Min D and Human Preadipocyte DifferentiationFigure 5. 1,25(OH)2D3 promoted the maturation phase of adipogenesis. Human preadipocytes were Title Loaded From File differentiated in the adipogenic cocktail for 3 days and then maintained in the maintenance media until harvest (d13?4). 1,25(OH)2D3 (1028 M) was added during the first 3 days of induction (09?d), maturation (3d-end), or continuously throughout (09-end). Expression levels of adipogenic markers [LPL (A, n = 6) and PPARc (B, n = 6) mRNA and FABP4 protein (C, n = 4)] were measured after differentiation. Data are presented as increase over vehicle control. *, p,0.05, **, p,0.01, vehicle control vs. 1,25(OH)2D3 treatment. doi:10.1371/journal.pone.0052171.gTZDs are potent stimulators of adipogenesis [19], we also tested the effects of 1,25(OH)2D3 in the absence of a TZD. As expected, without TZD fewer cells accumulated lipid (Fig. 6A). Notably however, the magnitude of induction of adipogenic markers by 1,25(OH)2D3 (fold stimulation) was greater in the absence of a TZD (Fig. 6B ).of 5 samples tested produced detectable amounts of 1,25(OH)2D3 (47 and 67 pg/106 cells).In 3T3-L1 Preadipocytes, 1,25(OH)2D3 Inhibited Adipogenesis while 25(OH)D3 had No EffectWe tested the effects of 1,25(OH)2D3 on 3T3-L1 adipogenesis to determine if we could confirm its reported inhibitory effects [3,4,20]. Previous studies had detected 1a-hydroxylase activity in 3T3-L1 preadipocytes [9], yet none had tested the effects of 25(OH)D3 on adipogenesis in 3T3-L1 cells. In 3T3-L1 1379592 cells, 1,25(OH)2D3 caused a dose- and Title Loaded From File time-dependent inhibition of adipogenesis (Fig. 7A B), as previously documented [3,4]. Additionally, in contrast to its pro-adipogenic effects in human preadipocytes, 25(OH)D3 did not affect adipogenesis in 3T3-L1 cells (as shown by the lack of change in FABP4 expression levels, Fig. 7A B).Activation of 25(OH)D3 in Human PreadipocytesBecause CYP27B1 expression was detectable and 25(OH)D3 induced CYP24A1 expression, we conducted preliminary studies to determine whether the enzyme was active. Preadipocytes incubated with 25(OH)D3 (1028 M, 24 h) produced detectable quantities of 1,25(OH)2D3 in the media. 4 samples tested produced 48620 pg/106 cells and one sample made much higher amounts, 1600 pg/106 cells. In newly-differentiated adipocytes, only 2 outVitamin D and Human Preadipocyte DifferentiationFigure 6. The pro-adipogenic effects of 1,25(OH)2D3 were independent of thiazolidinedione treatment. Human preadipocytes were differentiated in the differentiation cocktail with or without thiazolidinedione (TZD) for 7 days and maintained in maintenance media until harvest. 1,25(OH)2D3 or vehicle control was present throughout. Phase contrast image of adipocytes were taken at day 13 after differentiation (A). Expression levels of adipogenic markers [LPL (B) and PPARc (C) mRNA and FABP4 (D) protein] were measured after differentiation (d13?4). Lane 3 and 4 (differentiated in the presence of TZD) were intentionally under loaded to show the results in the same blot. *, p,0.05, **, p,0.01, vehicle control vs. 1,25(OH)2D3 treatment, n = 3 for 1028 and n = 5 for 1027 M. doi:10.1371/journal.pone.0052171.gTo evaluate the possibility that apparent species differences between human preadipocytes and 3T3-L1 cells were not merely related to the initial level of commitment to 18325633 the adipocyte cell fate, we also tested the effect of 1,25(OH)2D3 on primary mouse preadipocyte differentiation. 