Study design

The study design has been summarized in Fig. 1. For this study, the stable 3T3-L1 murine preadipocyte cell line and the limited self-renewal hAMSC human adipose-derived mesenchymal stem cells were used. Because of the rapid senescent of hAMSC the type and the number of experiments with these cells were limited.

Fig. 1: Representation of study design.

Mouse 3T3-L1 and human hAMSC cells were used for the study. VitD and BPA treatment, alone and in combination, was performed during differentiation in white adipocytes in both cell models. VDR protein expression was evaluated by WB in 3T3-L1 pre-adipocytes and in undifferentiated hAMSC and during differentiation in the presence and absence of VitD in both cell models. At the end of the differentiation, gene and protein expression of adipogenic markers were evaluated by RT-qPCR and WB in 3T3-L1 cells and only protein expression by WB in hAMSC with and without treatment. Adipogenesis was also evaluated by staining lipid droplets with Oil Red O dye in both cell models with and without VitD and BPA treatment. Bioinformatic analysis was performed to find conserved miRNAs able to regulate PPARγ in mice and humans. The expression of such miRNAs was investigated in 3T3-L1 treated with VitD and BPA and their role was explored in both cell models by using miRNA inhibitors and by evaluating lipid droplet accumulation using Oil Red O staining and adipogenic marker protein expression by WB.

Briefly, after assessing the VitD receptor (VDR) expression in pre-adipocytes and in mature white adipocytes, 3T3-L1 and hAMSC cells were treated with BPA and VitD every other day during differentiation. At the end of cell differentiation, gene and protein adipogenic markers expression were evaluated by RT-qPCR, in 3T3-L1, and WB, in both cell lines; lipid accumulation was evaluated by Oil-Red-O staining; miRNAs expression and silencing were estimated by RT-qPCR and by single-strand inhibitors of endogenous miRNAs, respectively, in order to confirm the BPA-induced pro-adipogenic effect and to explore the potential epigenetic protective effect of VitD.

Cell lines, differentiation, and treatment

3T3-L1 mouse fibroblasts were purchased by American Type Culture Collection (ATCC CL-173) and cultured in Dulbecco’s modified Eagle’s medium (DMEM (1X) + GlutaMAX) supplemented with 10% calf serum (CS) and 1 × 105 U/L penicillin and streptomycin (Gibco, Italy) to reach the confluence. Cultures were maintained in humidified atmosphere of 95% air and 5% CO2 at 37 °C. To achieve adipocyte differentiation, 3T3-L1 pre-adipocytes were incubated with differentiation medium (DMEM (1X) + GlutaMAX with 10% fetal bovine serum (FBS) and 1 × 105 U/L penicillin and streptomycin, containing insulin 10−6 M, dexamethasone 10−7 M and 3-isobutyl-1-methylxanthine 10−3 M to starting adipogenesis (day 0). At day 2 of differentiation process, the medium was replaced with the post-differentiation medium (DMEM, 10% FBS and 1 × 105 U/L penicillin and streptomycin, containing only insulin 10−6 M). Then, the post-differentiation medium was changed every 2 days until the mature adipocytes were obtained (day 10).

The primary Human Adipose-derived Mesenchymal Stem Cells (hAMSC) were purchased by PromoCell (hMSC-AT-c; C-12977) and cultured with MesenCult MSC Basal Medium (Stem Cell Technologies; #05401), MesenCult MSC Stimulatory Supplement (Stem Cell Technologies; #05401) and 1 × 105 U/L penicillin and streptomycin (Gibco, Italy) to reach the confluence. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. To achieve adipogenic differentiation, hAMSC were incubated with MesenCult MSC Basal Medium (Stem Cell Technologies; #05413), MesenCult 10X Adipogenic Differentiation Supplement (Stem Cell Technologies; #05414), MesenCult 500X Adipogenic Differentiation Supplement (Stem Cell Technologies; #05415), 1 × 105 L-Glutamine (Gibco, Italy) and 1 × 105 U/L penicillin and streptomycin (Gibco, Italy). According to the protocol, hAMSC were plated in a proliferating medium for 14 days, with a half-medium change on day 7. After 14 days, the medium was replaced with the adipogenic differentiation medium with a medium change every 2 days up to 14 days. Both cell lines were tested and resulted in mycoplasma-free.

For rigorous and reproducible experiments, different batches (3 for 3T3-L1, undergoing passages 1–15, and 2 for hAMSC, undergoing just 2 passages) were used.

