NBR1 is a critical step in the repression of thermogenesis of p62-deficient adipocytes through PPARγ

Figures

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  Fig. 1. Loss of NBR1 inhibits increased adiposity of Sqstm1-deficient mice. a–d Body mass (a) and fat tissue masses of eWAT (b), iWAT (c), and BAT (d) from WT and total body knockout mice at 10–12 weeks of age. WT (n = 18), Sqstm1–/– (n = 21), Nbr1–/– (n = 23), and Sqstm1–/–Nbr1–/– (n = 26). p = 0.0422 WT vs Sqstm1–/–, p = 0.0281 vs Nbr1–/– (a), p < 0.0001 WT vs Sqstm1–/–, p = 0.0142 WT vs Sqstm1–/–Nbr1–/–, p < 0.0001 Sqstm1–/– vs Sqstm1–/–Nbr1–/– (b), p < 0.0001 WT vs Sqstm1–/– and Sqstm1–/– vs Sqstm1–/–Nbr1–/– (c, d). e Representative H&E staining of BAT and eWAT (n = 5, per genotype). Scale bar: 100 μm (BAT) and 200 μm (eWAT). f, g Adipocyte size measurement from H&E staining of eWAT described above (n = 5 mice, per genotype). Distribution range and frequency (f) and mean diameter of adipocyte size (g) were shown. p = 0.045 Sqstm1–/– vs Sqstm1–/–Nbr1–/– (<30), p = 0.0214 Sqstm1–/– vs WT, p = 0.0071 vs Sqstm1–/–Nbr1–/– (30–40), p = 0.0136 Sqstm1–/– vs Sqstm1–/–Nbr1–/– (40–50), p = 0.0083 Sqstm1–/– vs WT, p = 0.0008 vs Sqstm1–/–Nbr1–/– (60–70), p = 0.0323 Sqstm1–/– vs WT, p = 0.0237 vs Sqstm1–/–Nbr1–/– (70–80) (f), p = 0.0243 Sqstm1–/– vs WT, p = 0.0191 vs Sqstm1–/–Nbr1–/– (g). h qPCR…

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  Fig. 2. Role of adipocyte NBR1 in the control of adiposity. a Recording body weight of adipocyte-specific knockout male mice and WT controls from 13 to 24 weeks of age. Sqstm1f/f (n = 20), Sqstm1AKO (n = 20), Nbr1f/f (n = 19), Nbr1AKO (n = 16), Sqstm1f/fNbr1f/f (n = 18), and Sqstm1AKONbr1AKO (n = 15). p = 0.0261 (13 w), p = 0.0041 (14 w), p = 0.0003 (15 w), p < 0.0001 (16–24 w) Sqstm1AKO vs Sqstm1f/f, p = 0.0177 (22 w), p = 0.012 (23 w), p = 0.0018 (24 w) Sqstm1AKONbr1AKO vs Sqstm1f/fNbr1f/f. b–d Body mass (b) and fat tissue weight of BAT (c) and eWAT, iWAT (d) from adipocyte-specific knockout male mice at 25–28 weeks of age. Sqstm1f/f (n = 14), Sqstm1AKO (n = 13), Nbr1f/f (n = 16), Nbr1AKO (n = 14). Sqstm1f/fNbr1f/f (n = 13) and Sqstm1AKONbr1AKO (n = 17). p = 4.73 × 10−7 Sqstm1AKO vs Sqstm1f/f, p = 3.08 × 10−5 vs Sqstm1AKONbr1AKO, p = 0.046 Sqstm1AKONbr1AKO vs Sqstm1f/fNbr1f/f (b), p = 1.81 × 10−5 Sqstm1AKO vs Sqstm1f/f, p = 0.001 vs Sqstm1AKONbr1AKO (c), p = 2.4 × 10−7 Sqstm1AKO vs Sqstm1f/f, p = 4.33 × 10−6 vs Sqstm1AKONbr1AKO, p = 0.014 Sqstm1AKONbr1AKO vs Sqstm1f/fNbr1f/f (d, left), p = 1.48 × 10−8 Sqstm1AKO vs Sqstm1f/f, p = 6.39 × 10−6 vs Sqstm1AKONbr1AKO, p = 0.01…

