Correlation of vitamin D levels with TNF-α and IL-6 expression in insulin-resistant type 2 diabetes mellitus patients

Abstract

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and chronic low-grade inflammation. Pro-inflammatory cytokines, particularly tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), contribute to impaired insulin signaling and metabolic dysfunction. Vitamin D possesses immunomodulatory properties and may influence inflammatory pathways associated with insulin resistance. This study aimed to evaluate the association between serum vitamin D levels, TNF-α and IL-6 expression, and glycemic control among insulin-resistant patients with T2DM. A hospital-based case–control study was conducted between September 2025 and February 2026 among 556 participants, including 426 insulin-resistant patients with T2DM and 130 age- and sex-matched healthy controls. Fasting blood samples were analyzed for serum vitamin D, TNF-α, IL-6, fasting blood glucose, insulin, and HbA1c. Insulin resistance was assessed using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). Serum cytokine concentrations were measured using enzyme-linked immunosorbent assay (ELISA), while adipose tissue TNF-α and IL-6 gene expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Correlation and multivariable regression analyses were performed to examine associations between vitamin D status, inflammatory markers, insulin resistance, and glycemic indices. Serum vitamin D levels were significantly lower among T2DM patients than controls (17.9 ± 6.8 ng/mL vs. 31.4 ± 7.6 ng/mL, p < 0.001). Conversely, serum TNF-α (18.4 ± 5.2 pg/mL vs. 9.8 ± 3.1 pg/mL, p < 0.001) and IL-6 (12.6 ± 4.7 pg/mL vs. 5.3 ± 2.0 pg/mL, p < 0.001) were significantly elevated in T2DM patients. Adipose tissue analysis demonstrated a 2.8-fold increase in TNF-α expression and a 3.2-fold increase in IL-6 expression among cases compared with controls (p < 0.001). Vitamin D levels were inversely correlated with TNF-α (r = − 0.42), IL-6 (r = − 0.38), HbA1c (r = − 0.35), and HOMA-IR (r = − 0.39) (all p < 0.001). Multivariable regression analysis demonstrated that vitamin D remained an independent negative predictor of TNF-α and IL-6 expression after adjustment for potential confounding variables. Vitamin D deficiency was significantly associated with increased TNF-α and IL-6 expression, greater insulin resistance, and poorer glycemic control among patients with T2DM. These findings support a potential role of vitamin D in metabolic inflammation; however, prospective studies and randomized controlled trials are needed to determine whether improving vitamin D status can favorably influence inflammatory and metabolic outcomes in T2DM.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia due to insulin resistance and relative insulin deficiency. It represents a major and escalating public health challenge worldwide. According to the International Diabetes Federation (IDF), the global prevalence of diabetes is projected to increase to 783 million by 2045, with the Middle East and North Africa (MENA) region among the worst affected^1,2. The economic and health burdens of T2DM include increased risks of cardiovascular disease, nephropathy, retinopathy, neuropathy, and reduced quality of life.In Saudi Arabia, the problem is particularly acute. Recent estimates suggest that 17.6% of Saudi adults live with diabetes, a sharp rise from 15.8% in 2016, and projections indicate a prevalence of nearly 18.2% by 2026^2,3. Alarmingly, diabetes often occurs at younger ages compared to global averages, with substantial implications for workforce productivity and healthcare costs. Contributing factors include rapid urbanization, dietary transitions towards high-calorie diets, physical inactivity, obesity, and genetic predisposition³. Parallel to this, vitamin D deficiency is highly prevalent in Saudi Arabia, despite abundant sunlight exposure. A large population study in the central region (2017–2021) found that 67.3% of individuals had insufficient vitamin D levels (< 30 ng/mL), with ~ 36% showing deficiency (< 20 ng/mL)^4,5. Another study in Jeddah reported deficiency in 60.9% of elderly participants and insufficiency in 29.9%⁵. These data underscore the dual burden of diabetes and vitamin D deficiency in Saudi Arabia.Pathophysiologically, insulin resistance is closely linked with chronic low-grade inflammation. Adipose tissue, especially visceral fat, is now recognized as an active endocrine organ that secretes numerous cytokines (adipokines) contributing to metabolic dysfunction. Among these, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) have been implicated as central mediators^6,7. TNF-α interferes with insulin signaling by promoting serine phosphorylation of insulin receptor substrates, thereby reducing glucose uptake and increasing lipolysis⁸. It also induces β-cell apoptosis, accelerating disease progression. IL-6, on the other hand, exerts a dual role: while it has beneficial acute effects during exercise, its chronic elevation is associated with hepatic insulin resistance, increased gluconeogenesis, and systemic inflammation⁸.Emerging evidence suggests that vitamin D has immunomodulatory properties beyond its classical role in bone and calcium metabolism. Vitamin D receptors (VDRs) are expressed in pancreatic β-cells, adipocytes, and immune cells, where active vitamin D [1,25(OH)₂D₃] regulates gene transcription. It has been shown to suppress pro-inflammatory cytokine production (including TNF-α and IL-6) and enhance anti-inflammatory cytokines such as IL-10^9,10. Mechanistically, vitamin D inhibits NF-κB activation, reduces Toll-like receptor expression, and modulates oxidative stress—all of which contribute to improved insulin sensitivity and reduced systemic inflammation^11,12.Several international studies have demonstrated an inverse relationship between vitamin D status and inflammatory cytokines. Individuals with vitamin D deficiency often exhibit higher serum TNF-α and IL-6 levels, correlating with poor glycemic control¹². Interventional studies of vitamin D supplementation in T2DM patients have reported improvements in insulin sensitivity, lipid profiles, and reductions in inflammatory markers, though findings remain inconsistent across populations^13,14. In Saudi Arabia, however, region-specific data investigating the interplay between vitamin D deficiency, cytokine expression, and T2DM pathogenesis remain limited. Given the unique epidemiological context of Saudi Arabia—high diabetes prevalence and widespread vitamin D deficiency—such studies are highly relevant for public health strategies^13,14.

