Accepted Manuscript

Title: 25-hydroxyvitamin D is associated with adiposity and

cardiometabolic risk factors in a predominantly vitamin
D-deficient and overweight/obese but otherwise healthy cohort

Author: Aya Mousa Negar Naderpoor Maximilian P.J. de

Courten Robert Scragg Barbora de Courten

PII: S0960-0760(16)30347-8
DOI: http://dx.doi.org/doi:10.1016/j.jsbmb.2016.12.008
Reference: SBMB 4842

To appear in: Journal of Steroid Biochemistry & Molecular Biology

Received date: 27-6-2016

Revised date: 14-12-2016
Accepted date: 17-12-2016

Please cite this article as: Aya Mousa, Negar Naderpoor, Maximilian P.J.de Courten,
Robert Scragg, Barbora de Courten, 25-hydroxyvitamin D is associated with
adiposity and cardiometabolic risk factors in a predominantly vitamin D-deficient and
overweight/obese but otherwise healthy cohort, Journal of Steroid Biochemistry and
Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.12.008

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Running title: Mousa, A., et al. Vitamin D is associated with cardiometabolic risk

25-hydroxyvitamin D is associated with adiposity and

cardiometabolic risk factors in a predominantly vitamin D-
deficient and overweight/obese but otherwise healthy cohort

Aya Mousaa, Negar Naderpoora,b, Maximilian P J de Courtenc, Robert Scraggd, Barbora de

Courtena,b

a
Monash Centre for Health Research and Implementation, School of Public Health and
Preventive Medicine, Monash University, MHRP, 43-51 Kanooka Grove, Clayton VIC 3168
Australia

b
Diabetes and Vascular Medicine Unit, Monash Health, Locked Bag 29, Clayton, VIC 3168,
Australia

c
Centre for Chronic Disease, Victoria University, Melbourne, Australia
d
School of Population Health, The University of Auckland, New Zealand

Corresponding author:
A/Prof Barbora de Courten, MD PhD MPH FRACP
Monash Centre for Health, Research and Implementation,
School of Public health and Preventive Medicine, Monash University
43-51 Kanooka Grove, Clayton
Melbourne, VIC 3168, Australia
Telephone: +61 3 857 22651
Email: barbora.decourten@monash.edu

1
MS ID SBMB-D-16-00226: 25-hydroxyvitamin D is associated with adiposity and
cardiometabolic risk factors in a predominantly vitamin D-deficient and overweight/obese but
otherwise healthy cohort

Highlights:

 25-hydroxyvitamin D levels were associated with cardiometabolic risk factors including

adiposity, glucose intolerance, insulin resistance, and inflammation in predominantly
overweight/obese but otherwise healthy adults.
 Most associations between 25-hydroxyvitamin D levels and cardiometabolic risk factors
were mediated by adiposity.
 The mediating effect of adiposity on the relationship between 25-hydroxyvitamin D and
cardiometabolic risk was only observed when using direct adiposity measures such as
percent body fat, but not when using indirect measures such as body mass index.

2
ABSTRACT
Vitamin D deficiency has reached epidemic proportions worldwide and has recently been linked to
cardiometabolic risk factors including obesity, insulin resistance, hypertension, dyslipidemia, as well
as type 2 diabetes and cardiovascular disease. The objective of this study was to examine the
associations between circulating 25-hydrovitamin D (25(OH)D) levels and cardiometabolic risk
factors using direct measures of adiposity, glucose intolerance, and insulin resistance, as well as lipids,
blood pressure, and plasma markers of inflammation. We measured circulating 25(OH)D, physical
activity (International Physical Activity Questionnaire- IPAQ), anthropometry (body mass index
(BMI), waist-to-hip ratio (WHR), % body fat (dual energy X-ray absorptiometry), metabolic
parameters (fasting and 2-hour plasma glucose levels during oral glucose tolerance test; insulin
sensitivity (M, hyperinsulinaemic-euglycaemic clamp), and cardiovascular and inflammatory profiles
(blood pressure (BP), pulse pressure (PP), mean arterial pressure (MAP), plasma lipid levels, white
blood cell count (WBC), and plasma high-sensitivity C-reactive protein levels (hsCRP)) in 111
healthy, non-diabetic adults (66 males/ 45 females; age 31.1±9.2 years; % body fat 36.0±10.2%).
Mean 25(OH)D was 39.8±19.8 nmol/L with no difference between genders (p=0.4). On univariate
analysis, 25(OH)D was associated with % body fat (r=-0.27; p=0.005), 2-hour glucose (r=-0.21;
p=0.03), PP (r=0.26; p=0.006), and insulin sensitivity (r=0.20, p=0.04), but not with age, BMI, WHR,
fasting glucose, BP, MAP, lipids, or inflammatory markers (all p>0.05). After adjusting for age and
sex, 25(OH)D remained associated with % body fat (β=-0.12%; p=0.003), 2-hour glucose (β=-0.13
mmol/L; p=0.02), PP (β=0.12 mmHg; p=0.009), and insulin sensitivity (β=0.22 mg/kg/min; p=0.03),
and became associated with fasting glucose (β=-0.04 mmol/L; p=0.04) and hsCRP (β=-0.51 mg/L;
p=0.04). After adjusting for age, sex, and % body fat, 25(OH)D was no longer associated with insulin
sensitivity, 2-hour glucose, or hsCRP, but remained associated with fasting glucose (β=-0.05 mmol/L;
p=0.03) and PP (β=0.10 mmHg; p=0.03). 25(OH)D remained associated with fasting glucose (β =-
0.06 mmol/L; p=0.02) after hsCRP and physical activity were added to the model with % body fat,
age, and sex. These cross-sectional data suggest that associations between vitamin D and
cardiometabolic risk among healthy, non-diabetic adults are largely mediated by adiposity. Large-
scale intervention and mechanistic studies are needed to further investigate whether vitamin D has an
independent role in the prevention and/or management of cardiometabolic risk and disease.

