Abstract

Background: Pancreatic β-cell mass, crucial for insulin secretion, decreases in Diabetes Mellitus (DM). β-cells possess receptors for vitamin D3, and low serum vitamin D3 levels are linked to an increased DM risk. This study explores the association between serum vitamin D3 levels and glycaemic status in DM individuals. Methodology: Subjects with Type 1 DM (T1DM), Type 2 DM (T2DM), and Gestational DM (GDM), diagnosed per ADA criteria, were sampled consecutively over a year. Glycaemic status (HbA1c) and serum vitamin D3 levels were measured. Data were analysed using SPSS, employing Chi-square and linear regression. A p-value of <0.05 was considered significant. Results: The study included 150 participants (50.74% males; mean age: 48.91 ± 14.87 years). Mean serum vitamin D3 was 20.52 ± 11.96 ng/mL, with 57.33% showing deficiency (<20 ng/mL). In T2DM individuals, there was a significant inverse correlation between poor glycaemic control (HbA1c >7.6%) and serum vitamin D3 levels (p=0.000001), and between disease duration and serum vitamin D3 (p=0.000571). No significant correlations were found in T1DM (p=0.5060, 0.0732) and GDM (p=0.6422). Conclusion: Serum vitamin D3 levels were inversely correlated with poor glycaemic control and disease duration in T2DM. No significant associations were observed in T1DM and GDM.

Keywords: Vitamin D3; Diabetes Mellitus; Type 1 Diabetes Mellitus; Type 2 Diabetes Mellitus; Gestational Diabetes Mellitus

Introduction

Diabetes Mellitus, a prevalent metabolic non-communicable disease, poses a significant global health threat with approximately 537 million adults affected [1,2]. This condition is associated with increased morbidity and mortality, leading to frequent hospitalizations and premature death. Individuals with Diabetes Mellitus, including Type 1 Diabetes Mellitus (T1DM), Type 2 Diabetes Mellitus (T2DM) and women with Gestational Diabetes Mellitus (GDM), are at risk of developing various complications.

The pancreatic β-cells possess receptors for active vitamin D3 (calcitriol), allowing vitamin D3 to regulate insulin and blood glucose level [1,3]. Calcitriol promotes β-cell biosynthetic capacity and facilitates the conversion of proinsulin to insulin [3]. Numerous studies have demonstrated an association between serum vitamin D3 level and Diabetes Mellitus. Observational studies consistently show that individuals with good glycaemic control in T1DM, T2DM and GDM have higher serum vitamin D3 level than those with poorer glycaemic control [4-6]. Serum vitamin D3 level estimation is cost-effective and easily accessible. If a correlation between serum vitamin D3 level and glycaemic status is established in individuals with Diabetes Mellitus, vitamin D3 supplementation may potentially aid in achieving adequate glycaemic control and preventing diabetic complications.

India, a country with a high prevalence of both Diabetes Mellitus and vitamin D3 deficiency, lacks sufficient data on the correlation between the two. Therefore, this study aimed to investigate serum vitamin D3 level in individuals with T1DM, T2DM, and GDM, along with exploring the relationship between serum vitamin D3 and glycaemic status.

Aim and Objectives

Aim

To measure serum vitamin D3 levels in patients with Diabetes Mellitus

Study objectives

1. To measure serum vitamin D3 level in patients with Type 1 Diabetes mellitus, Type 2 diabetes Mellitus and Gestational Diabetes Mellitus
2. To find out if there is any correlation of HbA1c level and duration of Diabetes Mellitus with serum vitamin D3 levels in these subtypes of Diabetes Mellitus.

Methodology Study population and sampling strategy

This prospective, hospital-based observational analytical crosssectional study was conducted over a one-year period, from January 2021 to January 2022, at a tertiary care hospital in Northeast India. The primary focus was on individuals diagnosed with various types of DM, including T1DM, T2DM, and women with GDM. The study was carried out in the General Medicine and Obstetrics & Gynaecology departments of the hospital, aiming to explore the relationship between diabetes and serum vitamin D3 levels, while accounting for various demographic, clinical, and biochemical parameters.

