Accepted Manuscript

Title: A randomised controlled trial of high dose vitamin D inrecent-onset type 2 diabetes

Author: Shirley Elkassaby Leonard C. Harrison NamitaMazzitelli John M. Wentworth Peter G. Colman TimothySpelman Spiros Fourlanos

PII: S0168-8227(14)00395-7DOI: http://dx.doi.org/doi:10.1016/j.diabres.2014.08.030Reference: DIAB 6152

To appear in: Diabetes Research and Clinical Practice

Received date: 2-3-2014Revised date: 15-7-2014Accepted date: 31-8-2014

Please cite this article as: S. Elkassaby, L.C. Harrison, N. Mazzitelli, J.M. Wentworth,P.G. Colman, T. Spelman, S. Fourlanos, A randomised controlled trial of high dosevitamin D in recent-onset type 2 diabetes, Diabetes Research and Clinical Practice(2014), http://dx.doi.org/10.1016/j.diabres.2014.08.030

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Highlights Vitamin D deficiency has been associated with impaired pancreatic beta-cell function. We conducted a randomised trial of high dose vitamin D3 in people with recent-onset type 2 diabetes. D3 was associated with transient improvement in glycaemia but not beta-cell function. High dose D3 appears to offer little or no therapeutic benefit in type 2 diabetes.

*Highlights (for review)

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A randomised controlled trial of high dose vitamin D in recent-onset type 2 diabetes

Shirley Elkassaby FRACP, PhD, Leonard C Harrison MD, FRACP, FRCPA, DSc, Namita

Mazzitelli BSc, John M Wentworth FRACP, PhD, Peter G Colman MD, FRACP, Timothy

Spelman MBBS, BSc, Spiros Fourlanos FRACP, PhD

Walter & Eliza Hall Institute of Medical Research (S.E., L.C.H., N.M., J.M.W., S.P.),

1G Royal Parade, Parkville 3052, Victoria, Australia; Department of Diabetes &

Endocrinology (J.M.W., P.G.C.), Royal Melbourne Hospital, Parkville 3050, Victoria,

Australia; Burnet Clinical Research Unit (S.E., L.C.H., N.M., S.F.), Royal Melbourne

Hospital, Parkville, 3050, Victoria, Australia; Burnet Institute (T.S.), 85 Commercial Road,

Melbourne 3004, Victoria, Australia

Abbreviated title: RCT of vitamin D in type 2 diabetes.

Author for correspondence and reprints:

L. C. Harrison

Walter & Eliza Hall Institute of Medical Research

1G Royal Parade Parkville Victoria 3052, Australia

Tel (61-3) 93452461

Fax (61-3) 93470852

harrison@wehi.edu.au

Word count: Text 3018 ; Abstract 250. Tables 2, Figures 2

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Abstract

Aims Vitamin D insufficiency has been associated with impaired pancreatic beta-cell

function. We aimed to determine if high dose oral vitamin D3 (D) improves beta-cell

function and glycaemia in type 2 diabetes.

Methods 50 adults with type 2 diabetes diagnosed less than 12 months, with normal baseline

serum 25-OH D (25D), were randomised to 6000 IU D (n=26) or placebo (n=24) daily for 6

months. Beta-cell function was measured by glucagon-stimulated serum C-peptide (delta C-

peptide [DCP], nmol/l). Secondary outcome measures were fasting plasma glucose (FPG),

post-prandial blood glucose (PPG), HbA1c and insulin resistance (HOMA-IR).

Results In the D group, median serum 25D (nmol/l) increased from 59 to 150 (3 months) and

128 (6 months) and median serum 1,25D (pmol/l) from 135 to 200 and 190. After 3 months,

change in DCP from baseline in D (+0.04) and placebo (-0.08) was not different (P=0.112).

However, change in FPG (mmol/l) was significantly lower in D (-0.40) compared to placebo

(+0.1) (P=0.007), as was change in PPG in D (-0.30) compared to placebo (+0.8) (P=0.005).

Change in HbA1c (%) between D (-0.20) and placebo (-0.10) was not different (P=0.459). At

6 months, changes from baseline in DCP, FPG, PPG and HbA1c were not different between

groups.