Powders of VitD (Selleck Chemicals (UK) and BPA (Sigma-Aldrich) were dissolved in EtOH and DMSO100%, respectively. Aliquots were stored at −80 °C and fresh aliquots were defrosted prior of each new experiment. Serial dilutions were prepared using EtOH and DMSO 40%, respectively, reaching vehicles concentrations of 0.04% in the final volume in each well. VitD was used at a concentration of 10−7 M, a concentration generally used in in vitro studies [22,23,24], and below the cholecalciferol Cmax reached in subjects in condition of VitD deficiency supplemented with different dosing schedules [25]. BPA was used at concentrations of 10−8 M and 10−9 M, concentrations considered biologically relevant [26] according to 0.2 ng BPA/kg bw per day established by EFSA in 2023 [27]. BPA and VitD were inoculated every other day in the medium changed during the 10 or 14 days of differentiation, for 3T3-L1 and hAMSC cells, respectively. BPA and VitD were inoculated as a single and combined treatment, while vehicles (DMSO 40% and EtOH 40%) were added in control wells.

Oil Red O staining

Lipid accumulation was measured using Oil Red O staining at the end of adipocyte differentiation in untreated and treated cells. After incubation, cells were washed three times with PBS 1X and fixed with 4% paraformaldehyde. After 30 min at room temperature, the fixed cells were washed twice with PBS 1X and then stained using Oil Red O, diluted 6/10 in distilled water, for 20 min at room temperature. From this stage on, the cells were no longer exposed to light. Excess Oil Red O was washed with PBS 1X. Stained oil droplets in 3T3-L1 mature adipocytes and differentiated hAMSC were viewed and photographed under the optical microscope Leica DMIL at ×40 magnification. Then, quantification of lipid droplets was performed by dissolving Oil Red O with isopropanol 100% and by measuring the absorbance of the extracts at 490 nm by Victor X4 Multilabel Plate Reader (Perkin Elmer).

Oil Red O staining was performed in 3T3-L1 cells on a 6-well plate in six independent technical experiments and in hAMSC cells on a 24-well plate in one independent technical experiment including four replicates.

RNA isolation and quantitative real‑time PCR

Total RNA was extracted from differentiated 3T3-L1 cells following the same protocol previously reported [28], with the only exception regarding the use of the QIAzol reagent (Qiagen) in the current study. cDNA was generated from 1 μg RNA retro-transcribed using the RT2 First Strand Kit (Qiagen) as previously described [28]. The cDNA obtained was used for quantification of mRNA levels of all investigated genes or the main markers involved in adipogenesis (PPARγ; C/EBPα; Adiponectin; Leptin; and LPL). Cyclophilin A was used as an internal control for normalization. The primers used (Invitrogen; Thermo Fisher Scientific, Inc.) were as follows: peroxisome proliferator-activated receptor-γ (PPARγ) forward 5’-CAAGAATACCAAAGTGCGATCAA-3’ and reverse 5’-GAGCTGGGTCTTTTCAGAATAATAAG-3’; CCAAT/enhancer-binding protein-α (C/EBP-α) forward 5’-GACCATTAGCCTTGTGTGTTAC-3’ and reverse 5’-TGGATCGATTGTGCTTCAAGTT-3’; Adiponectin forward 5’-TGCCGAAGATGACGTTACTA-3’ and reverse 5’-TCTCACCCTTAGGACCAAGA-3’; Leptin forward 5’-CTGGCAGTCTATCAACAGGTC-3’ and reverse 5’-TCCACCTCTGTGGAGTAGAG-3’; LPL forward 5’-GCTCTCAGATGCCCTACAAA-3’ and reverse 5’-GATGTCCACCTCCGTGTAAA-3’; Cyclophilin A forward 5’-GCAGACAAAGTTCCAAAGACAG-3’and reverse 5’CACCCTGGCACATGAATCC-3’.

cDNAs were amplified in Power Up SYBR Green Master mix (Applied Biosystems by Thermo Fisher Scientific) using the StepOne Plus Real-Time PCR System (Applied Biosystems, Life Technologies) running as previously reported [28]. The relative expression of target genes was calculated using the comparative threshold method, 2−ΔCt correction of target and reference gene transcripts [29]. PCR was performed in 3T3-L1 cells in six independent technical experiments.

Protein extraction and western blot

Protein expression of VDR was evaluated in both cell lines during the proliferation/differentiation period at different time points. Proteins were extracted from 3T3-L1 and hAMSC cells: (1) grown with proliferation medium for 24 h; (2) grown with differentiation medium for 48 h; (3) after 4, 6, and 10 days of post-differentiation medium for 3T3-L1 and after 4, 6 and 14 days of differentiation medium for hAMSC; (4) after 4, 6 and 10 days of post-differentiation medium plus VitD 10−7 M for 3T3-L1 and after 4, 6 and 14 days of differentiation medium plus VitD 10−7 M for hAMSC. Protein expression of the main adipogenic markers was evaluated on pellets collected by treated cells at the end of 10 and 14 days of differentiation, for 3T3-L1 and hAMSC, respectively.

