Aged mice exhibit decreased iWAT beiging in response to cold exposure or β3-agonist treatment.

(A) Young (9-week-old) and aged (57-week-old) C57BL/6 mice were acclimated to 30°C for 3 weeks, followed by two additional weeks remaining at 30°C (TN, thermoneutral controls), spending the last 3 days at 6°C (3D, acute cold) or last 14 days at 6°C (14D, chronic cold). (B) Relative mRNA levels of thermogenic marker genes in mouse iWAT from (A), n=5. (C) Immunofluorescence analysis of UCP1 (green) and DAPI (blue) in iWAT sections from mice in (A), LN = lymph node. Scale bar 100 μm. (D-F) Relative mRNA levels of Ucp1 and Cidea in iWAT from young and aged mice that were either: exposed to 6°C cold for 6 weeks (D), treated with CL-316,243 for 1 hour (E) or treated with CL 316,243 for 5 days (F). Data represent mean ± SEM, points represent biological replicates, 2 groups analyzed using a Student’s t-test, and multiple conditions analyzed with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001.

Aging blocks beige adipogenesis from fibroblastic ASPCs.

(A) Schematic of PdgfraCreERT2;R26RtdTomato reporter mouse model and lineage tracing paradigm. (B) Flow cytometry-based quantification showing proportion of tdTomato-expressing PDGFRα+cells (as % of total Live, Lin-, PDGFRα+ cells) in iWAT from young and aged Cre+ and Cre- (control) mice. n=6 young, 5 aged (Circles represent male mice, triangles represent female mice). (C) Immunofluorescence analysis of tdTomato (red), UCP1 (green), PLIN1 (white) and DAPI (blue) in iWAT from young and aged reporter mice after 14 days of 6°C cold exposure (chase). Scale bar 100 μm. (D) Representative stitched images of full length iWAT histology slices from samples in (C) showing quantification of traced tdTomato+;UCP1+ multilocular (beige) adipocytes (blue numbers). LN= lymph node, scale bar 500 μm. (E) Quantification of traced beige adipocytes from (D) presented as total cell number (left) or proportion of PLIN1+ area (right), n=7 (young), n=5 (aged). Data represent mean ± SEM, points represent biological replicates, 2 groups analyzed using a Student’s t-test, and multiple conditions analyzed with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001..

Single cell expression profiling of ASPCs during iWAT beiging.

(A) Fully integrated UMAP of gene expression in 54,987 stromal vascular cells (FACS depleted of CD45+ immune cells) from young and aged mouse groups detailed in Figure 1A. (B) UMAPs split by condition. (C) Violin plots showing the expression levels of representative marker genes for various cell clusters. y-axis = log-scale normalized read count. (D) Expression heatmap of the top differentially expressed genes in young vs. aged fibroblastic ASPCs (combined Dpp4+, preadipocytes and Cd142+ cells). Table shows expression of these genes in ASPC populations across temperature conditions (TN, cold 3D, cold 14D) from young and aged mice.

ASPCs from young and aged mice display similar beige adipogenic activity ex vivo.

(A, C) Phase contrast images of DPP4+, ICAM1+ and CD142+ cells from iWAT of young and aged mice that were induced to undergo adipocyte differentiation with minimal (MIN, A) or maximal (MAX, C) induction cocktail for 8 days. Scale bar 200 μm. (B, D) mRNA levels of adipocyte marker genes Adipoq and Fabp4 in cultures from (A, C). Data points represent separate wells, sorted from a pool of 5 mice (A) or sorted from two pools of 2-3 mice (C). (E) Stromal vascular fraction (SVF) cell cultures from the iWAT of young and aged mice were induced to differentiate for 8 days with MIN or MAX cocktail, followed by Bodipy (green) staining of lipid droplets and DAPI (blue) staining of nuclei. Scale bar 100 μm. (F) Relative mRNA levels of Adipoq and Fabp4 in cultures from (E). Data points represent wells from individual mice, n = 5. (G, H) Relative mRNA levels of Ucp1 in adipocyte cultures from (C, E) with or without treatment with isoproterenol for 4 hours. Data points represent wells sorted from two pools of 2-3 mice (G) or wells from individual mice, n=5 (H). Data represent mean ± SEM, 2 groups analyzed using a Student’s t-test, and multiple conditions analyzed with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001.