Participants were recruited based on specific inclusion and exclusion criteria. The inclusion criteria encompassed adults (both men and women) aged 18 years and above who had been diagnosed with T1DM, T2DM, or GDM according to the diagnostic criteria established by the American Diabetes Association (ADA) [2]. The diagnosis for GDM in pregnant women was confirmed through a 75g Oral Glucose Tolerance Test (OGTT), as recommended by ADA guidelines.

Participants were excluded if they had secondary forms of diabetes (e.g., resulting from pancreatitis, hormonal disorders, or medication-induced), or if they were suffering from chronic comorbid conditions that could independently affect bone metabolism, such as Chronic Kidney Disease (CKD), Chronic Liver Disease (CLD), or thyroid disorders. Moreover, individuals who were on medications or supplements known to influence bone metabolism, including vitamin D3 supplements, bisphosphonates, or other multivitamins and calcium supplements, were also excluded from the study to prevent confounding variables. This meticulous selection ensured that the study population was homogenous with respect to the factors influencing vitamin D3 levels, providing a more accurate evaluation of the association between diabetes and vitamin D3.

Sample size, Data Collection and Screening

A total of 150 participants, meeting the inclusion and exclusion criteria, were enrolled in the study. Participants were approached through the General Medicine and Obstetrics & Gynaecology outpatient departments. Upon enrolment, participants underwent a systematic and detailed data collection process, which began with the recording of demographic and clinical data, including age, gender, duration of diabetes, and relevant lifestyle factors such as smoking and alcohol use. A thorough clinical history was obtained. Physical examinations were performed meticulously, focusing on anthropometric measurements like height, weight, and waist-hip ratio, as well as vital signs including pulse, blood pressure, and respiratory rate. This provided a comprehensive baseline profile for each participant.

Biochemical evaluations were performed to assess the participants’ metabolic status. Blood samples were collected from all participants after an overnight fasting period of at least 8 hours. For women suspected of GDM, an additional 2-hour postprandial sample was collected following the administration of a 75g glucose load.

1. FBS: Measured using the hexokinase method to assess the basal glycaemic status.
2. PPBS: Measured 2 hours after a meal or glucose load to evaluate post-meal glucose control.
3. HbA1c: Measured using High-Performance Liquid Chromatography (HPLC) to provide an average of the participant’s glycaemic control over the past 3 months.
4. Serum Vitamin D3 Levels: Quantified using the Chemiluminescence Immunoassay (CLIA) method, which is highly sensitive and specific for detecting 1,25-dihydroxy vitamin D3 [1,25(OH)2D3]. Serum vitamin D3 levels were classified based on the criteria established by the US Endocrinology Association: values <20 ng/mL were categorized as deficient, 20-30 ng/mL as insufficient, and >30 ng/mL as sufficient [7,8].
5. Kidney function test (KFT): Assessed using serum creatinine and estimated Glomerular Filtration Rate (eGFR) to exclude participants with undiagnosed renal impairment.
6. Liver Function Test (LFT): Measured to exclude hepatic disorders that could potentially affect vitamin D3 metabolism.
7. Urinalysis was conducted to screen for microalbuminuria or proteinuria, which can be early indicators of diabetic kidney disease and cardiovascular risk.

Statistical analysis

A comprehensive data collection sheet was prepared to capture all relevant parameters, including socio-demographic data, clinical examination results, and biochemical findings. Demographic data included age, sex, educational status, occupation, and socioeconomic background. Clinical parameters recorded included BMI, waist-hip ratio, and blood pressure measurements. Biochemical data encompassed FBS, PPBS, HbA1c, serum vitamin D3, and kidney and liver function parameters.

For statistical analysis, MS Office 365 and IBM SPSS version 23.0 software were used. Continuous variables such as age, Body Mass Index (BMI), FBS, PPBS, HbA1c, and serum vitamin D3 levels were described using mean, median, mode, and standard deviation. Categorical variables, including sex, diabetes type, and vitamin D3 categorization, were presented as frequencies and percentages. One-way Analysis of Variance (ANOVA) was used to compare the means of continuous variables across more than two groups (e.g., vitamin D3 categories), while the Chi-Square test was employed for comparing proportions between groups (e.g., Gender vs. vitamin D3 status).

