EFFECTS OF VITAMIN D LEVELS ON GLUCOREGULATORY PARAMETERS IN

TYPE 2 DIABETES MELLITUS

ABSTRACT

Vitamin D affects the function of pancreatic beta cells, but the effects of vitamin D deficiency on glucoregulatory
mechanisms are still inconclusive. The aim of this study was to link vitamin D levels with insulin resistance and insulin
secretion parameters. The study included 65 male and female participants, 35 newly-diagnosed, therapy-naïve type 2
diabetics and 30 healthy controls. All participants were tested for fasting glucose, hemoglobin A1c, fasting insulin,
vitamin D levels, and the HOMA indexes were calculated using HOMA2 calculator. Fasting glucose levels,
insulinemia, hemoglobin A1c levels and HOMA IR were all significantly higher in the diabetic group (p<0,001), while
the vitamin D levels and HOMA S index were significantly lower (p<0,001). HOMA-B values did not differ between
the two groups (p=0.31). Vitamin D levels moderately correlated with HOMA S and HOMA B indexes (r=0,466,
p<0,001; r=0,394, p<0,001, respectively), whereas a negative correlation was found between vitamin D levels and
HOMA IR (r=−0,285; p<0,001). Multiple regression analysis showed that the vitamin D levels significantly predicted
the values of HOMA B index (p=0,001), but it had no predictive value on HOMA IR (p=0, 26). In conclusion, the
diabetic group showed statistically lower vitamin D values compared with the healthy control group. Connection
between vitamin D, glucose levels, hemoglobin A1c and insulin secretion index underline the role of this vitamin in
glucoregulation.

Key words: vitamin D; insulin resistance; insulin secretion; HOMA index

INTRODUCTION

Some of the most common chronic metabolic disorders of present time are insulin resistance and vitamin D deficiency, and

they are probably caused due to common risk factors, such as lack of physical activity, less sun exposure, bad dietary habits

based on increased intake of concentrated carbohydrates and lower intake of milk products and fibers, as well as the chronic

stress [1].

Insulin resistance and vitamin D deficiency are associated with chronic systemic inflammation, and although the exact

mechanism has not yet been completely clarified, there is increasing evidence in favor of the impact of vitamin D deficiency

on glycoregulatory mechanisms [1,2]. The published study have shown that vitamin D-deficient individuals have reduced

insulin secretion [3]. Vitamin D receptors (VDR) are found in pancreatic β cells which also possess 1-alpha hydroxylase

enyzme essential for the synthesis of the D hormone, providing an autocrine and paracrine biological effects [1,4].

Furthermore, there are studies which suggest that vitamin D affects an increased synthesis of the mRNA for the preproinsulin

of the pancreatic β cells [1]. The foregoing facts could indicate that vitamin D has a significant role in regulation of

physiological function of the endocrine pancreas. Additionally it is believed that vitamin D may also affect the sensitivity of

the peripheral insulin receptors due to existence of vitamin D receptors (VDR) in skeletal muscle, adipose tissue and the liver

cells. The presence of VDR may also increase the expression of insulin receptors on bone marrow cells. These mechanisms

are very complex and nowadays are intensively studied because they are considered to be very important, with no unified

position on this aspect of glyco-regulatory system.

However, published results considering the effects of vitamin D substitution on insulin resistance and secretion are still

inconclusive. In some studies, the vitamin D suplementation did not have an effect on insulin resistance in prediabetics and

type 2 diabetes mellitus patients (T2DM) [5,6]. On the other hand, some studies conducted on T2DM patients and patients

with impaired fasting glucose, showed that vitamin D substitution results in significant decrease of blood glucose, as well as

in improvement of calculated indexes of insulin resistance and / or insulin secretion [7-9].

Therefore the aim of this study was to estimate vitamin D status in newly-diagnosed type 2 diabetics, and to link vitamin

D levels with insulin resistance and insulin secretion parameters.

PATIENTS AND METHODS

This cross-sectional study was conducted in the Center of Laboratory Medicine in cooperation with the outpatient

department of Clinic for Endocrinology, Diabetes and Metabolic Disorders, Clinical center of Vojvodina, during summer

and autumn 2016. The study included 65 participants (55 men and 10 women). Based on the patient history, clinical

examination and laboratory analysis (fasting plasma glucose > 7 mmol/L and / or HbA1c > 48 mmol/mol) 35 participants

were classified into a group of T2DM [10]. All diabetics in this study were newly-diagnosed and therapy-naïve. The control

group consisted of 30 healthy subjects which correspond to the study group by age and gender (individuals with normal

fasting plasma glucose, insulin and glycated hemoglobin A1c).

