Association between anthropometric markers of adiposity, adipokines and vitamin D levels

Inverse association between serum levels of vitamin D and obesity has been pointed out in several studies. Our aim was to identify to the associations between vitamin D levels and a large panel of anthropometric markers and adipokines. Cross-sectional study including 6485 participants. Anthropometric markers included body mass index (BMI), % body fat, waist, waist-to-hip (WHR), waist-to-height (WHtR), conicity index, body roundness index (BRI) and a body shape index (ABSI). 55.7% of women and 60.1% of men presented with vitamin D deficiency. Vitamin D levels were negatively associated with most anthropometric markers, with correlation coefficients ranging between −0.017 (ABSI) and −0.192 (BMI) in women and between −0.026 (weight) and −0.130 (% body fat) in men. Vitamin D levels were inversely associated with leptin levels in both sexes and positively associated with adiponectin levels in women only. The likelihood of vitamin D deficiency increased with increasing adiposity levels, except for ABSI (women) and BMI (men). Total body fat, rather than localized or unevenly distributed body fat, is the adiposity marker most associated with decreased vitamin D levels. Monitoring vitamin D levels in people with overweight/obesity is essential.

Vitamin D levels. Vitamin D was assessed at baseline through an ultra-HPLC tandem-MS system. The calibrators, 3Plus1 Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2 (ChromoSystems), were standardized against the National Institute of Standards and Technology 972 reference material. Serum 25(OH)D3 and 3-epi-25(OH)D3 were expressed in nanomoles per liter (conversion factor: 1 nmol/L = 0.4006 μg/L). The interday CV% was 4.6% at 40 nmol/L 22 . Vitamin D levels were further categorized as normal (≥ 30 ng/mL or ≥ 75 nmol/l), insufficiency (21 to 29 ng/mL or 50-75 nmol/l) and deficiency (< 20 ng/mL or < 50 nmol/l) 23 . Hypovitaminosis D was defined for vitamin D levels < 30 ng/mL or < 75 nmol/l, encompassing insufficiency plus deficiency 24 . Other covariates. Educational level was categorized into university, high school, apprenticeship, and mandatory. Nationality as born in Switzerland or not. Smoking status was self-reported and categorized as never, former, and current. Physical activity was considered if the participant reported performing at least twice a week a minimum of 20 min of leisure-time physical activity 13 .
Adipokines (adiponectin and leptin) were assessed at baseline. Adiponectin was assessed by ELISA (R&D Systems, Inc, Minneapolis, USA), with a maximum inter-assay CV of 8.3% and a maximum intra-assay CV of 8.3%. Leptin was assessed by ELISA (American Laboratory Products Company, Windham, USA) with a maximum inter-assay CV of 12.8% and a maximum intra-assay CV of 5.8%. High sensitive C-reactive protein (CRP) was assessed by immunoassay and latex HS on a Modular P apparatus (Roche Diagnostics, Basel, Switzerland).
Exclusion criteria. Participants were excluded if they lacked any variable needed for the bivariate and the multivariate analyses. Hence, participants devoid of vitamin D data; without anthropometric measurements and any covariate needed for adjustment (education, smoking, BMI, or physical activity) were excluded. The exclusion procedure was conducted sequentially as follows: first, participants devoid of vitamin D data were excluded; of the remaining participants, those without anthropometric measurements were excluded; finally, of the remaining participants, those missing any covariate were excluded. Statistical analysis. Statistical  www.nature.com/scientificreports/ bioimpedance, CI, BRI and AABSI) were assessed as follows. First, comparison of vitamin D levels according to categories of anthropometric markers was performed using one-way (bivariate) or multivariate analysis of variance. Multivariate analysis was adjusted for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no) and the results were expressed as adjusted mean ± standard error. Second, bivariate nonparametric Spearman correlations and their 95% CIs were calculated between vitamin D levels and anthropometric markers as continuous variables. A stepwise multivariate linear regression analysis with age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no) as locked terms was conducted to identify the anthropometric marker most associated with vitamin D levels. For simplicity, all anthropometric markers were standardized (i.e., zero average and unit standard deviation) before the stepwise regression. Third, the association between vitamin D deficiency and categories of anthropometric markers was assessed using chi-square (bivariate analysis) and multivariate logistic regression with vitamin D deficiency (yes, no) as the dependent variable and adjusting for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no). Sensitivity analyses were conducted by excluding participants receiving medically prescribed vitamin D levels. Finally, the analysis of the associations between vitamin D levels and leptin and adiponectin levels as obesity markers was conducted using Spearman correlation and linear regression adjusting for the aforementioned covariates; results of the linear regression were expressed as standardized coefficients. Statistical significance was considered for a two-sided test with p < 0.05. Ethical statement. The institutional Ethics Committee of the University of Lausanne, which afterwards became the Ethics Commission of Canton Vaud (https:// www. cer-vd. ch) approved the baseline CoLaus study (reference 16/03, decisions of 13th January and 10th February 2003). The study was performed in agreement with the Helsinki declaration and its former amendments, and in accordance with the applicable Swiss legislation. All participants gave their signed informed consent before entering the study. Data analysis was conducted in Switzerland and no data was shared with outside groups.  Table 1. Excluded participants were younger, less frequently born in Switzerland, had a higher educational level, were less frequently former smokers, less physically active and took vitamin D supplements less frequently than included participants.