To investigate the potential predictors and associations between serum vitamin D3 levels and various clinical parameters (e.g., HbA1c, duration of diabetes, BMI), linear and multiple regression analyses were performed. These models aimed to identify independent predictors of serum vitamin D3 levels after adjusting for confounding variables such as age, sex, and glycaemic control. Correlation coefficients (Pearson’s or Spearman’s as appropriate) were calculated to evaluate the strength and direction of relationships between vitamin D3 and other continuous variables like HbA1c and BMI.

Statistical significance was defined at a p-value of less than 0.05. All statistical tests were two-tailed, and results were interpreted with a 95% confidence interval. This approach ensured robust and reliable conclusions, reducing the likelihood of type I and type II errors.

Ethical consideration

The study was conducted following a strict ethical framework, as mandated by the Ethics Committee of the hospital. Ethical approval was obtained before the commencement of the research, and all participants were thoroughly informed about the study’s objectives, methodology, potential risks, and benefits. Written informed consent was obtained from each participant, ensuring voluntary participation and compliance with ethical standards. The confidentiality and anonymity of the participants were maintained throughout the study, with all personal data being securely stored and used exclusively for research purposes. The study adhered to the principles of the Declaration of Helsinki, ensuring respect, dignity, and protection of the participants‘ rights throughout the research process.

Results Demographic structure of study population

The study enrolled a total of 150 individuals, ranging in age from 18 to 80 years, with a mean age of 48.91 ± 14.87 years and a male to female ratio of 1.03:1. Among them, 5 individuals had T1DM, 130 had T2DM, and 15 women had GDM. A subgroup of 40 individuals (26.7%) were newly diagnosed with Diabetes Mellitus, while 58 individuals (38 %) had been living with the condition for over 10 years. The HbA1c values ranged from 5.5 % to 16.5 %, with a mean of 8.87 ± 2.34 %. A substantial portion of individuals (71.53 %) had poorly controlled Diabetes Mellitus, predominantly those with T2DM (55.33 %). The serum vitamin D3 level ranged from 4.06 ng/ml to 68.54 ng/mL, with a mean of 20.52 ± 11.96 ng/mL. A majority of individuals (57.33 %) exhibited vitamin D3 deficiency, with no case of vitamin D3 toxicity observed. Baseline demographic characteristics were analysed in relation to serum vitamin D3 level. There was no significant correlation observed between age or sex and serum vitamin D3 level (p- value= 0.922 and 0.327 respectively). The following Table 1 explains the relation of various demographic variables with relation to serum vitamin D3 levels.

Table 1: Correlation of demographic parameters with serum vitamin D3 levels.

Correlation of duration of DM and HbA1c level with serum vitamin D3 levels in T1DM

Regarding the relationship between Diabetes Mellitus and serum vitamin D3 level, among individuals with T1DM, the majority exhibited vitamin D3 deficiency. There was no significant relation between disease duration and serum vitamin D3 level (p- value= 0.0732). Most individuals with poor glycaemic control had vitamin D3 deficiency, but there was no significant correlation between HbA1c level and serum vitamin D3 level (p- value=0.5060) (Table 2).

Table 2: Relation of serum vitamin D3 level in individuals with T1DM.

Correlation of duration of DM and HbA1c level with serum vitamin D3 levels in T2DM

All newly diagnosed individuals with T2DM had vitamin D3 deficiency, and a significant proportion of individuals with long-standing T2DM individuals also exhibited vitamin D3 deficiency (p- value=0.000571 and r- value=-0.388753) (Table 3 and Figure 1).

Table 3: Relation of serum vitamin D3 level in individuals with T2DM.

Individuals with poor glycaemic control were more likely to have vitamin D3 deficiency, as a significant inverse correlation observed between the two variables (p- value=0.000001 and r- value=-0.472) (Table 3 and Figure: 2).

Figure 1: Scatter diagram and regression analysis of Duration of T2DM in individuals with T2DM and serum vitamin D3 level.

Figure 2: Scatter diagram and regression analysis of glycaemic control in T2DM and serum vitamin D3 levels.

