Differentiating SGBS adipocytes respond to PPARγ stimulation, irisin and BMP7 by functional browning and beige characteristics

Figure 1. Browning of SGBS cells is induced by PPARγ-driven differentiation cocktail, irisin or BMP7 treatment. SGBS preadipocytes were differentiated to white (W) or brown (B) for two weeks; human recombinant irisin treatment at 250 ng/ml concentration (green bars) or BMP7 treatment at 50 ng/ml concentration (red bars) were applied to induce browning of SGBS cells from day 1. Expression of Ucp1 (a), Cidea, Elovl3 (b), Cyc1 (c), Tbx1, Cited1, Zic1, Pdk4 (e) genes in SGBS adipocytes. Gene expression was determined by RT-qPCR and target genes were normalized to Gapdh. Relative mitochondrial DNA content of preadipocytes and differentiated SGBS cells determined by quantitative PCR (d) n = 5 *p < 0.05; **p < 0.01.

Figure 2. Morphological features of browning SGBS adipocytes that were induced by PPARγ-driven differentiation cocktail, irisin or BMP7 treatment. UCP1 content, lipid droplets and nuclei are shown in gallery images (N = 9) of selected cells within the white and brown differentiated adipocytes (see an overview image of a sample of brown differentiation in Supplementary Fig. S6). SGBS preadipocytes were differentiated and treated as in Fig. 1. Images of differentiated samples were captured by a laser-scanning cytometer in 3 independent experiments. In every experiment 1000–2000 cells per each sample were recorded and measured. Applying image analysis, single cells were identified based on their nuclei and classified according to their UCP1 content and lipid droplet structure. (a) UCP1 protein content of browning induced adipocytes (PPARγ-driven differentiation cocktail, irisin or BMP7 treatment) as compared to white adipocytes. (b) Expression of UCP1 at protein level in SGBS adipocyte lysates detected by immunoblotting. (c) The blot was cropped. The original picture of the full-length blot is displayed in Supplementary Fig. S7. Density plot figures show texture sum variance and UCP1 protein…

Figure 3. Functional measurements detect high oxygen consumption and significant involvement of the creatine substrate cycle in Rosiglitazone, irisin and BMP7 differentiated browning adipocytes. SGBS preadipocytes were differentiated and treated as in Fig. 1. Mitochondrial oxygen consumption rate (OCR) of differentiated SGBS cells of one representative measurement determined by a Seahorse XF96 analyzer. (a) Basal, dibutyryl-cAMP-stimulated and oligomycin-inhibited oxygen consumption levels in SGBS cells, compared to basal OCR of white-directed adipocytes. (b) Extracellular acidification rate (ECAR) of differentiated SGBS cells measured by a Seahorse XF96 analyzer. (c) Inhibitory effect of ß-guanidiopropionic acid (ß-GPA) (at 100 mg/ml concentration) on the oxygen consumption of SGBS adipocytes. (d) n = 4 *p < 0.05; **p < 0.01. Expression of mitochondrial creatine kinases (Ckmt1a and Ckmt2) in SGBS adipocytes. (e) Gene expression was determined by RT-qPCR and target genes were normalized to Gapdh. n = 3 *p < 0.05.

Figure 4. PPARγ-driven differentiation can be maintained in long-term cultures of browning adipocytes. SGBS preadipocytes were differentiated to white (W) or brown (B) for two, three and four weeks (indicated by the number of days), respectively. At day 14, the PPARγ-driven browning cocktail was replaced by the white without rosiglitazone for additional one (B14,W7) or two weeks (B14,W14). Expression of UCP1 at mRNA and protein level (a). Normalized expression of Cidea, Elovl3 (b) and Lep (c) genes by RT-qPCR, n = 4. Density plot figures show texture sum variance and UCP1 protein content of differentiated cells detected by Laser-scanning cytometry in one representative SGBS replicate; n = 3, 1000–2000 cells per each sample (d). Expression of mitochondrial complex subunits detected by immunoblotting (e). Relative optical density was determined by Image J software. ß-actin was used as endogenous control, n = 3. All gels were run under the same conditions. The blots were cropped. The original pictures of the full-length blots are displayed in Supplementary Fig. S7. *p < 0.05; **p < 0.01.