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). MedChemExpress ��-Sitosterol ��-D-glucoside 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 22948146 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 25837696 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 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 effects of 1,25(OH)2D3 on adipogenic marker expression. Human preadipocytes were differentiated in the absence or presence of 1,25(OH)2D3 (1028 M, added continuously throughout). Expression levels of adipogenic markers [C/EBPb (A), C/EBPa (B), PPARc (C), and LPL (D)] were measured before (09) and at indicated time points during differentiation. Data are presented as of vehicle control after differentiation (d10?2; d10+) in each experiment. *, p,0.05, vehicle control vs. 1,25(OH)2D3 treatment, n = 4. E. MedChemExpress K162 Representative FABP4 and VDR blots from 3 independent experiments are presented. doi:10.1371/journal.pone.0052171.gSince 25(OH)D3 also increased CYP24A1 expression, the effects of 25(OH)D3 on adipogenesis were tested. 25(OH)D3 increased differentiation of human preadipocytes (Fig. 3). Interestingly, 1028 M 25(OH)D3 tended to be less effective than 1029 M at increasing LPL mRNA and FABP4 protein levels. Of note, we could not test higher concentrations of 25(OH)D3 ( 1027 M) as they were toxic to human preadipocytes, killing cells within 24 h of treatment. 25(OH)D3 (1028 M) significantly increased triglyceride accumulation by 72616 
compared to the vehic.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 22948146 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 25837696 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 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 effects of 1,25(OH)2D3 on adipogenic marker expression. Human preadipocytes were differentiated in the absence or presence of 1,25(OH)2D3 (1028 M, added continuously throughout). Expression levels of adipogenic markers [C/EBPb (A), C/EBPa (B), PPARc (C), and LPL (D)] were measured before (09) and at indicated time points during differentiation. Data are presented as of vehicle control after differentiation (d10?2; d10+) in each experiment. *, p,0.05, vehicle control vs. 1,25(OH)2D3 treatment, n = 4. E. Representative FABP4 and VDR blots from 3 independent experiments are presented. doi:10.1371/journal.pone.0052171.gSince 25(OH)D3 also increased CYP24A1 expression, the effects of 25(OH)D3 on adipogenesis were tested. 25(OH)D3 increased differentiation of human preadipocytes (Fig. 3). Interestingly, 1028 M 25(OH)D3 tended to be less effective than 1029 M at increasing LPL mRNA and FABP4 protein levels. Of note, we could not test higher concentrations of 25(OH)D3 ( 1027 M) as they were toxic to human preadipocytes, killing cells within 24 h of treatment. 25(OH)D3 (1028 M) significantly increased triglyceride accumulation by 72616 compared to the vehic.
