The authors have declared that no competing interests exist.
Conceived and designed the experiments: HN MH SF ML. Performed the experiments: HN ML. Analyzed the data: HN SF ML. Contributed reagents/materials/analysis tools: MH SF. Wrote the paper: HN MH SF ML.
1,25(OH)2D3 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)D3 to 1,25(OH)2D3, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)2D3, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)2D3 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)2D3 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)2D3 promoted maturation. 25(OH)D3 also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)2D3. Consistent with this possibility, incubation of preadipocytes with 25(OH)D3 led to 1,25(OH)2D3 accumulation in the media. 1,25(OH)2D3 also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.
In addition to its roles in regulating systemic calcium homeostasis and skeletal health, 1,25-dihydroxyvitamin D [1,25(OH)2D, D represents D2 or D3] regulates differentiation, proliferation and apoptosis of many cells types
The local production of 1,25(OH)2D from 25-hydroxyvitamin D [25(OH)D], catalyzed by 1α-hydroxylase (CYP27B1), modulates the cell and tissue specific regulation of this hormone’s action
The first objective of this study was to determine whether the VDR and 1α-hydroxylase genes are expressed in human adipose tissues, in which cell types (adipocytes vs. stromal cells), and to assess how they are influenced by differentiation. The second objective was to assess the effects of both 25(OH)D3 and 1,25(OH)2D3 on early and late markers of adipogenesis
Adipose tissues were obtained from a total of 13 subjects during abdominal surgeries for severe obesity, gynecological abnormalities or panniculectomy. All subjects were free of diabetes, endocrine, or inflammatory diseases by medical history. Surgeries took place at the University of Maryland, School of Medicine, Baltimore, MD and Boston University, Medical Center, Boston, MA. All subjects gave informed consent as approved by IRB of the University of Maryland, School of Medicine and the Boston University, Medical Center.
Aliquots of adipose tissues were either immediately frozen in the operating room or transferred to the lab in Medium 199. Omental and subcutaneous adipose tissues from 4 subjects (3 females and one male with a mean age of 37.5±6.8 years and BMI 42±4.5 kg/m2) were used to prepare isolated adipocytes and stromal vascular cells (SVC) by collagenase digestion
Abdominal subcutaneous adipose tissue samples from 9 subjects (8 females and one male) with a mean age of 44.8±3.5 years and BMI 32.8±8.2 kg/m2 (25.6–50.9) were used to prepare preadipocyte cultures by collagenase digestion
1,25(OH)2D3 (10−10, 10−8, 10−7 M), 25(OH)D3 (10−9, 10−8 M) or ethanol (vehicle) was added continuously, only during the induction phase, or only during the maintenance phase, as specified in the figure legends. Preadipocytes from different subjects were not pooled. Independent experiments using cultures derived from the same individual provided consistent results. All experiments were repeated on cultures derived from at least 3 different subjects. We did not notice any variations in the effects of vitamin D as a function of the BMI of the donor. In separate experiments, we also tested the effects of 1,25(OH)2D3 in the absence of a TZD in the differentiation cocktail.
3T3-L1 fibroblasts were cultured in 10% FBS supplemented DMEM. 2d post-confluent (day 0) cells were differentiated in DMEM with 10% FBS, 500 µM IBMX, 100 nM bovine insulin, and 1 µM dexamethasone. Medium was replenished with DMEM+10% FBS with 100 nM insulin on d2, and with DMEM+10% FBS on d4. 1,25(OH)2D3 (10−11, 10−10, 10−8 M), 25(OH)D3 (10−9 M), or vehicle (ethanol) was added to the media during differentiation at times specified in the figure legends.
Stromal vascular cells from the inguinal adipose tissue of C57BL/6J mice were prepared as described for human preadipocytes. Cells were grown and differentiated as described for 3T3-L1 cells, except that the differentiation cocktail with Rosiglitazone (1 µM) was added only during the initial 2d-induction period. 1,25(OH)2D3 or vehicle control (ethanol) was added continuously until harvest on day 7. Animal studies were conducted in conformity with PHS policy and approved by IACUC of Boston University Medical Campus.
