Absence of the vitamin D receptor gene in patients with severe deficiency: implications for inflammation and apoptosis in type 2 diabetes mellitus

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Introduction. Type 2 Diabetes Mellitus is a widespread metabolic disorder marked by chronic inflammation, insulin resistance, and oxidative stress, all of which contribute to progressive pancreatic β-cell dysfunction. In recent years, increasing attention has been given to the role of vitamin D in modulating these pathological processes. While vitamin D is obtained from dietary sources and synthesized in the skin under sunlight exposure, it must undergo activation in the liver and kidneys to exert its biological effects. These effects are mediated through the VDR, which is present in multiple tissues, including the pancreas, immune system, muscles, and bones.

However, vitamin D activity is not solely dependent on its circulating levels; it also requires functional VDRs to exert its influence on cellular processes. Genetic variations in the VDR gene can reduce receptor sensitivity or expression, limiting vitamin D’s regulatory impact. In cases where VDR expression is absent, even sufficient vitamin D levels may fail to protect against inflammation and apoptosis. This dysfunction is particularly relevant to T2DM, where uncontrolled apoptosis and cellular stress contribute to β-cell loss and disease progression.

Caspase-3, a key executioner of apoptosis, has been linked to increased β-cell destruction in diabetic patients, while HSP-70 serves a protective role by stabilizing cellular proteins and mitigating oxidative damage. The absence of functional VDR may disrupt this balance, exacerbating apoptotic pathways and weakening cellular defense mechanisms.

This study aims to investigate the connection between vitamin D levels, VDR gene expression, and the regulation of apoptosis in T2DM patients. By assessing caspase-3 and HSP-70 levels in individuals with varying degrees of vitamin D deficiency, we seek to clarify the potential consequences of impaired VDR signaling and its role in disease progression.

The aim. This study aims to evaluate the impact of severe vitamin D deficiency in T2DM patients who lack VDR expression. Specifically, we seek to investigate how the absence of VDR influences key apoptotic and stress-related biomarkers— netrin-1, caspase-3 and HSP-70. By analyzing these markers across different levels of vitamin D deficiency, with a focus on patients with undetectable VDR, we aim to determine the extent to which impaired vitamin D signaling contributes to increased apoptosis, reduced cellular protection, and the progression of T2DM-related complications.

Materials and Methods. The study analyzed clinical data from 160 individuals, including 130 patients diagnosed with type 2 diabetes mellitus and 30 participants in the control group. All patients received medical care at Voronezh City Clinical Polyclinic №18 and were divided into four groups based on their vitamin D levels: group 1 – patients with target vitamin D levels; group 2 – patients with insufficient vitamin D levels; group 3 – patients with vitamin D deficiency; group 4 – patients with severe vitamin D deficiency.

All patients received vitamin D therapy in the form of cholecalciferol oil solution. Patients with vitamin D deficiency and severe deficiency received 24,000 IU daily for eight weeks, followed by a maintenance dose of 6,000 IU per day.

The primary focus of the study was on Group 4, which included 12 patients with type 2 diabetes mellitus and severe vitamin D deficiency, aged 56 to 79 years (mean age 67 years). This group consisted of 4 men (mean age 67.5 years) and 8 women (mean age 67.6 years).

The study involved anthropometric measurements and laboratory analysis of blood serum samples. The levels of vitamin D, mutations in its receptor gene, glucose, HbA1c, netrin-1, caspase-3, and HSP-70 were determined. Comparisons were made across all groups; however, the primary emphasis was placed on patients with severe vitamin D deficiency. Statistical analysis was performed using the Mann-Whitney, Wilcoxon, and Kruskal-Wallis tests (p < 0.05). Median values were used as a measure of central tendency, and variability was assessed using the interquartile range. All data processing was conducted using Microsoft Excel 2007.

Results. The analysis of netrin-1, caspase-3, and HSP-70 levels was conducted in the control group and groups with varying degrees of vitamin D deficiency. Particular attention was given to Group 4, where patients exhibited a significant decrease in vitamin D levels and a lack of VDR expression. The dynamics of biomarker changes before and after therapy are presented in Tables 1, 2 and 3.

Table 1. Biomarkers levels before treatment

Parameter	Statistical indicators	Group					The value of the Karskal-Wallis test and the level of significance of differences (p)
		Control	1	2	3	4
Netrin-1	Ме	77,85	73,84	48,32	20,03	5,02	H=140,110, р<0,001 р0-2<0,001 р0-3<0,001 р0-4<0,001 р1-3<0,001 р1-4<0,001 р2-3<0,001 р2-4<0,001
	Q1 – Q3	75,5 – 79,8	71,07-71,01	41,27-54,3	16,59-22,03	4,27-5,43
Caspase-3	Ме	0,063	0,081	1,218	2,105	2,999	H=143,511, р<0,001 р0-2<0,001 р0-3<0,001 р0-4<0,001 р1-3<0,001 р1-4<0,001 р2-3<0,001 р2-4<0,001
	Q1 – Q3	0,057 – 0,066	0,079-0,083	1,109-1,290	1,906-2,226	2,965-3,010
HSP-70	Ме	1,66	1,48	1,26	1,24	0,56	H=106,241, р<0,001 р0-2<0,001 р0-3<0,001 р0-4<0,001 р1-2=0,006 р1-3=0,002 р1-4<0,001 р2-4<0,001 р3-4=0,004
	Q1 – Q3	1,58-1,75	1,58-1,66	1,34-1,48	1,41-1,51	1,46-1,51

