Bidirectional effect of vitamin D on brown adipogenesis of C3H10T1/2 fibroblast-like cells

Cell culture and induction of adipogenesis

C3H10T1/2-clone 8 cells (#IF050415) were obtained from the Health Science Research Resources Bank (Osaka, Japan). Cells were grown at 37°C in a 5% CO₂ humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). On reaching confluency, cells were induced to differentiate into brown adipocytes by supplementing the culture medium with 10 µg/mL insulin (Nacalai Tesque, Kyoto, Japan), 1 µM dexamethasone (DEX; Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), 125 µM indomethacin (Cayman Chemical, Ann Arbor, MI, USA), and 3 nM 3,3,5-triiodo-L-thyronine (T3; Sigma-Aldrich). Two days after stimulation, the medium was replaced with DMEM supplemented with 10% FBS, 10 µg/mL insulin, and 3 nM T3 as previously described (Kusudo et al., 2019). Thereafter, the medium was changed every two days for a period of seven to eight days. Cells were treated with 1,25(OH)₂D₃ (Sigma-Aldrich) at the indicated doses depending on the experiment. 3T3-L1 cells (Japanese Collection of Research Bioresources Cell Bank, Tokyo, Japan) were stimulated for differentiation using DMEM, containing 10% FBS, 10 µg/ml insulin, 1 µM DEX, and 0.5 mM IBMX, for two days, followed by DMEM supplemented with 10% FBS and 10 µg/ml insulin. The medium was changed every two days for eight days. 1,25(OH)₂D₃ or ethanol was added to the media during the experiment. For beige adipocyte differentiation, 3T3-L1 cells were differentiated with DMEM, supplemented with 10% FBS, 10 µg/ml insulin, 1 µM DEX, and 0.5 mM IBMX, 10 µM rosiglitazone (Sigma-Aldrich), and 3 nM T3, for 48 h (Asano et al., 2014). Subsequently, the medium was replaced with DMEM containing 10% FBS, 10 µg/mL insulin, 10 µM rosiglitazone and 3 nM T3 every two days for eight days. The cells were stimulated with 10 µM isoproterenol for 4 h before harvesting.

Vitamin D receptor (VDR) silencing in C3H10T1/2 cells

The VDR and negative control Stealth small interfering RNAs (siRNAs) (#MSS238646 and #12935200, respectively) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs were transfected into cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The following day, the medium was replaced with DMEM supplemented with 10% FBS. Three days after transfection, the medium was replaced with a differentiation medium for differentiation into brown adipocytes. In addition, to enhance UCP1 expression, C3H10T1/2 cells were stimulated with 1 µM all-trans retinoic acid (Sigma-Aldrich) for the last 24 h of differentiation.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH, USA), according to the manufacturer’s protocol. To quantify mRNA expression levels, qRT-PCR analysis was performed using a StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA) and PowerUp SYBR Green Master Mix (Thermo Fisher Scientific), as described previously (Kusudo et al., 2019). All gene expression data were normalized to the 36B4 expression levels. The respective sense and antisense oligonucleotide primers for the target genes were as follows: Cebpa, 5′-CAAGAACAGCAACGAGTACCG-3′ and 5′-GTCACTGGTCAACTCCAGCAC-3′; Cebpb, 5′-ACGACTTCCTCTCCGACCTCT-3′ and 5′-CGAGGCTCACGTAACCGTAGT-3′; Cebpd, 5′-CGACTTCAGCGCCTACATTGA-3′ and 5′-CTAGCGACAGACCCCACAC-3′; Plin, 5′-CTGTGTGCAATGCCTATGAGA-3′ and 5′-CTGGAGGGTATTGAAGAGCCG-3′; Vdr, 5′-GAATGTGCCTCGGATCTGTGG-3′ and 5′-ATGCGGCAATCTCCATTGAAG-3′; Cox7a1, 5′-AGAAAACCGTGTGGCAGAGA-3′ and 5′-CAGCGTCATGGTCAGTCTGT-3′; Cox8b, 5′-GCGAAGTTCACAGTGGTTCC-3′ and 5′-GAACCATGAAGCCAACGACT-3′; MT-CO1, 5′-CAAGAACAGCAACGAGTACCG-3′ and 5′-GTCACTGGTCAACTCCAGCAC-3′; and Ndfvu1, 5′-ACGACTTCCTCTCCGACCTCT-3′ and 5′-CGAGGCTCACGTAACCGTAGT-3′. All other oligonucleotide primer sets used in this study have been described previously (Kusudo et al., 2019; Hashimoto et al., 2019).

