Gene Expression Pattern in Response to Cholecalciferol Supplementation Highlights Cubilin as a Major Protein of 25(OH)D Uptake in Adipocytes and Male Mice White Adipose Tissue

Gene Expression Pattern in Response to Cholecalciferol Supplementation Highlights Cubilin as a... Abstract It is well established that the active form of vitamin D (i.e., 1,25-dihydroxyvitamin D [1,25(OH)2D]) regulates the expression of genes involved in its own metabolism and transport in the kidney and possibly in the liver. However, little is known about the transcriptional impact of cholecalciferol supplementation on white adipose tissue (WAT) and adipocytes, which are a major site of vitamin D and 25-hydroxyvitamin D [25(OH)D] storage in the organism. To fill this gap, we investigated the impact of cholecalciferol supplementation in WAT via a panel of genes coding for enzymes and proteins involved in vitamin D metabolism and uptake. Mice supplemented with cholecalciferol (15,000 IU/kg of body weight per day) for 4 days showed decreased messenger RNA (mRNA) levels of proteins involved in cholecalciferol metabolism (Cyp24a1, Cyp27a1) and decreased cubilin mRNA levels in WAT. These data were partly confirmed in 3T3-L1 adipocytes incubated with 1,25(OH)2D. The downregulation of cubilin mRNA observed in WAT and in 3T3-L1 was confirmed at the protein level in WAT and at the mRNA level in human primary adipocytes. Vitamin D receptor (VDR) agonist (EB1089) and RNA interference approaches demonstrated that VDR was involved in this regulation. Furthermore, chemical inhibitor and RNA inference analysis demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. This study established an overall snapshot of the genes regulated by cholecalciferol in mouse WAT and cell-autonomously in adipocytes. We highlighted that the regulation of cubilin expression was mediated by a VDR-dependent mechanism, and we demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. Vitamin D, or calciferol, is a hormone synthesized in the epidermis after exposure to UVB radiation, or it can be obtained from eating fatty fish (1, 2). After intestinal uptake (3), dietary vitamin D reaches the bloodstream. Both dietary and endogenous cholecalciferol undergoes its first hydroxylation in the liver by 25-hydroxylases including Cyp2r1—although Cyp27a1, Cyp3a11, and Cyp2j6 are also involved (4)—to produce 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D (5). 25-Hydroxylation is generally regarded as poorly regulated (6), even when 25-hydroxylase activity was decreased by 1,25-dihydroxyvitamin D [1,25(OH)2D] in rat liver (7). In plasma, calciferol and 25(OH)D are principally bound to the vitamin D‒binding protein (DBP; encoded by the Gc gene) [87% of 25(OH)D], their transport protein, and albumin [13% of 25(OH)D], although a small quantity remains unbound [<1% of 25(OH)D] (8, 9). 25(OH)D is taken up by the kidney, where a fraction is 1α-hydroxylated by Cyp27b1 to produce 1,25(OH)2D, the active form of vitamin D. This uptake is mediated by the megalin/cubilin complex (10), which involves other proteins such as disabled 2 (Dab2) (11) and amnionless (Amn) (12). Cyp27b1 activity in the kidney is positively regulated by parathyroid hormone and low calcium levels and deactivated by fibroblast growth factor 23 and 1,25(OH)2D itself through a negative feedback mechanism (13, 14). In target tissues, 25(OH)D and 1,25(OH)2D can be catabolized by 24-hydroxylases (Cyp24a1) to generate inactive metabolites (15). In the kidney, this step is autoregulated, and cholecalciferol supplementation induced Cyp24a1 expression (16, 17). The molecular mechanisms have been unraveled and shown to involve transcriptional regulation mediated by the vitamin D receptor (VDR), which binds 1,25(OH)2D with high affinity. After heterodimerization with the retinoic acid receptor, the resulting complex can bind to vitamin D response elements (VDREs) in the promoter region of regulated genes and can induce their transcriptional activation or repression (18). Vitamin D and 25(OH)D are stored mainly in white adipose tissue (WAT), plasma, and skeletal muscle (19). The uptake of vitamin D and its metabolites in preadipocytes and skeletal muscle cells was recently described (20) and involved megalin (21). In adipocytes, vitamin D and 25(OH)D not only are stored in lipid droplets (22) but could also be converted to active metabolites (23, 24) that were able to modulate adipocyte biology (25–27). Indeed, adipocytes have been shown to express most of the genes involved in vitamin D metabolism, such as 25-hydroxylases (23, 24), 1α-hydroxylase (24, 28), megalin (20), Cyp24a1, and Vdr (23, 29). Given that vitamin D regulates its own metabolism at a transcriptional level in the liver and kidney via its active metabolite 1,25(OH)2D, it is highly likely that similar regulations occur in WAT. Nevertheless, 1,25(OH)2 d-mediated transcriptional regulation of genes involved in vitamin D metabolism in adipocytes and in WAT has been only partly established so far. The main goal of this work was to study the overall impact of cholecalciferol supplementation on the regulation of genes involved in its own metabolism and uptake in vivo in WAT and in vitro in adipocytes. This approach provided key insights into the gene expression pattern of cubilin, and we went on to investigate the underlying molecular mechanism involved. We also demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. Materials and Methods Reagents Dulbecco’s modified Eagle medium (DMEM) was obtained from Life Technologies, and fetal bovine serum (FBS) was obtained from PAA Laboratories. Isobutylmethylxanthine, dexamethasone, and insulin were bought from Sigma-Aldrich. TRIzol reagent, random primers, and Moloney murine leukemia virus reverse transcription were obtained from Life Technologies. SYBR Green reaction buffer was purchased from Eurogentec (Liege, Belgium). [3H]-25(OH)D (161 Ci/mmol for specific activity) was sourced from PerkinElmer (Waltham, MA). Animal experiments The protocol received approval from the local ethics committee. Six-week-old male C57BL/6J mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France), were fed ad libitum with control food (chow diet A04 from SAFE Diets, Augy, France), and had full access to drinking water. Male mice were used to avoid the cyclic hormonal changes associated with the estrus cycle in female mice. Animals were maintained at 22°C under a 12-hour/12-hour light/dark cycle with a 20% humidity level. Mice were supplemented with cholecalciferol (15,000 IU/kg of body weight per day; Sigma-Aldrich, Saint-Quentin-Fallavier, France) for the cholecalciferol group (cholecalciferol; n = 6 mice) or with vehicle alone (olive oil) for the control group (control; n = 8 mice) for 4 days, by gavage (total volume of 200 µL), as previously described (30, 31). Weight gain was measured daily. After 4 days of treatment, the mice were fasted overnight, and blood was collected by cardiac puncture under anesthesia. After euthanasia, tissues [kidney, liver, and epididymal WAT (eWAT)] were collected, weighed, and stored at −80°C. Cell culture and treatment 3T3-L1 preadipocytes (American Type Culture Collection, VA) were seeded in 3.5 cm‒diameter dishes at a density of 15 × 104 cells per well and grown in DMEM supplemented with 10% FBS at 37°C in a 5% CO2-humidified atmosphere, as previously described (32, 33). After 2-day confluence, 3T3-L1 (day 0) was stimulated for 48 hours with 0.5 mM isobutylmethylxanthine, 0.25 μmol/L dexamethasone, and 1 μg/mL insulin in DMEM supplemented with 10% FBS to induce differentiation. The cultures were successively treated with DMEM supplemented with 10% FBS and 1 μg/mL insulin (all products were supplied by Life Technologies, Courtaboeuf, France). Human preadipocytes (isolated from female subcutaneous adipose tissue biopsies) supplied by Promocell (Heidelberg, Germany) were cultured and differentiated into adipocytes according to the company’s instructions. Briefly, cells were seeded at a density of 5000 cells/cm2 in Preadipocyte Growth Medium and grown until confluence, then allowed to differentiate for 3 days in Preadipocyte Differentiation Medium. Mature adipocytes were cultivated in Adipocyte Nutrition Medium for another 11 days, as previously reported (34). To examine the regulation of genes coding for proteins involved in cholecalciferol metabolism, both human and murine adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) dissolved in absolute ethanol for 24 hours or with EB1089 (10 or 100 nM; Life Technologies), a VDR agonist, for 24 hours. RNA interference 3T3-L1 differentiated cells seeded in 24- or 12-well plates were transfected with either targeted small interfering RNA (siRNA; against VDR or cubilin, respectively) or a nontargeting siRNA according to the manufacturer’s instructions (Dharmacon, Lafayette, CO) using INTERFERin (Polyplus Transfection, Illkirch, France) for 24 hours, as previously described (30). Uptake of 25(OH)D by adipocytes 3T3-L1 adipocytes were incubated with [3H]-25(OH)D (PerkinElmer, Villebon, France) at a concentration of 11.25 nCi/mL and with 50 nM of nonradiolabeled 25(OH)D (Sigma-Aldrich, St Louis, MO) in DMEM supplemented with 1 μg/mL insulin in the presence of either 1.9 µM DBP (Sigma Aldrich, St. Louis, MO), 0.125% bovine serum albumin (BSA), or ethanol (control condition). These 3T3-L1 adipocytes were treated with 10 or 100 nM of 1,25(OH)2D (Sigma Aldrich) or with 100 or 500 nM of receptor-associated protein (RAP) (Sigma Aldrich), an inhibitor of the megalin-cubilin complex. After 16 hours of incubation, cells were lysed, and radioactivity was measured by liquid scintigraphy. The