White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

Figures

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  Figure 1. Dynamic proteome remodeling occurred during cold adaptation in mouse subcutaneous adipose tissue. (A) Heat map (blue to yellow) representing all of the differentially expressed proteins during cold adaptation (n = 5, 2-way ANOVA, p < 0.03) clustered after z score normalization. Hierarchical clustering of proteins showing two distinct phases: acute vs chronic cold exposure and functionally related time points. (B) Fuzzy c-means clustering of all of the quantified proteins across the timespan using a tool developed by Schwämmle et al. [37]. Using the lowest value of the minimum centroid distance and the highest Xie-Beni index, proteins responding to cold acclimation in mouse scWAT were classified into 8 distinct clusters of regulation patterns (C1–C8). Proteins altered at early time points of cold exposure (≤24 h) are defined as “regulators” and proteins altered by chronic cold exposure (≥1 week) are referred to as “effectors.” (C–F) Gene ontology enrichment analysis was performed using GOrilla [79] and images were plotted using the REVIGO [80] bubble network for proteins upregulated in response to 3–24 h of cold exposure. (C) Proteins downregulated in response to 3–24 h of cold…

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  Figure 2. YBX1 abundance acutely increased in thermogenic adipose tissue upon cold exposure in mice. (A) Representative YBX1 and AKT immunoblots in various fat depots from mice kept at 5 °C for 0, 3, 8, and 24 s, 1 week, or 3 weeks. Quantification of the band intensities (n = 4 replicates per time point). Values are mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 analyzed by one-way ANOVA. (B) Immunoblots of YBX1 and AKT in various fat depots from mice kept at 29 °C for 3 weeks. Quantification of the band intensities (n = 3 replicates per time point). Values are mean ± SEM, ∗p < 0.05 analyzed by one-way ANOVA. (C) Histological analysis of scWAT from mice kept at either thermoneutrality (29 °C) or cold (5 °C) for 24 h. scWAT was stained with hematoxylin and eosin or YBX1 fluorescent antibody (red) together with DAPI (blue). Tissue autofluorescence is shown in white. Representative images (from n = 3 mice per group) are shown. Scale bars 200 μm and magnified scale bars 50 μm. (D)Ybx1 mRNA expression in the isolated mature adipocyte fraction (MAF) and stromal-vascular fraction (SVF) from scWAT of mice exposed to thermoneutrality (29 °C) or cold (5 °C) for 24 h (n = 5 replicates per…

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  Figure 3. YBX1 was necessary for the expression of thermogenic genes in adipocytes. Brown WT-1 preadipocytes and C3H/10T1/2 mesenchymal stem cells were transfected with Ybx1-specific (siYbx1, light bars) or negative control siRNA (siCon, dark bars) and induced to differentiate (d0). (A) The efficiency of Ybx1 knockdown (KD) in WT-1 cells is shown at the mRNA level at different days of differentiation following siRNA transfection. (B) The efficiency of Ybx1 knockdown (KD) in WT-1 cells is shown at the protein level by Western blotting on different days of differentiation following siRNA transfection. The blot quantification is on the right. YBX1 was normalized to vinculin (VCL) (C) Effects of Ybx1 KD on the expression of key thermogenic genes (Ppargc1a, Elovl3, Prdm16, Cidea, and Ucp1) across differentiation. (D) The effect of Ybx1 KD on UCP1 protein expression in WT-1 cells is shown by Western blotting, and the quantification is on the right. UCP1 was normalized to vinculin (VCL) (E) The efficiency of Ybx1 KD in C3H/10T1/2 mesenchymal cells is shown at mRNA levels on different days of differentiation following siRNA transfection. (F) The efficiency of Ybx1 KD in C3H/10T1/2 cells is shown…

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  Figure 4. YBX1 potentiated the thermogenic capacity of C3H/10T1/2 mesenchymal stem cells. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA (gRNA) directed to the promoter regions of Pparg2 (P) or Ybx1 (Y) as indicated. (A–B) mRNA expression levels of Ybx1 and Pparg2 post-transfection. (C) Gene expression analysis of thermogenic markers Ucp1, Cidea, and Elovl3. Data are based on n = 3 biological replicates. Two-way ANOVA; overall effects of the overexpression and differentiation day are shown for selected groups of interest (P vs EV; P vs P + Y). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (D–E) The effects of Ybx1 overexpression on the levels of YBX1 (D) and UCP1 (E) proteins in C3H/10T1/2 cells is shown by Western blotting and the quantification is added. UCP1 was normalized to vinculin (VCL). Four biological replicates were used, and the values are mean ± SEM. ∗p < 0.05 analyzed by unpaired t-test was applied for statistical analysis. (F) Oxygen consumption rates (OCR) in mature C3H/10T1/2-CRISPRa-SAM cells following injection with isoproterenol (ISO), FCCP, and rotenone/antimycin A (Rot/Anti). A representative experiment is shown.…

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  Figure 5. YBX1 induced early transcriptional changes to promote browning in C3H/10T1/2 mesenchymal stem cells. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA directed to the promoter region of Pparg2 (P) or Pparg2 and Ybx1 (PY) as indicated. (A–C) Volcano plots displaying transcripts significantly regulated by induction of Pparg2 expression (A) or combined induction of Pparg2 and Ybx1 expression compared to EV-transfected control cells (B) and a set of genes upregulated specifically by Ybx1(C). (D) Heat map of fold changes in relative abundance of the set of genes significantly regulated by Pparg2 and Ybx1 co-induction across days of differentiation. (E) Gene set enrichment analysis (GSEA) across different days of differentiation upon Ybx1 over-expression; GO terms with FDR < 0.1 were considered significantly enriched and the top 10 GO terms with the lowest FDR for each contrast are shown in the dot plot. The extended list is provided in Table S6. NES stands for normalized enrichment score for GSEA. Data represent the relative abundance based on counts per million (CPM) and 0.1 false discovery rate (FDR) cut-off.

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  Figure 6. JMJD1c contributed to YBX1-induced browning. C3H/10T1/2-CRISPRa-SAM cells were transfected with empty vector (EV) or vector delivering a single-guide RNA directed to the promoter region of Ybx1 as indicated. All of the transfections included the vector delivering a single-guide RNA directed to the promoter region of Pparg2. Furthermore, siRNA targeting Jmjd1c was used for knockdown. (A) Overexpression of Ybx1 in combination with siRNA targeting Jmj1dc. Transfection was performed two days pre-confluency (d2), the cells were harvested at d0, and gene expression levels of Ybx1 and Jmjd1c were assessed. (B–C) On day 6 of differentiation, the cells were harvested after 6 h of 10 μM NE stimulation or not, and the gene expression levels of Ucp1, Elovl3, Ppargc1a, and Cidea(B), and gene expression levels of Fasn, Fabp4, and Adipoq(C) were assessed. Data are based on n = 4 biological replicates and the values are the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 analyzed by 2-way ANOVA; overall effects of overexpression and knockdown are shown.