Western blot analysis

Western blotting was performed as previously described (Mukai et al., 2017). Briefly, C3H10T1/2 cells were lysed with radioimmunoprecipitation assay buffer, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). These membranes were incubated overnight at 4°C with primary antibodies against FABP4 (12802-1-AP; Proteintech, Rosemont, IL, USA), VDR (#12550; Cell Signaling Technology, Danvers, MA, USA), UCP1 (sc-518171; Santa Cruz Biotechnology, Inc., Dallas, TX. USA), CIDEA (13170-1-AP; Proteintech), PPARγ (#2435; Cell Signaling Technology), PPARα (15540-1-AP; Proteintech), FGF21 (ab171941; Abcam, Cambridge, UK) and α/β-tubulin (#2148, Cell Signaling Technology). The membranes were then washed and incubated with horseradish peroxidase–labeled secondary antibodies for 1 h at room temperature. Immunoreactivity was determined using Immobilon-P (Millipore) or Chemi-Lumi One L (Nacalai Tesque), and chemiluminescence was visualized using Amasham Imager 680 (GE Healthcare, Chicago, IL, USA).

Oil Red O staining

Cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and stained with Oil Red O (Merck Millipore, Billerica, MA, USA). Images were captured using a BZX-700 microscope (Keyence, Osaka, Japan). To quantify lipid levels, the stain was dissolved in isopropyl alcohol, and its absorbance was measured at 490 nm using an iMark microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA).

Cell viability

The viability of pre-differentiated and differentiated C3H10T1/2 cells was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer’s instructions. In addition, the absorbance at 570 nm was measured using an iMark microplate absorbance reader (Bio-Rad Laboratories).

Reporter assay

Reporter assays were performed as previously described (Kusudo et al., 2019). C3H10T1/2 cells were transfected with pGL4.21-UCP1 and pRL-TK. In addition, 24 h later, cells were stimulated with 100 pM 1,25(OH)₂D₃ for 24 h. Cells were lysed, and the luciferase activity was measured using the Dual-Luciferase System (Promega, Madison, WI, USA).

Quantification of relative mitochondrial copy number

C3H10T1/2 cells were differentiated for eight days. Total DNA was isolated using the NucleoSpin DNA RapidLyse (Takara Bio, Kyoto, Japan) according to the manufacturer’s instructions. To calculate relative mitochondrial DNA copy number, the expression of mitochondrially encoded cytochrome C oxidase I (MT-CO1) and NADH dehydrogenase [ubiquinone] flavoprotein 1 (Ndfvu1) genes was quantified using qRT-PCR (Amthor et al., 2007).

Statistical analysis

Data are expressed as mean ± standard error of the mean. The number of samples was three to six for RT-qPCR and four for Oil Red O staining, cell viability assay, and the reporter assay. The significance of differences between two groups was assessed using Student’s t-test. The significance of differences between multiple groups was assessed using a one-way analysis of variance followed by Dunnett’s post hoc test. Statistical significance was set at p < 0.05.

VDR is required for brown adipogenesis of C3H10T1/2 cells

To investigate the relationship between vitamin D signaling and brown adipocyte differentiation, we first examined the expression of VDR in C3H10T1/2 cells, an established model of brown adipogenesis (Brunmeir et al., 2016) (Fig. 1A). After differentiation, the mRNA expression of the adipogenic markers fatty acid-binding protein 4 (Fabp4) and peroxisome proliferator-activated receptor γ (Pparγ) and that of the brown adipogenesis-related genes uncoupling protein 1 (Ucp1), cell death-inducing DFFA-like effector A (Cidea), Pparα, and fibroblast growth factor 21 (Fgf21) significantly increased (p < 0.001). The expression of these genes was confirmed at the protein level (Fig. 1B). The mRNA expression of Vdr decreased transiently after stimulation (day 2) but recovered on day 4 and thereafter. However, at the protein level, VDR expression decreased after the differentiation (Fig. 1B).