results were expressed as counts per minute per well. RNA extraction and real-time quantitative polymerase chain reaction Total RNA was extracted from the liver, kidney, and eWAT or from cells using TRIzol reagent (Life Technologies, Courtaboeuf, France). One microgram of total RNA was used to synthesize complementary DNAs using random primers and Moloney murine leukemia virus reverse transcription (Life Technologies). Real-time quantitative polymerase chain reaction (PCR) analyses were performed using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA), as previously described (35). For each condition, expression was quantified in duplicate, and 18S ribosomal RNA was used as the endogenous control in the comparative cycle threshold method (36). Sequences of the primers used in this study are reported in the supplemental data (Supplemental Table 1). Mouse cubilin protein quantification The quantity of cubilin protein in WAT was determined using a specific enzyme-linked immunosorbent assay kit (mouse cubilin; Mybiosource, San Diego, CA) according to the manufacturer’s protocol. In silico promoter analysis The MatInspector software implemented in the Genomatix suite (www.genomatix.de) was used to perform in silico identification of VDREs within human and mouse cubilin promoter regions. Briefly, human and murine cubilin promoters were extracted directly from the Genomatix ElDorado Database (www.genomatix.de). These promoter regions correspond to loci identified by their ElDorado reference ID. Response elements were identified on these promoter regions using the MatInspector software workflow (37). Cholecalciferol, 25(OH)D, and 1,25(OH)2D quantification in plasma and WAT All quantifications were performed using liquid chromatography-mass spectrometry/mass spectrometry per the protocol described subsequently. Preparation of analytical and deuterated standards A working solution of deuterated analytes was prepared at 0.02 ng/mL of each internal standard [i.e., d3-cholecalciferol, d3-25(OH)D, and d3-1,25(OH)2D)]. Primary stock solutions of cholecalciferol, 25(OH)D, and 1,25(OH)2D standards were prepared at concentrations of 100, 50, and 10 ng/mL, respectively, in ethanol, and stored at −80°C in the dark. Calibration curves were prepared by serial dilution of the three analyte stock solutions to obtain calibration standards from 0 to 75 ng/mL, then the addition of 1.5 µL of the working solution of deuterated analytes to each dilution. After complete evaporation of solvent, derivatization was performed. A one-step derivatization was used to improve the ionization efficiency of the metabolites using Amplifex diene as a reagent (38). Amplifex (30 μL) was added to the previous dried sample, vortexed for 15 seconds, and incubated for 30 minutes at ambient temperature. Next, 30 μL of deionized water was added, vortexed for 15 seconds, and transferred for liquid chromatography injection. Calibration curves were plotted as peak area ratio of the vitamin D metabolite to the respective internal standard vs a range of analyte concentrations. Preparation of plasma Sample preparation was adapted from Wang et al. (39). Because cholecalciferol and its metabolites are light sensitive, the extraction procedure was conducted under low light. After thawing on ice, mice plasmas were centrifuged at 11,000 rpm for 15 minutes at 4°C, and then 100 µL of each sample was transferred to a glass test tube containing 10 µL of deuterated standard working solution. Proteins were precipitated by adding acetonitrile (ACN), vortex mixed, and centrifuged at 3000g for 10 minutes. The supernatant was moved to another glass tube, and the volume was reduced to half under a nitrogen stream. Then, 5 mL of ethyl acetate was added to the solution for liquid-liquid extraction. After being shaken vigorously, samples were centrifuged at 590g for 20 minutes, and the upper organic layer was transferred to a fresh glass tube and reduced under nitrogen stream. The samples were then derivatized as described previously. eWAT preparation Sample preparation was adapted from Lipkie et al. (40). First, 25 µL of deuterated standard working solution was added to tissue homogenates (50 mg of tissue ground within 1 mL of PBS) in a glass test tube. ACN was added, vortex mixed for 5 minutes, and centrifuged at 6000g for 5 minutes. Then, methyl tert-butyl ether was added, vortexed for 5 minutes, and centrifuged, and the upper organic layer was collected into a fresh glass tube. The extraction was repeated twice, and the combined supernatants were dried under nitrogen. Oasis HLB SPE cartridges (Waters, Guyancourt, France) were conditioned with ethyl acetate, methanol (MetOH), and H2O. The sample was reconstituted with 1 mL of MetOH and 1 mL of K2HPO4 (0.4 M) and added onto the cartridge. The cartridge was washed with H2O and 70% MetOH and then dried for 2 minutes under vacuum. Tips were washed with ACN, and analytes were eluted with ACN and dried under nitrogen. After complete evaporation of solvent, the samples were derivatized as described previously. Liquid chromatography-mass spectrometry/mass spectrometry analysis Accurate mass measurements were performed on the Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Heated Electrospray Ionization (H-ESI II) probe. Thermo Xcalibur 3.0.63 software was used for instrument setup, control of the selected reaction monitoring: adipose tissue (AT) system during acquisition, and data treatment. Tune Q Exactive Plus 2.5 software was used for direct control of the mass spectrometer. Samples were injected onto a Hypersil GOLD C18 column (2.1 × 100 mm; Thermo Scientific, Les Ulis, France). The flow rate was 0.4 mL/min, and the injection volume was 5 µL. The mobile phase was composed of A = ultrapure water with 0.1% formic acid (volume-to-volume ratio) and B = ACN with 0.1% formic acid (volume-to-volume ratio). Starting conditions were A = 70% and B = 30%, held for 4 minutes. A linear gradient was applied until 10.0 minutes, where A = 35% and B = 65%, which was held until 12.0 minutes; at 14 minutes, A = 0% and B = 100% until 16 minutes. Starting conditions were reimplemented at 18 minutes. The selected reaction monitoring transitions used for quantification for each analyte were 716.5→657.5 (cholecalciferol), 719.5→660.5 (d3-cholecalciferol), 732.5→673.4 [25(OH)D], 735.5→676.4 [d3-25(OH)D], 751.5→692.4 [d3-1,25(OH)2D], and 748.5→689.4 [1,25(OH)2D]. Data were validated for linearity and repeatability (Supplemental Table 2). Statistical analysis Data are presented as mean ± standard error of the mean. Significant differences between the control group and the treatment group were determined using a Student t test or analysis of variance followed by the Tukey-Kramer post hoc test, all using Statview software (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. Results Cholecalciferol supplementation modified expression of genes involved in cholecalciferol metabolism and uptake in WAT and adipocytes To examine the impact of cholecalciferol supplementation on the regulation of genes coding proteins involved in its own metabolism in WAT, wild-type C56BL/6J male mice were supplemented with cholecalciferol (15,000 IU/kg of body weight per day) for 4 days. Body weight and absolute and relative organ weights were not modified by the treatment (Table 1). As expected, supplemented mice showed higher plasma concentrations of cholecalciferol, 25(OH)D, and 1,25(OH)2D (26.22-, 6.32-, and 9.94-fold, respectively) (Table 1) and increased cholecalciferol and 25(OH)D in WAT (4.87-fold and 3.41-fold, respectively) (Table 1). WAT from supplemented mice also had a higher quantity of 1,25(OH)2D, but the difference compared with that of control mice did not reach statistical significance (Table 1). Table 1. Morphological and Biological Parameters of Mice   Control Mice  Cholecalciferol-Supplemented Mice  Body weight, g  21.9 ± 0.37  21.1 ± 0.90  Liver weight, mg  976.9 ± 29.09  955.5 ± 57.74  Liver weight/body weight ratio  0.0446 ± 0.0007  0.0451 ± 0.0009  Adipose tissue weight, mg  242.9 ± 10.12  281.0 ± 32.63  Adipose tissue weight/body weight ratio  0.0111 ± 0.0005  0.0136 ± 0.002  Serum cholecalciferol, ng/mL  2.58 ± 0.60  67.65 ± 9.66a  Serum 25(OH)D, ng/mL  16.79 ± 0.98  106.16 ± 18.60a  Serum 1,25(OH)2D, pg/mL  68.6 ± 16.95  682.2 ± 122.11a  AT cholecalciferol quantity, ng  158 ± 22.01  770.24 ± 175.28a  AT 25(OH)D quantity, ng  27.3 ± 1.05  93.13 ± 30.91a  AT 1,25(OH)2D quantity, pg  1.58 ± 0.31  2.77 ± 0.68    Control Mice  Cholecalciferol-Supplemented Mice  Body weight, g  21.9 ± 0.37  21.1 ± 0.90  Liver weight, mg  976.9 ± 29.09  955.5 ± 57.74  Liver weight/body weight ratio  0.0446 ± 0.0007  0.0451 ± 0.0009  Adipose tissue weight, mg  242.9 ± 10.12  281.0 ± 32.63  Adipose tissue weight/body weight ratio  0.0111 ± 0.0005  0.0136 ± 0.002  Serum cholecalciferol, ng/mL  2.58 ± 0.60  67.65 ± 9.66a  Serum 25(OH)D, ng/mL  16.79 ± 0.98  106.16 ± 18.60a  Serum 1,25(OH)2D, pg/mL  68.6 ± 16.95  682.2 ± 122.11a  AT cholecalciferol quantity, ng  158 ± 22.01  770.24 ± 175.28a  AT 25(OH)D quantity, ng  27.3 ± 1.05  93.13 ± 30.91a  AT 1,25(OH)2D quantity, pg  1.58 ± 0.31  2.77 ± 0.68  Values are reported as mean ± standard error of the mean. Student t test. a P < 0.05. View Large The expression of genes coding for vitamin D metabolism proteins (Supplemental Table 3) was measured by real-time PCR in liver, kidney, and eWAT. In liver, no major difference in gene expression was observed between the two groups except a decrease of messenger RNA (mRNA) expression of cubilin (Cubn) in cholecalciferol-supplemented mice (0.44-fold) (Fig. 1A). In kidney, Cyp24a1 and Vdr mRNA levels were increased, whereas Cyp27b1, Gc, and Cubn mRNA were decreased in cholecalciferol-supplemented mice (20.68-, 1.78-, 0.96-, 0.33-, and 0.18-fold, respectively) (Fig. 1B). Interestingly, in eWAT, a decrease in Cyp24a1, Cyp27a1, and Cubn gene expression was observed in cholecalciferol-supplemented mice compared with controls (0.59-, 0.34-, and 0.62-fold, respectively) (Fig. 1C). Note that genes not mentioned as regulated in the different tissues were not modified by cholecalciferol supplementation. Figure 1. View largeDownload slide Effect of cholecalciferol supplementation on the expression of cholecalciferol metabolism genes in liver, kidney, and adipose tissues. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA in the (A) liver, (B) kidney, and (C) eWAT of control mice or cholecalciferol-supplemented mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 1. View largeDownload slide Effect of cholecalciferol supplementation on the expression of cholecalciferol metabolism genes in liver, kidney, and adipose tissues. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA in the (A) liver, (B) kidney, and (C) eWAT of control mice or cholecalciferol-supplemented mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. To study the effect of 1,25(OH)2d-mediated cell-autonomous regulation on adipocyte gene expression, 3T3-L1 adipocytes were treated with different doses of 1,25(OH)2D (1, 10, and 100 nM) for 24 hours (Fig. 2; Supplemental Table 4). Cyp27a1 and Cubn mRNA levels were decreased in cells treated with 10 and 100 nM of 1,25(OH)2D compared with control cells (0.36- and 0.34-fold, respectively, for Cyp27a1 and 0.34 and 0.56-fold, respectively, for cubilin). Conversely, Cyp24a1 mRNA expression increased strongly with the higher dose (489.88-fold for 100 nM), similarly to Vdr mRNA (2.87- and 10.35-fold for 10 and 100 nM, respectively). Figure 2. View largeDownload slide Effect of 1,25(OH)2D incubation on expression of cholecalciferol metabolism genes in 3T3-L1 adipocytes. (A–D) 3T3-L1 adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) for 24 hours. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. rRNA, ribosomal RNA. Figure 2. View largeDownload slide Effect of 1,25(OH)2D incubation on expression of cholecalciferol metabolism genes in 3T3-L1 adipocytes. (A–D) 3T3-L1 adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) for 24 hours. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. rRNA, ribosomal RNA. The regulation of cubilin expression in adipocytes was VDR dependent To gain further insight into the mechanism of cholecalciferol uptake by WAT/adipocytes, we focused our analysis on the regulation of cubilin. The downregulation (0.28-fold) of Cubn mRNA was confirmed in human primary white adipocytes incubated with 1,25(OH)2D (100 nM for 24 hours) (Fig. 3A). The impact of cholecalciferol supplementation on cubilin protein was confirmed by enzyme-linked immunosorbent assay in mouse WAT, which showed 0.23-fold lower cubilin protein in cholecalciferol-supplemented mice (Fig. 3B). 3T3-L1 adipocytes were incubated with EB1089, a specific VDR agonist, for 24 hours (Fig. 4A). The mRNA level of Cubn was decreased in adipocytes treated with 10 and 100 nM of EB1089 (0.73- and 0.78-fold, respectively), thus supporting the putative role of VDR in this regulation. In addition, in silico analysis with MatInspector software unveiled the location of several VDREs in both human and mouse cubilin promoters (Table 2). To confirm the involvement of VDR in this regulation, 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide directed against VDR or a nonsilencing control for 24 hours. Quantitative PCR confirmed that the RNA interference was efficient, and a significant decrease of VDR expression was observed (0.83-fold) (Fig. 4B). In addition, transfection with siRNA-targeting VDR completely blunted the 1,25(OH)2d-mediated inhibition of Cubn mRNA level [100 nM of 1,25(OH)2D for 24 hours] (Fig. 4C). Figure 3. View largeDownload slide Cubilin mRNA level was downregulated in human adipocytes and at the protein level in mice adipose tissue. (A) Human primary white adipocytes were incubated with 100 nM of 1,25(OH)2D for 24 hours. Expression of cubilin relative to 18S rRNA. (B) Cubilin protein quantification performed by enzyme-linked immunosorbent assay tests in eWAT of mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 3. View largeDownload slide Cubilin mRNA level was downregulated in human adipocytes and at the protein level in mice adipose tissue. (A) Human primary white adipocytes were incubated with 100 nM of 1,25(OH)2D for 24 hours. Expression of cubilin relative to 18S rRNA. (B) Cubilin protein quantification performed by enzyme-linked immunosorbent assay tests in eWAT of mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 4. View largeDownload slide The regulation of cubilin expression was VDR dependent. (A) 3T3-L1 adipocytes were incubated with EB1089, a VDR agonist (10 and 100 nM) for 24 hours. (B) The 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide for VDR or a nonsilencing control for 24 hours. The efficiency of RNA interference against VDR was determined by quantitative polymerase chain reaction. (C) These cells were transfected with siRNA and incubated with 100 nM of 1,25(OH)2D for 24 hours. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA; siVDR, siRNA directed against VDR. Figure 4. View largeDownload slide The regulation of cubilin expression was VDR dependent. (A) 3T3-L1 adipocytes were incubated with EB1089, a VDR agonist (10 and 100 nM) for 24 hours. (B) The 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide for VDR or a nonsilencing control for 24 hours. The efficiency of RNA interference against VDR was determined by quantitative polymerase chain reaction. (C) These cells were transfected with siRNA and incubated with 100 nM of 1,25(OH)2D for 24 hours. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA; siVDR, siRNA directed against VDR. Table 2. VDRE Sequences and Location Identified With MatInspector Software Within Human and Mouse Cubilin Promoters   Sequence  ElDorado Reference ID  Position of the VDRE  Human promoter  gtttcaaaGGTCaaatagataatga  GXP_271874 (-)  17171654_17172330  Mouse promoter  tcaagagGATTcaaaggcaacttca  GXP_425459 (-)  13491712_13492424    Sequence  ElDorado Reference ID  Position of the VDRE  Human promoter  gtttcaaaGGTCaaatagataatga  GXP_271874 (-)  17171654_17172330  Mouse promoter  tcaagagGATTcaaaggcaacttca  GXP_425459 (-)  13491712_13492424  In silico analysis with MatInspector software of human and mouse cubilin promoters. The position of the VDRE (referred to by a start _ end number) corresponds to its location within the input sequence (ElDorado reference ID). View Large 25(OH)D endocytosis was mediated by cubilin and regulated by 1,25(OH)2D in 3T3-L1 adipocytes To highlight the functional role of cubilin for 25(OH)D uptake, we undertook a preliminary experiment to measure 25(OH)D uptake in 3T3-L1 adipocytes. Cells were incubated for 16 hours without (control) or with [3H]-25(OH)D and nonlabeled 25(OH)D (50 nM) under its free form (dissolved in ethanol) or complexed with different proteins: DBP or BSA. Counts per minute reflected the uptake of 25(OH)D. The highest uptake of 25(OH)D was obtained with BSA as vehicle (44-fold compared with control). A significant but lower uptake was obtained with free form and DBP conditions (22.8-fold and 4.4-fold, respectively) (Fig. 5A). Figure 5. View largeDownload slide 25(OH)D uptake in 3T3-L1 adipocytes was mediated by cubilin. (A) 3T3-L1 adipocytes were incubated with control (control) or with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (B–D) Cells were incubated with RAP (100 or 500 nM) and 25(OH)D was (B) solubilized in ethanol or complexed (C) to BSA or (D) to DBP for 16 hours. (E) 3T3-L1 cells were transfected with siRNA [nontargeted (siNT) or directed against VDR (siVDR)] and incubated with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (F–H) Cells were incubated with 1,25(OH)2D (10 and 100 nM), and 25(OH)D was (F) solubilized in ethanol or complexed (G) to BSA or (H) to DBP. In each experiment, 25(OH)D uptake was quantified by measuring the number of counts per minute (CPM) per well by liquid scintillation. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. siCubilin, siRNA designed against cubilin. Figure 5. View largeDownload slide 25(OH)D uptake in 3T3-L1 adipocytes was mediated by cubilin. (A) 3T3-L1 adipocytes were incubated with control (control) or with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (B–D) Cells were incubated with RAP (100 or 500 nM) and 25(OH)D was (B) solubilized in ethanol or complexed (C) to BSA or (D) to DBP for 16 hours. (E) 3T3-L1 cells were transfected with siRNA [nontargeted (siNT) or directed against VDR (siVDR)] and incubated with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (F–H) Cells were incubated with 1,25(OH)2D (10 and 100 nM), and 25(OH)D was (F) solubilized in ethanol or complexed (G) to BSA or (H) to DBP. In each experiment, 25(OH)D uptake was quantified by measuring the number of counts per minute (CPM) per well by liquid scintillation. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. siCubilin, siRNA designed against cubilin. To confirm the involvement of cubilin receptor in [3H]-25(OH)D endocytosis, cells were treated with RAP, a specific inhibitor of the megalin/cubilin complex. When [3H]-25(OH)D was solubilized in ethanol, there was no difference between RAP-treated conditions and controls (Fig. 5B). However, when [3H]-25(OH)D was bound to BSA or DBP, there was a strong decrease in [3H]-25(OH)D uptake (0.23-fold and 0-49 fold, respectively) (Fig. 5C and 5F). In a second set of experiments, 3T3-L1 adipocytes were transfected with either an siRNA directed against cubilin or a nontargeted siRNA used as control for 24 hours. [3H]-25(OH)D uptake in complexes with ethanol, BSA, or DBP was measured for 16 hours. [3H]-25(OH)D uptake was decreased in the presence of siRNA directed against cubilin compared with nontargeted siRNA when BSA was used as vehicle (0.63-fold) (Fig. 5E). To study the effect of modulating cubilin expression on [3H]-25(OH)D uptake, we incubated the cells with 1,25(OH)2D (10 or 100 nM) and measured the uptake of [3H]-25(OH)D solubilized in ethanol (Fig. 5F) or bound to BSA (Fig. 5G) or DBP (Fig. 5H). In all conditions, incubation with 100 nM of 1,25(OH)2D decreased uptake of [3H]-25(OH)D in adipocytes (0.04-, 0.19-, and 0.45-fold, respectively). Note that even if statistically significant, the 1,25(OH)2D-induced modification of ethanol-solubilized [3H]-25(OH)D uptake was quantitatively negligible (Fig. 5F). Discussion Here, we used targeted gene profiling to show that cholecalciferol regulates the expression of several genes involved in cholecalciferol metabolism and uptake in WAT in response to short-term cholecalciferol supplementation. Among regulated genes, we report the negative regulation of the Cubn gene. Additional experiments in 3T3-L1 adipocytes demonstrated that Cubn regulation was VDR dependent. Finally, we demonstrated that cubilin is involved in 25(OH)D uptake in adipocytes. To analyze the regulatory effect of cholecalciferol supplementation on gene expression in WAT, mice received cholecalciferol for 4 days by oral gavage. Cholecalciferol was diluted in olive oil to ensure better absorption of this lipophilic molecule (41). In this study, no difference in total body mass or organ mass (liver and AT) was detected between groups, but as expected, plasma concentrations of cholecalciferol, 25(OH)D, and 1,25(OH)2D in WAT concentrations of cholecalciferol and 25(OH)D strongly increased in cholecalciferol-supplemented mice. Despite a clear tendency to increase, the quantity of 1,25(OH)2D in the WAT of supplemented mice was not statistically different from that of control mice. In agreement with previously published data (42), cholecalciferol supplementation strongly induced kidney mRNA expression of Cyp24a1 and Vdr and decreased mRNA levels of Cyp27b1, thus validating our experimental conditions. Interestingly, we also observed a decrease of Gc and Cubn gene expression in the kidney. This regulation, if confirmed at the protein level, could result in a decrease in renal recycling of cholecalciferol and its metabolites, thus constituting a way to eliminate excess cholecalciferol from the plasma. In the liver, we observed only a decrease of Cubn expression, but there was no change in the expression of mRNA coding for other enzymes of hepatic cholecalciferol metabolism. This decrease of Cubn expression suggests that the uptake of these molecules could be regulated in the liver at a transcriptional level through a negative feedback mechanism, thus limiting their hepatic uptake. We also gained an overview of the transcriptional effect of cholecalciferol supplementation in eWAT. Our results showed a decrease of Cyp27a1 and Cyp24a1 mRNA levels, suggesting a putative decrease of 25-hydroxylation and inactivation of metabolites. Cyp24a1 induction has already been reported (28), but this is a report of Cyp27a1 repression. This finding will require further investigation, especially to confirm the real contribution of Cyp27a1 in the adipose metabolism of cholecalciferol. Interestingly, we also found a specific downregulation of Cubn mRNA levels, whereas other partners in the endocytosis complex (megalin, Dab2, and Amn) were not transcriptionally affected. To demonstrate the direct effect of 1,25(OH)2D on these regulations, we used murine 3T3-L1 adipocytes. In these cells, we observed an upregulation of Vdr and Cyp24a1, both of which are well-known VDR target genes (43), thus validating our experimental model. The fact that Cyp24a1 was decreased in vivo but strongly increased in vitro is surprising, but could be due to indirect regulations that simultaneously occurred in vivo, whereas induction in vitro resulted only from direct VDR-mediated induction (43). Interestingly, the patterns of Cyp27a1 and Cubn regulation were reproduced in vitro, confirming the direct and cell-autonomous nature of the regulation. Although it is well documented that most of the enzymes of cholecalciferol metabolism are expressed in adipocytes (25), including Vdr (29), 25-hydroxylation enzymes (23, 24), 1α-hydroxylation enzyme (24, 44), and megalin (20), here we report that certain putative actors of hepatic 25-hydroxylation (4) were not expressed in adipocytes. This is notably the case with Cyp2r1 and Cyp3a11, which were not detected in our conditions, in agreement with Zoico et al. (24), who did not detect Cyp2r1 in 3T3-L1 cells. The ability of adipocytes to produce 25(OH)D has been demonstrated (23, 24), but the enzyme involved has not been identified. On the basis of our results (i.e., downregulation of Cyp27a1, which could be considered a negative feedback), we could posit that Cyp27a1 is a major contributor to 25(OH)D production in adipocytes; however, further investigation is needed. The downregulation of Cubn mRNA levels in response to cholecalciferol was confirmed not only in 3T3-L1 adipocytes but also in human primary adipocytes and in mouse WAT. The cubilin protein is known to play a crucial role in 25(OH)D uptake, as mutations causing cubilin dysfunction lead to urinary excretion of 25(OH)D (10). Indeed, cubilin participates together with megalin (45), Dab2 (an intracellular adaptor protein), and Amn (a transmembrane protein) (11, 12), in the endocytosis of 25(OH)D, notably in proximal tubules of the glomerulus. We identified mRNA coding for megalin, Dab2, and Amn in adipocytes and WAT, but we did not observe any modification in expression levels. The detection of megalin mRNA does not fit with the report of Abboud et al. (20), who reported that megalin was expressed in preadipocytes but not in adipocytes. This discrepancy could be due to cell culture model specifics, but it nevertheless clearly demonstrates the existence of the megalin/cubilin complex in adipocytes. To investigate the molecular mechanism involved in Cubn regulation, several approaches were combined. First, the use of a specific VDR agonist (EB1089) led to downregulation of Cubn expression similar to that of 1,25(OH)2D, suggesting that the regulation described in vitro is mediated by VDR. This involvement was demonstrated by the RNA interference experiments implemented here using siRNA targeted against VDR. Furthermore, an in silico analysis (MatInspector in the Genomatix suite) confirmed the presence of putative VDREs within the murine and human promoters of cubilin. To study the involvement of cubilin in 25(OH)D uptake by adipocytes, experiments were undertaken using radiolabeled 25(OH)D. In the physiological context, plasma 25(OH)D is either bound to DBP [87% of total 25(OH)D] or albumin [13% of total 25(OH)D] or else considered unbound [“free form”; ˃1% of total 25(OH)D] (7). In preliminary experiments, the ability of different vehicles to deliver 25(OH)D to adipocytes was tested. We observed that the best vehicle for 25(OH)D was BSA, followed by free form (mimicked here by an ethanolic solution), and then DBP. These data suggest that BSA-complexed 25(OH)D is easily absorbed by cells. Note that the free form can also be internalized in adipocytes. Finally, it appears that the DBP is probably not the best way to deliver 25(OH)D to adipocytes but corresponds to a 25(OH)D storage site in plasma, as previously suggested (46). To confirm the involvement of cubilin in 25(OH)D uptake by adipocytes, two strategies were implemented. First, we used RAP [inhibitor of megalin/cubilin complex (47)], and second, we used an RNA interference approach. Interestingly, 25(OH)D uptake in complex with BSA or DBP was decreased by RAP and, to a lesser extent, siRNA directed against Cubn [especially for 25(OH)D-DBP complexes that were not impacted by siRNA]. Note that 25(OH)D uptake of the free form (in ethanol) was not impacted by RAP or siRNA, suggesting that the uptake of unbound 25(OH)D occurred independently of the megalin/cubilin pathway. Finally, to confirm that cubilin regulation is involved in 25(OH)D uptake, adipocytes were incubated with 1,25(OH)2D. Interestingly, this incubation led to a decrease of cubilin expression and was associated with a decrease of 25(OH)D uptake. Taken together, these data provide strong evidence that cubilin is involved in 25(OH)D uptake by adipocytes. From a physiological point of view, these data suggest that a negative feedback regulation occurs in WAT to control the uptake of cholecalciferol and its metabolites via modulation of cubilin expression. This kind of limitation of cholecalciferol and metabolite storage, which is generally assumed to be a passive mechanism because of lipophilicity, suggests that cholecalciferol and 25(OH)D storage in WAT is actually tightly controlled and regulated. Here, we demonstrated that there is a coordinated overall regulation of genes coding for enzymes involved in the cholecalciferol metabolism in WAT and in adipocytes. 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Gene Expression Pattern in Response to Cholecalciferol Supplementation Highlights Cubilin as a Major Protein of 25(OH)D Uptake in Adipocytes and Male Mice White Adipose Tissue

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00650
