The impact of vitamin D supplementation on VDR gene expression and body composition in monozygotic twins: randomized controlled trial

Vitamin D supplementation is widely used. However, there is no consensus on the use and dosage of this supplement and the existing recommendations arise from studies based on the benefits that this nutrient can facilitate in bones. In addition, individual genetics can influence the response to supplementation, therefore, research involving monozygotic twins aims to reduce these differences in phenotypic responses. The objective of this randomised controlled study is to examine the effect of vitamin D supplementation on body composition and the expression of the vitamin D receptor (VDR) mRNA. An intervention was performed through supplementation with cholecalciferol at the concentration of 2000 IU in 90 healthy adult monozygotic twins (male or female pairs) for 2 months. The findings showed that serum vitamin D concentration increased by 65% and VDR gene expression sixty times (p = 0.001). Changes in body composition parameters were observed regarding body fat and lean mass. Our results indicate that an increase in serum vitamin D concentration may have potential therapeutic implications.


Randomization. Randomization occurred by lot, performed centrally between 06 June 2016 and 21
December 2018 (at the Laboratory of movement at the Federal University of Rio Grande of Norte) using computer generated sequences stratified for twin 1 and twin 2, where each pair of twins received a numeric code and was separated, with one twin allocated to the control group (CG, without intervention) and the other to the supplemented group (SG, with intervention). The researcher responsible for the registration of the participants and for generating the randomisation and allocation of the assigned intervention were unique and different. The last follow-up occurred on February 21, 2019. In total, 94 eligible twins were recruited, and after being given information about the study, 90 individuals (73% women and 27% men) agreed to participate through written consent. There was sample loss only for analysis of gene expression of 40 individuals, since they did not return for collection regarding this analysis. However, to obtain a statistical power of 0.958, using the values of Mean and Standard Deviation, at least 25 individuals are required before supplementation and 25 individuals after supplementation in these analyzes. In the other variables evaluated the 90 individuals remained from beginning to end. The study complied with the Consolidated Standards of Reporting Trials-CONSORT (Fig. 1).
The twins included in the SG received 60 capsules containing cholecalciferol with a concentration of 2000 IU each, and were instructed to ingest one capsule per day for 60 days. This concentration does not exceed the maximum tolerable daily intake limit (UL), which is 4,000 IU/day 18 , in addition to being considered a dose for maintaining adequate levels of vitamin D in individuals after treatment for deficiency of this nutrient 4 .
At the beginning of the research, the primary endpoint was considered as the variation in the serum concentration of 25 (OH) D after 60 days of supplementation with cholecalciferol. Our secondary endpoint was the variation in gene expression and body composition after 60 days of cholecalciferol supplementation.
During the intervention period, the twins maintained their usual routine, without dietary, sports, or lifestyle changes. After 30 days of inclusion in the study, participants from both groups were interviewed by a phone call to confirm appropriate capsule intake and for routine maintenance. All participants received a follow-up form to daily record when they ingested each capsule. Sixty days after the start of supplementation, these forms were returned to the researchers. Body composition. Body composition was estimated by Dual Energy X-ray absorptiometry, performed using Lunar Prodigy Densitometer equipment, Model NRL 41990 (GE Lunar, Madison, WI, USA). The body mass index (BMI), total body fat and by body part, total lean mass and by body part, and bone mineral density (BMC) were measured on the day of inclusion in the study (T0, time 0) and 60 days after the first analysis (T60, time 60) in all participants in the CG and SG groups.
To calculate the BMI, height and body weight were measured using a stadiometer.
Sun exposure and skin phototype. The assessment of sun exposure was obtained through a score corresponding to the 7 days prior to the date of the bone densitometry evaluation 19 . The use of sunscreen was also recorded. The skin phototype was classified according to a questionnaire proposed by Fitzpatrick 20 . Biochemical and vitamin D measurement. Peripheral  RnA extraction. 4,0 mL of peripheral blood samples were collected from patients in tubes containing EDTA and leukocytes were isolated by centrifugation (1,340× g, 15 min) for gene expression analysis. Total RNA was extracted from leukocytes conserved in RNAlater stabilization solution (Invitrogen, California, USA) using Trizol reagent (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's protocol. RNA quantification and purity were assessed by spectrophotometry using a Nanodrop ND-1000. RNA samples were stored at − 80 °C until RT-qPCR analysis.