The ability of preadipocytes and newly-differentiated adipocytes to produce 1,25(OH)2D3 from 25(OH)D3 was tested. Upon reaching confluence, preadipocytes were incubated with 25(OH)D3 (10−8 M) for 24 h in α-MEM without FBS. Newly-differentiated human adipocytes were incubated with 25(OH)D3 (10−8 M) for 24 h in DMEM/F12 with no other additions. The quantity of 1,25(OH)2D3 in the incubation media was assayed with an enzyme immunoassay (Immunodiagnostic Systems Inc.). Data were expressed as picograms of 1,25(OH)2D3 produced per million cells.
Total RNA was extracted using Trizol (Invitrogen) and quantity and quality were assessed spectrophotometrically. 1 µg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and qPCR was performed with the Light Cycler 480 (Roche) with Taqman probes (Applied Biosystems). Cyclophilin A (PPIA) was used as a reference gene.
Cells were washed with ice-cold PBS and scraped in cell lysis buffer (Cell Signaling) supplemented with 5% SDS and protease inhibitors (Pierce). 5–10 µg total protein was resolved in 10 or 15% Tris-HCl gels (Biorad), transferred to PVDF membranes, and blocked in 5% milk in Tris buffered saline with 0.2% tween-20. Membranes were probed for FABP4 (a gift from Dr. Judith Storch at Rutgers University), VDR (D-6, Santa Cruz), adiponectin (BD Biosciences), CYP27B1 (C-12 and H-90, Santa Cruz), and loading controls [(α-tubulin (Santa Cruz) and total ERK (Cell Signaling)]. Chemiluminescence images were captured using an Imager (LAS 4000, Fuji) and quantified using software (Multi Guage, Fuji).
Total TG and DNA quantity in cell lysates were measured using a triglyceride determination kit (Sigma) and Quant-iT™ PicoGreen dsDNA reagent (Invitrogen).
Data are expressed as means±standard error mean (SEM). After log transformation, the differences between groups were determined by analysis of variance with repeated measures and 2-tailed Student t tests using GraphPad (GraphPad Software). Means were considered statistically different when p values were less than 0.05.
VDR and CYP27B1 (1α-hydroxylase) mRNA were easily detected in samples of both omental and sc human adipose tissues (
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).
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 (
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 both human 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.
To 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 (10−10, 10−9, 10−8 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 (
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.
Since 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 (
To determine whether 1,25(OH)2D3 affects early or late events in adipogenesis, we next assessed the time course effects of 1,25(OH)2D3 on mRNA levels of key transcription factors and adipocyte genes during differentiation
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.
To test whether 1,25(OH)2D3 affected the induction or maturation phase of adipogenesis, 1,25(OH)2D3 (10−8 M) was added continuously from the start of differentiation (0′-end), only during the initial 3d-induction period (0′–d3), or between day 3 to day 14 (d3-end). When added during the induction period (0′–3d), 1,25(OH)2D3 did not significantly affect the expression of any differentiation markers (
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.
Previous studies indicate that TZD partially ameliorate the inhibitory effects of vitamin D on adipogenesis
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.
Because 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 (10−8 M, 24 h) produced detectable quantities of 1,25(OH)2D3 in the media. 4 samples tested produced 48±20 pg/106 cells and one sample made much higher amounts, 1600 pg/106 cells. In newly-differentiated adipocytes, only 2 out of 5 samples tested produced detectable amounts of 1,25(OH)2D3 (47 and 67 pg/106 cells).
We tested the effects of 1,25(OH)2D3 on 3T3-L1 adipogenesis to determine if we could confirm its reported inhibitory effects
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.