Conclusion Oral D3 supplementation in type 2 diabetes was associated with transient

improvement in glycaemia, but without a measurable change in beta-cell function this effect is

unlikely to be biologically significant. High dose D3 therefore appears to offer little or no

therapeutic benefit in type 2 diabetes.

Clinical trial registration number: ACTRN 12611001023943

Key words: vitamin D, type 2 diabetes, beta-cell function, glycaemia, randomised trial.

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Introduction

With few dietary sources of vitamin D, sufficiency of this pleuripotent steroid depends on

its synthesis in the skin in response to ultraviolet B radiation. Indoor lifestyles and active

protection against sun exposure have contributed to an increased prevalence of vitamin D

insufficiency, even in sun-abundant countries [1]. Emerging evidence suggests that vitamin

D insufficiency may be a risk factor for both type 1 and type 2 diabetes [2-4] and associated

with reduced beta-cell function in type 2 diabetes (reviewed in ref 3). Pancreatic beta cells

not only express the nuclear vitamin D receptor (VDR) [5] but also 1-alpha-hydroxylase

which converts 25-hydroxyvitamin D (25D3) to the active metabolite 1,25-

dihydroxyvitamin D3 (1,25D3) [6]. Vitamin D supplementation may promote beta-cell

function [3,7] by a direct effect on insulin secretion or beta-cell survival ,or by an indirect

effect to inhibit production of inflammatory cytokines that impair beta-cell function [8] and

induce beta-cell apoptosis [9]. Despite indications that vitamin D insufficiency may increase

the risk of type 2 diabetes [3,4], the potential therapeutic effect of vitamin D has not been

widely tested in randomised controlled trials. We aimed to determine if high dose oral

vitamin D3 supplementation improves beta-cell function and glycaemic indices in recent-

onset type 2 diabetes.

Materials and Methods

Participants A randomised, double blind placebo-controlled clinical trial of oral vitamin D3

(D) was conducted in 50 Caucasians sequentially diagnosed with type 2 diabetes.

Participants gave written informed consent and the study was approved by the Royal

Melbourne Hospital Human Research Ethics Committee. Participants were recruited from

the community through a national diabetes register (National Diabetes Services Scheme,

Australia). They were eligible for enrolment if they had diabetes diagnosed according to

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World Health Organization criteria in the preceding 12 months, were aged 30-60 and not on

insulin treatment, and had a serum 25D between 28-85nmol/l and HbA1c < 8%

(64 mmol/mol). Those with very low serum 25D (<28nmol/l) were referred to their primary

care doctor as the Ethics Committee considered it unethical for them to participate in a

randomised controlled trial. The upper limit of serum 25D (85nmol/l) was an exclusion to

preserve study sensitivity, based on evidence that healthy adults with an outdoor lifestyle

have serum 25D levels above this value [10]. Exclusion criteria included insulin treatment,

liver impairment (serum transaminase concentration > 2-fold above the reference range),

renal impairment (eGFR <50ml/min), hyperparathyroidism, family or personal history of

renal calculi, history of recurrent falls, use of a gait aid, past history of a fragility fracture,

personal or family history of osteoporosis and current treatment with oral prednisolone,

methotrexate or other immunosuppressive drugs.

Protocol Standard dietary advice for both blood glucose control and optimal calcium

nutrition was provided to all participants. Participants were asked to avoid the sun and to

adopt usual sun protection measures such as sunscreen and wearing a hat when outdoors.

They continued their usual oral hypoglycaemic medications under the care of their family

physician, aiming for optimal glycaemic control, i.e. fasting plasma glucose (FPG) < 7

mmol/l and HbA1c < 7% (53 mmol/mol). If, at review during the trial, glycaemic control

had deteriorated (HbA1c > 8%, 64 mmol/mol), the oral hypoglycaemic regimen was

optimised and insulin treatment instituted if necessary.

Participants were block randomised without stratification across all seasons to oral D or

placebo by an off-site statistician, using random numbers generated with the STATA 8

program. D3 (2,000 IU) and placebo in tablet form (formulated by Cardinal Health,

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Braeside, Victoria, Australia) were identical in appearance and pre-packed in bottles. Coded

treatments were dispensed by independent pharmacists at the Royal Melbourne Hospital

Pharmacy, Clinical Trials Unit. Participants and investigators were blinded to treatment.

