Metabolism - Clinical and Experimental A new hope for obesity management: Boron inhibits adipogenesis in progenitor cells through the Wnt/β-catenin pathway  Ayşegül Doğan, Selami Demirci, Hüseyin Apdik, Omer Faruk Bayrak, Sukru Gulluoglu, Emre Can Tuysuz, Oleg Gusev, Albert A. Rizvanov, Emrah Nikerel, Fikrettin Şahin  Metabolism - Clinical and Experimental  Volume 69, Pages 130-142 (April 2017) DOI: 10.1016/j.metabol.2017.01.021 Copyright © 2017 Terms and Conditions

Fig. 1 Boric acid (BA) and sodium pentaborate pentahydrate (NaB) treatment inhibited adipogenic differentiation in 3T3-L1 cells in a concentration-dependent manner. (A) Oil red O staining of differentiated 3T3-L1 cells treated with or without various concentration of BA or NaB. (B) Quantification of lipid accumulation in boron-treated cells by measuring isopropanol-extracted Oil red O. (C) The inhibitory effects of boron chemicals were largely limited to the early phase of differentiation. Lipid accumulation in 3T3-L1 cells treated with 1mg/mL BA or NaB for 2days in various stages of differentiation was evaluated (C) microscopically and (D) quantitatively. The results are given as the percentage of differentiated control cells. MDI: 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; C: differentiated control cells; NC: undifferentiated negative control cells. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). *P<0.05 compared with C; #P<0.05 compared with NC. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 2 Boron treatment decreased the expression levels of adipogenesis-related genes at the end of the 8-day differentiation period. C: differentiated control cells, NC: undifferentiated negative control cells, BA: differentiating cells treated with 1mg/mL boric acid, NaB: differentiating cells treated with 1mg/mL sodium pentaborate pentahydrate. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). *P<0.05 compared with NC; #P<0.05 compared with C. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 3 Boron treatment decreased the expression of adipogenesis-related proteins at the end of the 8-day differentiation period. Adipogenesis-related protein expression in differentiated 3T3-L1 cells was repressed by 1mg/mL boric acid (BA) or sodium pentaborate pentahydrate treatments, as verified by (A) immunohistochemical or (B) Western blotting analyses. Data were normalized to the corresponding β-actin band intensity in Western blotting analysis. Expression levels of (C) adiponectin, (D) leptin, and (E) triglycerides and (F) glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity in 3T3-L1 cells treated with or without boron derivatives at the end of the differentiation process. MDI: 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; C: differentiated control cells; NC: undifferentiated negative control cells. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). *P<0.05 compared with NC; #P<0.05 compared with C. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 4 Boron treatment did not induce apoptosis in 3T3-L1 cells, but blocked mitotic clonal expansion in differentiating 3T3-L1 cells. (A) Apoptotic and necrotic cell fractions in 3T3-L1 cells treated with or without 1mg/mL boric acid (BA) and sodium pentaborate pentahydrate (NaB) during growth and differentiation. Cell cycle distributions of boron treated 3T3-L1 cells at (B) growth or (C) differentiation. NC: growth medium-treated cells, Post-conf.: postconfluent 3T3-L1 cells, C: differentiated control cells. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 5 The β-catenin pathway was responsible for the boron-dependent suppression of adipogenesis. (A) β-Catenin protein expression during the differentiation of 3T3-L1 cells treated with or without 1mg/mL boric acid (BA) or sodium pentaborate pentahydrate (NaB) for 2days. (B) Immunocytochemical analysis of β-catenin in 3T3-L1 cells at the end of the 8-day differentiation period. (C) Relative protein and mRNA levels of β-catenin in sh β-catenin or sh control virus-transduced 3T3-L1 cells. (D) Lipid content of sh β-catenin or sh control virus-transduced 3T3-L1 cells treated with or without 1mg/ml BA or NaB was evaluated by microscopy and spectrophotometric analysis at the end of the 8-day incubation period. (E) Expression of adipogenesis-related proteins in sh β-catenin or sh control virus-transduced 3T3-L1 cells treated with or without 1mg/mL BA or NaB. NC: undifferentiated control cells; C: differentiated control cells; MDI: 3-isobutyl-1-methylxanthine, dexamethasone, and insulin. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). *P<0.05. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 6 The expression levels of critical cytokines were regulated by boron treatment during the early phase of differentiation. Postconfluent 3T3-L1 cells treated with differentiation cocktail (MDI) with or without 1mg/mL boric acid (BA) or sodium pentaborate pentahydrate (NaB) for 48h. At the end of the incubation process, the levels of 144 cytokines were determined in all experimental groups. NC: postconfluent cells, C: postconfluent cells treated with MDI for 48h. The data are representative of three independent experiments and show the mean±SD. Comparisons were performed by ANOVA (Tukey's post hoc test). *P<0.05 compared with NC; #P<0.05 compared with C. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions

Fig. 7 Boron treatment regulated the gene expression profiles of differentiating 3T3-L1 cells. (A) Principal component analysis (PCA) of the expression profiles in normally differentiating (C, positive control) and boron-treated 3T3-L1 cells (1mg/mL boric acid [BA] or sodium pentaborate pentahydrate [NaB] for 48h). (B) Overlap in the expression of the genes upregulated in the first day of differentiation (24h) in the experimental groups. (C) Overlap in the expression of the genes downregulated in the first day (24h) of differentiation in the experimental groups. (D) Overlap in the expression of the genes upregulated in the second day (versus the first day) of differentiation in all experimental groups. NC: postconfluent 3T3-L1 cells. Metabolism - Clinical and Experimental 2017 69, 130-142DOI: (10.1016/j.metabol.2017.01.021) Copyright © 2017 Terms and Conditions