To 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 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 preadipocytes as determined by increases in FABP4 (
Our findings provide a number of novel insights into vitamin D actions on human adipose tissue. In contrast to its inhibitory effects in a mouse preadipocyte cell line, 3T3-L1, 1,25(OH)2D3 promoted adipogenesis in primary human preadipocytes as evidenced by the increased expression of adipogenic markers and lipid filling. In addition, we show that 25(OH)D3 can also promote the differentiation of human adipocytes, most likely via its activation to 1,25(OH)2D3. Furthermore, 1,25(OH)2D3 also had stimulatory effects on the differentiation of primary mouse preadipocytes. These results suggest that the local metabolism of vitamin D in adipose tissue may regulate the conversion of preadipocytes to adipocytes and hence support the healthy remodeling of human adipose tissue.
Addition of 1,25(OH)2D3 to the standard differentiation cocktail promoted the maturation of adipogenesis. Although 1,25(OH)2D3 did not affect the expression of C/EBPβ, an early marker of adipogenesis, it led to sustained increases in C/EBPα and PPARγ gene expression during the late phase of differentiation. Thus, 1,25(OH)2D3 may promote the differentiation of human preadipocytes by maintaining a high expression level of these key adipogenic transcription factors
Our data demonstrating that 1,25(OH)2D3 and 25(OH)D3 enhanced human preadipocyte differentiation are consistent with the findings that VDR−/− mice are leaner and resistant to diet induced obesity
Similar to our results that show an increase in lipid accumulation, Li et al showed that 1,25(OH)2D3 increases lipoprotein lipase expression in 3T3-L1 preadipocytes
Although 25(OH)D3 increased adipogenesis and induced CYP24A1 mRNA to a similar extent as 1,25(OH)2D3, our study cannot definitively establish whether this is due to the conversion of 25(OH)D3 to 1,25(OH)2D3. It is generally assumed that the induction of CYP24A1 mRNA, is due to the genomic actions of VDR, presumably by 1,25(OH)2D
The results of this study demonstrate for the first time that CYP27B1 mRNA, which encodes the 1α-hydroxylase that converts 25(OH)D to the biologically active 1,25(OH)2D, was present at significant levels in both omental and subcutaneous human adipose tissues. This gene was mainly expressed in the stromal vascular fraction of human adipose tissue that contains preadipocytes, macrophages and endothelial cells. CYP27B1 is known to be expressed in macrophages and endothelial cells, so this result is not unexpected
In preliminary experiments we found that intact human adipose tissue fragments produced easily detectable quantities of 1,25(OH)2D3 from 25(OH)D3 (HN and MJL, unpublished observation). Because adipose tissues of obese are infiltrated with macrophages, it seems likely that macrophages also contribute to the local activation of vitamin D. Further studies are needed to pinpoint the relative contribution of different cell type(s) expressing 1α-hydroxylase in human adipose tissues and to determine how vitamin D activation may change with pathophysiological states such as obesity. Nevertheless, the current results are consistent with the idea that 25(OH)D is activated locally within human adipose tissue and provide strong motivation for further studies directed at understanding the physiological and pathophysiological importance of local 1,25(OH)2D3 production in amplifying vitamin D action in human adipose tissues.
In contrast to our results that 1,25(OH)2D3 promoted human preadipocyte differentiation, Lorente-Cebrian recently noted that they could not find any effect on differentiation
The pro-adipogenic effect of 1,25(OH)2D3 in human preadipocytes is in contrast to its anti-adipogenic effect in the commonly used preadipocyte cell line, 3T3-L1
In conclusion, our studies provide evidence that 1,25(OH)2D3 as well as 25(OH)D3 can influence human adipocyte differentiation by acting during the maturation and lipid filling processes. Although the mechanisms by which 25(OH)D and 1,25(OH)2D influence human adipogenesis require further investigation, we speculate that vitamin D actions may promote the healthy remodeling of adipose tissue as dying adipocytes are replaced with newly-differentiated, insulin-sensitive ones