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  Fig. 3. Role of adipocyte NBR1 in the control of glucose intolerance and insulin resistance. a–c Glucose tolerance test (GTT) were performed in Sqstm1AKO (a), Nbr1AKO (b), Sqstm1AKONbr1AKO (c) male mice, and their respective controls at 25–28 weeks of age. Sqstm1f/f (n = 7), Sqstm1AKO (n = 5), Nbr1f/f (n = 7), Nbr1AKO (n = 6), Sqstm1f/fNbr1f/f (n = 5), and Sqstm1AKONbr1AKO (n = 6). Longitudinal graph: p < 0.0001, p = 0.002, p = 0.0319 (a), p = 0.0318 (c). Area under curves (AUC) were calculated from GTT. p = 0.0033 (a). d–f Insulin tolerance test (ITT) was performed in Sqstm1AKO (d), Nbr1AKO (e), Sqstm1AKONbr1AKO (f) male mice and their respective controls at 25–28 weeks of age. Results are presented as percent of the initial glucose levels. Sqstm1f/f (n = 7), Sqstm1Ako (n = 6), Nbr1f/f (n = 7), Nbr1AKO (n = 6), Sqstm1f/fNbr1f/f (n = 8), and Sqstm1AKONbr1AKO (n = 11). Longitudinal graph: p = 0.0273, p = 0.0002, p = 0.0035 (d). AUC was calculated from ITT. Data are presented as mean ± SEM (a–f). *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Two-way ANOVA followed by Bonferroni’s post-test (a–f longitudinal graphs). Two-tailed Student’s T-test (a–f AUC bar graphs). Source data are…

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  Fig. 4. Role of NBR1 in systemic energy expenditure. A metabolic characterization of the adipocyte-selective KO mice was performed by using an automated indirect calorimetry system (CLAMS) in male mice at 50–55 weeks of age. Sqstm1f/f (n = 7), Sqstm1AKO (n = 6), Nbr1f/f (n = 5), Nbr1AKO (n = 4), Sqstm1f/fNbr1f/f (n = 7), and Sqstm1AKONbr1AKO (n = 8). a Regression plots of Energy expenditure (EE) against body weight. ANCOVA test using body weight as covariate. p = 0.043 Sqstm1AKO vs Sqstm1f/f. b Predicted energy expenditure at the mean body weight of respective flox control mice. The mean values of body weight are 33.1 g in Sqstm1f/f, 33.46 g in Nbr1f/f, and 31.24 g in Sqstm1f/fNbr1f/f mice. Two-tailed Student’s T-test. p = 0.0004 Sqstm1AKO vs Sqstm1f/f. BW body weight. c Respiratory exchange rate (RER) was recorded and plotted. Two-way ANOVA followed by Bonferroni’s post-test. p = 0.0421 (4 h), p = 0.0196 (5 h), p = 0.0308 (28 h) Sqstm1AKO vs Sqstm1f/f, p = 0.0126 (48 h) Nbr1AKO vs Nbr1f/f. d Quantification of respective AUC from (c) was analyzed by two-tailed Student’s T-test. p = 0.0012 Sqstm1AKO vs Sqstm1f/f. Data are presented as mean ± SEM (b–d). *p < 0.05, **p < 0.01, ***p < 0.001.…

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  Fig. 5. Role of NBR1 in adaptive thermogenesis in BAT and inguinal WAT. a–c Male mice at 25 weeks of age were subjected to acute cold exposure (4 °C) for 7 h to stimulate brown thermogenesis. a Rectal core temperature was measured for consecutive 7 h. Sqstm1f/f (n = 10), Sqstm1AKO (n = 6), Sqstm1f/fNbr1f/f (n = 9), and Sqstm1AKONbr1AKO (n = 10). Two-way ANOVA followed by Bonferroni’s post-test. b Representative H&E staining in BAT of indicated mice (n = 3, per genotype). Scale bar = 100 μm. c qPCR analysis of thermogenesis-related genes in BAT of mice. Results are presented as change fold related to individual controls. Sqstm1f/f (n = 7), Sqstm1AKO (n = 5), Sqstm1f/fNbr1f/f (n = 6), and Sqstm1AKONbr1AKO (n = 6). Two-tailed Student’s T-test. d–g Male mice at 25 weeks of age were injected with CL316,243 or saline as control for consecutive 5 days. d Representative H&E staining in iWAT of indicated mice. Sqstm1f/f (n = 4), Sqstm1AKO (n = 4), Sqstm1f/fNbr1f/f (n = 3), and Sqstm1AKONbr1AKO (n = 3). Scale bar = 100 μm. e, f Immunoblot analysis of mitochondrial OXPHOS genes and UCP1 in BAT of Sqstm1AKO (e) and Sqstm1AKONbr1AKO (f) and their respective controls (n = 5, per genotype). g…

