25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ promote the differentiation of human subcutaneous preadipocytes

Abstract

1,25(OH)(2)D(3) inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D(3) to 1,25(OH)(2)D(3), were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)(2)D(3), as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)(2)D(3) enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)(2)D(3) was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0-3), suggesting that 1,25(OH)(2)D(3) promoted maturation. 25(OH)D(3) also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)(2)D(3). Consistent with this possibility, incubation of preadipocytes with 25(OH)D(3) led to 1,25(OH)(2)D(3) accumulation in the media. 1,25(OH)(2)D(3) also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.

Figure 1. Expression levels of VDR and CYP27B1 in human adipose tissues and primary cultures of human preadipocytes and adipocytes.

A and B. Expression levels of VDR and CYP27B1 mRNA were measured in adipose tissue (T), isolated fat cells (FC) and stromal vascular cells (SVC) from human omental and subcutaneous depots (n = 4). C and D. Expression levels of VDR and CYP27B1 mRNA were measured in human preadipocytes and newly-differentiated adipocytes (n = 5). **, p<0.01, preadipocytes vs. adipocytes. E. Protein levels of CYP27B1, VDR and adiponectin were measured with immunoblotting in 3 independent subjects before (preadipocytes; Pre) and 14d after differentiation (adipocytes: Adi).

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 (10⁻¹⁰, 10⁻⁹, 10⁻⁸ M), or 25(OH)D₃ (10⁻¹⁰, 10⁻⁹, 10⁻⁸ M) for 24 h and CYP24A1 mRNA expression was measured (n = 3). B. Differentiated adipocytes were treated with vehicle control, 1,25(OH)₂D₃ (10⁻⁸ M), or 25(OH)D₃ (10⁻⁸ M) for 24 h and CYP24A1 mRNA expression was measured (n = 5). **, p<0.01, control vs. treatments.

Figure 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 (10⁻¹⁰, 10⁻⁸ M) or 25(OH)D₃ (10⁻⁹, 10⁻⁸ 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 PPARγ 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.

Figure 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 (10⁻⁸ M, added continuously throughout). Expression levels of adipogenic markers [C/EBPβ (A), C/EBPα (B), PPARγ (C), and LPL (D)] were measured before (0′) and at indicated time points during differentiation. Data are presented as % of vehicle control after differentiation (d10–12; 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.

Figure 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 (d13–14). 1,25(OH)₂D₃ (10⁻⁸ M) was added during the first 3 days of induction (0′–3d), maturation (3d-end), or continuously throughout (0′-end). Expression levels of adipogenic markers [LPL (A, n = 6) and PPARγ (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.

Figure 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 PPARγ (C) mRNA and FABP4 (D) protein] were measured after differentiation (d13–14). 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 10⁻⁸ and n = 5 for 10⁻⁷ M.

Figure 7. Effects of 1,25(OH)₂D₃ on differentiation of 3T3-L1 preadipocytes (A & B) and mouse preadipocytes (C & D).

A&B. 3T3-L1 cells were grown and differentiated using a standard protocol. Vehicle control, 1,25(OH)₂D₃ or 25(OH)D₃ was added at indicated doses or periods of differentiation. FABP4 expression levels were measured as a late marker of differentiation. **, p<0.01, control vs. treatment, n = 2–3. C& D. 2d-post confluent mouse preadipocytes were differentiated in the presence of thiazolidinedione (1 µM Rosiglitazone during 2d-induction period). 1,25(OH)₂D₃ (10⁻⁸, 10⁻⁷ M) was added continuously and the degree of differentiation was determined by measuring FABP4 expression levels after differentiation. *, p<0.05, vehicle control vs. treatment, n = 4.