Figures and data in Aging impairs cold-induced beige adipogenesis and adipocyte metabolic reprogramming

Figures
Tables
Additional files

6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement

Download asset Open asset

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 either remaining at 30 °C (TN, thermoneutral), spending the last 3 days at 6 °C (3D, acute cold) or the 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 separate groups of young and aged mice that were either: exposed to 6 °C cold for 6 weeks (D), treated with CL-316,243 for 1 hr (E) or treated with CL 316,243 for 5 days (F). Data represent mean ± SEM, points represent biological replicates, two groups analyzed using a Student’s t-test, and multiple conditions analyzed using a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: not significant, p>0.05; * p<0.05 ** p<0.01; *** p<0.001.

Figure 1—figure supplement 1

Download asset Open asset

Aging impairs WAT beiging.

(**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) IF analysis of UCP1 (green) in iWAT, DAPI (nuclei, 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. (F) H&E staining of serial sections of iWAT from D (above) and Figure 1C, LN = lymph node. Scale bar 100 μm. Data represent mean ± SEM, points represent biological replicates, analyzed using a Student’s t-test with a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: not significant, p>0.05; * p<0.05 ** p<0.01; *** p<0.001.

Figure 2 with 1 supplement

Download asset Open asset

Aging blocks beige adipogenesis from fibroblastic ASPCs.

(A) Schematic of *Pdgfra-CreERT2;R26R-tdTomato* reporter mouse model and lineage tracing paradigm. (B) Flow cytometry-based quantification showing proportions of tdTomato-expressing cells (as % of total Live, Lin- (CD45-/CD31-, PDGFRα+ cells)) (left) and PDGFRα+ cells (as % of total Live, Lin- cells) (right) in iWAT from young and aged Cre- (control, +/+), and Cre+ (CER) mice. n=6 young, 5 aged (Circles represent male mice, triangles represent female mice). (C) IF 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, two groups analyzed using a Student’s t-test, and multiple conditions analyzed with a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: not significant, p>0.05; * p<0.05 ** p<0.01; *** p<0.001.

Figure 2—figure supplement 1

Download asset Open asset

Aging blocks beige adipogenesis from PDGFRa +ASPCs.

(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) IF 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.

Figure 3 with 1 supplement

Download asset Open asset

Single-cell expression profiling of ASPCs during iWAT beiging.

(A) 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 cell clusters. Y-axis=log-scale normalized read count. (D) Expression heatmap of the top differentially expressed genes in young vs. aged fibroblastic ASPCs (*Dpp4*+, *Icam1*+ 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.

Figure 3—figure supplement 1

Download asset Open asset

Single-cell expression profiling of ASPCs during iWAT beiging.

(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.

Figure 4

Download asset Open asset

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 five mice (A) or sorted from two pools of two to three 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 Minimal or Maximal 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 hr. Data points represent wells sorted from two pools of two to three mice (G) or wells from individual mice, n=5 (H). Data represent mean ± SEM, two groups analyzed using a Student’s t-test, and multiple conditions analyzed with a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: not significant, p>0.05; * p<0.05 ** p<0.01; *** p<0.001.

Figure 5 with 1 supplement

Download asset Open asset

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 4937 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.

Figure 5—figure supplement 1

Download asset Open asset

Single-nucleus expression profiling of iWAT during the beiging process.

Violin plot showing marker gene levels split by age, y-axis=log-scale normalized read count.

Figure 6 with 1 supplement

Download asset Open asset

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 plots showing *Npr3* mRNA levels in adipocyte populations at TN (T), and at 3 and 14 days of cold exposure, Y-axis=log-scale normalized read count. (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 their co-expression in adipocyte populations from young and aged mice. (H) Heatmap showing average expression of DNL 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, two groups analyzed using a Student’s t-test, and multiple conditions analyzed with a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: not significant, p>0.05; * p<0.05 ** p<0.01; *** p<0.001.

Figure 6—figure supplement 1

Download asset Open asset

Single-nucleus expression profiling of adipocytes during the beiging process.