Rationale and knowledge gap

While the roles of TNF-α and IL-6 in T2DM pathogenesis are well established, their relationship with vitamin D levels in insulin-resistant populations in Saudi Arabia has not been comprehensively studied. Understanding this correlation could help explain the high burden of diabetes in vitamin D-deficient populations and provide evidence for preventive or adjunctive interventions using vitamin D supplementation.

Study aim

Therefore, the present study aims to investigate the correlation between TNF-α and IL-6 expression with vitamin D levels in insulin-resistant T2DM patients in Saudi Arabia. By integrating biochemical analysis with gene expression profiling, this study seeks to elucidate the role of vitamin D in modulating inflammatory pathways involved in T2DM pathogenesis. Findings may support new therapeutic strategies and inform clinical practice and public health policies aimed at reducing the burden of diabetes and its complications in the Saudi population.

Figure 1 demonstrates significantly higher circulating TNF-α and IL-6 concentrations among T2DM patients compared with controls (p < 0.001 for both comparisons). These findings support the presence of a chronic pro-inflammatory state in insulin-resistant individuals (Fig. 1).

Fig. 1.

Comparison of serum TNF-α and IL-6 levels between healthy controls and T2DM patients.

Methodology

Study design and setting

This hospital-based case–control study was conducted between September 2025 and February 2026 at a tertiary care center in Saudi Arabia. The study aimed to evaluate the association between serum vitamin D levels, inflammatory cytokines (TNF-α and IL-6), and insulin resistance among patients with type 2 diabetes mellitus. The study was designed to assess the correlation of TNF-α and IL-6 expression with serum vitamin D levels in insulin-resistant type 2 diabetes mellitus (T2DM) patients, and to explore the role of vitamin D in inflammation and disease pathogenesis.

Study population and sample size

A total of 556 participants were enrolled using simple random sampling. The study population comprised 426 insulin-resistant patients with type 2 diabetes mellitus (T2DM) and 130 age- and sex-matched healthy controls. The larger number of cases reflected the availability of eligible T2DM patients attending the study center during the study period and was intended to improve the precision of correlation and regression analyses examining the relationship between vitamin D status, inflammatory markers, and insulin resistance.