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Abstract

Abstract It is well established that the active form of vitamin D (i.e., 1,25-dihydroxyvitamin D [1,25(OH)2D]) regulates the expression of genes involved in its own metabolism and transport in the kidney and possibly in the liver. However, little is known about the transcriptional impact of cholecalciferol supplementation on white adipose tissue (WAT) and adipocytes, which are a major site of vitamin D and 25-hydroxyvitamin D [25(OH)D] storage in the organism. To fill this gap, we investigated the impact of cholecalciferol supplementation in WAT via a panel of genes coding for enzymes and proteins involved in vitamin D metabolism and uptake. Mice supplemented with cholecalciferol (15,000 IU/kg of body weight per day) for 4 days showed decreased messenger RNA (mRNA) levels of proteins involved in cholecalciferol metabolism (Cyp24a1, Cyp27a1) and decreased cubilin mRNA levels in WAT. These data were partly confirmed in 3T3-L1 adipocytes incubated with 1,25(OH)2D. The downregulation of cubilin mRNA observed in WAT and in 3T3-L1 was confirmed at the protein level in WAT and at the mRNA level in human primary adipocytes. Vitamin D receptor (VDR) agonist (EB1089) and RNA interference approaches demonstrated that VDR was involved in this regulation. Furthermore, chemical inhibitor and RNA inference analysis demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. This study established an overall snapshot of the genes regulated by cholecalciferol in mouse WAT and cell-autonomously in adipocytes. We highlighted that the regulation of cubilin expression was mediated by a VDR-dependent mechanism, and we demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. Vitamin D, or calciferol, is a hormone synthesized in the epidermis after exposure to UVB radiation, or it can be obtained from eating fatty fish (1, 2). After intestinal uptake (3), dietary vitamin D reaches the bloodstream. Both dietary and endogenous cholecalciferol undergoes its first hydroxylation in the liver by 25-hydroxylases including Cyp2r1—although Cyp27a1, Cyp3a11, and Cyp2j6 are also involved (4)—to produce 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D (5). 25-Hydroxylation is generally regarded as poorly regulated (6), even when 25-hydroxylase activity was decreased by 1,25-dihydroxyvitamin D [1,25(OH)2D] in rat liver (7). In plasma, calciferol and 25(OH)D are principally bound to the vitamin D‒binding protein (DBP; encoded by the Gc gene) [87% of 25(OH)D], their transport protein, and albumin [13% of 25(OH)D], although a small quantity remains unbound [<1% of 25(OH)D] (8, 9). 25(OH)D is taken up by the kidney, where a fraction is 1α-hydroxylated by Cyp27b1 to produce 1,25(OH)2D, the active form of vitamin D. This uptake is mediated by the megalin/cubilin complex (10), which involves other proteins such as disabled 2 (Dab2) (11) and amnionless (Amn) (12). Cyp27b1 activity in the kidney is positively regulated by parathyroid hormone and low calcium levels and deactivated by fibroblast growth factor 23 and 1,25(OH)2D itself through a negative feedback mechanism (13, 14). In target tissues, 25(OH)D and 1,25(OH)2D can be catabolized by 24-hydroxylases (Cyp24a1) to generate inactive metabolites (15). In the kidney, this step is autoregulated, and cholecalciferol supplementation induced Cyp24a1 expression (16, 17). The molecular mechanisms have been unraveled and shown to involve transcriptional regulation mediated by the vitamin D receptor (VDR), which binds 1,25(OH)2D with high affinity. After heterodimerization with the retinoic acid receptor, the resulting complex can bind to vitamin D response elements (VDREs) in the promoter region of regulated genes and can induce their transcriptional activation or repression (18). Vitamin D and 25(OH)D are stored mainly in white adipose tissue (WAT), plasma, and skeletal muscle (19). The uptake of vitamin D and its metabolites in preadipocytes and skeletal muscle cells was recently described (20) and involved megalin (21). In adipocytes, vitamin D and 25(OH)D not only are stored in lipid droplets (22) but could also be converted to active metabolites (23, 24) that were able to modulate adipocyte biology (25–27). Indeed, adipocytes have been shown to express most of the genes involved in vitamin D metabolism, such as 25-hydroxylases (23, 24), 1α-hydroxylase (24, 28), megalin (20), Cyp24a1, and Vdr (23, 29). Given that vitamin D regulates its own metabolism at a transcriptional level in the liver and kidney via its active metabolite 1,25(OH)2D, it is highly likely that similar regulations occur in WAT. Nevertheless, 1,25(OH)2 d-mediated transcriptional regulation of genes involved in vitamin D metabolism in adipocytes and in WAT has been only partly established so far. The main goal of this work was to study the overall impact of cholecalciferol supplementation on the regulation of genes involved in its own metabolism and uptake in vivo in WAT and in vitro in adipocytes. This approach provided key insights into the gene expression pattern of cubilin, and we went on to investigate the underlying molecular mechanism involved. We also demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. Materials and Methods Reagents Dulbecco’s modified Eagle medium (DMEM) was obtained from Life Technologies, and fetal bovine serum (FBS) was obtained from PAA Laboratories. Isobutylmethylxanthine, dexamethasone, and insulin were bought from Sigma-Aldrich. TRIzol reagent, random primers, and Moloney murine leukemia virus reverse transcription were obtained from Life Technologies. SYBR Green reaction buffer was purchased from Eurogentec (Liege, Belgium). [3H]-25(OH)D (161 Ci/mmol for specific activity) was sourced from PerkinElmer (Waltham, MA). Animal experiments The protocol received approval from the local ethics committee. Six-week-old male C57BL/6J mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France), were fed ad libitum with control food (chow diet A04 from SAFE Diets, Augy, France), and had full access to drinking water. Male mice were used to avoid the cyclic hormonal changes associated with the estrus cycle in female mice. Animals were maintained at 22°C under a 12-hour/12-hour light/dark cycle with a 20% humidity level. Mice were supplemented with cholecalciferol (15,000 IU/kg of body weight per day; Sigma-Aldrich, Saint-Quentin-Fallavier, France) for the cholecalciferol group (cholecalciferol; n = 6 mice) or with vehicle alone (olive oil) for the control group (control; n = 8 mice) for 4 days, by gavage (total volume of 200 µL), as previously described (30, 31). Weight gain was measured daily. After 4 days of treatment, the mice were fasted overnight, and blood was collected by cardiac puncture under anesthesia. After euthanasia, tissues [kidney, liver, and epididymal WAT (eWAT)] were collected, weighed, and stored at −80°C. Cell culture and treatment 3T3-L1 preadipocytes (American Type Culture Collection, VA) were seeded in 3.5 cm‒diameter dishes at a density of 15 × 104 cells per well and grown in DMEM supplemented with 10% FBS at 37°C in a 5% CO2-humidified atmosphere, as previously described (32, 33). After 2-day confluence, 3T3-L1 (day 0) was stimulated for 48 hours with 0.5 mM isobutylmethylxanthine, 0.25 μmol/L dexamethasone, and 1 μg/mL insulin in DMEM supplemented with 10% FBS to induce differentiation. The cultures were successively treated with DMEM supplemented with 10% FBS and 1 μg/mL insulin (all products were supplied by Life Technologies, Courtaboeuf, France). Human preadipocytes (isolated from female subcutaneous adipose tissue biopsies) supplied by Promocell (Heidelberg, Germany) were cultured and differentiated into adipocytes according to the company’s instructions. Briefly, cells were seeded at a density of 5000 cells/cm2 in Preadipocyte Growth Medium and grown until confluence, then allowed to differentiate for 3 days in Preadipocyte Differentiation Medium. Mature adipocytes were cultivated in Adipocyte Nutrition Medium for another 11 days, as previously reported (34). To examine the regulation of genes coding for proteins involved in cholecalciferol metabolism, both human and murine adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) dissolved in absolute ethanol for 24 hours or with EB1089 (10 or 100 nM; Life Technologies), a VDR agonist, for 24 hours. RNA interference 3T3-L1 differentiated cells seeded in 24- or 12-well plates were transfected with either targeted small interfering RNA (siRNA; against VDR or cubilin, respectively) or a nontargeting siRNA according to the manufacturer’s instructions (Dharmacon, Lafayette, CO) using INTERFERin (Polyplus Transfection, Illkirch, France) for 24 hours, as previously described (30). Uptake of 25(OH)D by adipocytes 3T3-L1 adipocytes were incubated with [3H]-25(OH)D (PerkinElmer, Villebon, France) at a concentration of 11.25 nCi/mL and with 50 nM of nonradiolabeled 25(OH)D (Sigma-Aldrich, St Louis, MO) in DMEM supplemented with 1 μg/mL insulin in the presence of either 1.9 µM DBP (Sigma Aldrich, St. Louis, MO), 0.125% bovine serum albumin (BSA), or ethanol (control condition). These 3T3-L1 adipocytes were treated with 10 or 100 nM of 1,25(OH)2D (Sigma Aldrich) or with 100 or 500 nM of receptor-associated protein (RAP) (Sigma Aldrich), an inhibitor of the megalin-cubilin complex. After 16 hours of incubation, cells were lysed, and radioactivity was measured by liquid scintigraphy. The results were expressed as counts per minute per well. RNA extraction and real-time quantitative polymerase chain reaction Total RNA was extracted from the liver, kidney, and eWAT or from cells using TRIzol reagent (Life