Abstract word count: 368 words

Keywords: Vitamin D, 25-hydroxyvitamin D, diabetes, cardiovascular disease, cardiometabolic risk,

obesity

3
1. INTRODUCTION

Vitamin D deficiency is very common worldwide and has recently been linked to multiple

cardiometabolic risk factors including obesity, insulin resistance, hypertension, dyslipidaemia

and chronic low-grade inflammation [1]. These risk factors contribute to the development

and/or progression of type 2 diabetes and cardiovascular disease, the leading causes of

morbidity and mortality in the developed world [2]. Knowledge of the associations between

vitamin D deficiency and cardiometabolic risk before the development of disease is therefore

important in order to establish if vitamin D supplementation can be used for prevention of

these conditions [3].

Previous cross-sectional studies have found inverse associations between 25-hydroxyvitamin

D (25(OH)D) levels and cardiometabolic risk factors [4-6], while prospective studies suggest

that low 25(OH)D levels may predict the development of cardiometabolic risks [7-9] and

diseases [10, 11]. However, it is argued that observed associations between vitamin D

deficiency and cardiometabolic risks are mediated by underlying adiposity, since 25(OH)D is

fat-soluble and is therefore lower with greater adiposity [12]. Importantly, only few studies

have used direct methods such as dual X-ray absorptiometry to measure adiposity or the

hyperinsulinaemic-euglycaemic clamp to accurately capture cardiometabolic risk [13, 14].

Thus, a significant gap in knowledge remains with regard to the relationship between

25(OH)D and cardiometabolic risk as measured by direct, gold-standard methods. The aim of

the present study was therefore to investigate the relationship between 25(OH)D level and

cardiometabolic risk factors such as glucose tolerance, insulin sensitivity, lipids, blood

pressure, and inflammation, and to assess whether these relationships are mediated by

adiposity.

2. METHODS

2.1. Design and Participants:

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Participants were recruited from the community via online and poster advertising in

Melbourne, Australia, and were part of our ongoing studies investigating risk factors for

cardiometabolic syndrome. Cross-sectional analyses were performed on data from 111

healthy, non-diabetic adults (66 males and 45 females) aged 18 to 57 years (31.1±9.2).

Participants underwent a detailed physical examination and medical history, routine blood

analyses, anthropometric assessment, and an oral glucose tolerance test (OGTT) to exclude

diabetes based on World Health Organisation (WHO) criteria [15]. All participants were non-

smokers, not using illicit drugs or taking medications or supplements, and had no clinical or

laboratory signs of acute or chronic inflammation.

Ethical approval was obtained from the Alfred Hospital Ethics Committee and the Monash

University Human Research Ethics Committee and complied with the Declaration of Helsinki

(2004). All participants provided written informed consent prior to participation.

2.2. Analyses of Anthropometric Parameters:

Body mass index (BMI) was calculated from weight (kg)/height (m)2, and waist to hip ratio

(WHR) was derived from waist and hip circumferences and calculated as WHR= waist (mm)

/ hip (mm). Dual energy x-ray absorptiometry (DEXA) scans were performed to determine

body fat composition (% body fat) (Alfred Hospital and Monash Medical Centre, Melbourne

Australia). Physical activity information was derived from self-reported responses to the short

version of the International Physical Activity Questionnaires (IPAQ) [16]. This questionnaire

referred preceding 7 days and asked participants to report the number of days and

hours/minutes spent doing vigorous or moderate activity, or walked for at least 10 minutes.

Responses were computed based on the IPAQ scoring protocol and reported as MET-minutes

(multiples of the resting metabolic rate-minutes) [16].