Single nucleus expression profiling of adipocytes during the beiging process in young and aged mice.

(A) Fully integrated UMAP of mRNA levels in 11,905 nuclei from iWAT of mouse groups detailed in Figure 1A, n=2 mice per condition. (B) UMAPs split by condition. (C) Violin plots showing expression patterns of cell cluster-selective marker genes, Y-axis = log-scale normalized read count. (D) UMAP of gene expression in re-integrated adipocyte clusters including 4,937 nuclei from (A) identifying four populations: Npr3-high, beige, DNL-low, and DNL-high. (E) Adipocyte UMAPs split by condition. (F) Violin plots showing expression patterns of selected genes in adipocyte populations, Y-axis = log-scale normalized read count. (G) Adipocyte nuclei numbers in each sample, plotted as percent of total adipocytes captured for that sample.

Aging blocks activation of the lipogenic gene program in adipocytes

(A) Expression heatmap of the top aging-regulated genes in DNL-high adipocytes at TN (left) and after 14 days of cold exposure (right). (B) Expression heatmap of the top aging-regulated genes in beige adipocytes after 14 days of cold exposure. (C) UMAP of Npr3 mRNA levels in adipocyte populations (from Figure 5D). (D) Violin plot showing Npr3 mRNA levels in adipocyte populations, Y-axis = log-scale normalized read count, first bar in beige is ‘Young 3D’. (E) Npr3 mRNA levels in iWAT from mouse groups described in Figure 1A, n=5. (F) Npr3 mRNA levels in isolated adipocytes from TN-acclimated young and aged mice, n=6. (G) UMAPs of Ucp1, Acly, and co-expression mRNA levels in adipocyte populations from all young and aged mice. (H) Heatmap showing average expression of lipogenic genes in all nuclei from DNL-high and beige adipocytes per condition indicated in the top table. Data represent mean ± SEM, points represent biological replicates, 2 groups analyzed using a Student’s t-test, and multiple conditions analyzed with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001.

(A-B) Body mass and iWAT mass of mice described in Figure 1A, n=5. (C) Mouse dissection with lymph node (LN) orientation showing the dorsolumbar and inguinal regions of the iWAT pad. (D) Immunofluorescence analysis of iWAT with UCP1 (green) and DAPI (blue). LN=lymph node. Scale bar 100 μm. (E) mRNA levels of Ucp1 and Cidea in BAT of young and aged mice housed at TN, and either maintained at TN or exposed to cold for 2 weeks. Data represent mean ± SEM, points represent biological replicates, analyzed using a Student’s t-test with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001.

(A) Representative flow cytometry plots showing expression of tdTomato in gated Live, Lin-; PDGFRα+ stromal vascular cells isolated from young and aged reporter mice (described in Figure 2) immediately after treatment with tamoxifen (tmx, pulse). (B) Immunofluorescence analysis of iWAT from young and aged reporter mice with tdTomato (red), PLIN1 (white) and DAPI (blue) after the tmx pulse, scale bar 100 μm.

(A) Violin plot showing expression of ARC marker genes in cell clusters split by age, Y-axis = log-scale normalized read count. (B) Expression heatmap of top ASPC marker genes across age and housing conditions.

(A) Violin plot showing expression levels of ARC marker genes split by age, y-axis = log-scale normalized read count. (B) Expression heatmap of the top aging-regulated genes in DNL-high adipocytes. (C) Expression heatmap of the top aging-regulated and cold-regulated genes in beige adipocytes. (D-E) Npr1 and Npr2 mRNA levels in (D) iWAT from mouse groups described in Figure 1A, n=5 and (E) isolated adipocytes from iWAT from TN-acclimated young and aged mice, n=6. (E-F) UMAP of Ucp1 (E) and Acly (F) mRNA levels in adipocyte populations (from Figure 5D). (H) Acly mRNA levels in iWAT from mouse groups described in Figure 1A, n=5. (G) Enrichment analysis displaying the top six Reactome pathways in DNL high adipocytes downregulated in aged at 14 days. Data represent mean ± SEM, points represent biological replicates, 2 groups analyzed using a Student’s t-test, and multiple conditions analyzed with a Holm-Šidák correction for multiple comparisons. Significance: not significant, P > 0.05; * P < 0.05 ** P < 0.01; *** P < 0.001.