Cytes were incubated with 25(OH)D3 (1028 M) for 24 h in a-MEM without FBS. Newly-Figure 2. 1,25(OH)2D3 and 25(OH)D3 increased CYP24A1 mRNA in preadipocytes and newly-differentiated adipocytes. A. Preadipocytes were treated with vehicle control, 1,25(OH)2D3 (10210, 1029, 1028 M), or 25(OH)D3 (10210, 1326631 1029, 1028 M) for 24 h and CYP24A1 mRNA expression was measured (n = 3). B. Differentiated adipocytes were treated with vehicle control, 1,25(OH)2D3 (1028 M), or 25(OH)D3 (1028 M) for 24 h and CYP24A1 mRNA expression was measured (n = 5). **, p,0.01, control vs. treatments. doi:10.1371/journal.pone.0052171.gVitamin D and Human Preadipocyte DifferentiationFigure 3. 25(OH)D3 and 1,25(OH)2D3 promoted the differentiation of human preadipocytes. Human preadipocytes were differentiated in the presence of vehicle control, 1,25(OH)2D3 (10210, 1028 M) or 25(OH)D3 (1029, 1028 M) and expression levels of adipogenic markers were measured on d14. A. Representative immunoblots of FABP4 protein (left panel) and quantification (right panel) are shown (n = 6). Expression levels of LPL (B; n = 7) and PPARc mRNA (C; n = 6) and TG accumulation (D; n = 4) were presented as increase over vehicle control. *, p,0.05, **, p,0.01, vehicle control vs. treatments. doi:10.1371/journal.pone.0052171.gResults VDR and CYP27B1 Genes are Expressed in Omental and Subcutaneous Human Adipose Tissues and Primary Preadipocytes and AdipocytesVDR and CYP27B1 (1a-hydroxylase) mRNA were easily detected in PF-04554878 site samples of both omental and sc human adipose tissues (Fig. 1A,B). Expression levels of these mRNAs were similar between the two depots and were enriched in the stromal vascular cell SCH 727965 supplier compared to mature adipocyte fraction. We next determined whether VDR and CYP27B1 expression levels varied with preadipocyte differentiation. VDR mRNA levels did not change after differentiation, while VDR protein levels decreased (Fig. 1C,E). CYP27B1 mRNA levels decreased after differentiation, but due to low expression in some samples, we were unable to demonstrate consistent changes in CYP27B1 protein levels (Fig. 1D,E). To determine whether human preadipocytes and adipocytes respond to 1,25(OH)2D3, we tested whether it increased the expression of a known vitamin D target gene, CYP24A1. 1,25(OH)2D3 markedly increased CYP24A1 mRNA in bothhuman preadipocytes and newly-differentiated adipocytes (Fig. 2). In addition, 25(OH)D3 induced CYP24A1 mRNA expression in both human preadipocytes and newly-differentiated adipocytes.Both 1,25(OH)2D3 and 25(OH)D3 Increased the Differentiation of Human PreadipocytesTo test the effects of 1,25(OH)2D3 on human preadipocyte differentiation, 2d-post confluent preadipocytes were differentiated in the absence or presence of 1,25(OH)2D3 (10210, 1029, 1028 M, added continuously throughout). 1,25(OH)2D3 dose-dependently enhanced adipogenesis as determined by significant increases in the expression levels of adipogenic markers (FABP4 protein and LPL mRNA) and TG accumulation (Fig. 3). 1,25(OH)2D3 (1028 M) also tended to increase PPARc mRNA levels in this dose-response experiment (p = 0.06, n = 6). A statistically significant effect of 1,25(OH)2D3 (1028 M) to increase PPARc mRNA levels was clear when these 18325633 data and those from other experiments, also conducted at 1028 M with the identical protocol, were combined (n = 9, p = 0.02). 1,25(OH)2D3 treatment did not affect the number of cells per well (not shown).Vitamin D and Human Preadipocyte DifferentiationFigure 4. Time-course effe.Cytes were incubated with 25(OH)D3 (1028 M) for 24 h in a-MEM without FBS. Newly-Figure 2. 