2.3. Analyses of Cardiometabolic Parameters:

5
Resting systolic and diastolic blood pressure (SBP/ DBP) was measured using an automated

oscillometric measurement system (M6 Automatic BP monitor, Omron, Japan) after a 20

minute rest. The average BP derived from three measurements was recorded to calculate the

mean BP. Pulse pressure (PP) was calculated as the difference between SBP and DBP. Mean

arterial pressure (MAP) was calculated as DBP + (PP/3). Lipid profile-related parameters

including total cholesterol, triglycerides, low-density lipoprotein (LDL) and high-density

lipoprotein (HDL) cholesterol were measured using a standard commercial enzymatic assay

(Beckman Coulter LX20PRO Analyser and SYNCHRON Systems Lipid and Multi

Calibrators, Beckman Coulter Diagnostics, Australia).

A two-hour 75-g OGTT was performed after a 12 hour overnight fast and glucose tolerance

status was determined by WHO criteria [17]. A hyperinsulinaemic-euglycaemic clamp was

performed to determine insulin sensitivity. After an overnight fast, a primed continuous

intravenous insulin infusion (9 mU.kg-1) was administered for approximately 120 min at a

constant rate of 40 mU per m2 body surface area per minute. Plasma glucose was measured

every 5 minutes and the variable infusion rate of glucose was adjusted to maintain blood glucose

at a constant value of 5 mmol/L. This infusion achieved steady state plasma insulin

concentrations. The rate of total insulin-stimulated glucose disposal (M) was calculated for the

last 40 min of the insulin infusions. Plasma glucose concentrations for both OGTT and clamp

were determined by the glucose oxidase method (ELM 105 Radiometer, Copenhagen, and YSI

2300 STAT Glucose & Lactate Analyzer, YSI Inc., OH, USA).

2.4. Laboratory Analyses:

Blood samples were drawn for measurement of inflammatory markers and 25(OH)D,

respectively. White blood cell counts were measured using a quantitative automated

haematology analyser (Coulter Counter model LH750, Beckman Coulter Inc., Australia), and

high sensitivity C-reactive protein (hsCRP) was measured using highly sensitive near infrared

6
particle immunoassay rate methodology on an immunoturbidimetric assay (R&D Systems,

Minneapolis, MN, USA; and Beckman Coulter Synchron LX system chemistry analyser,

Beckman Coulter Inc., Australia). Circulating 25(OH)D concentrations were analysed on a

DiaSorin Liaison Analyser (DiaSorin Inc. USA), using the direct competitive chemiluminescent

immunoassay method (CLIA) to quantitatively determine the total 25(OH)D in plasma. Inter-

and intra-assay coefficients of variation (CV) using the Diasorin method were <10% and <4%,

respectively.

2.5. Statistical Analyses:

Statistical analyses were performed using Stata statistical software version 12.0 (StatCorp LP,

USA). Results are provided as means and standard deviations for participant characteristics

and overall cardiometabolic and inflammatory measures. 25(OH)D levels and all outcome

measures which were not normally distributed were logarithmically transformed to the base

10 to approximate normal distribution prior to analysis. Gender differences were assessed

using unpaired Student’s t tests. Pearson correlation coefficients were used to assess

univariate relationships between 25(OH)D and cardiometabolic risk factors. All associations

were analysed in partial correlations to account for age and sex and were examined for

interactions by age and sex. Variables considered clinically relevant to the outcome of interest

or which were statistically significant on univariate analysis were included as covariates in

multiple linear regression models. Findings were considered statistically significant at a level

of p<0.05.

3. RESULTS

3.1. Sample Characteristics:

Participant characteristics are presented in Table 1. A total of 111 participants (66 males and

45 females) were included in the study, aged 18- 57 years with no age difference between

genders (p=0.9). Mean circulating 25(OH)D was 39.8±19.8 nmol/L with no difference

7
 between genders (p=0.4), with 84% of participants (n=93) being classified as vitamin D

deficient (25(OH)D≤50 nmol/L), 11% (n=12) as sufficient (25(OH)D >50 to ≤75 nmol/L),

and 5% as replete (25(OH)D>75 nmol/L). BMI ranged from 20.6 to 52.9 kg/m2 (IQR= 27- 33

kg/m2), with 15 normal weight (13% BMI<25), 43 overweight (39% BMI 25<30), and 53

obese individuals (48% BMI ≥30) in our sample. Percent body fat ranged from 11.1- 56.8%

(IQR= 29.1- 44.5%). On average, male participants had a greater WHR than females

(p=0.0002), but females had a higher % body fat than males (p<0.0001); however, BMI did

not differ between genders (p=0.9), nor did physical activity (p=0.5). Males had a higher

fasting glucose than females (p=0.008) but there were no gender differences in 2-hour

glucose (p=0.4) or insulin sensitivity (M) values (p=0.3). Males had higher SBP (p=0.0003),

PP (p=0.0001), MAP (p=0.03), total cholesterol (p=0.02), and triglycerides (p<0.0001)

compared to females, and females had greater levels of HDL cholesterol (p=0.001) than

males. There were no gender differences in inflammatory markers.