Characteristics of participants.
The characteristics of the 6485 participants by sex are summarized in Table 1. Women were older, had a lower educational level, smoked less, and took vitamin D supplements more frequently. Women also had higher levels of vitamin D and presented less frequently with vitamin D deficiency. Regarding anthropometric markers, women had higher body fat percentage levels but lower levels for all other anthropometric markers than men (Table 1).

Associations between vitamin D levels and anthropometric markers. The levels of vitamin D
according to categories of anthropometric markers are summarized in Table 2. On bivariate analysis, and regardless of the anthropometric markers considered, participants with high adiposity levels had lower vitamin D levels than participants with normal adiposity measures. Those findings were further confirmed by multivariate analysis, where a linear trend for a decrease in vitamin D levels with increasing adiposity was found, except for BMI in men (Table 2). Those findings were further confirmed after excluding participants taking vitamin D supplements (supplementary Table 2).
The bivariate Spearman correlations between vitamin D levels and anthropometric markers are summarized in Fig. 1. Negative correlations were found for all anthropometric markers. With the exception of the correlations between vitamin D and ABSI (in women) and weight (in men), all correlations were statistically significant (p < 0.05). Similar findings were obtained after excluding participants taking vitamin D supplements (Fig. 1).
The results of the stepwise linear regression analysis are summarized in Table 3. After adjusting for major confounders, % body fat was consistently and negatively associated with vitamin D levels overall and for both sexes. Associations were also found for WHtR (overall), BMI (women) and conicity index (men). Similar findings were obtained after excluding participants taking vitamin D supplements (supplementary Table 3) or adjusting for CRP levels (not shown).

Association between vitamin D deficiency levels and anthropometric markers.
There were 1895 (55.7%) of women and 1854 (60.1%) men presenting with vitamin D deficiency. Table 4 presents the bivariate and multivariate analysis of the associations between vitamin D deficiency and the different measures of adiposity. The likelihood of vitamin D deficiency increased with increasing adiposity levels; the sole exceptions were no associations with ABSI (women) and BMI (men). Similar findings were obtained after excluding participants taking vitamin D supplements (supplementary Table 4).

Association between vitamin D levels and adipokines. The associations between vitamin D, leptin
and adiponectin levels are summarized in Fig. 1  www.nature.com/scientificreports/ findings were obtained after excluding participants taking vitamin D supplements ( Fig. 1 and supplementary Table 5).