RnA expression by Rt-qpcR. cDNA was synthesized with 50 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA), in a MyCycler Thermal Cycler (Bio-Rad, Philadelphia, PA, USA), according to the manufacturer's protocol, and stored at − 20 °C.
The qPCR assays were performed in a Quant Studio 12 K Flex Real-Time PCR System (Applied Biosystems, Forest City, USA). mRNAs with Ct-values > 35 were excluded from the statistical analysis. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), 18S rDNA, and ACTB were used as reference genes. According to the NormFinder algorithm, ACTB was the most stable gene under our experimental conditions, and thus it was used as the endogenous control. Relative expression was calculated by the 2 −ΔΔCt method 21 , using the nonsupplemented group as the control.
Statistical analysis. Distribution of variables was analyzed using the Kolmogorov-Smirnov test. Medians and quartiles are used to describe non-normally distributed data and absolute number and percentages to describe categorical data. The Mann-Whitney test was utilized to analyze the difference in independent group samples and the Wilcoxon test for dependent samples to verify intragroup differences. For categorical samples, the Chi-Square test was performed.
Statistical analysis was performed using the statistical software IBM SPSS version 20.0 for Windows (SPSS Inc., Chicago, IL, USA). All differences were considered significant when p ≤ 0.05. Socioeconomic data, including schooling and family income, were not significantly different between the CG and SG ( Table 1). The most prevalent phototype of skin in this study was phototype 3 (light brunette skin, moderately burning and tanning, normal sensitivity to the sun). The sun exposure score was 16 at time 0 and time 60 in both groups. Over 70% of the total subjects did not use sunscreen daily, and those who did restricted use to the face.

Body composition and biomarkers. No variables analyzed showed intergroup differences at T0
(p > 0,05). After supplementation there were changes in the results of body composition in the SG. In the CG, total body fat did not vary in relation to time 0 and time 60 (p = 0.639), and in the SG there was a decrease in the final median (p = 0.259). The percentage of total fat, android fat, and gynoid fat decreased significantly in the SG. The gynoid lean mass increased in both groups, however this increase was significant in the SG (p = 0.007). Intragroup bone mineral density did not change from time 0 to time 60 (CG, p = 0.233; SG, p = 0.433) ( Table 2).
Three subjects in the CG presented deficiency in serum concentrations of 25 (OH) D at time 0, and after 60 days one additional subject presented insufficiency. The subject received guidance on the use of vitamin D supplementation after completion of the research. Regarding the SG, only two subjects were classified as deficiency in 25(OH) D at the beginning of supplementation (60 days), and at the end of supplementation, with the dose used in this study (2000 IU cholecalciferol), no subjects presented insufficient serum concentrations of 25 (OH) D. No subject presented potentially toxic concentrations of 25-hydroxy-vitamin D.
The concentration of 25 (OH) D in the serum increased in the SG in relation to the initial value (> 50 µg/d). Comparing the groups separately, the total cholesterol, triglycerides, ALT, uric acid, and creatinine demonstrated analytical and biological variation after 2 months, without pathologic significance. However, when evaluating the difference in biomarkers between the CG and SG, no significant variations were found, with the exception of 25 (OH) D (Table 3).
Gene expression. The mRNA expression was performed in 50 MZ twins, 25 in the CG and 25 in the SG, as shown in (Fig. 2). Of the 3 genes analyzed in this study, mRNA expression of VDR increased significantly after 2 months of supplementation with 2000 IU cholecalciferol in the SG when compared with the CG (p = 0.001 and p = 0.023, respectively) (Supplementary Table 1 for sequence).     www.nature.com/scientificreports/ The main objective of clinical research involving human beings is to improve prophylactic, diagnostic, and therapeutic procedures and to understand the etiology and pathogenesis of certain diseases. This studie provide us with greater understanding of these interactions, which may contribute to the development of strategies to combat diseases and promote health.
In Brazil, blood values of 25 (OH) D greater than 20 ng/mL are considered sufficient 22 , however, worldwide, there are several parameters considered as sufficient values, varying from > 10 ng/mL, > 30 ng/mL, and > 40 ng/ mL 23 . These recommendations were established based on studies on bone health, and, to date, there are no recommendations that take into account other benefits that vitamin D may bring to the individual.