Males Females
Variable n All Sample P
(n=66) (n=45)
25(OH)D (nmol/L) 111 38 (26-48) 39 (27-48) 34 (26-46) 0.5
Age (yrs) 111 28 (23-37) 29 (24-37) 28 (23-41) 0.9
Weight (kg) 111 85 (76-94) 86.8 (80.0-101.4) 79 (74.1-87.5) 0.0005
BMI (kg/m2) 111 29.6 (27-33) 29.1 (26.9-32.6) 30.2 (27.6-33.0) 1.0
WHR (cm) 111 0.91 (0.86-0.97) 0.95 (0.91-0.99) 0.87 (0.83-0.92) 0.0001
% body fat 110 36.0 ± 10.2 31.3 ± 8.7 43.1 ± 8.0 <0.0001
IPAQ (METs score) 110 1866.5 (1093-3546) 1836 (891-3813) 1908 (1236-3351) 0.7
Systolic BP (mmHg) 111 120.3 ± 12.5 123.8 ± 12.2 115.2 ± 11.1 0.0003
Diastolic BP (mmHg) 111 77.6 ± 9.5 78.3 ± 9.7 76.5 ± 9.1 0.3
Pulse pressure (mmHg) 111 42.7 ± 9.5 45.5 ± 9.0 38.7 ± 8.7 0.0001
Mean arterial pressure (mmHg) 111 91.8 ± 9.6 93.5 ± 9.8 89.4 ± 8.9 0.03
Total cholesterol (mmol/L) 111 4.7 ± 0.9 4.9 ± 1.0 4.5 ± 0.7 0.02
HDL cholesterol (mmol/L) 110 1.2 (1.0-1.4) 1.1 (0.9-1.3) 1.3 (1.1-1.4) 0.001
LDL cholesterol (mmol/L) 110 2.9 ± 0.7 3.0 ± 0.8 2.7 ± 0.6 0.08
Triglycerides (mmol/L) 111 1.2 (0.85-1.9) 1.5 (1.0-2.3) 1 (0.8-1.3) <0.0001
Fasting glucose (mmol/L) 111 4.6 (4.3-4.9) 4.8 ± 0.5 4.5 ± 0.5 0.008
2-hour glucose (mmol/L) 111 5.3 (4.3-6.2) 5.2 (4.3-5.9) 5.3 (4.3-6.3) 0.4
Insulin sensitivity (mg/kg/min) 111 7.2 (4.6-9.6) 6.9 (4.5-9.0) 7.7 (5.2-9.8) 0.3
hsCRP (mg/L) 99 1.4 (0.7-3.0) 1.1 (0.6-2.2) 2.2 (0.9-4.3) 0.06
WBC (x109/L) 111 6.2 (5.3-7.5) 6.2 (5.3-7.6) 6.0 (5.4-7.3) 0.9
Table 1. Sample characteristics:
Data presented as mean ± standard deviation, or median (interquartile range) for non-normally distributed variables, unless
otherwise specified. P represents significance of difference between genders using Student’s paired t-tests. Abbreviations:

8
BMI, body mass index; IPAQ-METS, international physical activity questionnaire (multiples of the resting metabolic rate);
25(OH)D, 25-hydroxyvitamin D; BP, blood pressure; HDL, high density lipoprotein; LDL, low-density lipoprotein; hsCRP,
high sensitivity C-reactive protein; WBC, white blood cell.

3.2. Univariate and partial correlations between 25(OH)D and cardiometabolic risk factors:

On univariate analysis [Table 2], 25(OH)D was significantly associated with % body fat (r= -

0.27; p=0.005), but not with BMI, WHR, physical activity, or age (all p>0.1). 25(OH)D was

not associated with fasting glucose (p=0.09), but was inversely associated with 2-hour

glucose (r=-0.21, p=0.03), and positively associated with insulin sensitivity (r=0.20, p=0.04)

and PP (r=0.26, p=0.006). There were no associations between 25(OH)D and BP, MAP,

lipids, or inflammatory markers (all p>0.05) [Table 2]. Partial correlations controlling for age

and sex did not significantly alter any of the associations, except for hsCRP which became

significantly associated with vitamin D after controlling for age (p=0.02) [Table 2].