Sample size justification and power analysis

The sample size was primarily determined by feasibility and the availability of eligible participants during the study period rather than by a formal a priori sample size calculation. Nevertheless, the adequacy of the final sample size was evaluated using post-hoc power analysis based on the primary study outcome, namely the association between serum vitamin D levels and inflammatory cytokines in patients with T2DM.Using the observed correlation between vitamin D and TNF-α (r = − 0.42), a two-sided significance level of α = 0.05, and a sample size of 426 T2DM patients, the achieved statistical power exceeded 99%, indicating that the study was adequately powered to detect clinically meaningful associations. Furthermore, based on previous studies reporting moderate correlations between vitamin D status and inflammatory markers in individuals with T2DM, an expected effect size ranging from r = 0.25 to r = 0.35 would require approximately 123–194 participants to achieve 80% statistical power at α = 0.05. Therefore, the final study sample substantially exceeded the minimum sample size required for adequate statistical power.The case-to-control ratio of approximately 3.3:1 was selected to maximize the inclusion of eligible T2DM patients while maintaining an adequate comparison group of age- and sex-matched healthy controls. This approach increased the precision of estimates for inflammatory and metabolic markers within the primary study population.

Inclusion criteria

• Adults aged 35–65 years.

• Confirmed diagnosis of T2DM according to ADA criteria.

• Duration of diabetes ≥ 1 year.

• Evidence of insulin resistance based on HOMA-IR assessment.

• Availability of complete clinical and biochemical data.

Exclusion criteria

• Type 1 diabetes mellitus.

• Newly diagnosed diabetes (< 1 year duration).

• Acute infections or inflammatory disorders.

• Autoimmune diseases.

• Chronic kidney disease or chronic liver disease.

• Malignancy.

• Pregnancy.

• Vitamin D supplementation within the previous 6 months.

• Corticosteroid or immunomodulatory therapy.

• Recent hospitalization (< 3 months).

Ethical considerations

Ethical approval was obtained from the Institutional Research Ethics Committee of the University of Hail, Saudi Arabia Approval No.: HCM/IRB/2024/019).. Administrative and academic collaboration was provided by Symbiosis International (Deemed University), Pune, India. The study was not multicentric. Written informed consent was obtained from all participants prior to data collection. Confidentiality and anonymity were ensured throughout the study. Participants were informed of their right to withdraw at any time without penalty.

Data collection

Demographic and clinical data were collected, including age, gender, medical history, duration of diabetes, and relevant laboratory findings such as vitamin D levels, HbA1c, fasting blood glucose, lipid profile, and uric acid levels.

Sample collection and processing

• Blood samples: Collected after overnight fasting. Serum was separated by centrifugation at 12,000 × g for 15 min at 4 °C and stored at − 70 °C until analysis.

• Adipose tissue biopsies: Subcutaneous adipose tissue samples (50–100 mg) were collected from the abdominal region using a needle-based biopsy technique. Samples were snap-frozen in liquid nitrogen and stored at − 80 °C until RNA extraction.

Adipose tissue sampling

To evaluate local inflammatory gene expression, subcutaneous adipose tissue samples were obtained from a representative subset of study participants who provided separate written informed consent for tissue biopsy. Adipose tissue was selected because it is an active endocrine organ and a major source of pro-inflammatory cytokines, including TNF-α and IL-6, which are implicated in insulin resistance and the pathogenesis of type 2 diabetes mellitus. Tissue samples (50–100 mg) were collected from the abdominal subcutaneous region using a minimally invasive needle-biopsy technique under local anesthesia and aseptic conditions. Following collection, samples were immediately snap-frozen in liquid nitrogen and stored at − 80 °C until RNA extraction and gene expression analysis.

Biochemical analysis

• Serum Vitamin D: Measured using Mybiosource ELISA kit (Catalogue # MBS704497).

• TNF-α: Measured using Invitrogen Human TNF-α ELISA Kit (Catalogue # KHC 3011).

• IL-6: Measured using Invitrogen Human IL-6 ELISA Kit (Catalogue # EH2IL6).

Other parameters

Uric acid, lipid profile, HbA1c, and fasting blood glucose were analyzed using AMP diagnostic kits.