 

Table 2. Biomarkers levels after treatment

Parameter	Statistical indicators	Group					The value of the Karskal-Wallis test and the level of significance of differences (p)
		Control	1	2	3	4
Netrin-1	Ме	77,85	74,85	73,92	57,03	43,01	H=93,688, р<0,001 р1-3<0,001 р1-4<0,001 р2-3<0,001 р2-4<0,001
	Q1-Q3	75,5-79,8	74,44-76,52	72,65-75,81	54,77-60,26	41,17-45,33
Caspase-3	Ме	0,063	0,068	0,126	1,528	2,009	H=103,300, р<0,001 р1-3<0,001 р1-4<0,001 р2-3<0,001 р2-4<0,001
	Q1-Q3	0,057-0,066	0,068-0,069	0,095-0,204	1,205-1,807	1,549-2,193
HSP-70	Ме	1,66	1,63	1,43	1,46	1,49	H=34,520, р<0,001 р1-2<0,001 р1-3<0,001
	Q1 – Q3	1,58-1,75	1,58-1,66	1,34-1,48	1,41-1,51	1,46-1,51

 

Table 3. Before-after comparisons of biomarkers in Group 4

Parameter	Statistical indicators	Before treatment	After treatment	Wilcoxon test value and significance level of differences (p)
Netrin-1	Me	5,02	43,01	Z=-3,059, р=0,002
	Q1-Q3	4,27-5,43	41,17 – 45,33
Caspase-3	Me	2,999	2,009	Z=-3,059, р=0,002
	Q1-Q3	2,965-3,0105	1,549 – 2,193
HSP-70	Me	0,56	1,49	Z=-3,061, р=0,002
	Q1-Q3	0,48-0,63	1,46 – 1,51

 

The study results demonstrate a significant impact of vitamin D deficiency on the levels of the investigated biomarkers, particularly in Group 4, where patients exhibited a lack of VDR expression. Before the initiation of therapy, netrin-1 levels in this group were minimal (Me = 5.02, Q1-Q3: 4.27-5.43), indicating severe impairments in regenerative processes and cellular stress response. At the same time, extremely high levels of caspase-3 were observed (Me = 2.999, Q1-Q3: 2.965-3.010), indicating enhanced apoptosis activation. HSP-70 levels in Group 4 were the lowest among all studied groups (Me = 0.56, Q1-Q3: 0.48-0.63), confirming a deficiency in cellular protective mechanisms.

Following the course of therapy, positive changes were observed in all groups with vitamin D insufficiency and deficiency. In Group 4, netrin-1 levels increased more than eightfold (Me = 43.01, p < 0.001), suggesting partial restoration of regenerative processes. HSP-70 levels also significantly increased (Me = 1.49, p < 0.001), indicating the activation of cellular defense mechanisms. At the same time, caspase-3 concentrations decreased (Me = 2.009, p < 0.001), reflecting a reduction in the intensity of apoptotic processes.

However, despite the positive dynamics, in Group 4, post-therapy HSP-70 levels did not reach those of the control group, and caspase-3 levels remained above normal. This supports the hypothesis that vitamin D deficiency, combined with the absence of VDR expression, contributes to chronic inflammation and cellular stress even after partial correction. Thus, the study results highlight the importance of further research into additional treatment strategies and the possibility of more intensive vitamin D deficiency correction in this patient group.

Conclusions. The study results confirm the significant impact of vitamin D deficiency on the levels of the examined biomarkers, especially in the absence of VDR expression. Patients with severe deficiency exhibited extremely low levels of netrin-1 and HSP-70, along with a high concentration of caspase-3, indicating impaired regenerative processes, enhanced apoptosis, and weakened cellular defense mechanisms.

After therapy, a significant increase in netrin-1 and HSP-70 levels, as well as a decrease in caspase-3, was observed, suggesting partial restoration of regeneration and a reduction in apoptotic processes. However, despite the positive dynamics, in patients with severe vitamin D deficiency and lack of VDR expression, HSP-70 levels remained below normal, while caspase-3 levels remained elevated, indicating persistent chronic inflammation and cellular stress.

Thus, the study highlights the need for early detection and correction of vitamin D deficiency, as well as the development of additional therapeutic strategies for patients with type 2 diabetes, particularly in cases of absent VDR expression.

About the authors

Valeria Alexandrovna Dugusheva

Voronezh State Medical University named after N. N. Burdenko

Email: semikolenovaval@yandex.ru
ORCID iD: 0000-0002-7342-2218

Assistant, Department of Clinical Laboratory Diagnostics

Russian Federation, 394036, Russia, Voronezh, 10 Studencheskaya St.

Viktoria Evgenievna Shchegoleva

Voronezh State Medical University named after N. N. Burdenko

Author for correspondence.
Email: vikaut0vaz@list.ru
ORCID iD: 0009-0008-3336-5758

Student

Russian Federation, 394036, Russia, Voronezh, 10 Studencheskaya St.

Yulia Alexandrovna Kotova

Voronezh State Medical University named after N. N. Burdenko

Email: kotova_u@inbox.ru
ORCID iD: 0000-0003-0236-2411

Head of the Department of Clinical Laboratory Diagnostics, Doctor of Medical Sciences, Associate Professor

Russian Federation, 394036, Russia, Voronezh, 10 Studencheskaya St.

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