Circulating levels of 25 (OH) D correlate negatively with the body mass index and percentage of fat mass, since the higher the BMI and percentage of fat mass the lower the serum vitamin D concentration 11,24 . Obese healthy adults presented increased blood levels of 25 (OH) D after weight loss over a 12 month period 25 . In addition, although there is no clear evidence on the beneficial effect of vitamin D supplementation on metabolic parameters in obese individuals and weight loss 26 , our findings indicate a likely benefit for patients who need to decrease fat mass.
We present new evidence regarding the concentration of vitamin D and body composition, indicating that an increase in serum 25 (OH) D concentration and expression of the VDR gene without weight loss, since the BMI did not change during the period of 2 months, contributes to the reduction in body fat and a slight increase in gynoid lean mass. Therefore, studying its molecular bases, metabolic pathways and interaction with tissues, such as adipose tissue, is needed to assist in future practical and clinical actions for the control of diseases that affect a large part of the population 27 .
A study that supplemented vitamin D for 6 months at 2,000 IU/day in 40 overweight and obese adults found no alterations in BMI and body fat percentage; however the analysis was performed using bioimpedance 28 . We evaluated body composition with the reference standard for the assessment of body composition 29 . On the other hand, after 24 weeks of supplementation with 60,000 IU/week of cholecalciferol, subcutaneous truncal fat reduced in overweight and obese pre-diabetic Asian women, with vitamin D deficiency 1 .
Fuller and collaborators 30 showed that adults over the age of 45 who received supplements to increase lean mass only gained more strength when vitamin D levels were above 30 ng/mL. After supplementation with 420 IU cholecalciferol/day for 12 months, men and women presented significantly increased lean body mass (from 43.8 ± 9.6 to 44.3 ± 9.8 kg) 31 .
These findings may be related to the fact that the vitamin D receptor is present in almost all human tissues and mediates the vitamin D biological actions in adipose tissue, where it can be activated or inactivated 32,33 . VDR is a transcription factor that plays an important role in inducing the expression of several genes involved in the pathogenesis of obesity 34 .
Vitamin D and its metabolites belong to a small set of dietary compounds that have direct effects on gene regulation 35 . VDRs are critical for vitamin D to perform its functions, since the active form of this micronutrient 1,25-dihydroxy-vitamin D (1,25(OH)2D), penetrates the cell membrane and binds to it. This complex interacts with the retinoic acid receptor (RXR) and forms heterodimers that act on target gene promoter regions, called vitamin D response elements, thus initiating a cascade of molecular interactions that regulate the transcription and suppression of specific genes 36 . VDRs have direct effects on the epigenome and expression of over 1,000 genes 37 . This gene and its ligands antagonize proinflammatory transcription factors, such as NF-AT, AP-1, and NF-κB in T cells, which results in decreased expression of cytokines such as IL2 and IL12 38 .
A cohort study of 335 adults (age > 18 years old) with sickle cell anemia, followed at the Chicago Hospital in the United States, found that low vitamin D levels were associated with decreased VDR expression in peripheral blood mononuclear cells 39 . In white adipose tissue of obese rats, VDR gene expression increased after vitamin D supplementation of 50, 1,000, or 10,000 IU/kg for 12 weeks 40 . In 110 patients with oral neoplastic lesions, premalignant lesions, and oral cancer, VDR expression was increased, while insufficient or deficient blood levels of this Studies demonstrate that cholecalciferol can regulate gene expression by direct binding to VDR and that its affinity for VDR is several times higher than the active form of vitamin D (1,25-OH vitamin D) when bound to the vitamin D binding protein 44,45 . In humans, the Vitamin-D receptor (VDR) interacts with 1α,25(OH)2D3 in the cell nucleus to generate genomic responses. ChIP-chip, ChIP-seq analyses and FAIRE-qPCR analyses have shown that, less than 1,000 sites are generally occupied by the VDR in the absence of 1α,25(OH)2D3 and nearly 9,000 sites are changed following 24 h treatment with the vitamin-D. In effect stimulation with active vitamin-D increases the chromatin accessibility for VDR and its signalling 15,16,46 .