3T3-L1 and hAMSC cells were plated at a density of 3 × 105 in 60 mm dishes and treated with BPA and VitD. Then the cells were washed two times with PBS 1X. Then, lysis buffer (1% NP-40, 10% Glycerol, 137 mM NaCl, 20 mM Tris pH7.6, 20 mM NaF, 2 μg/mL aprotinin, 2 μg/mL leupeptin, 2 μg/mL pepstatin, 200 μM Na3VO4, 1 mM PMSF) was added and cells were stored at 4 °C for 30 min. The homogenate was centrifuged for 15 min, at 12,000 × g and 4 °C and the supernatant was collected and stored at −80 °C until use. Protein concentrations were measured using Pierce BCA Protein Assay (Thermo Scientific) and 40 μg of protein was subjected to SDS-PAGE. The loaded gel was then electroblotted onto a nitrocellulose membrane for 1 h and half in a TransBlot Amersham membrane. After a blocking treatment for 1 h with 5% of milk, the membrane was incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The specific primary antibodies were as follows: VDR (1:1000; #sc-13133, Santa Cruz), peroxisome proliferator-activated receptor-γ (PPARγ; 1:1000; #2443, Cell Signaling); CCAAT/enhancer-binding protein-α (C/EBP-α; 1:1000; #2295, Cell Signaling); Adiponectin (1:1000; #2789, Cell Signaling); Leptin (1:500; #PA1-051; Invitrogen); Lipoprotein Lipase (1:1000; # MA5-18055, Invitrogen); β-actin (1:10,000; #A5441, Sigma-Aldrich). The secondary antibodies goat anti-mouse IgG, HRP Conjugate and donkey anti-rabbit IgG, HRP Conjugate (1:2000; #GtxMu-003-DHRPX and #RbxGt-003-DHRPX, respectively) were purchased from ImmunoReagents.

The proteins were detected using the ECL chemiluminescent HRP substrate (Millipore) and imaged by ImageQuant™ LAS 4000 mini luminescent image analyzer (GE Healthcare).

WB was performed in 3T3-L1 cells in five independent technical experiments to assess adipogenic markers expression and in three independent technical experiments to assess VDR expression and adipogenic markers after VitD and miR-27 inhibitors treatment.

WB was performed in hAMSC cells in two independent technical experiments to assess adipogenic markers expression and in three independent technical experiments to assess VDR expression and adipogenic markers with and without VitD and miR-27 inhibitors treatment.

Bioinformatic miRNAs prediction

Using the algorithm Target Scan, a computational analysis was performed to find miRNA families broadly conserved among the vertebrates and targeting PPARγ both in humans and mice. The analysis revealed miR-27-3p as a modulator of the target PPARγ, thus with a potential role in adipogenesis.

miRNAs isolation and quantitative real‑time PCR

For miRNA analysis, total RNA was extracted using a miRNeasy Mini Kit (#217004; Qiagen). After treatment with BPA 10−8 and 10−9 M and VitD 10−7 M, cells were lysed in QIAzol Lysis Reagent. miRNAs isolation and RT-qPCR have been performed according to the previously used protocol [30]. After washings with buffers and centrifugations for several minutes, miRNeasy Mini spin columns were used to inhibit RNases and to remove cellular DNA and proteins from the samples. Obtained RNA samples were eluted in 30 μL of RNAse-free water. Then the total RNA was used to generate cDNAs via reverse transcription with TaqMan® MicroRNA Reverse Transcription Kit (#4366596, Applied Biosystem). cDNAs derived from the reverse-transcription reaction were pre-amplified using TaqMan® PreAmp Master Mix (#4488593, Applied Biosystem) and specific pre-formulated TaqMan® MicroRNA Assay 20X (TaqMan® probe and primer set) miR-27a-3p and miR-27b-3p (#4427975, assay ID:00408 and assay ID:00409, respectively).

Once samples were ready, RT-qPCR was performed to detect the mRNA expression levels of miR-27a-3p and miR-27b-3p using 2 µl of cDNA that was added to 10 µl of PCR mix prepared by adding 0.5 µl of TaqMan® MicroRNA assay 20X, 5 µl PCR master mix (TaqMan® Fast Advanced Master mix, #4444964, Applied Biosystem), and 4.5 µl of sterile water. The miR-27a-3p and miR-27b-3p levels were normalized to U6 small nuclear RNA (#4427975, assay ID: 001973). PCR was performed in 3T3-L1 cells in ten independent technical experiments.

Statistical analysis

The GraphPad Prism 9 software was used to perform statistical analysis. The experiments were expressed as the means and standard errors of the mean (S.E.M.). For statistical comparison, analysis of variance (ANOVA) or Student’s t tests with post hoc testing, Tukey’s multiple comparison test and Mann–Whitney test two-tailed for parametric and nonparametric data, were used. A p value < 0.05 with a 95% confidence interval (CI) was considered significant.