(A) Expression heatmap of the top aging-regulated genes in DNL-high adipocytes. (B) Expression heatmap of the top aging- and cold-regulated genes in beige adipocytes. (**C, D**) *Npr1* and *Npr2* mRNA levels in iWAT (from mouse groups in Figure 1A) (C), n=5 and in isolated adipocytes from iWAT (D), n=6. (**E, F**) UMAP of *Ucp1* (E) and *Acly* (F) mRNA levels in adipocyte groups (from Figure 5D). (G) Enrichment analysis of age-regulated genes in DNL high adipocytes. (H) Acly mRNA levels in iWAT (from groups in Figure 1A) n=5. Data are mean ± SEM, points represent biological replicates, two groups analyzed using a Student’s t-test, and multiple conditions analyzed with a two-way ANOVA with a Tukey correction for multiple comparisons. Significance: *p<0.05; **p<0.01; ***p<0.001.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (M. musculus)	C57BL/6 J	The Jackson Laboratory, Bar Harbor, ME	RRID:IMSR_JAX:000664
Genetic reagent (M. musculus)	C57BL/6JN	NIA, Bethesda, MD	NA
Genetic reagent (M. musculus)	Rosa26 loxp-stop-loxp tdTomato Reporter (Ai14)	The Jackson Laboratory, Bar Harbor, ME	RRID:IMSR_JAX:007914
Genetic reagent (M. musculus)	Pdgfra^CreERT2	The Jackson Laboratory, Bar Harbor, ME	RRID:IMSR_JAX:032770
Antibody	Rabbit polyclonal anti–red fluorescent protein (RFP)	Rockland, Pottstown, PA	600-401-379, RRID:AB_2209751	1:500
Antibody	Rabbit polyclonal anti-Perilipin (D418)	Cell Signaling, Denvers, MA	3470, RRID:AB_2167268	1:200
Antibody	Rabbit polyclonal anti- UCP1	Specially made by AstraZeneca, Cambridge, UK	NA	1:2000
Antibody	Rabbit polyclonal Anti-mouse CD142	Sino Biological, Chesterbrook, PA	R001	1:100
Antibody	Goat polyclonal Anti-mouse CD142	R & D Systems, Minneapolis, MN	AF3178, RRID:AB_2278143	1:50
Antibody	Rat monoclonal Anti-mouse CD140a-(PDGFRɑ)-PECy7	Biolegend, San Diego, CA	135912, RRID:AB_2715974	1:100
Antibody	Rat monoclonal Anti-mouse-CD31 (APC-Fire)	Biolegend, San Diego, CA	102528, RRID:AB_2721491	1:1000
Antibody	Rat monoclonal Anti-mouse CD45-allophycocyanin (APC/Cy7)	Biolegend, San Diego, CA	103116, RRID:AB_312981	1:1000
Antibody	Rat monoclonal Anti-mouse ICAM1-phycoerythrin (PE/Cy7)	Biolegend, San Diego, CA	116122, RRID:AB_2715950	1:100
Antibody	Rat monoclonal Anti-mouse CD26 (DPP-4)- fluorescein isothiocyanate (FITC)	Biolegend, San Diego, CA	137806, RRID:AB_10663402	1:200
Sequence-based reagent	mTbp	PMID:24703692	NA	F-GAAGCTGCGGTACAATTCCAG R-CCCCTTGTACCCTTCACCAAT
Sequence-based reagent	mAdipoq	PMID:24703692	NA	F-GCACTGGCAAGTTCTACTGCAA R-GTAGGTGAAGAGAACGGCCTTGT
Sequence-based reagent	mFabp4	PMID:24703692	NA	F-ACACCGAGATTTCCTTCAAACTG R-CCATCTAGGGTTATGATGCTCTTCA
Sequence-based reagent	mCidea	PMID:24703692	NA	F-TGCTCTTCTGTATCGCCCAGT R-GCCGTGTTAAGGAATCTGCTG
Sequence-based reagent	mPgc1a	PMID:24703692	NA	F-CCCTGCCATTGTTAAGACC R-TGCTGCTGTTCCTGTTTTC
Sequence-based reagent	mUcp1	PMID:24703692	NA	F-ACTGCCACACCTCCAGTCATT R-CTTTGCCTCACTCAGGATTGG
Sequence-based reagent	mDio2	PMID:24703692	NA	F-CAGTGTGGTGCACGTCTCCAATC R-TGAACCAAAGTTGACCACCAG
Sequence-based reagent	mAcly	PMID:31141698	NA	F-GAGTGCTATTGCGCTTCCC R-GGTTGCCGAAGTCACAGGT
Sequence-based reagent	mNpr3	This Paper	NA	F-TTTTCAGGAGGAGGGGTTGC R-ACACATGATCACCACTCGCT
Sequence-based reagent	mNpr1	MGH PrimerBank	Primer Bank ID: 113930717 c1	F-GCTTGTGCTCTATGCAGATCG R-CCTCGACGAACTCCTGGTG
Sequence-based reagent	mNpr2	MGH PrimerBank	Primer Bank ID: 118129825 c2	F-CATGACCCCGACCTTCTGTTG R-CGAACCAGGGTACGATAATGCT
Commercial assay or kit	ABI High-Capacity cDNA Synthesis kit	Applied Biosystems, Waltham, MA	4368813
Commercial assay or kit	Purelink RNA Mini columns	Invitrogen, Waltham, MA	LT-12183018
Commercial assay or kit	TSA TMR Tyramide Reagent Pack	Akoya Biosciences, Marlborough, MA	NEL742001KT