RNA extraction and cDNA synthesis

Total RNA was extracted from adipose tissue samples using Trizol reagent. RNA quality and concentration were assessed spectrophotometrically. cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lithuania) following manufacturer’s protocol.

Quantitative real-time PCR (qPCR)

Relative gene expression levels of TNF-α and IL-6 were quantified using qPCR on a Bio-Rad C1000 Thermal Cycler. GAPDH was used as a housekeeping gene. Thermocycling conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The relative expression of target genes was calculated using the 2^–ΔΔCt method. Primer sequences are listed in Table 1.

Table 1.

Baseline characteristics of study participants.

Parameter	Controls (n = 130)	T2DM cases (n = 426)	p-value
Age (years, mean ± SD)	50.2 ± 7.9	51.9 ± 8.6	0.18
Male, n (%)	68 (52.3%)	222 (52.1%)	0.96
BMI (kg/m²)	25.1 ± 3.4	29.4 ± 4.8	< 0.001
FBG (mg/dL)	94.6 ± 8.9	168.3 ± 34.7	< 0.001
HbA1c (%)	5.6 ± 0.4	8.2 ± 1.5	< 0.001
HOMA-IR	1.86 ± 0.71	5.84 ± 1.92	< 0.001
Total Cholesterol (mg/dL)	172.1 ± 21.5	214.8 ± 34.9	< 0.001
Triglycerides (mg/dL)	118.7 ± 30.5	198.2 ± 49.1	< 0.001
Uric Acid (mg/dL)	4.9 ± 1.2	6.3 ± 1.5	< 0.001
Vitamin D (ng/mL)	31.4 ± 7.6	17.9 ± 6.8	< 0.001

HOMA-IR assessment

Insulin Resistance Assessment: “Fasting plasma glucose and fasting serum insulin concentrations were used to calculate insulin resistance using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) according to the following formula:

Participants with HOMA-IR values greater than 2.5 were considered insulin resistant.”

Statistical analysis

Data were analyzed using SPSS version 22.0. Descriptive statistics (mean ± SD) were calculated.

• Independent t-test: To compare mean differences between groups.

• Pearson correlation coefficient (r): To assess associations between TNF-α, IL-6, vitamin D levels, glycemic control markers, and lipid profile.

• ANOVA: To analyze associations between cytokine expression levels and disease severity (HbA1c categories).

• A p-value ≤ 0.05 was considered statistically significant.

Results

Baseline characteristics of the study population

A total of 556 participants were included, comprising 426 insulin-resistant T2DM patients (cases) and 130 healthy controls. The mean age of participants was 51.6 ± 8.4 years, with 52% males and 48% females. Among the T2DM patients, the mean duration of diabetes was 8.3 ± 3.9 years.Compared with controls, T2DM patients showed significantly higher BMI, fasting blood glucose (FBG), HbA1c, total cholesterol, triglycerides, and uric acid levels (p < 0.001 for all). Serum vitamin D levels were significantly lower in T2DM patients compared to controls (p < 0.001).

Insulin resistance assessment

As expected, insulin resistance was significantly greater among T2DM patients compared with healthy controls. The mean HOMA-IR value was 5.84 ± 1.92 in T2DM patients and 1.86 ± 0.71 in controls (p < 0.001), confirming marked insulin resistance in the diabetic cohort. Serum Levels of TNF-α and IL-6 :The mean serum TNF-α level was significantly higher in T2DM patients (18.4 ± 5.2 pg/mL) compared to controls (9.8 ± 3.1 pg/mL, p < 0.001). Similarly, IL-6 levels were elevated in T2DM patients (12.6 ± 4.7 pg/mL) versus controls (5.3 ± 2.0 pg/mL, p < 0.001) (Table 1).

Gene expression of TNF-α and IL-6 in adipose tissue

Quantitative PCR analysis revealed a significant upregulation of TNF-α and IL-6 mRNA expression in adipose tissue samples of T2DM patients compared to controls (Fig. 2).

Fig. 2.

Comparison of TNF-α and IL-6 levels between cases and controls.

• TNF-α expression was 2.8-fold higher (p < 0.001).

• IL-6 expression was 3.2-fold higher (p < 0.001).