Correlation of duration of DM and HbA1c level with serum vitamin D3 levels in GDM

All women with GDM were newly diagnosed, with a notable portion exhibiting vitamin D3 deficiency (Table 2). While women with good glycaemic control were seen to have sufficient vitamin D3 level, however there was no significant correlation between HbA1c and serum vitamin D3 level (p- value=0.6422) (Table 4).

Table 4: Relation of serum vitamin D3 level in individuals with GDM.

Discussion

In this study, the majority of individuals were aged over 40years. Most of the individuals had Diabetes Mellitus for more than 10 years. The majority of individuals had vitamin D3 deficiency, which was widely distributed among different age groups. However, there was no significant relation (p- value= 0.327) between the gender distribution and serum vitamin D3 level. A similar study conducted in Nigeria by Anyanwu AC, et al. concluded that there was no significant difference (p- value= NS) in the distribution of vitamin D3 deficiency status by age or sex in both individuals with T2DM and control groups [9]. This might be due to similar amount of sunlight exposure, less disparity in dietary habits, compensation by sex hormones and environmental factors.

Relation between T1DM and serum vitamin D3

In our study, there was no significant correlation of serum vitamin D3 level in individuals with T1DM. A randomized control trial conducted by Manousaki D, et al. inferred similar result (OR=1.09, 95% CI and p=0.48) [10]. This non-significant correlation may be attributed to genetic variability, vitamin D3 receptor polymorphism, autoimmune nature of T1DM and chronic inflammation. Immune dysregulation in T1DM might overshadow the effects of vitamin D3 on immune modulation, leading to apparent lack of association.

Another prospective non-randomized control trial conducted by Alijabri KS, et al. in Saudi Arabia, showed that, there was an inverse correlation (p- value= 0.001 and r- value= -0.4) between glycaemic control and serum vitamin D3 level [11]. This study is not comparable with our study, as there was less sample size in our study.

Relation between T2DM and serum vitamin D3

All newly diagnosed individuals with T2DM had vitamin D3 deficiency. Similar findings were established by a case control study conducted by Anwar T, et al. and a cross sectional study conducted by Alam MS et al., [12,13]. There might be a complex interplay between vitamin D3 deficiency and newly diagnosed T2DM. The possible mechanisms involve impaired insulin secretion and sensitivity, chronic inflammation, life style factors, dietary habits, genetic conditions, and comorbidity. Vitamin D3 plays a role in enhancing insulin sensitivity by affecting insulin receptor expression and signalling. Deficiency of the same may impair the body’s response to insulin contributing to insulin resistance, which is a hallmark of T2DM. Also, vitamin D3 involves in pancreatic β-cell function, and deficiency of the same impairs insulin secretion.

In our study, there was an inverse relation between duration of T2DM and serum vitamin D3 level. A similar cross-sectional study, conducted by Ghavam S, et al. in Iran showed a linear inverse correlation (p-value<0.01 and r- value= -0.164) between the disease duration and serum vitamin D3 level, which is comparable to our study [14]. Chronic inflammation, impaired kidney function, obesity, medication effects, lifestyle factors and endothelial dysfunction play a complex role in this inverse relation.

There was also an inverse relation between glycaemic control and serum vitamin D3 level in individuals with T2DM. Some studies by Anyanwu AC, et al., Makadia MG, et al., Karau, et al. showed the similar results [9,15,16]. Chronic inflammation leads to increased production of inflammatory cytokines, which impair the metabolism of vitamin D3 and reduce its serum concentration. Inflammation can also affect the carrier protein binding to vitamin D3, reducing its availability in serum.