Criteria for exclusion from the study were: kidney diseases, psychiatric disorders and the presence of metabolic bone diseases,

as well as supplementation with vitamin D and / or calcium.

Anthropometric measurements

All participans had their body height (TV) and body weight (BW) measured. Body height was measured using Martin

anthropometer and is expressed in centimeters (cm). Body weight was determined on a decimal scale in kilograms (kg). Body

mass index is calculated using the formula: BMI (kg / m²) = BW (kg) / TV² (m²) [11].

Laboratory assay

All laboratory analysis were performed before implementing any treatment of T2DM. After an eight-hour overnight fast and

rest for 15 minutes before blood sampling, blood samples were taken in order to determine fasting plasma glucose (FPG) and

insulin (FPI) concentration, vitamin D level (25(OH)D) and glycated hemoglobin A1c (HbA1c). Glucose was determined by

a specific enzyme, GOD-perid method (reference range from 4,0 to 6,1 mmol/L) and the insulin was measured on an

automated system ADVIA Centaur XP (reference value is 3,0 to 25,0 mIU/L). 25(OH)D was measured by direct

chemiluminescent technology on platform ADVIA Centaur XP. Vitamin D status was defined according to blood level of

25(OH)D as follows: vitamin D deficiency is defined as a 25(OH)D below 30 nmol/L, and vitamin D insufficiency as a

25(OH)D of 30 to 50 nmol/L, normal vitamin D status as a 25(OH)D concentration greater than 50 nmol/L [12]. HbA1c was
determined by immunoturbidimetric method of inhibition of agglutination on the microparticles, on automated system Abbott

Architect ci 4100.

HOMA indexes were estimated using HOMA 2 calculator: HOMA index for esimation of insulin resistance (HOMA-IR),

insulin sensitivity (HOMA-S) and secretory capacity of pancreatic β cells (HOMA-B) [13].

Data were presented using descriptive statistical methods for continuos variables as mean (x±s.d.). In order to evaluate

differences in parametric variables we used Student T-test. Relations among variables were assessed using Pearson's

correlation coefficient. Using linear corelation analysis we estimated relationship between all examinated parameters and

25(OH)D. Multiple regression analysis was performed in order to estimate the independent relation between 25(OH)D levels

and calculated parameters of insulin resistance/sensitivity as well as, insulin secretion. A 2-tailed p value of less than 0,05

was considered statistically significant.Statistical analysis was performed using the Data Analysis Excel (Microsoft Corp.,

Redmond, WA)and MedCalc 12.1.4.0 statistical software (MedCalc Software, Mariakerke, Belgium).

RESULTS

Comparing type 2 diabetics with healthy controls, statistically significant difference was found for all tested parameters

except for HOMA-B, (p=0,31). Compared with control group, examined diabetic group had significantly lower vitamin D

level (p<0,001) and HOMA-S (p<0,001) index, and significantly higher HOMA-IR index, BMI, FPG, FPI and HbA1c

(p<0,001) (Table 1). In the diabetic group 77% of participans were D vitamin insufficient - 27 of 35 diabetics (D vitamin

below 50nmol/l), while all of the healthy controlls had vitamin D level above 50nmol/l (p<0,001) (Chart 1).

Using linear correlation analysis on both examinated groups together (N=65), vitamin D level moderately correlated with

HOMA S and HOMA B indexes (r=0,466, p<0,001; r=0,394, p<0,001, respectively), whereas a negative correlation was

found between vitamin D levels and HOMA IR (r=−0,285; p<0,001). Negative correlations were also determined between

25(OH)D and FPG (r =-0,600; p<0,001) and HbA1c (r = -0,616; p<0,001). However we did not established a correlation

between 25(OH)D and FPI (r=-0,201; p>0,05) (Table 2).

Using multiple regression analysis we found that 25(OH)D has a significant predictive value towards HOMA-B%, while

BMI showed no significant prediction (Table 3). BMI and 25(OH)D have a significant inverse prediction with HOMA-S.
The above statistical analysis showed that level of 25(OH)D has no statistically significant prediction over HOMA-IR,

opposite to BMI.