Correlation between vitamin D and inflammatory markers

Pearson correlation analysis demonstrated significant inverse associations between serum vitamin D levels and inflammatory markers. Vitamin D levels were negatively correlated with TNF-α (r = − 0.42, p < 0.001) and IL-6 (r = − 0.38, p < 0.001). Furthermore, vitamin D levels showed significant negative correlations with HbA1c (r = − 0.35, p < 0.001), BMI (r = − 0.29, p = 0.004), and HOMA-IR (r = − 0.39, p < 0.001), indicating that lower vitamin D concentrations were associated with greater insulin resistance, poorer glycemic control, and heightened inflammatory activity (Table 2).

Table 2.

Correlation of vitamin D with inflammatory and metabolic markers in T2DM patients.

Variable	Correlation coefficient (r)	p-value
TNF-α	–0.42	< 0.001
IL-6	–0.38	< 0.001
HbA1c	–0.35	< 0.001
BMI	–0.29	0.004
HOMA-IR	–0.39	< 0.001

Further analysis revealed positive correlations between inflammatory cytokines and insulin resistance. TNF-α showed a significant positive correlation with HOMA-IR (r = 0.45, p < 0.001), while IL-6 was also positively correlated with HOMA-IR (r = 0.41, p < 0.001), suggesting that increasing inflammatory activity is associated with worsening insulin resistance Fig. 3. Association Between Cytokine Levels and GlycemicContro. When T2DM patients were stratified based on HbA1c levels (< 7%, 7–9%, > 9%), both TNF-α and IL-6 levels showed a progressive increase with worsening glycemic control (p < 0.001, ANOVA (Table 2).

Fig. 3.

Relationship between HbA1c categories and cytokine levels.

Significance of the results

The findings of this study provide compelling evidence that vitamin D deficiency is closely associated with heightened inflammatory activity and poor glycemic control in patients with insulin-resistant type 2 diabetes mellitus (T2DM). Several significant implications emerge from these observations: 1. Strengthening the Inflammatory Hypothesis of Insulin Resistance : The marked elevation of TNF-α and IL-6 in both serum and adipose tissue of T2DM patients reinforces the concept that chronic low-grade inflammation is a central mechanism in insulin resistance and diabetes pathogenesis. The upregulation of these cytokines supports their established roles in impairing insulin receptor signaling and promoting β-cell dysfunction^14,15. 2. Vitamin D as a Modulator of Inflammation: The strong inverse correlations between vitamin D levels and inflammatory cytokines (TNF-α and IL-6) suggest that vitamin D plays a protective, anti-inflammatory role in metabolic regulation. This finding aligns with mechanistic studies demonstrating that vitamin D suppresses NF-κB activation and modulates cytokine gene transcription. It highlights the potential of maintaining optimal vitamin D status to mitigate inflammation-driven insulin resistance^16,17. 3. Clinical Relevance for Glycemic Control: The progressive increase in TNF-α and IL-6 concentrations across worsening HbA1c categories demonstrates that poor glycemic control is directly linked to intensified inflammatory activity. This observation supports the need for integrated therapeutic approaches targeting both metabolic regulation and inflammation in diabetic management^18,19. 4. Population-Specific Importance for Saudi Arabia: Given the high prevalence of both T2DM and vitamin D deficiency in the Saudi population, these results hold particular public health significance. The dual burden of these conditions may create a self-perpetuating cycle of metabolic dysfunction and inflammation. Addressing vitamin D deficiency through supplementation or fortification could serve as a simple, low-cost adjunctive strategy to reduce inflammation and improve glycemic outcomes in this region^20,21. 5. Translational and Preventive Implications: This study underscores the potential for vitamin D optimization as a preventive or adjunctive therapy in insulin-resistant individuals. Routine screening for vitamin D deficiency in diabetic and pre-diabetic patients could help identify those at risk of severe inflammation and poor metabolic control. 6. Contribution to the Scientific Literature: This research contributes valuable region-specific evidence from Saudi Arabia, where data linking vitamin D status, inflammatory cytokines, and T2DM have been limited (Fig. 4). The integration of biochemical assays with gene expression analysis strengthens the biological plausibility of the observed associations, offering a comprehensive model of vitamin D’s role in metabolic inflammation^22,23. Study demonstrates that vitamin D deficiency exacerbates inflammation (via TNF-α and IL-6 overexpression) and contributes to poor glycemic control in insulin-resistant T2DM patients. These findings highlight the therapeutic potential of vitamin D in reducing systemic inflammation and improving metabolic outcomes, particularly in populations with high rates of both diabetes and vitamin D deficiency^24,25.