Table 2. Univariate and partial correlations between 25-hydroxyvitamin D and cardiometabolic

Correlation Partial correlation Partial correlation

Variable n (%) †
Coefficients (p-value) coefficients1 (p-value) coefficients2 (p-value)
Anthropometric Risk Factors
BMI* (kg/m2) 111 (100) -0.04 (0.7) -0.04 (0.6) -0.03 (0.8)
WHR (cm) 111 (100) -0.07 (0.5) -0.11 (0.2) -0.10 (0.3)
% body fat 110 (99) -0.27 (0.005) -0.28 (0.003) -0.27 (0.004)
Physical activity* (IPAQ METs) 110 (99) 0.07 (0.5) 0.09 (0.3) 0.1 (0.3)
Metabolic Risk Factors
Fasting glucose* (mmol/L) 111 (100) -0.16 (0.09) -0.17 (0.07) -0.18 (0.05)
2-hour glucose* (mmol/L) 111 (100) -0.21 (0.03) -0.22 (0.02) -0.20 (0.03)
Insulin sensitivity* (mg/kg/min) 111 (100) 0.20 (0.04) 0.20 (0.04) 0.21 (0.03)
Cardiovascular Risk Factors
Systolic BP (mmHg) 111 (100) 0.18 (0.05) 0.17 (0.08) 0.17 (0.08)
Diastolic BP (mmHg) 111 (100) -0.02 (0.9) -0.04 (0.7) -0.03 (0.8)
Pulse pressure (mmHg) 111 (100) 0.26 (0.006) 0.26 (0.006) 0.25 (0.008)
Mean arterial pressure (mmHg) 111 (100) 0.06 (0.5) 0.05 (0.6) 0.05 (0.6)
Cholesterol* (mmol/L) 111 (100) -0.08 (0.4) -0.13 (0.2) -0.09 (0.3)
HDL cholesterol* (mmol/L) 110 (99) 0.03 (0.8) 0.009 (0.9) 0.05 (0.6)
LDL cholesterol (mmol/L) 110 (99) -0.12 (0.2) -0.16 (0.09) -0.13 (0.2)
Triglycerides* (mmol/L) 111 (100) -0.03 (0.7) -0.04 (0.7) -0.07 (0.5)
Inflammatory Markers
hsCRP* (mg/L) 99 (89) -0.19 (0.07) -0.23 (0.02) -0.17 (0.1)
WBC* (x109/L) 111 (100) -0.16 (0.08) -0.16 (0.1) -0.16 (0.08)
risk factors
†
*Non-normally distributed variables were log-transformed prior to analyses; Coefficients presented from Pearson
correlation analyses; Partial correlation coefficients1 adjusted for age; Partial correlation coefficients2 adjusted for
sex. Abbreviations: BMI, body mass index; WHR, waist-to-hip ratio; IPAQ-METS, international physical activity

9
questionnaire-multiples of the resting metabolic rate; BP, blood pressure; HDL, high density lipoprotein; LDL, low-
density lipoprotein; hsCRP, high sensitivity C-reactive protein; WBC, white blood cell.

3.3. Multiple linear regression model of 25(OH)D and cardiometabolic risk factors:

After adjusting for age and sex, 25(OH)D remained associated with % body fat (β=-12.0 %;

p=0.003), 2-hour glucose (β=-0.13 mmol/L; p=0.02), insulin sensitivity (β=0.22 mg/kg/min;

p=0.03), and PP (β=0.12 mmHg; p=0.009), and became associated with fasting glucose (β=-

0.04 mmol/L; p=0.04) and hsCRP (β=-0.51 mg/L; p=0.04) [Table 3]. After additional

adjustment for % body fat to the above model, 25(OH)D was no longer associated with

insulin sensitivity, 2-hour glucose, or hsCRP, but remained associated with fasting glucose

(β=-0.05 mmol/L; p=0.03) and PP (β=0.10 mmHg; p=0.03). 25(OH)D remained associated

with fasting glucose (β=-0.06 mmol/L; p=0.02) after hsCRP and IPAQ were added to the

model with % body fat, age, and sex. There were no interactions by age or sex on any of the

observed associations (all p>0.05) and all associations were unchanged when % body fat was

replaced with fat mass (calculated as weight (kg) x % body fat) (data not shown).

Table 3. Multiple linear regression analysis for relationship between 25-hydroxyvitamin D