1,25(OH)2D3 increased the differentiation of mouse prea.Min D and Human Preadipocyte DifferentiationFigure 5. 1,25(OH)2D3 promoted the maturation phase of adipogenesis. Human preadipocytes were differentiated in the adipogenic cocktail for 3 days and then maintained in the maintenance media until harvest (d13?4). 1,25(OH)2D3 (1028 M) was added during the first 3 days of induction (09?d), maturation (3d-end), or continuously throughout (09-end). Expression levels of adipogenic markers [LPL (A, n = 6) and PPARc (B, n = 6) mRNA and FABP4 protein (C, n = 4)] were measured after differentiation. Data are presented as increase over vehicle control. *, p,0.05, **, p,0.01, vehicle control vs. 1,25(OH)2D3 treatment. doi:10.1371/journal.pone.0052171.gTZDs are potent stimulators of adipogenesis [19], we also tested the effects of 1,25(OH)2D3 in the absence of a TZD. As expected, without TZD fewer cells accumulated lipid (Fig. 6A). Notably however, the magnitude of induction of adipogenic markers by 1,25(OH)2D3 (fold stimulation) was greater in the absence of a TZD (Fig. 6B ).of 5 samples tested produced detectable amounts of 1,25(OH)2D3 (47 and 67 pg/106 cells).In 3T3-L1 Preadipocytes, 1,25(OH)2D3 Inhibited Adipogenesis while 25(OH)D3 had No EffectWe tested the effects of 1,25(OH)2D3 on 3T3-L1 adipogenesis to determine if we could confirm its reported inhibitory effects [3,4,20]. Previous studies had detected 1a-hydroxylase activity in 3T3-L1 preadipocytes [9], yet none had tested the effects of 25(OH)D3 on adipogenesis in 3T3-L1 cells. In 3T3-L1 1379592 cells, 1,25(OH)2D3 caused a dose- and time-dependent inhibition of adipogenesis (Fig. 7A B), as previously documented [3,4]. Additionally, in contrast to its pro-adipogenic effects in human preadipocytes, 25(OH)D3 did not affect adipogenesis in 3T3-L1 cells (as shown by the lack of change in FABP4 expression levels, Fig. 7A B).Activation of 25(OH)D3 in Human PreadipocytesBecause CYP27B1 expression was detectable and 25(OH)D3 induced CYP24A1 expression, we conducted preliminary studies to determine whether the enzyme was active. Preadipocytes incubated with 25(OH)D3 (1028 M, 24 h) produced detectable quantities of 1,25(OH)2D3 in the media. 4 samples tested produced 48620 pg/106 cells and one sample made much higher amounts, 1600 pg/106 cells. In newly-differentiated adipocytes, only 2 outVitamin D and Human Preadipocyte DifferentiationFigure 6. The pro-adipogenic effects of 1,25(OH)2D3 were independent of thiazolidinedione treatment. Human preadipocytes were differentiated in the differentiation cocktail with or without thiazolidinedione (TZD) for 7 days and maintained in maintenance media until harvest. 1,25(OH)2D3 or vehicle control was present throughout. Phase contrast image of adipocytes were taken at day 13 after differentiation (A). Expression levels of adipogenic markers [LPL (B) and PPARc (C) mRNA and FABP4 (D) protein] were measured after differentiation (d13?4). Lane 3 and 4 (differentiated in the presence of TZD) were intentionally under loaded to show the results in the same blot. *, p,0.05, **, p,0.01, vehicle control vs. 1,25(OH)2D3 treatment, n = 3 for 1028 and n = 5 for 1027 M. doi:10.1371/journal.pone.0052171.gTo evaluate the possibility that apparent species differences between human preadipocytes and 3T3-L1 cells were not merely related to the initial level of commitment to 18325633 the adipocyte cell fate, we also tested the effect of 1,25(OH)2D3 on primary mouse preadipocyte differentiation. 1,25(OH)2D3 increased the differentiation of mouse prea.