Participants were instructed to take an initial loading dose of 5 tablets daily (10,000 IU D or

placebo) for 2 weeks then 3 tablets daily (6,000 IU D daily or placebo) for the remaining 6

months of the trial. Compliance was ascertained by interview at follow-up visits and by

tablet counts.

Study visits and monitoring Study visits were conducted at the Royal Melbourne Hospital at

baseline, 3 and 6 months. At each visit participants were interviewed, examined including

blood pressure measurement with a mercury sphygmomanometer in the supine position after

resting for 5 minutes, and had blood drawn for biochemistry. Beta-cell function was

assessed by i.v. glucagon-stimulated secretion of C-peptide (a surrogate for insulin), a

measure of beta-cell function reasonably comparable to the more physiological mixed meal

stimulation test [11]. Participants were fasted for 10 hours overnight, withholding oral

hypoglycaemic drugs. Blood samples were taken between 8 and 10 am via an i.v. cannula in

an antecubital vein, providing fasting blood glucose was <10 mM (no participant exceed

this limit). Blood was collected for serum C-peptide just before (baseline) and 6 minutes

after administering glucagon 1mg i.v. Delta C-peptide (DCP) is the difference between 6

minute and baseline serum concentrations. Participants were requested to measure with a

glucometer and record their capillary blood glucose levels 2 hours after meals for 2 days

prior to each study visit. Post-prandial glucose was an average of these 6 readings. Dual

energy X-ray absorbiometry (DEXA) for assessment of body fat was performed at baseline

and 6 months.

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At 1 and 3 months after randomisation, an independent clinician unblinded to treatment

allocation adjusted the dose in the D group to maintain serum 25D in the range 100-165

nmol/l. This clinician contacted study participants but had no contact with the investigators.

If a participant required an increase or decrease in the number of D tablets based on serum

25D results 1 month from baseline,, extra blood was taken for measurement of serum 25D

and calcium after another month. For any adjustment in the number of tablets for a D group

participant, the same change was made in a randomly selected placebo participant.

Laboratory assays Serum 25D3 was assayed using the Diasorin 125

I radioimmunoassay Kit.

The CVs for the assay of serum 25D3 of 31, 56 and 107 nmol/l were 13%, 10% and 9%,

respectively. Serum 1,25D3 was assayed using the Immunodiagnostics Systems 125

I

immunoassay Kit. The CVs for the assay of serum 1,25D3 at 47, 53 and 149 pmol/l were

10%, 9% and 10%, respectively. Serum C-peptide and serum insulin were batch analyzed by

commercial chemiluminescence assays (Immulite and Abbot Imx, respectively).

Statistics The primary outcome measure was DCP. Secondary outcomes were measures of

glycaemia (FPG, PPG, HbA1c) and insulin resistance (HOMA-IR). HOMA-IR was

calculated as fasting insulin (mU/l) × fasting glucose (mmol/l)/22.5 [12]. Power calculations

were based on the median and variance for DCP, and an expectation that D supplementation

would improve DCP by 50% [13, 14]. The sample size per arm was determined to be 23

(power 85%, 5% level of significance).

Categorical variables were summarised using frequency and percentage and compared using a

Fisher’s Exact Test. Continuous variables were first assessed for significant skew. As all

continuous variables demonstrated significant departures from normality they were

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summarised using median and inter-quartile range and compared using a Wilcoxon rank-sum

or signed-rank test as appropriate. Where a study hypothesis was explicitly unidirectional,

one-tailed p values were used. For each analysis a P < 0.05 was considered significant. Prism

version 5 (GraphPad Software, San Diego, CA) was used for all analyses. As a sensitivity

analysis, Generalized Estimating Equations were used to model differences in longitudinal

outcome trends by treatment arm.

Results

Recruitment Of the 50 participants, 26 were randomly allocated to the D group and 24 to

placebo, with males and females being equally represented. Only 3 required modification of

their diabetes therapy due to hyperglycaemia during the trial, with one requiring insulin.