Chart 1. Vitamin D insufficiency among two groups

p<0,001

100%
25%
80%

60% 100%

40% 75%

20%

0%
Diabetic group Control group

Vitamin D insufficiency D vitamin above 50 nmol/l

Table 1: Significance of the differences of the determined parameters

Determined T2DM Control P

parameters group group value
X±SD X±SD
N=35 N=30
BMI (kg/m²) 29,94±5,77 22,05±1,89 p<0,001
FPG (mmol/L) 7,86±1,98 4,77±0,39 p<0,001
FPI (mIU/L) 17,24±7,07 7,40±4,64 p<0,001
HbA1c (mmol/mol) 47,9±17,72 31,1±4,01 p<0,001
HOMA-IR 2,82±1,12 0,96±0,58 p<0,001
HOMA-S (%) 42,07±18,98 135,86±70,88 p<0,001
HOMA-B (%) 88,15±40,19 98,45±28,74 p=0,31
25(OH)D (nmol/L) 45,17±20,65 70,7±16,33 p<0,001

Legend: BMI-body mass index, izražen; FPG-fasting plasma glucose; FPI- fasting plasma insulin; HOMA-IR- index for
estimation of insulin resistance; HOMA-S- index for estimation of insulin sensitivity; HOMA-B-index for estimation
secretory capacity of pancreatic β cells; 25(OH)D-25 hydroxyvitamin D; p-value
Table 2: Linear correlation analysis: 25(OH)D

25(OH)D
N=65
r P
BMI(kg/m²) -0,261 p<0,001
FPG (mmol/L) -0,600 p<0,001
FPI (mIU/L) -0,201 p>0,05
HbA1c (mmol/mol) -0,616 p<0,001
HOMA-IR -0,285 p<0,001
HOMA-S (%) 0,466 p<0,001
HOMA-B% (%) 0,394 p<0,001

Legend: BMI-body mass index, izražen; FPG-fasting plasma glucose; FPI- fasting plasma insulin; HOMA-IR- index for
estimation of insulin resistance; HOMA-S- index for estimation of insulin sensitivity; HOMA-B-index for estimation
secretory capacity of pancreatic β cells; 25(OH)D-25 hydroxyvitamin D; r- correlation coefficient; p-value

Table 3: Multiple regression analysis

Dependent
variable Adjusted R² P value Predictors
N=65
t p(t)
BMI 6,986 <0,001
HOMA-IR 0,484 p<0,001
25(OH)D -1,144 0,257

BMI -4,693 <0,001

HOMA-S (%) 0,412 p<0,001
25(OH)D 3,316 0,001

BMI 0,532 0,596

HOMA-B (%) 0,13 0,006
25(OH)D 3,297 0,001

Legend: BMI-body mass index, izražen; FPG-fasting plasma glucose; FPI- fasting plasma insulin; HOMA-IR- index for
estimation of insulin resistance; HOMA-S- index for estimation of insulin sensitivity; HOMA-B-index for estimation
secretory capacity of pancreatic β cells; 25(OH)D-25 hydroxyvitamin D; Adjusted R²-the coefficient of determination that
is consistent with the number of independent variables included in the model; p-value
DISCUSSION

The main purpose of this study was to compare the two of our examined groups in vitamine D level, and to find relationship

between vitamine D and insulin secretion and resistance indexes. The results showed that our diabethic group had

significantly lower vitamin D level comared with control group. Also, 75% of our diabetic participants were D vitamin

insufficient.

It has not been clarified yet if low vitamin D status is responsible for insulin resistance, or it is just consequence of prediabetic

state, including obesity, which exist together with insulin resistance. One of the major cohort studies concluded that reduced

vitamin D levels lead to an increased risk of T2DM [14]. Also, published results showed that people who have been

substituted vitamin D or calcium, had a lower risk of T2DM [15]. The absence of an antiinflammatory effect, which is

attributed to reduced level of vitamin D, can contribute to the development of insulin resistance, and therefore disrupt the

glyco-regulatory mechanisms, as well as the onset of T2DM [1]. On the other hand, it is known that people with higher BMI

have lower level of vitamine D due to sequestration of liposolubile vitamine D in the larger adipose compartment, so this

vitamine would be low in serum [16]. Also, the study [17] came to another conslusion about low vitamin D status in obese

patients. It is said that simple volumetric dilution in obese patients can be explanation for the low vitamin D status in obesity.

Our data showed that our diabetic participants had significantly lower values od 25(OH) D, even though mean value of BMI

was under 30 in that group.

Compared to the control group, diabetic group had statistically higher HOMA-IR and insulin level and lower HOMA-S

values. The obtained results are expected and are in accordance with previously known literature data [18]. The

pathophysiological basis of T2DM refers to development of insulin resistance, and therefore, a significantly higher fasting

insulin in our diabetic group represents an attempt to overcome the present insulin resistance. On the other hand, diabetic

group had a lower HOMA-B than the control group (88.15% ± 40.19 vs. 98.45% ± 28.14), but without statistical significance.

