Fig 2: Impaired energy expenditure and BAT thermogenesis in obese p62Δ69-251 mice.Energy expenditure (EE) plotted against body weight and ANCOVA predicted energy expenditure at a given body weight of 40 g in obese 32-wk-old male C57Bl/6J wt or p62Δ69-251 mice fed a regular chow diet. When considering body weight as covariate, the ANCOVA genotype effect is p = 0.016 (a). Predicted energy expenditure (b) and locomotor activity during the dark phase (c) of 26-wk-old male mice at different environmental temperatures. Energy expenditure of male mice during a norepinephrine (NE) challenge at 30 °C (d). PET/MRI imaging of male 8-mths-old male mice with standardized uptake values (SUV); white arrow indicates location of BAT (e). Cytochrome c oxidase (COX) activity normalized to protein concentration (f), thermogenic gene expression profiling in BAT (g) and iWAT (h), and protein levels of p-p38, p38, ATF2, p-ATF2 (Thr69/71), and GAPDH (i) in BAT of obese 33-wk-old male mice. Samples sizes are (a) n = 8/7 mice, (b, c) n = 8/6 mice, (d) n = 8/8 mice, (e) n = 5/7 mice, (f) n = 7/7 mice, (g) Ucp1 n = 8/7 mice; Pgc-1α n = 7/7 mice, Prdm16 n = 8/7 mice, Dio2 n = 7/6 mice, Cidea n = 8/7 mice, (h) Ucp1 n = 8/6 mice, Pgc-1α n = 8/7 mice, Prdm16 n = 8/7 mice, Cidea n = 8/7 mice, (i) representative western blot of n = 4 (out of n = 6) p62 wt and n = 4 (out of n = 6) p62Δ69-251 mice and quantification of pATF2/GAPDH in n = 6 mice each genotype. The full blot with n = 6 mice each genotype is provided in the Source data file. Data represent mean ± SEM. Data in panels (a) and (b) were analyzed using ANCOVA using body weight as covariate. Bar graphs in panels (a), (b), (c), (e), (f), (g), (h), and (i) were analyzed using two-side two-tailed t-test. Data in (d) were analyzed using two-way ANOVA using time and genotype as covariates and with Bonferroni post hoc multiple comparison test for each individual time-point. *p < 0.05; **p < 0.01; ***p < 0.001. Exact p-values are given in the Source data file. Hprt hypoxanthine guanine phosphoribosyltransferase, Prdm16 PR/SET domain 16, Cidea cell death-inducing DNA fragmentation factor alpha subunit-like effector A, Dio2 deiodinase 2, GAPDH Glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: Cell autonomous regulation of energy expenditure by p62.Oxygen consumption rate (a), as well as basal and proton leak respiration (b) in BAT primary cells harvested from 6- to 8-wk-old male wt or p62Δ69-251 mice. Western blot analysis of p-ATF2 (Thr69/71) and UCP1 (c) and gene expression of Ucp1 and Pgc-1α (d) in 6 d differentiated BAT primary cells harvested from 6- to 8-wk-old male wt or p62Δ69-251 mice and which have been stimulated with saline (vhcl) or isoproterenol (0.5 μM) for 6 h. For measurement of oxygen consumption (a), basal respiration (b) and proton leak respiration (c), BAT primary cells were harvested from p62 wt or p62Δ69-251 mice, plated with equal number of cells in n = 4/5 wells each genotype and individually differentiated into mature brown adipocytes. Oxygen consumption was then measured in n = 4/5 individually differentiated wells each genotype with n = 3 technical replicates each well. For data in panel (c), BAT primary cells were harvested from p62 wt or p62Δ69-251 mice, plated with equal number of cells in n = 3 wells each treatment group and individually differentiated into mature brown adipocytes. Protein level of p-ATF2, GAPDH and UCP1 was then measured in n = 3/3/3/3 individually differentiated wells. For data in panel (d), BAT primary cells were harvested from p62 wt or p62Δ69-251 mice, plated with equal number of cells in n = 3 wells each treatment group and individually differentiated into mature brown adipocytes. Target gene expression was then measured in n = 3/3/3/3 individually differentiated wells with n = 2 technical replicates each well. Panels (a) and (d) are representative examples of two independently performed studies, each yielding similar results. Data in panel (a) have been analyzed using two-way ANOVA with time and genotype as co-variant and using Bonferroni post hoc multiple comparison to test for differences in individual time points. Bar graphs have been analyzed using two-sided two-tailed t-test. Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Exact p-values are given in the Source data file.

Fig 4: p62 regulation of ATF2 nuclear target activation.Co-immunoprecipitation (IP) in HEK293T cells transfected with Flag-p62 wt, Flag-p62Δ69-251, or HA-ATF2, respectively, and stimulated with isoproterenol as indicated. Flag-IP was performed to pulldown p62. Western blot analysis of pulldown (upper image) and input fraction (lower image) (a). Western blot analysis of ATF2 and p-ATF2 upon p62 overexpression in primary brown adipocytes isolated from 8-wk-old male p62Δ69-251 mice (b). Nuclear/membrane and cytosolic protein level of ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3), and UCP1 in BAT of 12-wk-old male wt and p62Δ69-251 mice upon injection of norepinephrine (1 mg kg−1) for 20 min (c). Nuclear/membrane and cytosolic protein level of p38, p-p38, GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 in BAT of male wt and p62Δ69-251 mice upon injection of norepinephrine (1 mg kg−1) for 20 min (d). Nuclear/membrane and cytosolic protein level of ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 upon 4 h stimulation with 1 µM isoproterenol in immortalized brown adipocytes from 6- to 8-wk-old male p62flx/flx Ucp1-Cre− or p62flx/flx Ucp1-Cre+ mice (e). Nuclear/membrane and cytosolic protein level of p38, p-p38, ATF2, p-ATF2 (Thr69/71), GAPDH, Histone 3 (H3) as well as total lysates showing UCP1 upon 4 h stimulation with 10 µM isoproterenol in immortalized brown adipocytes from wt or p62−/− mice (f). Panel (a) represents a representative example of three independently performed studies, each yielding similar results. Panels (b), (c), (d), and (f) are representative examples of two independently performed studies, each yielding similar results.