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  Fig. 6. Role of NBR1 in adaptive thermogenesis in primary brown adipocytes. a qPCR analysis of Ucp1 expression in primary brown adipocytes differentiated from SVF of four indicated genotypes (n = 5 biological replicates). b Immunoblot analysis of UCP1 in primary brown adipocytes of four indicated genotypes, treated with ISO (0.5 µM) for indicated time. Representative blots (left) and quantification (right) from three independent experiments were shown. c, d Immortalized SVF from WT, Sqstm1–/–, and Sqstm1–/–Nbr1–/– mice were differentiated into brown adipocytes for Seahorse analyses. c Oxygen consumption rate (OCR) were determined (n = 6 biological replicates). Left: plot of OCR to time measured by Seahorse. Right: calculated ISO-stimulated respiration levels after subtracting levels of basal proton leak. d Extracellular acidification rate (ECAR) were measured (n = 6 biological replicates). Left: plot of ECAR to time measured by Seahorse. Right: calculated ISO-stimulated ECAR values after subtracting oligomycin-treated levels. Data are presented as mean ± SEM (a–d). *p < 0.05, **/##p < 0.01, ***p < 0.001, ****/####p < 0.0001. Two-tailed Student’s T-test (a, c, d bar graphs). Two-way ANOVA…

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  Fig. 7. p62 and NBR1 interact with PPARγ in brown adipocytes. a Representative immunoblotting of p62 and NBR1 levels in cytoplasmic/nuclear fractions in primary brown adipocytes. Cells were treated with 0.5 µM isoproterenol (ISO) for indicated time. Densitometric quantification of nuclear NBR1 and p62 levels was also shown (n = 3 independent experiments). b, c Endogenous interaction of PPARγ with p62 and NBR1. p62 (b) or PPARγ (c) immunoprecipitates from nuclear lysates extracted from ISO and rosiglitazone-treated brown adipocytes were analyzed for the levels of specified proteins. Representative blots and densitometric quantification were shown (n = 3 independent experiments for both). d, e Recombinant FLAG-p62 (d) or FLAG-NBR1 (e) was incubated with GST and GST-PPARγ proteins separately and the interactions were analyzed by immunoblotting in glutathione-beads pull-down. Representative blots and densitometric quantification were shown (n = 4 independent experiments for d and n = 3 independent experiments for e). f HEK293T cells were transfected with cDNA vectors expressing WT/mutants of HA-p62 or HA-NBR1, and GST-PPARγ. The interacting proteins were pulled down using glutathione-beads…

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  Fig. 8. p62 and NBR1 regulate PPARγ-RXRα heterodimerization to control thermogenesis in brown adipocytes. a FLAG-RXRα, GST-PPARγ, WT/mutants of HA-p62, and HA-NBR1 were overexpressed in HEK293T cells and the interaction of RXRα with PPARγ was analyzed by immunoblotting in pull-downs using glutathione-beads against GST-PPARγ, in the present of NBR1 and/or p62. Representative immunoblotting and densitometric quantification were shown (n = 3 independent experiments). EV empty vector, wt wild-type, mu mutant. b Endogenous interaction of PPARγ with RXRα in BAT of mice exposed to cold for 7 h. PPARγ immunoprecipitates were analyzed by immunoblotting. Densitometric quantification was shown (n = 6, per Sqstm1f/f, Sqstm1AKO, Sqstm1f/fNbr1f/f, and Sqstm1AKONbr1AKO). c Endogenous interaction of PPARγ with RXRα in double KO iBAs (sgSqstm1sgNbr1) reconstituting p62, NBR1, or both. PPARγ immunoprecipitates were analyzed by immunoblotting. Densitometric quantification was shown (n = 3 independent experiments). d Luciferase assay determining transcriptional activity of PPARγ in iBAs transfected with indicated cDNA vectors, cells were treated with ISO (1 µM) and rosiglitazone (1 µM) for 48 h (n = 6…

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  Fig. 9. Model for the role of p62 and NBR1 in the regulation of PPARγ:RXRα heterodimerization and PPARγ-mediated thermogenic program in brown adipocytes. p62 and NBR1 translocate into the nucleus of brown adipocytes upon adrenergic stimulation and form a multi-protein complex with the nuclear receptor PPARγ and its obligate co-activator RXRα. p62 separately interacts with PPARγ and RXRα, increasing their nuclear proximity and facilitates their heterodimerization to drive thermogenic gene expression. NBR1 emerges as an opposite player that impairs PPARγ:RXRα complex formation and decreases PPARγ activity through direct interaction with PPARγ. The fact that p62 and NBR1 synergistically bind to PPARγ allows p62 to restrain NBR1 from its inhibitory binding to PPARγ. Nuclear p62 serves as a thermogenic promoter contributing to PPARγ activation in brown adipocytes.