There were no adverse events related to the study treatments, in particular no participant

developed hypercalcemia. Similar numbers of D and placebo participants were enrolled in

all seasons. Randomisation commenced in January 2008 and the last participant completed

the trial July 2009. The D3 dose remained at 6000 IU/day in almost all participants. Four

participants in the D group required dose adjustment on only 8 occasions over the 6 month

period, to maintain serum 25D >100 nmol/l. The largest dose administered was 8000

IU/day. No participant was excluded during the trial due to a serum 25D below 28 nmol/l.

All participants completed the trial and tablet counts indicated full compliance.

Baseline characteristics Overall, participants were obese (median [interquartile range (IR)]

BMI 31 kg/m2 [27-36]), middle-aged (median age 54 [48-58]) and had well-controlled

diabetes (median HbA1c 6.1% [5.6-6.6]) 43mmol/mol [38- 49] (Table 1). More than half

were on oral hypoglycaemic medication at baseline. Two participants (one in each group)

were taking calcium supplementation, which continued during the study. Groups were well

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matched at baseline (Table 1) and differed significantly only in diastolic blood pressure,

which was lower in the D group (median 77vs. 82 mmHg, P=0.009) and FPG, which was

higher in the D group (median 6.8 vs. 6.4 mmol/l, P=0.024) (Table 1). Differences between

the number of D and placebo participants on all oral hypoglycaemic medications, metformin

alone, anti-hypertensive and lipid lowering agents were not significant (Fisher’s exact tests)

at all time-points. Adiposity measured by DEXA did not differ between the D and placebo

groups at baseline, 3 or 6 months and no relationship was apparent between body fat mass

and the required dose of D.

Serum 25D and calcium Serum 25D and 1,25D increased significantly from baseline in the

D group and were unchanged in the placebo group (Figure 1A & 1B). Median serum 25D at

baseline, 1, 3 and 6 months in the D group was 59, 115, 150 and 128 nmol/l and in the

placebo group 61, 54, 61 and 60 nmol/l, respectively. Median serum 1,25D at baseline, 3

and 6 months in the D group was 135, 200, 190 pmol/l and in the placebo group 145, 150,

160 pmol/l, respectively.

Median plasma calcium remained steady at 2.4 mmol/l in both groups at baseline, 3 and 6

months mmol/l. Median serum PTH at baseline, 3 and 6 months was 3.8, 2.7 and 2.4 pmol/l

in the D group and 3.0, 4.0 and 3.8 pmol/l in the placebo group. The differences in serum

PTH between D and placebo groups were significant at 3 (P=0.001) and 6 (P=0.006)

months.

Primary outcome Median DCP (nmol/l) at baseline, 3 and 6 months in D vs. placebo groups

was 0.80, 0.89 and 0.83 vs. 0.83, 0.65 and 0.80, with differences at 3 months being

significant (P=0.040) (Table2). Alternatively, we measured the absolute change in DCP

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between baseline and 3 months in D and placebo groups. The median difference between

this change in the D group (+0.04 nmol/l) was not significantly different than in the placebo

group (-0.08 nmol/l) (P=0.112) (Figure 2).

Secondary outcomes Median FPG (mmol/l) at baseline, 3 and 6 months in D and placebo

groups was 6.8, 6.5 and 7.0 and 6.4, 6.6 and 6.4 (Table 2). The FPG change from baseline at

3 months was significant for D (-0.40) vs. placebo (+0.1) groups (P=0.006) (Figure 2B).

Median PPG (mmol/l) at baseline, 3 and 6 months in D and placebo groups was 7.3, 7.2 and

7.5 and 7.3, 7.7 and 7.8. The PPG change from baseline at 3 months was significant for D (-

0.30) vs. placebo (+0.8) groups (P=0.008) (Figure 2C). HbA1c (%) at baseline, 3 and 6

months in D and placebo groups was 6.2, 5.9 and 6.1 and 6.1, 6.1 and 6.0 (44mmol/mol, 41

and 43 and 43, 43 and 42). HOMA-IR at baseline, 3 and 6 months in D and placebo groups

was 3.2, 2.7 and 2.2 and 4.2, 3.5 and 3.1. In the case of both HbA1c and HOMA-R,

differences between D and placebo groups at any time point and changes from baseline were

not significant (Table 2, Figure 2D). At 6 months, differences between D and placebo groups

in DCP, FPG, PPG or HbA1c were not significant (Table 2, Figure 2). Although limited

possibly by sample size, analysis by Generalised Estimating Equations did not reveal

significant associations in any of the outcomes by treatment arm.