Technologies, Courtaboeuf, France). One microgram of total RNA was used to synthesize complementary DNAs using random primers and Moloney murine leukemia virus reverse transcription (Life Technologies). Real-time quantitative polymerase chain reaction (PCR) analyses were performed using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA), as previously described (35). For each condition, expression was quantified in duplicate, and 18S ribosomal RNA was used as the endogenous control in the comparative cycle threshold method (36). Sequences of the primers used in this study are reported in the supplemental data (Supplemental Table 1). Mouse cubilin protein quantification The quantity of cubilin protein in WAT was determined using a specific enzyme-linked immunosorbent assay kit (mouse cubilin; Mybiosource, San Diego, CA) according to the manufacturer’s protocol. In silico promoter analysis The MatInspector software implemented in the Genomatix suite (www.genomatix.de) was used to perform in silico identification of VDREs within human and mouse cubilin promoter regions. Briefly, human and murine cubilin promoters were extracted directly from the Genomatix ElDorado Database (www.genomatix.de). These promoter regions correspond to loci identified by their ElDorado reference ID. Response elements were identified on these promoter regions using the MatInspector software workflow (37). Cholecalciferol, 25(OH)D, and 1,25(OH)2D quantification in plasma and WAT All quantifications were performed using liquid chromatography-mass spectrometry/mass spectrometry per the protocol described subsequently. Preparation of analytical and deuterated standards A working solution of deuterated analytes was prepared at 0.02 ng/mL of each internal standard [i.e., d3-cholecalciferol, d3-25(OH)D, and d3-1,25(OH)2D)]. Primary stock solutions of cholecalciferol, 25(OH)D, and 1,25(OH)2D standards were prepared at concentrations of 100, 50, and 10 ng/mL, respectively, in ethanol, and stored at −80°C in the dark. Calibration curves were prepared by serial dilution of the three analyte stock solutions to obtain calibration standards from 0 to 75 ng/mL, then the addition of 1.5 µL of the working solution of deuterated analytes to each dilution. After complete evaporation of solvent, derivatization was performed. A one-step derivatization was used to improve the ionization efficiency of the metabolites using Amplifex diene as a reagent (38). Amplifex (30 μL) was added to the previous dried sample, vortexed for 15 seconds, and incubated for 30 minutes at ambient temperature. Next, 30 μL of deionized water was added, vortexed for 15 seconds, and transferred for liquid chromatography injection. Calibration curves were plotted as peak area ratio of the vitamin D metabolite to the respective internal standard vs a range of analyte concentrations. Preparation of plasma Sample preparation was adapted from Wang et al. (39). Because cholecalciferol and its metabolites are light sensitive, the extraction procedure was conducted under low light. After thawing on ice, mice plasmas were centrifuged at 11,000 rpm for 15 minutes at 4°C, and then 100 µL of each sample was transferred to a glass test tube containing 10 µL of deuterated standard working solution. Proteins were precipitated by adding acetonitrile (ACN), vortex mixed, and centrifuged at 3000g for 10 minutes. The supernatant was moved to another glass tube, and the volume was reduced to half under a nitrogen stream. Then, 5 mL of ethyl acetate was added to the solution for liquid-liquid extraction. After being shaken vigorously, samples were centrifuged at 590g for 20 minutes, and the upper organic layer was transferred to a fresh glass tube and reduced under nitrogen stream. The samples were then derivatized as described previously. eWAT preparation Sample preparation was adapted from Lipkie et al. (40). First, 25 µL of deuterated standard working solution was added to tissue homogenates (50 mg of tissue ground within 1 mL of PBS) in a glass test tube. ACN was added, vortex mixed for 5 minutes, and centrifuged at 6000g for 5 minutes. Then, methyl tert-butyl ether was added, vortexed for 5 minutes, and centrifuged, and the upper organic layer was collected into a fresh glass tube. The extraction was repeated twice, and the combined supernatants were dried under nitrogen. Oasis HLB SPE cartridges (Waters, Guyancourt, France) were conditioned with ethyl acetate, methanol (MetOH), and H2O. The sample was reconstituted with 1 mL of MetOH and 1 mL of K2HPO4 (0.4 M) and added onto the cartridge. The cartridge was washed with H2O and 70% MetOH and then dried for 2 minutes under vacuum. Tips were washed with ACN, and analytes were eluted with ACN and dried under nitrogen. After complete evaporation of solvent, the samples were derivatized as described previously. Liquid chromatography-mass spectrometry/mass spectrometry analysis Accurate mass measurements were performed on the Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Heated Electrospray Ionization (H-ESI II) probe. Thermo Xcalibur 3.0.63 software was used for instrument setup, control of the selected reaction monitoring: adipose tissue (AT) system during acquisition, and data treatment. Tune Q Exactive Plus 2.5 software was used for direct control of the mass spectrometer. Samples were injected onto a Hypersil GOLD C18 column (2.1 × 100 mm; Thermo Scientific, Les Ulis, France). The flow rate was 0.4 mL/min, and the injection volume was 5 µL. The mobile phase was composed of A = ultrapure water with 0.1% formic acid (volume-to-volume ratio) and B = ACN with 0.1% formic acid (volume-to-volume ratio). Starting conditions were A = 70% and B = 30%, held for 4 minutes. A linear gradient was applied until 10.0 minutes, where A = 35% and B = 65%, which was held until 12.0 minutes; at 14 minutes, A = 0% and B = 100% until 16 minutes. Starting conditions were reimplemented at 18 minutes. The selected reaction monitoring transitions used for quantification for each analyte were 716.5→657.5 (cholecalciferol), 719.5→660.5 (d3-cholecalciferol), 732.5→673.4 [25(OH)D], 735.5→676.4 [d3-25(OH)D], 751.5→692.4 [d3-1,25(OH)2D], and 748.5→689.4 [1,25(OH)2D]. Data were validated for linearity and repeatability (Supplemental Table 2). Statistical analysis Data are presented as mean ± standard error of the mean. Significant differences between the control group and the treatment group were determined using a Student t test or analysis of variance followed by the Tukey-Kramer post hoc test, all using Statview software (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. Results Cholecalciferol supplementation modified expression of genes involved in cholecalciferol metabolism and uptake in WAT and adipocytes To examine the impact of cholecalciferol supplementation on the regulation of genes coding proteins involved in its own metabolism in WAT, wild-type C56BL/6J male mice were supplemented with cholecalciferol (15,000 IU/kg of body weight per day) for 4 days. Body weight and absolute and relative organ weights were not modified by the treatment (Table 1). As expected, supplemented mice showed higher plasma concentrations of cholecalciferol, 25(OH)D, and 1,25(OH)2D (26.22-, 6.32-, and 9.94-fold, respectively) (Table 1) and increased cholecalciferol and 25(OH)D in WAT (4.87-fold and 3.41-fold, respectively) (Table 1). WAT from supplemented mice also had a higher quantity of 1,25(OH)2D, but the difference compared with that of control mice did not reach statistical significance (Table 1). Table 1. Morphological and Biological Parameters of Mice   Control Mice  Cholecalciferol-Supplemented Mice  Body weight, g  21.9 ± 0.37  21.1 ± 0.90  Liver weight, mg  976.9 ± 29.09  955.5 ± 57.74  Liver weight/body weight ratio  0.0446 ± 0.0007  0.0451 ± 0.0009  Adipose tissue weight, mg  242.9 ± 10.12  281.0 ± 32.63  Adipose tissue weight/body weight ratio  0.0111 ± 0.0005  0.0136 ± 0.002  Serum cholecalciferol, ng/mL  2.58 ± 0.60  67.65 ± 9.66a  Serum 25(OH)D, ng/mL  16.79 ± 0.98  106.16 ± 18.60a  Serum 1,25(OH)2D, pg/mL  68.6 ± 16.95  682.2 ± 122.11a  AT cholecalciferol quantity, ng  158 ± 22.01  770.24 ± 175.28a  AT 25(OH)D quantity, ng  27.3 ± 1.05  93.13 ± 30.91a  AT 1,25(OH)2D quantity, pg  1.58 ± 0.31  2.77 ± 0.68    Control Mice  Cholecalciferol-Supplemented Mice  Body weight, g  21.9 ± 0.37  21.1 ± 0.90  Liver weight, mg  976.9 ± 29.09  955.5 ± 57.74  Liver weight/body weight ratio  0.0446 ± 0.0007  0.0451 ± 0.0009  Adipose tissue weight, mg  242.9 ± 10.12  281.0 ± 32.63  Adipose tissue weight/body weight ratio  0.0111 ± 0.0005  0.0136 ± 0.002  Serum cholecalciferol, ng/mL  2.58 ± 0.60  67.65 ± 9.66a  Serum 25(OH)D, ng/mL  16.79 ± 0.98  106.16 ± 18.60a  Serum 1,25(OH)2D, pg/mL  68.6 ± 16.95  682.2 ± 122.11a  AT cholecalciferol quantity, ng  158 ± 22.01  770.24 ± 175.28a  AT 25(OH)D quantity, ng  27.3 ± 1.05  93.13 ± 30.91a  AT 1,25(OH)2D quantity, pg  1.58 ± 0.31  2.77 ± 0.68  Values are reported as mean ± standard error of the mean. Student t test. a P < 0.05. View Large The expression of genes coding for vitamin D metabolism proteins (Supplemental Table 3) was measured by real-time PCR in liver, kidney, and eWAT. In liver, no major difference in gene expression was observed between the two groups except a decrease of messenger RNA (mRNA) expression of cubilin (Cubn) in cholecalciferol-supplemented mice (0.44-fold) (Fig. 1A). In kidney, Cyp24a1 and Vdr mRNA levels were increased, whereas Cyp27b1, Gc, and Cubn mRNA were decreased in cholecalciferol-supplemented mice (20.68-, 1.78-, 0.96-, 0.33-, and 0.18-fold, respectively) (Fig. 1B). Interestingly, in eWAT, a decrease in Cyp24a1, Cyp27a1, and Cubn gene expression was observed in cholecalciferol-supplemented mice compared with controls (0.59-, 0.34-, and 0.62-fold, respectively) (Fig. 1C). Note that genes not mentioned as regulated in the different tissues were not modified by cholecalciferol supplementation. Figure 1. View largeDownload slide Effect of cholecalciferol supplementation on the expression of cholecalciferol metabolism genes in liver, kidney, and adipose tissues. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA in the (A) liver, (B) kidney, and (C) eWAT of control mice or cholecalciferol-supplemented mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 1. View largeDownload slide Effect of cholecalciferol supplementation on the expression of cholecalciferol metabolism genes in liver, kidney, and adipose tissues. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA in the (A) liver, (B) kidney, and (C) eWAT of control mice or cholecalciferol-supplemented mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. To study the effect of 1,25(OH)2d-mediated cell-autonomous regulation on adipocyte gene expression, 3T3-L1 adipocytes were treated with different doses of 1,25(OH)2D (1, 10, and 100 nM) for 24 hours (Fig. 2; Supplemental Table 4). Cyp27a1 and Cubn mRNA levels were decreased in cells treated with 10 and 100 nM of 1,25(OH)2D compared with control cells (0.36- and 0.34-fold, respectively, for Cyp27a1 and 0.34 and 0.56-fold, respectively, for cubilin). Conversely, Cyp24a1 mRNA expression increased strongly with the higher dose (489.88-fold for 100 nM), similarly to Vdr mRNA (2.87- and 10.35-fold for 10 and 100 nM, respectively). Figure 2. View largeDownload slide Effect of 1,25(OH)2D incubation on expression of cholecalciferol metabolism genes in 3T3-L1 adipocytes. (A–D) 3T3-L1 adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) for 24 hours. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. rRNA, ribosomal RNA. Figure 2. View largeDownload slide Effect of 1,25(OH)2D incubation on expression of cholecalciferol metabolism genes in 3T3-L1 adipocytes. (A–D) 3T3-L1 adipocytes were incubated with 1,25(OH)2D (1, 10, and 100 nM) for 24 hours. Expression of genes coding for proteins involved in cholecalciferol metabolism relative to 18S rRNA. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. rRNA, ribosomal RNA. The regulation of cubilin expression in adipocytes was VDR dependent To gain further insight into the mechanism of cholecalciferol uptake by WAT/adipocytes, we focused our analysis on the regulation of cubilin. The downregulation (0.28-fold) of Cubn mRNA was confirmed in human primary white adipocytes incubated with 1,25(OH)2D (100 nM for 24 hours) (Fig. 3A). The impact of cholecalciferol supplementation on cubilin protein was confirmed by enzyme-linked immunosorbent assay in mouse WAT, which showed 0.23-fold lower cubilin protein in cholecalciferol-supplemented mice (Fig. 3B). 3T3-L1 adipocytes were incubated with EB1089, a specific VDR agonist, for 24 hours (Fig. 4A). The mRNA level of Cubn was decreased in adipocytes treated with 10 and 100 nM of EB1089 (0.73- and 0.78-fold, respectively), thus supporting the putative role of VDR in this regulation. In addition, in silico analysis with MatInspector software unveiled the location of several VDREs in both human and mouse cubilin promoters (Table 2). To confirm the involvement of VDR in this regulation, 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide directed against VDR or a nonsilencing control for 24 hours. Quantitative PCR confirmed that the RNA interference was efficient, and a significant decrease of VDR expression was observed (0.83-fold) (Fig. 4B). In addition, transfection with siRNA-targeting VDR completely blunted the 1,25(OH)2d-mediated inhibition of Cubn mRNA level [100 nM of 1,25(OH)2D for 24 hours] (Fig. 4C). Figure 3. View largeDownload slide Cubilin mRNA level was downregulated in human adipocytes and at the protein level in mice adipose tissue. (A) Human primary white adipocytes were incubated with 100 nM of 1,25(OH)2D for 24 hours. Expression of cubilin relative to 18S rRNA. (B) Cubilin protein quantification performed by enzyme-linked immunosorbent assay tests in eWAT of mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 3. View largeDownload slide Cubilin mRNA level was downregulated in human adipocytes and at the protein level in mice adipose tissue. (A) Human primary white adipocytes were incubated with 100 nM of 1,25(OH)2D for 24 hours. Expression of cubilin relative to 18S rRNA. (B) Cubilin protein quantification performed by enzyme-linked immunosorbent assay tests in eWAT of mice (control, n = 9; cholecalciferol, n = 6). Values are reported as mean ± standard error of the mean. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA. Figure 4. View largeDownload slide The regulation of cubilin expression was VDR dependent. (A) 3T3-L1 adipocytes were incubated with EB1089, a VDR agonist (10 and 100 nM) for 24 hours. (B) The 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide for VDR or a nonsilencing control for 24 hours. The efficiency of RNA interference against VDR was determined by quantitative polymerase chain reaction. (C) These cells were transfected with siRNA and incubated with 100 nM of 1,25(OH)2D for 24 hours. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA; siVDR, siRNA directed against VDR. Figure 4. View largeDownload slide The regulation of cubilin expression was VDR dependent. (A) 3T3-L1 adipocytes were incubated with EB1089, a VDR agonist (10 and 100 nM) for 24 hours. (B) The 3T3-L1 adipocytes were transfected with either an siRNA oligonucleotide for VDR or a nonsilencing control for 24 hours. The efficiency of RNA interference against VDR was determined by quantitative polymerase chain reaction. (C) These cells were transfected with siRNA and incubated with 100 nM of 1,25(OH)2D for 24 hours. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. rRNA, ribosomal RNA; siVDR, siRNA directed against VDR. Table 2. VDRE Sequences and Location Identified With MatInspector Software Within Human and Mouse Cubilin Promoters   Sequence  ElDorado Reference ID  Position of the VDRE  Human promoter  gtttcaaaGGTCaaatagataatga  GXP_271874 (-)  17171654_17172330  Mouse promoter  tcaagagGATTcaaaggcaacttca  GXP_425459 (-)  13491712_13492424    Sequence  ElDorado Reference ID  Position of the VDRE  Human promoter  gtttcaaaGGTCaaatagataatga  GXP_271874 (-)  17171654_17172330  Mouse promoter  tcaagagGATTcaaaggcaacttca  GXP_425459 (-)  13491712_13492424  In silico analysis with MatInspector software of human and mouse cubilin promoters. The position of the VDRE (referred to by a start _ end number) corresponds to its location within the input sequence (ElDorado reference ID). View Large 25(OH)D endocytosis was mediated by cubilin and regulated by 1,25(OH)2D in 3T3-L1 adipocytes To highlight the functional role of cubilin for 25(OH)D uptake, we undertook a preliminary experiment to measure 25(OH)D uptake in 3T3-L1 adipocytes. Cells were incubated for 16 hours without (control) or with [3H]-25(OH)D and nonlabeled 25(OH)D (50 nM) under its free form (dissolved in ethanol) or complexed with different proteins: DBP or BSA. Counts per minute reflected the uptake of 25(OH)D. The highest uptake of 25(OH)D was obtained with BSA as vehicle (44-fold compared with control). A significant but lower uptake was obtained with free form and DBP conditions (22.8-fold and 4.4-fold, respectively) (Fig. 5A). Figure 5. View largeDownload slide 25(OH)D uptake in 3T3-L1 adipocytes was mediated by cubilin. (A) 3T3-L1 adipocytes were incubated with control (control) or with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (B–D) Cells were incubated with RAP (100 or 500 nM) and 25(OH)D was (B) solubilized in ethanol or complexed (C) to BSA or (D) to DBP for 16 hours. (E) 3T3-L1 cells were transfected with siRNA [nontargeted (siNT) or directed against VDR (siVDR)] and incubated with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (F–H) Cells were incubated with 1,25(OH)2D (10 and 100 nM), and 25(OH)D was (F) solubilized in ethanol or complexed (G) to BSA or (H) to DBP. In each experiment, 25(OH)D uptake was quantified by measuring the number of counts per minute (CPM) per well by liquid scintillation. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. siCubilin, siRNA designed against cubilin. Figure 5. View largeDownload slide 25(OH)D uptake in 3T3-L1 adipocytes was mediated by cubilin. (A) 3T3-L1 adipocytes were incubated with control (control) or with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (B–D) Cells were incubated with RAP (100 or 500 nM) and 25(OH)D was (B) solubilized in ethanol or complexed (C) to BSA or (D) to DBP for 16 hours. (E) 3T3-L1 cells were transfected with siRNA [nontargeted (siNT) or directed against VDR (siVDR)] and incubated with 25(OH)D solubilized in ethanol (free form) or complexed with BSA or DBP. (F–H) Cells were incubated with 1,25(OH)2D (10 and 100 nM), and 25(OH)D was (F) solubilized in ethanol or complexed (G) to BSA or (H) to DBP. In each experiment, 25(OH)D uptake was quantified by measuring the number of counts per minute (CPM) per well by liquid scintillation. Values are reported as mean ± standard error of the mean. Bars not sharing the same letter were significantly different in a Tukey-Kramer post hoc test at P < 0.05. *P < 0.05 for an unpaired Student t test. siCubilin, siRNA designed against cubilin. To confirm the involvement of cubilin receptor in [3H]-25(OH)D endocytosis, cells were treated with RAP, a specific inhibitor of the megalin/cubilin complex. When [3H]-25(OH)D was solubilized in ethanol, there was no difference between RAP-treated conditions and controls (Fig. 5B). However, when [3H]-25(OH)D was bound to BSA or DBP, there was a strong decrease in [3H]-25(OH)D uptake (0.23-fold and 0-49 fold, respectively) (Fig. 5C and 5F). In a second set of experiments, 3T3-L1 adipocytes were transfected with either an siRNA directed against cubilin or a nontargeted siRNA used as control for 24 hours. [3H]-25(OH)D uptake in complexes with ethanol, BSA, or DBP was measured for 16 hours. [3H]-25(OH)D uptake was decreased in the presence of siRNA directed against cubilin compared with nontargeted siRNA when BSA was used as vehicle (0.63-fold) (Fig. 5E). To study the effect of modulating cubilin expression on [3H]-25(OH)D uptake, we incubated the cells with 1,25(OH)2D (10 or 100 nM) and measured the uptake of [3H]-25(OH)D solubilized in ethanol (Fig. 5F) or bound to BSA (Fig. 5G) or DBP (Fig. 5H). In all conditions, incubation with 100 nM of 1,25(OH)2D decreased uptake of [3H]-25(OH)D in adipocytes (0.04-, 0.19-, and 0.45-fold, respectively). Note that even if statistically significant, the 1,25(OH)2D-induced modification of ethanol-solubilized [3H]-25(OH)D uptake was quantitatively negligible (Fig. 5F). Discussion Here, we used targeted gene profiling to show that cholecalciferol regulates the expression of several genes involved in cholecalciferol metabolism and uptake in WAT in response to short-term cholecalciferol supplementation. Among regulated genes, we report the negative regulation of the Cubn gene. Additional experiments in 3T3-L1 adipocytes demonstrated that Cubn regulation was VDR dependent. Finally, we demonstrated that cubilin is involved in 25(OH)D uptake in adipocytes. To analyze the regulatory effect of cholecalciferol supplementation on gene expression in WAT, mice received cholecalciferol for 4 days by oral gavage. Cholecalciferol was diluted in olive oil to ensure better absorption of this lipophilic molecule (41). In this study, no difference in total body mass or organ mass (liver and AT) was detected between groups, but as expected, plasma concentrations of cholecalciferol, 25(OH)D, and 1,25(OH)2D in WAT concentrations of cholecalciferol and 25(OH)D strongly increased in cholecalciferol-supplemented mice. Despite a clear tendency to increase, the quantity of 1,25(OH)2D in the WAT of supplemented mice was not statistically different from that of control mice. In agreement with previously published data (42), cholecalciferol supplementation strongly induced kidney mRNA expression of Cyp24a1 and Vdr and decreased mRNA levels of Cyp27b1, thus validating our experimental conditions. Interestingly, we also observed a decrease of Gc and Cubn gene expression in the kidney. This regulation, if confirmed at the protein level, could result in a decrease in renal recycling of cholecalciferol and its metabolites, thus constituting a way to eliminate excess cholecalciferol from the plasma. In the liver, we observed only a decrease of Cubn expression, but there was no change in the expression of mRNA coding for other enzymes of hepatic cholecalciferol metabolism. This decrease of Cubn expression suggests that the uptake of these molecules could be regulated in the liver at a transcriptional level through a negative feedback mechanism, thus limiting their hepatic uptake. We also gained an overview of the transcriptional effect of cholecalciferol supplementation in eWAT. Our results showed a decrease of Cyp27a1 and Cyp24a1 mRNA levels, suggesting a putative decrease of 25-hydroxylation and inactivation of metabolites. Cyp24a1 induction has already been reported (28), but this is a report of Cyp27a1 repression. This finding will require further investigation, especially to confirm the real contribution of Cyp27a1 in the adipose metabolism of cholecalciferol. Interestingly, we also found a specific downregulation of Cubn mRNA levels, whereas other partners in the endocytosis complex (megalin, Dab2, and Amn) were not transcriptionally affected. To demonstrate the direct effect of 1,25(OH)2D on these regulations, we used murine 3T3-L1 adipocytes. In these cells, we observed an upregulation of Vdr and Cyp24a1, both of which are well-known VDR target genes (43), thus validating our experimental model. The fact that Cyp24a1 was decreased in vivo but strongly increased in vitro is surprising, but could be due to indirect regulations that simultaneously occurred in vivo, whereas induction in vitro resulted only from direct VDR-mediated induction (43). Interestingly, the patterns of Cyp27a1 and Cubn regulation were reproduced in vitro, confirming the direct and cell-autonomous nature of the regulation. Although it is well documented that most of the enzymes of cholecalciferol metabolism are expressed in adipocytes (25), including Vdr (29), 25-hydroxylation enzymes (23, 24), 1α-hydroxylation enzyme (24, 44), and megalin (20), here we report that certain putative actors of hepatic 25-hydroxylation (4) were not expressed in adipocytes. This is notably the case with Cyp2r1 and Cyp3a11, which were not detected in our conditions, in agreement with Zoico et al. (24), who did not detect Cyp2r1 in 3T3-L1 cells. The ability of adipocytes to produce 25(OH)D has been demonstrated (23, 24), but the enzyme involved has not been identified. On the basis of our results (i.e., downregulation of Cyp27a1, which could be considered a negative feedback), we could posit that Cyp27a1 is a major contributor to 25(OH)D production in adipocytes; however, further investigation is needed. The downregulation of Cubn mRNA levels in response to cholecalciferol was confirmed not only in 3T3-L1 adipocytes but also in human primary adipocytes and in mouse WAT. The cubilin protein is known to play a crucial role in 25(OH)D uptake, as mutations causing cubilin dysfunction lead to urinary excretion of 25(OH)D (10). Indeed, cubilin participates together with megalin (45), Dab2 (an intracellular adaptor protein), and Amn (a transmembrane protein) (11, 12), in the endocytosis of 25(OH)D, notably in proximal tubules of the glomerulus. We identified mRNA coding for megalin, Dab2, and Amn in adipocytes and WAT, but we did not observe any modification in expression levels. The detection of megalin mRNA does not fit with the report of Abboud et al. (20), who reported that megalin was expressed in preadipocytes but not in adipocytes. This discrepancy could be due to cell culture model specifics, but it nevertheless clearly demonstrates the existence of the megalin/cubilin complex in adipocytes. To investigate the molecular mechanism involved in Cubn regulation, several approaches were combined. First, the use of a specific VDR agonist (EB1089) led to downregulation of Cubn expression similar to that of 1,25(OH)2D, suggesting that the regulation described in vitro is mediated by VDR. This involvement was demonstrated by the RNA interference experiments implemented here using siRNA targeted against VDR. Furthermore, an in silico analysis (MatInspector in the Genomatix suite) confirmed the presence of putative VDREs within the murine and human promoters of cubilin. To study the involvement of cubilin in 25(OH)D uptake by adipocytes, experiments were undertaken using radiolabeled 25(OH)D. In the physiological context, plasma 25(OH)D is either bound to DBP [87% of total 25(OH)D] or albumin [13% of total 25(OH)D] or else considered unbound [“free form”; ˃1% of total 25(OH)D] (7). In preliminary experiments, the ability of different vehicles to deliver 25(OH)D to adipocytes was tested. We observed that the best vehicle for 25(OH)D was BSA, followed by free form (mimicked here by an ethanolic solution), and then DBP. These data suggest that BSA-complexed 25(OH)D is easily absorbed by cells. Note that the free form can also be internalized in adipocytes. Finally, it appears that the DBP is probably not the best way to deliver 25(OH)D to adipocytes but corresponds to a 25(OH)D storage site in plasma, as previously suggested (46). To confirm the involvement of cubilin in 25(OH)D uptake by adipocytes, two strategies were implemented. First, we used RAP [inhibitor of megalin/cubilin complex (47)], and second, we used an RNA interference approach. Interestingly, 25(OH)D uptake in complex with BSA or DBP was decreased by RAP and, to a lesser extent, siRNA directed against Cubn [especially for 25(OH)D-DBP complexes that were not impacted by siRNA]. Note that 25(OH)D uptake of the free form (in ethanol) was not impacted by RAP or siRNA, suggesting that the uptake of unbound 25(OH)D occurred independently of the megalin/cubilin pathway. Finally, to confirm that cubilin regulation is involved in 25(OH)D uptake, adipocytes were incubated with 1,25(OH)2D. Interestingly, this incubation led to a decrease of cubilin expression and was associated with a decrease of 25(OH)D uptake. Taken together, these data provide strong evidence that cubilin is involved in 25(OH)D uptake by adipocytes. From a physiological point of view, these data suggest that a negative feedback regulation occurs in WAT to control the uptake of cholecalciferol and its metabolites via modulation of cubilin expression. This kind of limitation of cholecalciferol and metabolite storage, which is generally assumed to be a passive mechanism because of lipophilicity, suggests that cholecalciferol and 25(OH)D storage in WAT is actually tightly controlled and regulated. Here, we demonstrated that there is a coordinated overall regulation of genes coding for enzymes involved in the cholecalciferol metabolism in WAT and in adipocytes. 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Published: Feb 1, 2018

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