Fig. 4.

Global trends in vitamin D deficiency and inflammatory cytokine levels in type 2 diabetes mellitus.

Interpretation: The present study is consistent with global evidence demonstrating significant associations between vitamin D deficiency, increased inflammatory cytokine expression, insulin resistance, and poorer glycemic control. However, due to the observational nature of these studies, causal relationships cannot be established, and randomized controlled trials are needed to evaluate the effects of vitamin D supplementation on metabolic and inflammatory outcomes (Table 3).

Table 3.

Comparison of present study findings with global evidence.

Study/region	Population studied	Key findings on vitamin D and inflammation in T2DM	Relevance/comparison with present study
Present Study (Saudi Arabia)	426 T2DM patients, 130 controls	Vitamin D levels significantly lower in T2DM (17.9 ± 6.8 ng/mL); TNF-α and IL-6 significantly elevated; inverse correlations between vitamin D and TNF-α (r = − 0.42), IL-6 (r = − 0.38), HbA1c (r = − 0.35), and HOMA-IR (r = − 0.39).	Demonstrates a significant association between vitamin D deficiency, inflammation, insulin resistance, and poor glycemic control in Saudi patients with T2DM.
Gökhan S. Hotamisligil et al., 1993 (USA)26	Obese adults with insulin resistance	Identified TNF-α as a key mediator of insulin resistance through impairment of insulin receptor signaling.	Supports the elevated TNF-α expression observed among T2DM patients in the present study.
Atul Pradhan et al., 2001 (USA)27	Women in the Nurses’ Health Study	Elevated IL-6 and CRP levels predicted future development of T2DM.	Consistent with the higher IL-6 levels and poorer glycemic control observed in the current study.
Kenneth C. Chiu et al., 2004 (USA)28	Healthy adults	Low vitamin D levels were associated with reduced insulin sensitivity and impaired β-cell function.	Aligns with the inverse association between vitamin D, HbA1c, and insulin resistance observed in our cohort.
Nita G. Forouhi et al., 2012 (UK)29	EPIC-Norfolk cohort	Vitamin D concentrations were inversely associated with fasting glucose and HOMA-IR.	Similar to the negative correlation between vitamin D and HOMA-IR reported in the present study.
Hanieh Mazahery et al., 2017 (New Zealand)30	T2DM and pre-diabetic adults	Vitamin D supplementation reduced inflammatory markers and improved metabolic parameters.	Supports the hypothesis that vitamin D may influence inflammatory pathways associated with T2DM.
Ulla Kampmann et al., 2020 (Denmark)31	Overweight adults	Vitamin D3 supplementation was associated with reduced IL-6 levels and modest improvements in insulin sensitivity.	Reinforces the observed association between vitamin D status and inflammatory activity.
India and China (multiple studies)32–34	Adults with T2DM	High prevalence of vitamin D deficiency associated with elevated TNF-α, IL-6, and poor glycemic control.	Findings are comparable to those observed in the present study.
Gulf Region (Kuwait, UAE, Qatar)35–37	General population and T2DM cohorts	Vitamin D deficiency highly prevalent despite abundant sunlight exposure and associated with obesity and insulin resistance.	Reflects similar environmental and lifestyle determinants affecting the Saudi population.

T2DM, Type 2 Diabetes Mellitus; TNF-α, Tumor Necrosis Factor-Alpha; IL-6, Interleukin-6; HOMA-IR, Homeostatic Model Assessment of Insulin Resistance; CRP, C-Reactive Protein.