To examine the contribution of VDR to brown adipogenesis in C3H10T1/2 cells, we performed knockdown experiments using siRNA for Vdr in C3H10T1/2 cells (Fig. 2A). VDR knockdown significantly inhibited the differentiation of C3H10T1/2 cells (Fig. 2B). In addition, the expression of Ucp1 and adipogenic markers Fabp4 and perilipin (Plin) were significantly reduced in VDR knockdown cells compared to the control cells (all p < 0.001, Fig. 2C). Decreased UCP1 expression was also confirmed at the protein level (Fig. 2D). These results suggest that VDR plays a vital role in brown adipocyte differentiation.

Effect of 1,25(OH)₂D₃ concentration on brown adipocyte differentiation

To explore the role of vitamin D signaling in brown adipocyte differentiation, we investigated the effects of 1,25(OH)₂D₃ on brown adipocyte differentiation in C3H10T1/2 cells. As shown in Figs. 3A and 3B, high concentrations of 1,25(OH)₂D₃ suppressed lipid accumulation in the C3H10T1/2 cells cultured in adipogenic medium. In addition, consistent with Oil Red O staining, the expression of Plin (adipogenic and lipid droplet accumulation marker) decreased depending on the concentration of 1,25(OH)₂D₃ (Fig. 3C). Figure 3D shows the cytotoxic effect of 1,25(OH)₂D₃ on pre-differentiated or differentiated C3H10T1/2 cells. While 100 pM 1,25(OH)₂D₃ did not exert cytotoxic effects on either stage of C3H10T1/2 cells, 1,000 pM 1,25(OH)₂D₃ showed significant cytotoxic effects on both cell types (p < 0.001).

Furthermore, we examined Ucp1 mRNA expression in 1,25(OH)₂D₃-treated cells. Similar to the lipid accumulation results, at a high concentration of 1,25(OH)₂D₃, Ucp1 expression decreased in a dose-dependent manner (Fig. 4A). However, Ucp1 mRNA expression increased at low concentrations of 1,25(OH)₂D₃ (50–250 pM). UCP1 expression was confirmed at the protein level (Fig. 4B). Analysis of the effects of high (1,000 pM) and low (100 pM) concentrations of 1,25(OH)₂D₃ on the expression of different adipocyte differentiation markers revealed that the PR domain containing 16 (Prdm16), a master regulator of BAT differentiation, was significantly elevated in cells treated with 100 pM 1,25(OH)₂D₃ when compared with control cells (p = 0.030, Fig. 4C). Notably, 100 pM 1,25(OH)₂D₃ did not affect the expression of the white adipocyte-specific markers resistin (Retn) and angiotensinogen (Agt) or the adipogenic genes Fabp4 and Plin when compared with the control cells. Except for Prdm16, the expression of all these genes was suppressed following treatment with 1,000 pM 1,25(OH)₂D₃ (p < 0.001), suggesting that a high concentration of 1,25(OH)₂D₃ inhibits the differentiation of C3H10T1/2 cells into brown and white adipocytes. At 100 pM, 1,25(OH)₂D₃ did not increase mitochondrial copy number (Fig. 4D) but significantly increased the mRNA expression of cytochrome c oxidase polypeptide (Cox)7a1 (p = 0.05) and Cox8b (p <0.001), which are brown fat-selective mitochondrial genes (Fig. 4E). The blood concentration of 1,25(OH)₂D₃ is typically within 2–350 pM (Ryan et al., 2013), suggesting that a low concentration of 1,25(OH)₂D₃ stimulates the brown adipogenic program at physiological concentrations.