(25(OH)D) and cardiometabolic risk factors

10
 Co-efficient
Dependent Variable Model R2 t p- value
β (95% CI)
25(OH)D -13.3 (-22.5, -4.1) -2.86 0.005
% body fat
+ age and sex 0.38 -12.0 (-19.7, -4.3) -3.10 0.003
25(OH)D -0.04 (-0.1, 0.006) -1.70 0.09
Fasting glucose* (mmol/L) + age and sex 0.10 -0.04 (-0.09, -0.002) -2.07 0.04
+ % body fat 0.11 -0.05 (-0.1, -0.006) -2.23 0.03
25(OH)D -0.12 (-0.2, -0.01) -2.21 0.03
2-hour glucose* (mmol/L) + age and sex 0.06 -0.13 (-0.2, -0.02) -2.28 0.02
+ % body fat 0.08 -0.10 (-0.2, 0.02) -1.66 0.10
25(OH)D 0.21 (0.01, 0.4) 2.09 0.04
Insulin sensitivity* + age and sex 0.05 0.22 (0.02, 0.4) 2.16 0.03
(mg/kg/min) + % body fat 0.34 0.05 (-0.1, 0.2) 0.59 0.6
25(OH)D 12.2 (3.6, 20.7) 2.82 0.006
Pulse Pressure (mmHg) + age and sex 0.18 11.2 (3.0, 19.3) 2.71 0.008
+ % body fat 0.20 9.4 (1.0, 17.8) 2.23 0.03
25(OH)D -0.46 (-0.9, 0.03) -1.86 0.07
hsCRP* (mg/L) + age and sex 0.12 -0.51 (-1.0, -0.03) -2.10 0.04
+ % body fat 0.20 -0.3 (-0.8, 0.2) -1.29 0.2

*Non-normally distributed variables were log-transformed prior to analyses; independent variable: logarithm of
25(OH)D; Abbreviations: hsCRP, high sensitivity C-reactive protein.

4. DISCUSSION & CONCLUSIONS:

We found that lower circulating 25(OH)D levels were related in univariate analyses to greater

cardiometabolic risk factors including degree of adiposity, glucose intolerance, insulin

resistance, and inflammation. However, the associations between 25(OH)D and these risk

factors, with the exception of fasting glucose and pulse pressure, were no longer significant

after adjustment for % body fat, suggesting that associations between vitamin D deficiency

and cardiometabolic risks are driven by underlying adiposity.

In our study, we found that 25(OH)D was inversely associated with % body fat but not with

BMI. This is consistent with previous cross-sectional studies, which measured % body fat

using DEXA, skin-fold thickness, or computed tomography (CT) scans and showed inverse

associations between body fat and 25(OH)D [12, 14, 18-24]. Percent body fat is a more

accurate method for measurement of adiposity, which is supported by studies where both

11
BMI and DEXA measures were used and only % body fat emerged as a significant predictor

of vitamin D levels [12, 21, 25, 26]. The direction and causality of the association between

vitamin D and obesity/adiposity, and the effects of vitamin D on weight loss remain unclear

[27, 28]. Obesity may be associated with decreased sunlight exposure resulting from

decreased outdoor activity or clothing habits that limit cutaneous vitamin D synthesis. Second,

because vitamin D is fat soluble, it may be sequestered and stored in fat tissues, thus the

bioavailability of endogenously produced vitamin D in the circulation may decrease with

increasing % body fat [14, 29, 30]. Lastly, moderate to severe vitamin D deficiency could

promote greater adiposity via elevated parathyroid hormone (PTH), which in turn stimulates

calcium influx into adipocytes and enhances lipogenesis, thereby promoting weight gain [14,

21, 31, 32]. Consistent with this notion, a positive association between PTH and BMI, and a

reduction in PTH following weight loss have been reported [33, 34].

We observed that 25(OH)D levels were inversely associated with fasting and 2-hour glucose

and positively associated with insulin sensitivity. This is consistent with a review by

Teegarden and Donken [35], where only one out of nine cross-sectional studies showed no

association between vitamin D and fasting glucose [36]. The remaining seven studies, some

of which had 10,000+ subjects reported inverse associations between vitamin D and both

fasting and 2-hour glucose [35]. Large-scale nationally representative cross-sectional studies

such as the third National Health and Nutrition Examination Survey (NHANES III) [4, 6]

have demonstrated significant positive relationships between vitamin D and insulin sensitivity.

However, most existing studies have used surrogate measures of insulin sensitivity such as

fasting insulin, quantitative insulin-sensitivity index (QUICKI), homeostatic model

assessment of insulin resistance (HOMA-IR), and insulin sensitivity index (ISI) [6, 37-40],

with only few using the hyperinsulinaemic-euglycaemic clamp [13, 41]. Most studies have

also not adjusted for confounders such as physical activity or have used BMI as the covariate

to adjust for obesity, which, as mentioned, may not accurately reflect adiposity. In accordance

12
with this, we have shown that the association between vitamin D and insulin sensitivity was

attenuated by adjusting for adiposity. Previous studies that have used % body fat as a

covariate instead of BMI have found similar results [14, 42]. Intervention studies are yet to

determine if vitamin D deficiency is associated with development of insulin resistance [43].

With regard to the mechanisms, vitamin D enhances transcription and expression of the

human insulin gene promoter, upregulates the insulin receptor, and facilitates basal- and

insulin-mediated glucose oxidation and transport, all of which improve glucose tolerance and

insulin sensitivity [44-48]. Vitamin D also regulates extracellular calcium which optimizes

insulin signal transduction and insulin action on insulin-responsive tissues [49], and elevates

intracellular calcium which is vital for β-cell glycolysis and glucose signalling [50].