The figure illustrates the worldwide prevalence of vitamin D deficiency (map, shaded by percentage) and compares relative TNF-α and IL-6 levels among T2DM patients across regions. The Middle East and Saudi Arabia show the highest deficiency rates (> 50%) and elevated inflammatory cytokine levels, emphasizing a regional health concern consistent with global findings linking vitamin D deficiency to inflammation and insulin resistance (Fig. 1).

Discussion

The present study investigated the interrelationship between vitamin D status, inflammatory cytokines (TNF-α and IL-6), and glycemic control in patients with insulin-resistant type 2 diabetes mellitus (T2DM). The results demonstrated significantly lower serum vitamin D levels and higher concentrations of TNF-α and IL-6 among T2DM patients compared with healthy controls. Moreover, vitamin D levels exhibited a strong negative correlation with TNF-α, IL-6, and HbA1c, suggesting that vitamin D deficiency is closely linked to enhanced systemic inflammation and poor glycemic regulation.Vitamin D Deficiency and Diabetes Pathogenesis: Vitamin D deficiency was highly prevalent in the studied T2DM cohort, despite abundant sunlight exposure in Saudi Arabia. This paradox reflects the influence of lifestyle, clothing, dietary habits, and limited outdoor activity, which collectively restrict cutaneous vitamin D synthesis. The finding aligns with prior studies across the Gulf region, which report deficiency rates exceeding 60% in adults^1,2. Vitamin D plays a crucial role in pancreatic β-cell function, insulin sensitivity, and immune modulation. Its deficiency may impair insulin secretion and promote insulin resistance through direct and indirect mechanisms, including increased oxidative stress and chronic low-grade inflammation. Inflammatory Cytokines and Insulin Resistance: In this study, serum and gene expression levels of TNF-α and IL-6 were significantly upregulated in T2DM patients compared with controls. These findings are consistent with the well-established role of pro-inflammatory cytokines in the pathogenesis of insulin resistance. TNF-α interferes with insulin signaling by promoting serine phosphorylation of insulin receptor substrate (IRS-1), thereby attenuating insulin-mediated glucose uptake³. Similarly, IL-6 contributes to hepatic insulin resistance and impairs glucose metabolism via modulation of JAK/STAT pathways⁴. Elevated cytokine levels observed in the current study reinforce the concept of T2DM as an inflammatory metabolic disorder rather than a purely endocrine condition.Vitamin D and Inflammation: Interconnected Pathways: The observed inverse association between vitamin D and inflammatory cytokines supports the anti-inflammatory and immunomodulatory properties of vitamin D. Through its receptor (VDR) present on immune cells, vitamin D inhibits NF-κB activation and downregulates the expression of TNF-α and IL-6^5,6. This mechanism may explain the negative correlations between vitamin D levels and inflammatory markers found in the present study. Previous interventional trials have demonstrated that vitamin D supplementation can reduce circulating TNF-α and IL-6 concentrations and modestly improve insulin sensitivity, particularly among vitamin D-deficient individuals^7–9. Although supplementation outcomes vary globally, the biological plausibility of vitamin D’s anti-inflammatory action remains widely supported. Comparison with Global Context: The results of this study are in close agreement with global findings. Studies from the U.S., U.K., Europe, and Asia consistently show that vitamin D deficiency correlates with higher inflammatory cytokine levels and poor glycemic control^10–13. Notably, while deficiency is a global concern, its prevalence in Middle Eastern populations—including Saudi Arabia—is among the highest worldwide, despite abundant sunlight. This discrepancy underscores the role of sociocultural factors such as conservative clothing, indoor lifestyles, and limited dietary vitamin D intake. Compared with Western populations, the Saudi cohort exhibited a more pronounced degree of deficiency and inflammation, suggesting a stronger interplay between environmental and metabolic determinants in this region.

Clinical implications

The findings highlight the potential role of vitamin D optimization as an adjunctive therapeutic strategy in managing insulin resistance and inflammation in T2DM. Routine assessment of vitamin D status and correction of deficiency through supplementation or safe sun exposure may provide metabolic benefits and mitigate inflammatory burden. However, the effectiveness of such interventions depends on baseline vitamin D status, dosage, duration, and individual genetic variability in VDR expression.