Discussion

High dose D supplementation substantially increased both 25D and 1,25D concentrations

without toxicity. It has been shown previously that oral D increases serum 25D in people

with diabetes [8, 15-17], but its effect on serum 1,25D has not been reported. The significant

increase in 1,25D in response to oral D challenges the traditional view that 1,25D

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production is tightly regulated by renal 1-alpha hydroxylase to maintain calcium

homeostasis. It indicates that oral D has broader potency to increase 1,25D systemically.

The primary outcome of beta-cell function, measured as DCP in response to intravenous

glucagon, was significantly higher in the D compared to placebo group at 3 months. This

reflected predominantly the decrease in DCP in the placebo group at 3 months. The decrease

in DCP in the placebo group is unlikely to be due to the natural decline in insulin secretion

because it did not continue to 6 months and was similar in the D and placebo groups at 6

months. Moreover the change in DCP from baseline to 3 months was not significantly

different between the groups. It is possible that C-peptide stimulation with glucagon may

not have been sufficiently sensitive to detect an effect of D supplementation to sustain beta-

cell function. While the glucagon stimulation test is convenient and has been frequently

employed in the past, a more recent multi-center study [11] demonstrated that the mixed

meal tolerance test has superior sensitivity and precision. Fasting plasma glucose and post-

prandial glucose were significantly reduced by 3 months in the D group. However, these

changes were not reflected by significant changes in HbA1c. The effect of D on glucose was

associated with no measurable change in insulin resistance as estimated by HOMA-IR.

These changes in glucose were only observed at 3 months. Serum 25D peaked at 3 months

then decreased in 18/26 participants in the D group by 6 months. Although the median

decrease of 25D from150 at 3 months to 128 nmol/l at 6 months was not significant

(P=0.08, paired t test, 2-tail) if the effect of D is dose-dependent it remains possible that

failure to detect an effect on glycemic endpoints at 6 months was related to lower

concentrations of serum 25D achieved. We found no evidence of non-compliance to account

for the decrease in serum 25D at 6 months, but this cannot be excluded. An alternative

possibility is that daily oral D and a sharp rise in serum 25D rather than episodic synthesis

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in the skin might have lead to homeostatic adaptation, for example downregulation of the

nuclear receptor, thereby decreasing the action of D. Calcium intake independent of D could

influence glucose metabolism (reviewed in ref. 3) and a possible limitation of our trial is

that dietary calcium intake was not recorded.

Despite the contemporary interest in the extra-skeletal effects of vitamin D, it is difficult to

find published randomised trials of D supplementation in people with type 2 diabetes or

impaired glucose tolerance (IGT). In IGT, Pittas et al [18] found that low dose D (700 IU

daily for 3 years) stabilised FPG compared to an increase in FPG in the placebo group.

However, this finding arose from a post-hoc analysis in a trial designed to measure the impact

of D and calcium supplementation on bone endpoints. In a follow-up randomised trial [19],

the same investigators gave 46 predominantly Caucasian individuals with IGT 2000 IU D

daily for 4 months. Despite an increase of only 15.5 nmol/l in mean serum 25D from a

baseline of 59.9 nmol/l, the insulin secretory response to intravenous glucose increased

overall, although HbA1c remained similar to the placebo group. In a small study [16] in which

32 individuals with type 2 diabetes were randomised to oral D3 (40,000 IU weekly) or

placebo for 6 months there was no effect on FPG, HbA1c, fasting C-peptide, HOMA-IR,

lipids or blood pressure. However, in contrast to the present study, participants had a longer

duration of diabetes (>1 year), were receiving subcutaneous insulin and had sub-optimal

glycaemic control at baseline (HbA1c 8.0%), and serum 25D was not documented. Witham et

al [20] randomised what appear to be predominantly Caucasians with type 2 diabetes with

normal serum 25D to placebo or single dose D3, either 100,000 or 200,000 IU. After 8 weeks,

HbA1c and HOMA-IR were unchanged. Harris et al [21] randomised 89 overweight or obese

