Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy

Figure 1. Cold exposure induces weight loss and browning in obese mice. C57BL/6 mice on either normal chow (10% kcal fat) or HFD (60% kcal fat) were maintained at thermoneutrality (30°C, Warm) or exposed to 4°C (Cold) after adaptation at 18°C. (A) The design of the experiment. (B) VAT and SCAT weights after 10-days 4°C cold exposure (N ≥ 18). *P < 0.05, ***P < 0.001 by Student’s t-test. Data are shown as mean ± SEM. (C) Representative IF for UCP1 (red) in lean SCAT and VAT. (D) Western blot analysis for UCP1 expression in lean mice maintained at thermoneutrality or exposed to 4°C for an indicated time as in Fig. 1A. HSP90 serves as loading control. (E) Representative IF for UCP1 (red) in HFD-fed SCAT and VAT. DAPI (blue) stains cell nuclei. (F) Western blot analysis for UCP1 expression in obese mice maintained at thermoneutrality or exposed to 4°C for an indicated time as in Fig. 1A. HSP90 serves as loading control. (G) H&E staining in lean SCAT and VAT. (H) H&E staining in HFD-fed SCAT and VAT. CLS in Warm VAT are indicated by triangles. Inlets highlight separate areas within the same section exhibiting multilocular beige adipocytes.

Figure 2. Cold exposure induces VAT remolding in obese mice. HFD-fed mice were maintained at 30°C (Warm) or exposed to 4°C for 10 days (Cold) as indicated in Fig. 1A. Representative IF in HFD-fed SCAT (A) and VAT (A–H) were shown. (A) CD206 (green) and F4/80 (red) IF. CLS are indicated by triangles. (B) Collagen I (red) and Arg1 (green) IF. (C) Picro Sirius Red staining imaged with polarized light microscopy, visualizing collagen fibers type I (thick fibers, yellow-orange) and type III (Thin fibers, green). (D) CD206 (red) and Lyve1 (green) IF. Red blood cells (arrow), Lyve1+ ATM2 (Mac), Lyve1+ lymphatic vessels (LV) are indicated. (E) Hyaluronan fibers (HABP-stain, red) and Lyve1 (green) IF. Arrows indicate ATM2 (F) aSMA (red) and Lyve (green) IF. (G) TUBB3 (red) IF. Sympathetic nerves (SN) are indicated by triangles, surrounding blood vessel (BV). Higher magnification image is shown at side. (H) Lyve1 (green) and TH (left) or UCP1 (right) (red) IF in adjacent sections (right two). Higher magnification images of the inlets (yellow dashed boxes) are also shown at left side. Note TH+ nerves (triangles) were closely associated with beige adipocytes (red) and blood vessels (BV). NB, nerve…

Figure 3. Cold exposure recruits newly differentiated ATM2. HFD-fed mice were maintained at 30°C (W: Warm) or exposed to 4°C for 10 days (C: Cold) as indicated in Fig. 1A. (A) Total SVF cell count per fat pad. (B) Frequencies of CD45+ cells in live SVF. (C) Frequencies of CD45+/F4/80+/CD11b+/Gr1−/Fcer1−/siglec-f− total ATM in total SVF. (D) Representative plots for M2/M1 ATM populations. (E) Ratio of the frequencies of ATM2 (CD11c−/CD206+) over ATM1 (CD11c+/CD206−). (F) Frequencies of CD45−/CD31−/PDGFRα+/Sca1+/CD29+ AP in SVF. (G–I) The effects of anti-CSF1R antibody treatment in HFD-fed mice during cold exposure, using the same gating strategy as above. The frequencies of ATM1, ATM2, AP, and BEC in SCAT (G) and VAT (H) in SVF. (I) Cytokine concentrations in VAT lysate. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test. Data are shown as mean ± SEM.

Figure 4. ATM and AP gene expression after cold exposure. Gene expression analysis in obese VAT ATM and AP: HFD-fed mice were maintained at 30°C (Warm) or acclimated at 18°C for 7 days before exposure to 4°C for 8 days (Cold). ATM1, ATM2 and AP were FACS sorted from VAT for RNA-Seq analysis. (A) tSNE analysis. (B) The expression of Itgax, Emr1, Lyve1, and Th. (C) The expression of adrenergic receptor genes. (D) Gene Ontology (GO) enrichment analysis of differentially expressed genes between warm and cold conditions in ATM1, ATM2, and AP. (E) Heatmap showing ATM expression of various genes in indicated GO pathways (ECM organization: GO 0030198; PPAR signaling pathway: KEGG 03320; Inflammatory response: GO 0006954). For expression heatmaps, data (log2(RPKM)) are standardized along each gene (row), and color scale corresponds to the relative expression level. (F) Expression of ECM-related genes that were regulated by cold-exposed in ATM1. (G) Induction of transcription activator genes implicated in beige/brown adipogenesis in AP. **P < 0.01 by Student’s t-test. Data are shown as mean ± SEM (N = 3 samples, 3 mice pooled as one sample).

Figure 5. The potential role of GDF/BMP ligands in AP differentiation. (A) ATM Genes regulated by cold exposure. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test. (B) The expression of BMP receptor and Smad genes in AP. (C) Western blot analysis in sorted CD45−/CD31− cells from HFD-fed VAT treated with the indicated ligand at 500 ng/ml for 30 min. HSP90 expression serves as loading control (N = 5). (D) qPCR analysis of Scd1 and Ppargc1a expression in FACS sorted AP from HFD-fed VAT after adipocyte differentiation. Cells were differentiated for 14 days in the presence or absence of the indicated ligand at 250 ng/ml. (E) GAL-ELK1 luciferase assay in HEK293T cells to monitor RET stimulation. Cells were transfected to express the appropriate luciferase reporter construct and an appropriate GFRA coreceptor as indicated with (right) or without (left) RET. Transfected cells were stimulated with the indicated ligand. Results shown as mean fold induction ± SEM (N = 3). (F) Expression of Gfra and Ret receptor genes in warm and cold conditions in FACS sorted ATM1, ATM2, and AP. Data are shown as mean ± SEM (N = 3).

Figure 6. Administration of FGF21-mimetic antibody phenocopies cold exposure in obese VAT. HFD-fed male mice received bFKB1 (10 mg/kg i.p.) or vehicle once on day 0 at thermoneutral condition. Tissues were harvested on day 14 after bFKB1 injection for histology and FACS analysis (A–F), and day 8 for gene expression analysis (G–J). (A) H&E staining in HFD-fed SCAT. (B) H&E staining in HFD-fed VAT. (C) Representative plots for M2/M1 macrophage populations. (D) Ratio of the frequencies of ATM2 (CD11c−/CD206+) over ATM1 (CD11c+/CD206−). (E) CD206 (green) and Arg1 (red) IF. (F) Lyve1 (green) and Arg1 (red) IF. DAPI (blue) stains cell nuclei. (G) tSNE analysis of RNA-Seq results from ATM1 and ATM2 from obese VAT. (H) 4-way differential analysis revealed bFKB1-induced gene expression profile in ATM2 mimics cold exposure. Grey dots indicate all the expressed genes. Red dots indicate genes of interest. (I) Expression of genes regulated by bFKB1 in ATM1 or ATM2. Data are shown as mean ± SEM (N = 3 samples, 5 mice pooled as one sample). (J) Heatmap showing the expression of bFKB1-regulated genes in ATM2. The gene set was selected based on a 2-Way comparison between bFKB1-treated samples and control…