Discussion
To our knowledge, this is one of the most comprehensive study in a large population-based cohort of apparently healthy subjects associating vitamin D levels with a wide array of obesity markers. Our results show a negative association between various adiposity measurements and vitamin D levels, and that total body fatness is the adiposity marker most associated with vitamin D levels. Our results add further evidence to the hypothesis that low vitamin D levels might be explained by the sequestration of the vitamin by excess body fat stores. www.nature.com/scientificreports/

Associations between vitamin D levels and anthropometric markers. Negative associations
between vitamin D levels and all markers of adiposity were found, the sole exception being BMI in men, where underweight participants had vitamin D levels close to those of participants with obesity. A possible explanation is that underweight men might have reduced vitamin D dietary intake and/or be affected by pathophysiological conditions affecting vitamin D metabolism, but this hypothesis remains to be assessed. Still, our findings are consistent with the previous literature. Inverse associations have been reported between vitamin D levels and abdominal and visceral fat 26 ; waist circumference and BRI 27 ; the visceral adiposity index 28 , and with both total and regional adiposity 29 . Also, De Pergola et al., found that vitamin D circulating levels were progressively lower with the increase of fat mass in a cohort of healthy overweight and subjects with obesity 5 . WC, WHR and WHtR are traditional indicators of abdominal obesity. BRI and ABSI have recently been proposed as novel anthropometric measurements and are positively correlated with visceral adiposity 10,21,27 . Conversely, CI is rarely used as a measure to assess adiposity, despite being a good predictor of abdominal adiposity 8 . Notwithstanding, total body fat was the sole anthropometric marker consistently associated with low vitamin D levels, suggesting that it is the total amount of body fat, rather than localized or unevenly distributed body fat, that are associated with vitamin D levels. Our results thus add further evidence to the hypothesis that low levels of vitamin D in individuals with obesity can be explained by the sequestration of vitamin D by adipose tissue or by simple volumetric dilution in adipose tissue 30 . Table 2. Bivariate and multivariate comparisons of total vitamin D levels according to adiposity categories, overall and stratified by sex, CoLaus|PsyCoLaus study, Lausanne. *p-value for linear trend. BRI, body roundness index; ABSI, a body shape index. Results are expressed in nmol/L of vitamin D and as mean standard ± deviation for bivariate analyses or as adjusted mean ± standard error for multivariate analyses. Statistical analysis using ANOVA. Multivariate analysis adjusting for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no); for the overall analysis, adjustment on sex (men, women) was also performed. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Results of the stepwise linear regression to assess the anthropometric markers most associated with vitamin D levels, overall and stratified by sex, CoLaus|PsyCoLaus study, Lausanne. Results are expressed as slope and (95% confidence interval) for the markers retained. All anthropometric markers were standardized (i.e., zero average and unit standard deviation) before the stepwise regression. -, not retained.  Table 4. Bivariate and multivariate associations between vitamin D deficiency (i.e. defined as < 30 ng/mL or < 50 nmol/l) and adiposity measures, overall and stratified by sex, CoLaus|PsyCoLaus study, Lausanne. *p-value for linear trend for multivariate analysis. BRI, body roundness index; ABSI, a body shape index.
Results are expressed as number of participants and row (%) of vitamin D deficiency for bivariate analyses or as adjusted odds ratio (95% confidence interval) for vitamin D insufficiency. Bivariate analysis using chisquare. Multivariate analysis using logistic regression adjusting for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no); for the overall analysis, adjustment on sex (men, women) was also performed. www.nature.com/scientificreports/ Association between vitamin D deficiency levels and anthropometric markers. Over half of the sample presented with vitamin D deficiency, hypovitaminosis D (i.e. insufficiency or deficiency) being more frequent in men than in women. Our findings do not replicate recent studies showing that hypovitaminosis D is more prevalent in women than in men 10,31,32 . Possible explanations include the fact that the women in this study were less frequently were less frequently diagnosed with obesity than men, or an unhealthier dietary intake in men.
Overall, our results show that individuals with higher levels of adiposity are more predisposed to reduced serum concentrations of vitamin D. This reduced bioavailability would trigger a hypothalamic action, stimulating food intake and reducing energy expenditure 33 . Chronic inflammation linked to obesity could also explain hypovitaminosis D 34 due to adipose tissue macrophage infiltration and production of proinflammatory adipokines 35 . It has also been postulated that the increase in adiposity would be promoted by resistance to stimulation of lipolysis by catecholamines and natriuretic peptide in people with obesity 36 . Furthermore, this imbalance may lead to reduced release of vitamin D from fat depots, given the fat-soluble nature of vitamin D 36 .
Importantly, it is unclear whether increasing vitamin D supplementation to correct low vitamin D levels in subjects with increased adiposity is the best option. One study demonstrated that adapting supplementation to both vitamin D deficit and adiposity levels would be more efficient 37 , but further studies are needed to confirm this finding. Besides, improvements in vitamin D levels have been associated with more weight loss, a result which can be partly attributed to the anti-inflammatory effects of vitamin D 33 .