HOMA-B value depends on the level of insuline resistance, and the functional ability of pancreatic β cells. Thus, the reduction

in insulin secretion can result from the preserved sensitivity of the peripheral tissues, and lower need for insulin secretion.

On the other hand, after the hyperinsulinemic phase in T2DM, the insulin secretion becomes insufficient and reduced in

relation to the level of current glycemia. So we can expect lower HOMA B values in healthy subjects, and in type 2 diabetics
as a consequence of the relative exhaustion of pancreatic β-cell, and a gradual decline in insulin secretion. Also, knowing

that HOMA B is calculated using FPI and FPG, this index shows whether this insulin secretion is capable of maintaining

fasting glucose in a normal range. We can see that in our controll group HOMA B is near the value of 100% which means

that insulin secretion is optimal for that glucose level, whereas in diabetic group, although fasting insulin is higher than in

control group, that insulin level is not capable of maintaining glucose level in euglicamic range, and that is the reason for

lower HOMA B index in this group.

Results of this study also showed a statistically significant relationship between 25(OH)D and HOMA-B which is in

accordance with the previously published study results [19]. Using mutiple regression analysis we have found that D vitamin

is a predictor of insulin secretion estimated by HOMA B index. It is known that the insulin secretion is glucose dependent

due to activation of GLUT 2 transporters, ATP synthesis, blockage of ATP-dependent potassium channels, as well as

consequent depolarization of the cell membrane and influx of calcium ions. Cell influx of Ca ions directly stimulates

exocytosis and the release of insulin molecules into the circulation. In addition to this mechanism, the second pathway of

regulation of insulin secretion is also indicated, via cAMP [20,21]. Besides the glucose-dependent, there are also glucose

independent pathways of insuline secretion (for example the effects of incretin, amino acids or glucagon on β cells) [22,23].

The presence of VDR receptors and 1α-hydroxylase in the pancreatic β cells suggest the possibility of regulatory role of

vitamin D on the intracellular concentration of Ca ions, as well as the effect of vitamin D on the transcription of the

preproinsulin gene [1,24]. The second theory is based on the fact that insulin secretion can be indipendently induced by

initiators (glucose, Ca) as well as potentiators (glucagon, acetylcholine, β-adrenergic receptor agonists, cAMP) which

enhance insulin secretion, but only in the presence of glucose as the most dominant initiator. According to this hypothesis,

vitamin D can affect insulin secretion by acting on the efficacy of the initiator and / or potentiator, modulating the effects of

the initiator of insulin secretion [24]. Vitamin D deficiency causes a change in the efficiency of the glucose-dependent cAMP

pathway and, consequently, its role in the regulation of insulin secretion [25]. As a confirmation to this theory numerous

studies on animals have been published. Those study results showed the influence of vitamin D on the cAMP signaling

pathway, as well as changes in the activity of the cAMP dependent protein kinase in vitamin D deficient animals [25-27].On

the other hand, clinical study which observed changes in the level insulin secretion during parenteral administration of vitamin

D concluded that an increase in insulin secretion was found only in people with elevated glycemia above the euglikemic

range, whereas in subjects with normal glycemia, no increase occurred [28-30]. Jeddi and associates had similar results on

experimental animals [31].

According to results, significant negative correlation was found between 25(OH)D and HOMA-IR which is in accordance

with previously published data [19,30,32]. However, multiple linear regression analysis has shown that level of vitamin D

influence on HOMA-IR is statistically insignificant. This finding is in line with the latest findings concerning relation between

vitamin D and insulin resistance [1]. Also, multiple linear regression showed that vitamin D is a significant predictor of

HOMA-S, an index that represents the sensitivity of peripheral tissues to insulin. The fact is that both, HOMA-IR and HOMA-

S indexes are relatively close and point to the similar occurrences from a different perspective. However, the parameters used

to calculate these indexes are different. In the HOMA-IR equation only the basal values of glucose and insulin are used,

whereas the estimation of HOMA-S also includes a deviation factor from normal values characteristic for healthy population

(100%). Different models of these indexes also point to different aspects of insulin effects on peripheral tissues [13]. The role

which vitamin D potentially achieves on insulin sensitive tissues is still not well known and explained in the literature. The

local concentration of vitamin D and the production of hormone D in tissues, which directly affect the level of insulin

resistance or sensitivity, can not be fully identified with the measured level of vitamin D in systemic circulation.

CONCLUSION

Diabetic group showed statistically lower vitamin D values compared with the healthy control group. Connection between

vitamin D and glucoregulatory parameters such as glucose levels, hemoglobin A1c and insulin secretion index (HOMA B)

underline the role of this vitamin in glucoregulation, primarily acting on the secretory function of β-pancreatic cells.
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