Limitations

The findings of this study should be interpreted in light of several limitations. First, the case–control design is observational in nature and therefore does not permit conclusions regarding causality between vitamin D deficiency, inflammatory cytokine expression, insulin resistance, and glycemic control. Second, this was a single-center study conducted in Saudi Arabia, which may limit the generalizability of the findings to other populations with different demographic, genetic, environmental, and lifestyle characteristics.Third, serum 25-hydroxyvitamin D [25(OH)D] concentrations were measured using an ELISA-based immunoassay. Although this method is widely used and clinically accepted, liquid chromatography–tandem mass spectrometry (LC-MS/MS) is considered the reference standard for vitamin D assessment and may provide greater analytical accuracy and specificity. Fourth, detailed information regarding medication use, including metformin, insulin, statins, and other glucose-lowering agents, was not systematically collected. As some of these medications possess anti-inflammatory effects, their potential influence on cytokine levels could not be evaluated.Fifth, several biological factors that may influence vitamin D metabolism and inflammatory responses were not measured, including parathyroid hormone (PTH), serum calcium, magnesium, phosphate levels, and vitamin D receptor (VDR) gene polymorphisms. These variables may act as important modifiers of the relationship between vitamin D status and metabolic inflammation. In addition, lifestyle-related factors such as dietary vitamin D intake, sunlight exposure, physical activity, smoking status, and seasonal variation were not comprehensively assessed and therefore could not be included in the analysis.Finally, adipose tissue gene-expression analysis was performed in a subset of participants who consented to biopsy procedures, which may limit the generalizability of the molecular findings. Future multicenter prospective studies and randomized controlled trials incorporating detailed pharmacological, genetic, biochemical, and lifestyle assessments are needed to better elucidate the role of vitamin D in inflammation, insulin resistance, and type 2 diabetes mellitus.

Conclusion

The present study demonstrated that patients with type 2 diabetes mellitus exhibited significantly lower serum vitamin D levels and higher TNF-α and IL-6 concentrations compared with healthy controls. Furthermore, vitamin D levels were inversely associated with inflammatory markers, insulin resistance, and glycemic indices. These findings are consistent with a relationship between vitamin D deficiency, systemic inflammation, and metabolic dysregulation in T2DM.The observed associations support the hypothesis that vitamin D status may be linked to inflammatory pathways involved in insulin resistance and diabetes progression. However, due to the observational case–control design, causal relationships cannot be established. Therefore, it cannot be concluded that vitamin D deficiency directly causes increased cytokine expression or worsened glycemic control.The findings contribute region-specific evidence from Saudi Arabia, where both vitamin D deficiency and T2DM are highly prevalent. Future prospective cohort studies and randomized controlled trials are needed to determine whether improving vitamin D status can influence inflammatory markers, insulin resistance, and clinical outcomes in individuals with T2DM.

Author contributions

Fahmida Khatoon: Conceptualization, Methodology, Data Curation, Formal Analysis, Writing—Original Draft. Farida Habib Khan: Methodology, Investigation, Data Collection, Writing—Review & Editing. Ahmed Aljadani: Statistical Analysis, Software, Validation, Visualization.Abdullah D. Alotaibi: Resources, Supervision, Funding Acquisition, Project Administration Jyoti Pushkar Deshpande: Conceptualization, Supervision, Critical Review, Writing—Review & Editing, Correspondence.

Funding

Open access funding provided by Symbiosis International (Deemed University). The authors received financial support for the research, authorship and/or publication of this article.scientific research Deanship at the University of Ha’il in Saudi Arabia, through the project number RG23-156. Open access funding provided by Symbiosis International (Deemed University). Symbiosis International University Pune India.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The study protocol was reviewed and approved by the Institutional Review Board (IRB) of the College of Medicine, University of Hail, Saudi Arabia (Approval No.: HCM/IRB/2024/019). All participants provided written informed consent prior to participation. The research was performed in accordance with the ethical standards of the Declaration of Helsinki (2013 revision).

Informed consent

Written informed consent was obtained from all participants prior to data collection and very human participant was provide their consent. Participants were informed about the objectives of the study, their voluntary participation, and their right to withdraw at any time without any impact on their medical care. All participants agreed to take part in the study.