Studies with MZ are of the utmost importance, since each pair comes from a single zygote and they present genetically identical elements, i.e., have the same genotype and constitute the only isogenic series in the human species. In twin studies, to assess the importance of genotype in determining the phenotype, the following assumptions are accepted, 1. Twins are a sample of the general population, which is to say that among twins all characters are distributed in the same way as in the general population. 2. The elements of each pair of twins are subject to the same environmental influences. 3. The environment of the twins is, on average, equal to that of the elements of the general population. By accepting these assumptions, one also accepts that the quantitative characters are determined solely by environmental factors in MZ 14 . Each pair of twins in this research had the same conditions and lifestyle as their sibling.
The supplementation dose used by the MZ in the current study is the recommended daily dose to maintain adequate serum values in adults (2000 IU/day), unlike most studies on vitamin D supplementation, which have been performed at doses of 1,000 IU/day for a period of 12 months 47 , 2000 IU/day for 3 to 6 months 48,49 , 2,500 IU/ day for 4 or 6 months 50,51 , 5,000 IU/day for 3 months 52 , 40,000 IU/weekly for a period of 2 months 53 , 50,000 IU/ weekly in studies ranging from 5 to 6 months 54,55 , and 100,000 IU/monthly from 2 to 12 months 56,57 . A study developed with a maintenance dose of 2000 IU/day of vitamin D3 for 3 months in men and women who had been treated with 50,000 IU of vitamin D3 weekly for 3 months showed that this dose is not sufficient to maintain elevated levels of 25 (OH) D, however, serum values remained above 20 ng/mL 58 .
Experiments with vitamin D are aimed at population groups with 25(OH)D deficiency and some pathologies, such as diabetes, hypertension, insulin resistance, musculoskeletal disorders, cardiovascular disease, and kidney disease, as there is an association between low vitamin D levels and an increased risk of developing and worsening cardiovascular disease 59 , different types of cancers 60 , diabetes 61 , autoimmune diseases 62 , and overall mortality 63 . In addition, studies which used lower dosages of cholecalciferol were performed for periods longer than 2 months. For example, Lopez 64 supplemented individuals with pre-diabetes with 2000 IU of cholecalciferol for 4 months and found a beneficial metabolic response, reducing circulating miR-7 and miR-192, accompanied by increased miR-152, which are useful biomarkers in prevention studies for diabetes.
Vitamin D has regulatory effects on innate and adaptive immunity and its receptor VDR is also expressed in cells of the immune system 65 . Therefore, the use of vitamin D3 and its analogues with other immunosuppressants has been studied synergistically or in isolation, in cases of autoimmune diseases, transplants, type 2 diabetes mellitus, and neoplasms 66,67 .
Pathogenic stimuli induce TNFa expression, which in turn induces other mediators and proteases responsible for the inflammatory response 68 . Omidian 69 , supplementing diabetic patients with 4,000 IU/day of vitamin D for 3 months, found a significant difference in serum TNFa levels, showing the possible protective effect that this micronutrient can have on inflammation when used in larger dosages and for a prolonged period. Borges 70 showed an association between vitamin D deficiency and reduced beta-oxidation markers, including PPARa. However, in the current research no significant association was found between 2000 IU cholecalciferol supplementation with TNFa or PPARa mRNA expression in MZ, possibly owing to the healthy population used.
The major limitations of the present study were the use of a single concentration of the supplement and evaluations performed with only 2 months of use, which did not enable us to address the long-term effects of this supplementation. The small sample size, as our pilot study population consisted of same-sex MZ twin siblings and the decrease in the sample number for analysis of gene expression. However, this was the first clinical trial with vitamin D supplementation evaluating gene expression in this study population. Thus, further studies with different concentrations and different supplementation times are necessary to confirm our hypothesis that the increase in the serum concentration of vitamin D possibly contributes to the fight against cardiovascular diseases through the reduction in body fat.
In conclusion, increasing serum 25 (OH) D to values above 50 ng/mL yields health benefits through increased expression of VDR mRNA, reduced body fat, and increased lean mass, however, supplementation with 2000 IU of cholecalciferol for 2 months did not alter TNFa or PPARa mRNA expression in twins.

Data availability
All datasets generated for this study are available from the corresponding author on direct request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/.