Conversely, vitamin D deficiency increases PTH, which has been shown to interfere with

insulin action and signalling, supress insulin release, and decrease insulin-mediated glucose

uptake by reducing the number of glucose transporters (GLUT-1 and -4) in several cell

membranes [35]. Vitamin D may also mitigate the harmful effects of advanced glycation

products which have recently been associated with the development of insulin resistance [51].

We report no associations between 25(OH)D and BP or lipid levels. This is consistent with

some [36, 52, 53], but not all previous studies [5, 54]. Importantly, those studies that found an

association between 25(OH)D and cardiovascular risk factors analysed data from large

samples such as NHANES III [5], which included participants with dyslipidaemia,

hypertension, type 2 diabetes, and a range of vitamin D levels, and where analyses were not

adjusted for confounders including medication use and existing co-morbidities [5, 54]. In

contrast, those studies that found no association included predominantly healthy participants

[52] without vitamin D deficiency [36, 53], and adjusted for multiple confounders including

BMI, season, and physical activity [36, 52, 53]. Our study included primarily vitamin D

deficient but otherwise healthy participants, with only 8% and 12% having mildly elevated

systolic (140-150 mmHg) and diastolic (90-100 mmHg) BP levels, respectively, and mostly

13
normal lipid levels. It is therefore possible that our sample did not have sufficient variation in

either vitamin D levels nor in lipids or BP to detect significant differences. Clinical trials

including the VITAL trial in the United States, FIND trial in Finland, D-Health Study in

Australia, and the ViDA trial in New Zealand are currently underway and may provide

answers to key questions regarding the role of vitamin D in cardiovascular risk and disease

[55]. With regard to dyslipidaemia, vitamin D appears to act on the VDR to prevent foam cell

formation [56]; reduce acetylated LDL cholesterol uptake [57]; promote HDL particle

formation, and regulate serum apolipoprotein A-1 levels, all of which enhance cholesterol

transport and improve overall lipids [57-59]. Anti-hypertensive effects of vitamin D are

believed to occur primarily via down-regulation of the renin-angiotensin-aldosterone system

(RAAS) [60-63], which reduces angiotensin II levels in plasma, thereby reducing angiotensin

II- induced vasoconstriction [60-63]. Vitamin D also enhances endothelial vasodilatation and

modulates the flux of calcium, leading to reduced renin secretion by juxtaglomerular cells of

vascular smooth muscle tissue [60-62]. Finally, vitamin D reduces advanced glycation

products and oxidative stress [64], which may contribute to its anti-hypertensive properties

[65].

We found that 25(OH)D was positively associated with PP. Previous studies have reported

predominantly negative associations between 25(OH)D and PP [66-68]; however these

studies included older participants or those with existing heart disease hence they were likely

to have high PP due to established atherosclerosis [66-68]. However, our study participants

were young and healthy, and most likely had normal vascular resistance due to absence of

atherosclerosis. PP is an indirect measure of arterial resistance, and our results suggest that

high circulating 25(OH)D levels may improve vascular compliance [69]. Further

interventional and mechanistic studies in young, healthy cohorts are needed to explain the

etiological pathways by which 25(OH)D may be associated with PP.

14
We observed an inverse association between 25(OH)D and hsCRP after adjusting for age and

sex, which was no longer significant after adjustment for % body fat. CRP is an important

marker of chronic low-grade inflammation, and has been linked to obesity,

hypertriglyceridaemia, hypertension, insulin resistance, and endothelial dysfunction [70].

Studies involving participants with a wide range of 25(OH)D levels have shown variable

associations with regard to CRP, many of which reported no relationship [71-75].

Interestingly, a study using NHANES III data reported an inverse relationship between

25(OH)D and CRP levels in adults with low 25(OH)D levels, which was reversed so that

25(OH)D levels above the population median of 52 nmol/L were associated with higher CRP

levels [74]. Our study participants were mostly vitamin D deficient hence our data are

consistent with the NHANES III study [74]. This suggests that both vitamin D deficiency and

supraphysiologically high vitamin D levels may be pro-inflammatory. By acting on the VDR,

which is present in most inflammatory cells, vitamin D modulates cytokine and chemokine

secretion [76], inhibits the proliferation and stimulatory abilities of T-cells and monocytes,

increases circulating adiponectin- a peptide hormone with favourable effects on chronic low-

grade inflammation, and glucose and lipid metabolism [64, 77], and down-regulates pro-

inflammatory cytokines, including CRP, while up-regulating anti-inflammatory cytokines

such as IL-10 [78]. Absence of the VDR has been associated with increased nuclear factor-

kappa B (NFkB) activity, a transcription factor which has a key role in regulating immune

responses to infection and which has been implicated in the pathophysiology of insulin

resistance and type 2 diabetes [79, 80].