Relation between GDM and serum vitamin D3

Among women with GDM, there was no significant correlation between HbA1c and serum vitamin D3 level. Unrelated to our study, there are some studies by Soheilykhah S, et al., Wang L, et al. and Iqbal, et al. which showed some correlation between serum vitamin D3 level and increased risk of GDM [17-19]. Low vitamin D3 levels during pregnancy can have several adverse effects on both the mother and the developing foetus. Vitamin D3 plays a critical role in calcium absorption and bone metabolism, which is essential for foetal skeletal development. Deficiency can lead to complications such as gestational diabetes, preeclampsia, preterm birth, and low birth weight. For the foetus, low maternal vitamin D3 levels can increase the risk of neonatal rickets, impaired bone growth, and delayed physical development. Additionally, inadequate vitamin D3 during pregnancy may be associated with a higher risk of childhood asthma and autoimmune diseases. Ensuring adequate vitamin D3 levels is vital for promoting maternal health and foetal development. Women with GDM are more prone for vitamin D3 deficiency because of less sunlight exposure, increased calcium demand, obesity, malabsorption issues, ethnicity, skin pigmentations and decreased renal function [18]. Our study showed a contradicting result may be because of less sample size or may be attributed to a combination of adequate sunlight exposure in Indian women, proper dietary intake, adequate supplementations, efficient absorption and metabolism, optimal pre-pregnancy health, pro-active healthcare, favourable ethnic and skin pigmentation factors, and absence of comorbidities. So, patients with GDM should be evaluated for serum vitamin D3 and supplemented with the same, when it is required.

Possible explanations of the study findings

Summarising the findings of our study and existing evidences suggest that vitamin D3 may help in delaying the onset of T2DM and controlling the blood sugar, which might prevent further complications of T2DM. But there is no role of serum vitamin D3 in individuals with T1DM and GDM. In individuals with T2DM, there is insulin secretion defect and insulin resistance. Pancreatic β-cell expresses both vitamin D3 receptor transcript (VDR) and 1-alpha hydroxylase (Cyp27b1), which catalyses the activation of 25 (OH) D3 into 1,25 (OH)2 D3 consistent with cell intrinsic role of VDR [20]. Presence of VDR in the human insulin receptor gene promoter region suggests a potential role of vitamin D3 in influencing insulin action [21]. Vitamin D3 regulates insulin synthesis and secretion via variable mechanisms. The active form of vitamin D3 binds to VDR and induces genes related to glucose transport, and insulin secretion. It also indirectly regulates insulin secretion by impacting intracellular calcium. Calcium triggers insulin release by promoting mobilization of insulin vesicles and their exocytosis [7,9,21,22]. In T1DM, there is an intrinsic deficiency of insulin production, so correlation with vitamin D3 is doubtful. In GDM also, there is a temporary excessive requirement of insulin and temporary peripheral resistance of insulin, which mostly does not progress to Diabetes Mellitus. So, the correlation of serum vitamin D3 with GDM is also doubtful.

Therapeutic Considerations of Measuring Serum Vitamin D3 in T1DM, T2DM, and GDM

Measuring serum vitamin D3 levels in individuals with T1DM, T2DM, and GDM is clinically relevant due to the growing evidence linking vitamin D3 deficiency to the pathophysiology, progression, and complications of these conditions. In T1DM, low levels of vitamin D have been associated with increased risk of autoimmunity and higher levels of pro-inflammatory cytokines, which can exacerbate β-cell destruction. By measuring and correcting vitamin D3 levels, clinicians may potentially mitigate these inflammatory processes, thereby preserving residual β-cell function and delaying disease progression. Supplementation could also enhance immune regulation, reducing the risk of further autoimmune attacks.

In T2DM, vitamin D3 deficiency has been linked to impaired insulin secretion and reduced peripheral insulin sensitivity. This deficiency may contribute to both the onset and worsening of insulin resistance, a hallmark of T2DM. Low vitamin D3 levels are believed to disrupt the function of the VDR and its expression in pancreatic β-cells and muscle tissue, leading to suboptimal insulin release and increased insulin resistance. Therapeutic supplementation in deficient patients could improve glycaemic control by enhancing insulin sensitivity and promoting better glucose uptake in peripheral tissues. Additionally, adequate vitamin D3 levels may help reduce chronic inflammation, which is a key contributor to the metabolic dysfunction seen in T2DM. Regular monitoring of vitamin D3 and timely correction could thus serve as an adjunctive strategy in managing T2DM, potentially lowering the risk of complications such as cardiovascular disease, nephropathy, and neuropathy.