African Americans with ‘pre-diabetes’ (FPG ≥ 5.5 mmol/l and HbA1c < 7%) to oral D3

(4,000 IU/day) or placebo for 3 months. The mean 25D at baseline in both groups was low at

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40 nmol/l and increased to 81 nmol/l after supplementation. In the D group the C-peptide

response to oral glucose increased whereas insulin sensitivity decreased and measures of

glycaemia were unchanged, leading the investigators to conclude that short-term D

supplementation did not alter the pathophysiology of type 2 diabetes. A similar more recent

study in people with pre-diabetes, did not find an effect of high dose vitamin D (average daily

dose of 12,695 IU) on FPG and insulin secretion but there was a small effect on HbA1c (-

0.2%) over the 12 months of the study [22]. One trial that did demonstrate significant

improvements in glycemic endpoints including HbA1c in people with type 2 diabetes, was

unblinded and was powered for a change in serum vitamin D not glycemic endpoints [23].

Our study, in Caucasians with recent-onset, well-controlled type 2 diabetes and normal serum

25D, employed a high dose of supplemental D. It was distinguished by significant increases in

both serum 25D and 1,25D and an early endpoint assessment at 3 months. If adaptive changes

occur with tonic oral vitamin D supplementation, then trials that assess glycaemia at 6 months

or beyond may fail to detect an effect. While our findings show that high dose D

supplementation leads to short-term, transient improvement in glycaemia the biological

significance of this effect is questionable. Our findings are consistent with a recent meta-

analysis that concluded vitamin D intervention trials have failed to demonstrate improvement

in a number of diseases including diabetes [24]. Larger longer term randomised trials of

supplemental D will be necessary to confirm this view, as well as studies to determine if

adaptive responses in D metabolism occur in this context.

Acknowledgements The authors thank Diabetes Australia for assistance in recruiting

participants and Professor John Wark and Dr Mark Stein, Royal Melbourne Hospital, for

advice on vitamin D dosing and comments on the manuscript.

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Funding This work was funded by grants from The Munro Foundation, Melbourne, and

Diabetes Australia Research Trust, and supported by Victorian State Government

Operational Infrastructure Support and Australian Government NHMRC IRIIS. SE was a

Postgraduate Scholar of the National Health and Medical Research Council of Australia

(NHMRC) and received a Basser Family Scholarship from the Royal Australasian College

of Physicians. LCH is a Senior Principal Research Fellow of the NHMRC.

Conflict of interest. There is no conflict of interest for any of the authors.

Contribution statement Study concept and design LCH, SF, SE; acquisition of data: SE,

NM, PGC, JMW; analysis and interpretation of data: SE, SF, TS, LCH; drafting of manuscript

SE, SF, LCH.

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23. Nikooyeh B, Neyestani TR, Farvid M, Alavi-Majd H, Houshiarrad A, Kalayi A et al.

Daily consumption of vitamin D– or vitamin D + calcium–fortified yogurt drink

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improved glycemic control in patients with type 2 diabetes: a randomized clinical trial.

Am J Clin Nutr 2011; 93:764–71.

24. Autier P, , Mathieu B, Pizot C, Mullie P. Vitamin D status and ill health: a systematic

review, The Lancet Diabetes & Endocrinology, 2014; 2: 76 – 89.

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Figure legends

Fig. 1 Outcome measures in D (filled circles) and placebo (open circles) groups at 0, 3 and 6

months. A) Serum 25D, B) Serum 1,25D. Median values are indicated by the horizontal lines.

Fig. 2 Change in glycaemic and insulin secretion parameters in D (filled bars) versus placebo

(unfilled bars) groups at 3 and 6 months. A) Delta C-peptide, B) Fasting plasma glucose, C)

Post-prandial capillary glucose and D) HbA1c. Data shown are median + IQR.