Commercial assay or kit	TSA Fluorescein Tyramide Reagent Pack	Akoya Biosciences, Marlborough, MA	NEL741001KT
Commercial assay or kit	Bulls Eye Decloaking Buffer	Biocare, Pacheco, CA	BULL1000 MX
Commercial assay or kit	AbC Total Antibody Compensation Bead Kit	BioLegend,San Diego, CA	A10497
Commercial assay or kit	Biotium Mix-n-Stain CF647	Sigma, Burlington, MA	MX647S100
Commercial assay or kit	PicoPure RNA Isolation Kit	Invitrogen, Waltham, MA	KIT0204
Commercial assay or kit	Qubit dsDNA High Sensitivity assay kit	ThermoFisher, Waltham, MA	Q32851
Commercial assay or kit	DNA High Sensitivity Bioanalyzer Chip (Agilent)	Agilent, Santa Clara, CA	5067–4626
Software, algorithm	Graphpad Prism	Graphpad, San Diego, CA	RRID:SCR_002798
Software, algorithm	Adobe Illustrator	Adobe, San Jose, CA	RRID:SCR_010279
Software, algorithm	Adobe Photoshop	Adobe, San Jose, CA	RRID:SCR_014199
Software, algorithm	Image J	PMID:22743772	RRID:SCR_003070
Software, algorithm	Cell Ranger	10 x Genomics	RRID:SCR_017344
Software, algorithm	Seurat	PMID:34062119	RRID:SCR_016341
Software, algorithm	bcl2fastq	Illumina	RRID:SCR_015058
Software, algorithm	Cumulus	PMID:32719530	RRID:SCR_021644
Software, algorithm	FACSDiva Softward	Becton Dickinson, Franklin Lakes, NJ	RRID:SCR_001456
Other	Tamoxifen (Free Base)	Sigma, Burlington, MA	T5648	Synthetic estrogen receptor antagonist used to activate Cre.
Other	Corn Oil	Sigma, Burlington, MA	C8267	Vehicle solution for tamoxifen.
Other	16% Paraformaldehyde	EMS, Hatfield, PA	15710	Fixative used for tissue histology
Other	TRIzol	Invitrogen, Waltham, MA	15596018	Phenol-based solution used for nucleic acid extraction
Other	CL-316,243	Sigma, Burlington, MA	C5976	Agonist of Beta3-adrenergic receptor
Other	4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI), 1:10,000	Roche, Basel, Switzerland	10236276001	Fluorescent stain for DNA/nuclei
Other	Bovine Serum Albumin, fraction V, fatty-acid free	Gold Biotechnology, St. Louis, MO	A-421–250	Protein carrier for small molecules
Other	DMEM/F12	Fisher Scientific, Waltham, MA	11320033	Basal cell culture medium
Other	Fetal Bovine Serum	Omega Scientific, Tarzana, CA	FB-11, Lot 401714	For cell culture
Other	Primocin	InvivoGen, San Diego, CA	ant-pm-2	Anti-microbial for cell culture
Other	PCR Master Mix, Power SYBR Green	Applied Biosystems, Waltham, MA	4367659	Kit for qRT-PCR
Other	HBSS, 1 X	Fisher Scientific, Waltham, MA	14175079	Hank’s Balanced Salt Solution
Other	Dispase II	Roche, Basel, Switzerland	4942078001	Enzyme used for adipose tissue digestion
Other	Collagenase, Type 1	Worthington, Lakewood, NJ	LS004197	Enzyme used for adipose tissue digestion
Other	Red Blood Cell Lysis Buffer, 10 x	BioLegend, San Diego, CA	420302	For lysing red blood cells during cell isolations
Other	Human Insulin, Novolin	Novo Nordisk, Bagsvaerd, Denmark	183311	Used for cell culture studies
Other	Dexamethasone	Sigma-Aldrich, Burlington, VT	D4902	Glucorticoid Receptor agonist
Other	3-isobutyl-1-methylxanthine (IBMX)	Sigma-Aldrich, Burlington, VT	I7018	Chemical used to Increase cAMP levels, used in adipocyte differentiation cocktail
Other	Rosiglitazone	Cayman Chemical, Ann Arbor, MI	11884	Synthetic PPARgamma activator
Other	Indomethacin	Sigma-Aldrich, Burlington, VT	I8280	Chemical used in adipocyte differentiation cocktail
Other	3,30,5-Triiodo-L-thyronine sodium salt (T3)	Sigma-Aldrich, Burlington, VT	T6397	Thyroid Receptor agonist
Other	isoproterenol	Sigma-Aldrich, Burlington, VT	I6504	Pan beta-adrenergic receptor agonist
Other	Bodipy 493/503	Invitrogen, Waltham, MA	D3922	Fluorescent dye for neutral lipids
Other	Hoechst 33342	Thermo Fisher, Waltham, MA	62249	DNA stain
Other	Protector RNase Inhibitor	Roche, Basel, Switzerland	3335399001	Used for RT-PCR