For women with GDM, ensuring optimal vitamin D3 status is particularly crucial given the dual impact on both maternal and foetal health. Vitamin D3 plays an essential role in glucose metabolism and placental function. Deficiency during pregnancy has been associated with adverse outcomes, including increased risk of preeclampsia, preterm birth, and macrosomia. Moreover, low vitamin D3 levels may exacerbate insulin resistance during pregnancy, complicating glycaemic control and increasing the risk of both maternal hyperglycaemia and fetal hyperinsulinemia. Measuring serum vitamin D3 in GDM allows for early identification of deficiency, enabling prompt intervention to reduce these risks. Supplementation in deficient pregnant women has shown potential benefits, such as improved insulin sensitivity, better pregnancy outcomes, and a decreased likelihood of developing T2DM postpartum.

From a therapeutic standpoint, the measurement of serum vitamin D3 levels in patients with T1DM, T2DM, and GDM should be considered a routine part of clinical management, particularly in populations at high risk for deficiency, such as those residing in regions with limited sunlight exposure, older adults, and individuals with higher BMI. Early detection of deficiency can guide targeted interventions, such as vitamin D3 supplementation or lifestyle modifications, thereby addressing a modifiable risk factor that could influence disease outcomes. Furthermore, therapeutic correction of vitamin D3 deficiency should be personalized, taking into account factors like baseline serum levels, body weight, dietary intake, and comorbid conditions, to achieve optimal serum concentrations of 1,25 Dihydroxy vitamin D3 [1,25 (OH)2D3] that are associated with better glycemic control and reduced complication rates.

Overall, the therapeutic value of measuring and optimizing serum vitamin D3 levels extends beyond bone health, offering a strategic advantage in the comprehensive management of diabetes and its associated complications. By maintaining adequate vitamin D3 status, clinicians may be able to support better metabolic health, reduce inflammation, and improve long-term outcomes in patients with T1DM, T2DM, and GDM.

Limitations

This study has few limitations that need to be acknowledged. Firstly, it is a single-centered study, which may limit the generalizability of the findings to the broader population. The sample size was relatively small and not highly diversified, which restricts the applicability of the results to larger and more varied populations. Additionally, the study was conducted in a specific geographical region in Northeast India, which might influence the outcomes due to regional variations in lifestyle, diet, and sun exposure affecting vitamin D3 level. Furthermore, the study design was observational and cross-sectional, which only allows for the identification of associations rather than causation. Longitudinal studies would be more effective in establishing causal relationships between serum vitamin D3 level and glycaemic control in individuals with Diabetes Mellitus. Another limitation is the lack of comprehensive data on potential confounding factors such as dietary habits, physical activity level, and sun exposure, which are known to influence vitamin D3 level. Lastly, while the study suggests a correlation between vitamin D3 deficiency and poor glycaemic control in T2DM individuals, it does not explore the underlying mechanisms in depth. Future research should aim to elucidate the biological pathways linking vitamin D3 and insulin regulation to better understand the potential therapeutic implications.

Conclusion

This study highlights a significant association between serum vitamin D3 level and glycaemic control in individuals with T2DM. The findings indicate that vitamin D3 deficiency is prevalent among individuals with T2DM, and lower serum vitamin D3 levels are correlated with poor glycaemic control and longer disease duration. Maintaining adequate vitamin D3 level may be beneficial in managing blood glucose level and potentially delaying the onset of complications associated with T2DM. However, this association was not observed in individuals with T1DM or GDM. The lack of significant correlation in these groups suggests that the role of vitamin D3 may vary depending on the type of Diabetes Mellitus, possibly due to different underlying pathophysiological mechanisms. Additionally, exploring the biological mechanisms linking vitamin D3 and insulin regulation could provide deeper insights into the potential therapeutic role of vitamin D3 in diabetes management.

Author Contribution Statement

Designed research: PKB, DPS, BB, MJ; Performed research: DPS, PKB, BB, MJ, GW, AAR, NS, VS, AG; Analysed data: DPS, VS; Data acquisition: DPS, PKB; Writing original draft: PKB, DPS; Review & editing: DPS, PKB, BB, MJ, GW, AAR, NS, VS, AG. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest: Authors Declare No conflict of Interest

Ethical Guideline

Ethical clearance for the study was obtained from the Institute Ethics Committee (NEIGR/IEC/M14/T13/2021), before the commencement of the research. The study was conducted with adherence to the Ethics Committee Proposal.

Data Availability Statement

Data generated during this study are available from the corresponding author on reasonable request.

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