1,25(OH)₂D₃ affects the differentiation phase of brown adipogenesis

To determine whether 1,25(OH)₂D₃ affects the early or late phase of brown adipogenesis, C3H10T1/2 cells were treated with 1,25(OH)₂D₃ specifically during the differentiation phase (first 48 h) and cultured without 1,25(OH)₂D₃ (days 2 to 7). As shown in Fig. 5A, treatment with 100 pM 1,25(OH)₂D₃ for the first two days increased Ucp1 and Fabp4 mRNA expression in C3H10T1/2 cells compared to that in the control cells (p = 0.019 and p = 0.049, respectively). Plin expression, however, remained unchanged. We then investigated the effect of 1,25(OH)₂D₃ on the maturation phase of brown adipogenesis (days 2 to 7). To achieve this, C3H10T1/2 cells were initially differentiated in the absence of 1,25(OH)₂D₃ and then treated with 100 pM 1,25(OH)₂D₃ from days 2 to 7. The addition of 1,25(OH)₂D₃ did not affect the expression of Ucp1, Fabp4, or Plin. UCP1 protein expression under both conditions showed the same tendency as the mRNA expression (Fig. 5B). Therefore, a physiological concentration of 1,25(OH)₂D₃ enhanced brown adipocyte differentiation of C3H10T1/2 cells by acting during the early stages of brown adipogenesis. To further understand the molecular mechanism by which 1,25(OH)₂D₃ stimulates the brown adipocyte differentiational program, we examined the expression of transcription factors 48 h after induction of differentiation. As shown in Fig. 5C, 1,25(OH)₂D₃ treatment significantly increased the mRNA expression of Prdm16 and peroxisome proliferator-activated receptor γ coactivator-1α (Pgc1α) and slightly decreased the expression of CCAAT-enhancer-binding protein α (Cebpa) (p < 0.001, p = 0.003, and p < 0.001, respectively). Pparα, Pparγ, and Cidea were unaffected by 1,25(OH)₂D₃ treatment. Studies have reported that at an early stage of differentiation, the CEBP family and PPARγ play an essential role in brown and white adipogenesis (Wu, Bucher & Farmer, 1996; Yubero et al., 1994; Manchado et al., 1994). Therefore, we further investigated the expression levels of Pparγ, Cebpa, Cebpb, and Cebpd 12 h after the initiation of differentiation. However, the addition of 1,25(OH)₂D₃ did not alter the mRNA expression of these genes (Fig. 5D). Malloy & Feldman (2013) reported that VDR directly modulates UCP1 expression. The effect of 1,25(OH)₂D₃ on Ucp1 promoter activity was examined. As shown in Fig. 5E, Ucp1 promoter activity was not affected by adding 1,25(OH)₂D₃.

1,25(OH)₂D₃ inhibits white adipocyte differentiation in a dose-dependent manner but enhances Ucp1 expression in 3T3-L1 beige adipogenesis

We also examined whether the effect of 1,25(OH)₂D₃ on differentiation in the physiological range is specific to brown adipocyte differentiation. To investigate the effect of 1,25(OH)₂D₃ on white adipocyte differentiation, 3T3-L1 cells, a widely used white adipocyte differentiation model, were differentiated with 1,25(OH)₂D₃. As shown in Fig. 6A, treatment with 1,000 pM 1,25(OH)₂D₃ inhibited the differentiation of 3T3-L1 cells. At 100 pM, there was no apparent morphological difference in 3T3-L1 cells compared to that in the control, but the Plin mRNA level was slightly decreased (p = 0.025, Fig. 6B). In addition, the expression of Plin and adiponectin (Adipoq) was significantly reduced at 1,000 pM 1,25(OH)₂D₃ (p < 0.001 and p = 0.011, respectively). Thus, 1,25(OH)₂D₃ inhibits white adipocyte differentiation in a dose-dependent manner. To investigate the inhibitory effect of 1,25(OH)₂D₃ on white adipogenesis of 3T3-L1, we measured the expression of Pparγ, Cebpa, and Cebpb after 48 h of initiation of differentiation. At 1,000 pM, 1,25(OH)₂D₃ significantly downregulated these genes (p < 0.001, p < 0.001, and p = 0.010, respectively, Fig. 6C), which suggested that the inhibitory effect of 1,25(OH)₂D₃ on 3T3-L1 differentiation is exerted through suppression of regulator gene expression. 3T3-L1 cells were reported to differentiate into beige adipocytes with long-term treatment with rosiglitazone, T3, and IBMX (Asano et al., 2014). Therefore, we verified the effect of 1,25(OH)₂D₃ on beige adipogenesis of 3T3-L1. As shown in Fig. 6D, 1,25(OH)₂D₃ significantly increased Ucp1 mRNA expression in a dose-dependent manner at 100 pM (p = 0.011) or higher (p < 0.001).