RNA Binding Protein Ybx2 Regulates RNA Stability During Cold-Induced Brown Fat Activation

Ybx2 is an essential regulator of brown adipocyte differentiation in vitro. A: Primary brown preadipocytes were infected by retroviral shRNAs targeting RBPs, Ybx2, and Akap1, followed by induction of differentiation for 5 days. Oil Red O staining was used to assess lipid accumulation. B–D: Real-time PCR was used to measure the knockdown efficiency (left), panadipogenic marker expression (right), and BAT-selective marker expression (bottom) in cultured primary brown adipocytes (day 5) infected by retroviral shRNAs targeting Ybx2 (B), Akap1 (C), and Rbpms2 (D) (n = 3). *P < 0.05 by one-way ANOVA. F: Representative metabolic flux curves from cultured brown adipocytes (day 5) infected by retroviral shRNA targeting Ybx2. Cells were sequentially treated with oligomycin, FCCP, and rotenone. OCRs are normalized by protein concentration (n = 5). *P < 0.05 by Student t test. F: Western blot was used to examine the protein levels of Ybx2 during primary brown and white adipocyte differentiation in culture. The error bars in the graphs are mean ± SEM. Sh-1, short hairpin RNA-1; Sh-2, short hairpin RNA-2; Sh-3, short hairpin RNA-3.

Ybx2 can promote BAT-selective gene expression in white and brown adipocyte cultures. A: Western blot was used to confirm the overexpression of Ybx2 in primary white adipocyte culture. B: Representative photomicrograph of boron-dipyrromethene staining for lipids in primary white adipocytes infected by Ybx2-expressing or empty vector. C: Real-time PCR was used to examine marker gene expression during the time course of white adipocyte cultures expressing Ybx2 or vector (n = 4). D–F: Same as in A–C, but in primary brown adipocyte culture (n = 4). The error bars are mean ± SEM. *P < 0.05. G: Representative metabolic flux curves from cultured brown adipocytes (day 3) infected by retroviral-overexpressing Ybx2. Cells were sequentially treated with oligomycin, FCCP, and rotenone. OCRs are normalized by protein concentration (n = 5). H: Western blot was used to detect the protein levels of Ucp1 and two fatty acid oxidation components, Cpt1a and Mcad, at day 3.

Ybx2 is needed for cold-induced BAT activation. A: Western blot was used to detect Ybx2 expression in eWAT, BAT, and iWAT from WT and KO mice. B: Body weight and BAT organ weight of WT (n = 6) and KO (n = 7) male mice at 8–9 weeks old. C: Representative photomicrograph of hematoxylin and eosin staining of WT and KO BAT. D: Distribution of the diameters of lipid droplets from panel C measured by ImageJ software. E: Body weight and BAT weight of WT and KO animals (8–9 weeks old, n = 5) after 6 h at 4°C exposure. F: Representative picture and photomicrograph with hematoxylin and eosin staining of BAT from WT and KO mice after cold exposure. G: Distribution of the diameters of lipid droplets from panel F. H: Body temperature was measured by rectal probe at the indicated times at 4°C (n = 5). Error bars are the mean ± SEM. *P < 0.05.

The effect of Ybx2 KO on cold-induced gene expression in BAT. A: Heat map of the gene expression in WT and KO BAT after cold exposure for 6 h. Heat map showed the row mean-centered abundance. FPKM, fragments per kilobase of exon per million fragments mapped. B: Five top nonredundant GO terms enriched among mRNAs that showed significantly low (top) or high (bottom) expression (P < 0.05, Cuffdiff) in KO vs. WT BAT. C: Real-time PCR to confirm gene expression of BAT-selective genes in WT and KO BAT (n = 5). D: Western blot to confirm gene expression of BAT markers in WT and KO BAT. E: Real-time PCR to confirm the expression of genes involved in lipogenesis and glucose uptake (n = 5). Error bars are the mean ± SEM. *P < 0.05.

Ybx2 stabilizes mRNA targets encoding proteins enriched for mitochondria functions. A: Western blot to confirm immunoprecipitation (IP) of Ybx2 protein by Ybx2 antibody; 10% IP cell lysate was used as the input. B: Targets of Ybx2 were selected based on their enrichment in the Ybx2 IP vs. IgG control. Venn diagram shows the overlapping of candidates from brown and white adipocytes. C: Bubble chart shows the GO terms enriched in the common targets. The x-axis indicates P values, and the y-axis indicates the enrichment score. The bubble size indicates the number of targets in that GO category. D: Relative abundance of each mRNA was calculated in anti-Ybx2 vs. IgG RIP-seq. The cumulative fraction of mRNAs involved in mitochondrion and all other detectable genes were plotted. The Kolmogorov-Smirnov test was performed to determine the distribution difference. E: Relative expression of each gene in KO vs. WT BAT at room temperature based on RNA-seq data. The cumulative fraction curves were plotted for 414 common target mRNAs and other genes detectable in the RIP-seq assays. F: The cumulative fraction curves were plotted for common target mRNAs and other genes after cold exposure. The Kolmogorov-Smirnov test was performed to determine the statistical significance of the difference in the distributions.