Association between vitamin D levels and adipokines.
Our study showed that vitamin D levels were inversely associated with leptin and positively associated with adiponectin. Our findings are in agreement with those of Gangloff et al. 38 , who reported a negative correlation vitamin D levels and leptin. The authors also reported that a decrease in visceral adipose tissue volume and corresponding decrease in leptin levels was associated with an increase in plasma vitamin D concentrations.
Studies have shown that vitamin D affects energy homeostasis by directly regulating leptin expression. However, the exact in vivo effect of vitamin D on leptin expression in humans is controversial 11 . Some studies have suggested that vitamin D can improve adipose tissue inflammation and suppress expression of leptin 39 . A recent study demonstrated that vitamin D acts through its receptor (VDR) to inhibit inflammatory pathways and the expression of adipokines in human adipocytes 12 . Overall, these findings suggest that improving vitamin D status in people with obesity may decrease adipose inflammation, which may contribute to reduce risks of obesityassociated pathophysiological processes. Although more recent studies point to CRP as a marker of inflammation associated with vitamin D levels, our results remained unchanged after adjusting for CRP.

Strengths and limitations.
This study was carried out in a representative sample of the population and included a large panel of adiposity markers. Some limitations should be acknowledged. First, the analysis was conducted in an urban Swiss population and the participants were mostly Caucasian. The results might not be generalizable to other countries. Still, our results are concordant with ones reported in other populations. Second, fat mass was measured by bioimpedance with the known limitations of this technique to accurately quantify adipose tissue particularly using a single time point measurement. Third, our cross-sectional study does not allow to draw any potential causality relationships between the investigated variables (i.e. whether it is obesity that causes vitamin D deficiency or vice versa). However, a prospective study is envisaged. Fourthly, we did not use different cutoff points for vitamin D according to seasonality as indicated in other studies 40 . Still, we adjusted the analysis considering the month. We chose this methodology as changing the threshold according to season would be difficult to apply in clinical practice.

Conclusions
Our data show a negative association between vitamin D levels and most adiposity markers and that people with excess adiposity are more likely exhibit to present with vitamin D deficiency as measured by total circulating 25-hydroxyvitamin D. Total fat mass is the adiposity marker most consistently associated with decreased vitamin D levels. Monitoring the levels of vitamin D in people with overweight/obesity is essential.

Data availability
The CoLaus|PsyCoLaus cohort data used in this study cannot be fully shared as they contain potentially sensitive patient information. As discussed with the competent authority, the Research Ethic Committee of the Canton of Vaud, transferring or directly sharing this data would be a violation of the Swiss legislation aiming to protect the personal rights of participants. Non-identifiable, individual-level data are available for interested researchers, who meet the criteria for access to confidential data sharing, from the CoLaus Datacenter (CHUV, Lausanne, Switzerland). Instructions for gaining access to the CoLaus data used in this study are available at https:// www. colaus-psyco laus. ch/ profe ssion als/ how-to-colla borate/.