The present study is limited by the use of cross-sectional data, which precludes inference of a

causal relationship between vitamin D and cardiometabolic risk factors. Our sample

comprised mostly of healthy overweight/obese participants, hence our results may not be

generalizable to other populations such as lean adults or those with co-morbidities.

Recruitment was achieved via voluntary participation by interested subjects who were

15
potentially more health-conscious and thus may not be nationally representative. We did not

include specific information on sun exposure, or time spent outdoors, and although the

physical activity index was used as a surrogate for solar radiation, this does not fully reflect

sun exposure. Use of the IPAQ questionnaire is also a limitation as it tends to over-estimate

physical activity in comparison to more objective measures [81]. Moreover, it has been

suggested that ethnic variations affect the relationships between vitamin D and

cardiometabolic risks [82]. We did not collect ethnicity information were therefore unable to

adjust for ethnicity in our study. Due to facility constraints, we were unable to measure

25(OH)D using the gold-standard liquid chromatography mass spectrometry method. Finally,

we measured risk factors for cardiometabolic disease, hence longitudinal studies would be

needed to ascertain if changes in these risk factors translate into decreased incidence of

disease.

This study has several strengths which add to the current body of knowledge. Our cohort was

predominantly vitamin D-deficient and overweight/obese but otherwise healthy, thus we

could examine the associations between vitamin D and cardiometabolic risk factors in a high-

risk group where there was no confounding by disease status or medication use. We

employed direct, gold-standard measures of cardiometabolic risk including DEXA for body

fat and hyperinsulinaemic- euglycaemic clamp for insulin sensitivity. We were able to show

that % body fat, a more accurate measure of actual adiposity than BMI, may be a potential

driver for the observed associations between vitamin D and cardiometabolic risk, a factor that

has seldom been reported in previous studies.

In summary, we report that 25(OH)D levels are associated with cardiometabolic risk factors

and that these associations are mediated in large part by adiposity. Randomised clinical trials

employing sensitive measures of adiposity and gold-standard measures of cardiometabolic

risks are needed in order to establish causality between vitamin D and cardiometabolic risk

and disease, and to tease out whether these effects are independent, or modulated by adiposity.

16
5. COMPETING INTERESTS

The authors declare that they have no competing interests.

6. AUTHOR CONTRIBUTIONS

Ms. Aya Mousa wrote the first draft of the manuscript and is involved in data acquisition and

analysis. Dr Negar Naderpoor contributed to writing and editing the manuscript and is

involved in data acquisition and analysis. A/Prof Barbora de Courten is the principal

investigator of the trials which collected the data used in this study, obtained funding for

these trials, and contributed to writing and editing the manuscript. Prof Maximilian de

Courten, and Prof Robert Scragg contributed to the study design and obtaining of funding and

to writing and editing the manuscript. All authors read and approved the final manuscript, and

all authors meet the ICMJE criteria for authorship.

7. ACKNOWLEDGEMENTS

Ms. Aya Mousa and Dr Negar Naderpoor are recipients of the Australian Postgraduate Award

Scholarships provided by Monash University. A/Prof Barbora de Courten is supported by

National Heart Foundation Future Leader Fellowship (100864), Royal Australasian College

of Physicians, Foundation for High Blood Pressure Research, and is a recipient of a National

Health and Medical Research Council (NHMRC) grant (APP: 1047897).

8. ABBREVIATIONS
25(OH)D, 25-hydroxyvitamin D; BMI, body mass index; WHR, waist-to-hip ratio; DEXA,
dual energy x-ray absorptiometry; CT, computed tomography; OGTT, oral glucose tolerance
test; IPAQ, international physical activity questionnaire; BP, blood pressure; SBP, systolic
blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; MAP, mean arterial
pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; WBC, white blood
cell; hsCRP, high-sensitivity C-reactive protein; IQR, interquartile range; UV, ultraviolet;
CLIA, chemiluminescent immunoassay; VDR, vitamin D receptor; PTH, parathyroid
hormone; RAAS, renin-angiotensin-aldosterone system; NFkB, nuclear factor-kappa B;

17
GLUT, glucose transporter; QUICKI, quantitative insulin-sensitivity index; HOMA-IR,
homeostatic model assessment of insulin resistance; ISI, insulin sensitivity index; NHANES
III, third National Health and Nutrition Examination Survey; VITAL, vitamin D and omega-3
trial; FIND, Finnish vitamin D trial; ViDA, vitamin D assessment study; WHO, World Health
Organization; NHMRC, National Health and Medical Research Council of Australia; MCHRI,
Monash Centre for Health Research and Implementation; ICMJE, International Committee of
Medical Journal Editors.

18
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