Figure legend(s)

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Figure

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-0.6

-0.4

-0.2

0.0

0.2

0.4

3 6 3 6

Ch

ang

ein

DC

P(n

mo

l/l)

-1.0

-0.5

0.0

0.5

1.0

3

3 6 3 6

P=0.006

Ch

ang

ein

FP

G(m

mo

l/l)

-1.0

-0.5

0.0

0.5

1.0

1.5

Ch

ang

ein

PP

G(m

mo

l/l)

3 6 3 6

P=0.008

-0.4

-0.2

0.0

0.2

0.4

3 6 3 6C

han

ge

inH

bA

1c(%

)

Month

Month

A B

C D

Figure 2

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Table 1. Baseline characteristics of trial participants Vitamin D3 Placebo

Number (n) 26 24

Age 53 51

Male (n) 16 13

Female (n) 10 11

BMI (kg/m2) 30.6 [26.6-36.4]* 31.1 [26.7-38.0]

Systolic BP (mmHg) 135 [124-140] 139 [128-147]

Diastolic BP (mmHg) 77 [65-80]a 82 [74- 90]

25-D3 (nmol/l) 59 [42-75] 62 [40-80]

1-25D3 (pmol/l) 135 [100-165] 145 [130-178]

Serum calcium (mmol/l) 2.44 [2.35-2.49] 2.43 [2.36-2.50]

Serum PTH (pmol/l) 3.8 [2.5-4.3] 3.0 [2.4- 5.1]

Creatinine (μmol/l) 0.09 [0.08-0.10] 0.08 [0.08-0.09]

HbA1C %, mmol/mol 6.2 % [6.0-6.6], 44mmol/mol [42-49] 6.1% [5.8-6.4], 43 mmol/mol [40- 46]

Fasting plasma glucose (mmol/l) 6.8 [6.4-7.3]b 6.4 [6.0-6.9]

Post prandial glucose (mmol/l)c 7.3 [6.6-8.3] 7.3 [6.5-8.3]

Delta C-peptide (nmol/l) 0.80 [0.58-1.2] 0.83 [0.43-1.2]

HOMA-IR 3.2 [1.7-5.4] 4.2 [1.8-5.7] *Median [interquartile range]. The only significant differences between the vitamin D3 and placebo groups were diastolic blood pressure (P=0.009)a and fasting plasma glucose (P=0.024)b. Post-prandial blood glucose was calculated as the mean of 6 home capillary blood glucose readings over the 2 days before each follow-up visit.c

Table

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Table 2. Outcome measures

*Median [interquartile range]. D compared to placebo at same time-points: a) P<0.05; b) P< 0.01; c) P<0.001. D compared to baseline: d) P<0.05; e) P< 0.001; f) P<0.0001. Placebo compared to baseline: g) P<0.05; h) P<0.01; i) P<0.001.

Vitamin D Baseline

Vitamin D 3 months

Vitamin D 6 months

Placebo Baseline

Placebo 3 months

Placebo 6 months

25-D3 (nmol/l) 59 [41-74]* 150 [120-172]c,f 128 [111-146] c,f 62 [40-80] 62 [37-77] 61 [45-87]

1,25-D3 (pmol/l) 135 [100-165] 200 [153-240]b,f 190 [140-235]b,e 145 [130-178] 150 [120-170] 160 [130-200]g

DCP (nmol/l) 0.80 [0.6-1.2] 0.89 [0.5-1.4]a 0.83 [0.5- 1.1] 0.83 [0.4-1.2] 0.65 [0.4 -1.1] 0.80 [0.5-1.1]

FPG (mmol/l) 6.8 [6.4-7.3]a 6.5 [6-7.2]d 7.0 [6.2-7.2] 6.4 [6.0-6.9] 6.6 [6.1-7.2] 6.4 [5.8-7.1]

PPG (mmol/l) 7.3 [6.6-8.3] 7.2 [6.5-7.8]a 7.5 [6.4-8.4] 7.3 [6.5-8.3] 7.7 [7.2-8.9]h 7.8 [6.9-8.3]g

HbA1c (%) 6.2 [6.0-6.6] 5.9 [5.7-6.5]d 6.1 [5.7-6.7] 6.1 [5.8-6.4] 6.1 [5.7-6.5] 6.0 [5.8-6.6]

HbA1c (mmol/mol) 44 [42-49] 41 [39-48]d 43 [39-50] 43 [40-46] 43 [39-48] 42 [40- 49]

HOMA-IR 3.2 [1.7-5.4] 2.7 [1.8-4.8] 2.2 [1.4-5.4] 4.2 [1.8-5.7] 3.5 [1.7-6.1] 3.1 [1.4-5.8]

Table