1,25(OH)2D3 and 25(OH)D3 increased CYP24A1 mRNA in preadipocytes and newly-differentiated adipocytes. A. Preadipocytes were treated with vehicle control, 1,25(OH)2D3 (10210, 1029, 1028 M), or 25(OH)D3 (10210, 1326631 1029, 1028 M) for 24 h and CYP24A1 mRNA expression was measured (n = 3). B. Differentiated adipocytes were treated with vehicle control, 1,25(OH)2D3 (1028 M), or 25(OH)D3 (1028 M) for 24 h and CYP24A1 mRNA expression was measured (n = 5). **, p,0.01, control vs. treatments. doi:10.1371/journal.pone.0052171.gVitamin D and Human Preadipocyte DifferentiationFigure 3. 25(OH)D3 and 1,25(OH)2D3 promoted the differentiation of human preadipocytes. Human preadipocytes were differentiated in the presence of vehicle control, 1,25(OH)2D3 (10210, 1028 M) or 25(OH)D3 (1029, 1028 M) and expression levels of adipogenic markers were measured on d14. A. Representative immunoblots of FABP4 protein (left panel) and quantification (right panel) are shown (n = 6). Expression levels of LPL (B; n = 7) and PPARc mRNA (C; n = 6) and TG accumulation (D; n = 4) were presented as increase over vehicle control. *, p,0.05, **, p,0.01, vehicle control vs. treatments. doi:10.1371/journal.pone.0052171.gResults VDR and CYP27B1 Genes are Expressed in Omental and Subcutaneous Human Adipose Tissues and Primary Preadipocytes and AdipocytesVDR and CYP27B1 (1a-hydroxylase) mRNA were easily detected in samples of both omental and sc human adipose tissues (Fig. 1A,B). Expression levels of these mRNAs were similar between the two depots and were enriched in the stromal vascular cell compared to mature adipocyte fraction. We next determined whether VDR and CYP27B1 expression levels varied with preadipocyte differentiation. VDR mRNA levels did not change after differentiation, while VDR protein levels decreased (Fig. 1C,E). CYP27B1 mRNA levels decreased after differentiation, but due to low expression in some samples, we were unable to demonstrate consistent changes in CYP27B1 protein levels (Fig. 1D,E). To determine whether human preadipocytes and adipocytes respond to 1,25(OH)2D3, we tested whether it increased the expression of a known vitamin D target gene, CYP24A1. 1,25(OH)2D3 markedly increased CYP24A1 mRNA in bothhuman preadipocytes and newly-differentiated adipocytes (Fig. 2). In addition, 25(OH)D3 induced CYP24A1 mRNA expression in both human preadipocytes and newly-differentiated adipocytes.Both 1,25(OH)2D3 and 25(OH)D3 Increased the Differentiation of Human PreadipocytesTo test the effects of 1,25(OH)2D3 on human preadipocyte differentiation, 2d-post confluent preadipocytes were differentiated in the absence or presence of 1,25(OH)2D3 (10210, 1029, 1028 M, added continuously throughout). 1,25(OH)2D3 dose-dependently enhanced adipogenesis as determined by significant increases in the expression levels of adipogenic markers (FABP4 protein and LPL mRNA) and TG accumulation (Fig. 3). 1,25(OH)2D3 (1028 M) also tended to increase PPARc mRNA levels in this dose-response experiment (p = 0.06, n = 6). A statistically significant effect of 1,25(OH)2D3 (1028 M) to increase PPARc mRNA levels was clear when these 18325633 data and those from other experiments, also conducted at 1028 M with the identical protocol, were combined (n = 9, p = 0.02). 1,25(OH)2D3 treatment did not affect the number of cells per well (not shown).Vitamin D and Human Preadipocyte DifferentiationFigure 4. Time-course effe.