mice. n = 6 mice per group. *p = 0.048. c, d Representative western blot and quantification of pIRF3 upon incubation of recombinant IRF3 with or without active ASK1. n = 6 biological replicates. ***p = 0.0006. e, f Western blot and quantification of pIRF3 protein levels in subcutaneous adipocytes transfected with lentivirus expressing wild-type (pLV-CMV-ASK1; ASK1 wt) or kinase-negative (pLV-CMV-ASK1-K716R; ASK1 mut) ASK1. n = 3 biological replicates. #p = 0.070. g, h Representative western blot and quantification of pIRF3 protein levels in subcutaneous adipocytes transfected with control siRNA (Co siRNA) or siRNA targeting ASK1 (ASK1 siRNA) treated with or without 100 ng/ml LPS for 6 h. n = 4 biological replicates. ****p < 0.0001. i Ucp1 mRNA expression in subcutaneous adipocytes transfected with control shRNA lentivirus (shLuc) or shRNA lentivirus targeting IRF3 (shIRF3) pre-treated with 100 ng/ml LPS for 24 h followed by stimulation with 0.1 µM isoproterenol for 6 h. n = 6 biological replicates. *p = 0.019. Values are…