Functional and Genetic Characterization of Porcine Beige Adipocytes

Figures

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  Figure 1. The Establishment of an in vitro model of porcine beige adipogenesis. (A) Schematic illustration of the in vitro differentiation of porcine beige and white adipocytes. (B) Oil Red-O staining (left panel), bright field imaging (middle panel), BODIPY 493/503 fluorescence staining (right panel) of white and beige adipocytes (scale bars are 200 μm, 50 μm and 10 μm, respectively). (C) Frequency statistical data of lipid droplet size measurements in white and beige adipocytes based on the bright field images shown in (B) (white: n = 5943; beige: n = 5974, pixel: 1 μm). (D) The oxygen consumption rates (OCRs) of white and beige adipocytes were measured by the Seahorse Mito Stress Test assay. Oligomycin (1.5 μM), FCCP (0.5 μM) and antimycin A/rotenone (0.5 μM) were added at the indicated time points. The data were normalized to the total DNA content. (E–G) The basal respiration rate (E), ATP production (F) and proton leak (G) were calculated based on the data presented in D (n = 10). All data are shown as the mean ± SEM. The p value was calculated by unpaired two-tailed Student’s t test. *** p < 0.001.

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  Figure 2. Distinct transcriptional profiles of porcine white and beige adipocytes. (A) Principal component analysis (PCA) plots representing SVFs, and white and beige adipocytes based on transcriptome data (n = 4 for each group). (B–D) Volcano plots showing a global overview of the gene expression profiles of SVF cells and white and beige adipocytes; well-known adipocyte marker genes are highlighted. Up: upregulated genes; Down: downregulated genes. (E–G) Gene set enrichment analysis (GSEA) of the RNA-seq data. GSEA showing the enriched pathways in white adipocytes compared with SVF cells (E). Comparison of a signature from beige adipocytes to genes upregulated in SVF cells as described; the GSEA results showed enrichment for brown fat cell differentiation (F). GSEA of beige adipocyte signatures compared to the list of ranked upregulated genes from white adipocytes revealed that the oxidative phosphorylation pathway was enriched (G). (H) A heatmap was constructed based on the top 15 genes involved in adipogenesis (E), brown fat cell differentiation and the oxidative phosphorylation (G) pathway. (I) Relative expression levels of adipocyte marker genes in three groups of cells (n = 4–6).…

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  Figure 3. Identification of beige adipogenesis-related candidate genes. (A,D). Venn diagram showing the overlapping genes that were specifically and significantly upregulated (A) or downregulated (D) in the beige/SVF and beige/white groups (FC > 1.5, p < 0.05). Note that a total of 1228 upregulated and 1376 downregulated genes were included based on the criteria. (B,E). The above 1228 upregulated and 1376 downregulated genes were subjected to GO analysis, and the dot plot shows the most significantly enriched pathways. The color of the dot represents the p value, and the size of the dot represents the number of differentially expressed transcripts. (C,F). Heatmap of the select genes among the 1228 (C) and 1376 (F) genes. The color scale shows the z score read counts representing the mRNA level of each gene in the blue (low expression)—white–red (high expression) scheme. (G,H). The relative expression levels of the significantly upregulated genes among the 1228 genes identified (G) and of the significantly downregulated genes among the 1376 genes identified (H) in SVF cells and white and beige adipocytes were validated by qPCR (n = 4–6). (I,J). Heatmap of all transcription factors among the…

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  Figure 4. Comparative analysis of differentially expressed genes in beige adipocytes from different samples. (A). Venn diagram showing the overlapping genes that are specific and significant in the beige/white and Tibetan pig comparisons after cold stimulation (FC >1.5, p < 0.05). Note that a total of 95 upregulated genes and 68 downregulated genes were included based on the established criteria. (B) Expression profiles of the top 20 genes enriched in vitro and in vivo. The color scale shows the z score read counts representing the mRNA level of each gene in the blue (low expression)-white–red (high expression) scheme. RT: room temperature, cold: 4 °C for 4 h. (C) GO analysis of 95 upregulated genes overlapping in (A). The dot plot shows the most significantly enriched pathways. The color of the dot represents the p value, and the size of the dot represents the number of DEGs. (D) Venn diagram showing the beige adipocyte-related genes specific for pigs and conserved across the species (FC > 1.5, p < 0.05). Note that a total of 18 upregulated genes and 14 downregulated genes were conserved across humans, mice and pigs. Human and mouse core brown fat-selective genes: List of core brown…

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  Figure 5. ITAG2 and CNN1 are involved in porcine beige adipocyte differentiation and thermogenesis. (A) Schematic of si-ITGA2 or si-CNN1 treatment during adipogenic induction. (B) The knockdown efficiency of si-ITGA2 was examined by qPCR on day 6. (C) Expression of beige adipocyte markers in cells transfected with si-ITGA2. (D) Effect of si-ITGA2 transfection on beige adipogenesis, as determined by Oil Red-O staining (top, scale bar is 200 μm) and fluorescence staining (bottom, scale bar is 100 μm) on day 6. (E) Quantitative analysis of the Oil Red-O staining data shown in D via OD measurements. F-H. OCR in differentiated porcine beige adipocytes transfected with si-NC or si-ITGA2 (F); the basal cellular respiration rate (G) and proton leak (H) were calculated (n = 10). (I,J) The knockdown efficiency of si-CNN1 was examined by qPCR and Western blotting on day 6. (K) Expression of beige adipocyte markers in cells transfected with si-CNN1. (L). Effect of si-CNN1 transfection on beige adipogenesis, as determined by Oil Red-O staining (top, scale bar is 200 μm) and fluorescence staining (bottom, scale bar is 100 μm) on day 6. (M) Quantitative analysis of the Oil Red-O staining data shown in L…