Circulating CXCL16 in Type 2 Diabetes Mellitus Egyptian Patients

Introduction

Type 2 Diabetes mellitus (T2DM) is considered to be a rapidly increasing medical concern in Egypt with a substantial effect on morbidity and mortality. Currently the incidence of type 2 diabetes Mellitus (T2DM) in Egypt is around 15.6% of all adults aged 20 to 79. The International Diabetes Federation (IDF) has classified Egypt as the ninth leading state globally for the number of patients with T2DM. The incidence of T2DM in Egypt has approximately trebled over the last two decades [1].

Normal regulation of glucose metabolism is determined by a feedback loop comprising the islet β-cell and insulin-sensitive tissues. Tissue sensitivity to insulin influences the amount of the β-cell response. When insulin resistance occurs, the β-cell maintains a normal glucose tolerance by increasing insulin output. Glucose levels rise whenever β-cells are incapable of releasing sufficient insulin in the presence of insulin resistance. Although β-cell dysfunction is thought to have a strong genetic element, environmental factors also play a crucial role in the development of T2DM.

Modern studies have also stressed the importance of hexoses, vitamin D, amino acids, fatty acids and the potential role of changes in the microbiome in shaping insulin resistance and β-cell dysfunction [2]. Recent discoveries have suggested that inflammation plays a key role in the pathogenesis of T2DM. Several reports have also demonstrated that low grade inflammation and activation of innate immunity are two of the main mechanisms involved in the pathogenesis of T2DM [3].

The understanding about vitamin D and its relationship to T2DM was motivated by primary animal studies identifying a particular vitamin D receptor in pancreatic tissue. Vitamin D seems to have many physiological roles due to the fact that most tissues such as brain, breast, prostate, colon, pancreatic tissue, white blood cells carry vitamin D receptors [4]. Vitamin D and its by-products appear to have dynamic effects on insulin synthesis, secretion and action [5], as well as components of inflammation [6] all of which may have an effect on the etiology of T2DM.

A protein called C-X-C chemokine ligand 16 (CXCL16) has been isolated, which combines the scavenger receptor functions with the properties of an inflammatory chemokine. This transmembrane protein is made up of an extracellular chemokine domain united with a trans-membrane mucin stalk [7]. The chemokine domain not only acts as a recruiter for cells expressing the CXCR6 receptor, but also as a scavenger receptor facilitating the uptake of oxidized low-density lipoprotein (ox-LDL).

Pro-inflammatory stimuli enhance CXCL16 expression increasing the uptake of ox-LDL and hastening foam cell formation in the vascular endothelium [8]. CXCL16 has been linked to a range of inflammatory diseases. The evidence has demonstrated that CXCL16 promotes cancer [9] fatty liver [10] kidney damage [11] and coronary artery disease [12]. The role of CXCL16 in diabetes mellitus type 2 is not yet fully understood.

The aim of the present study was to explore whether serum CXCL16 and active vitamin D in addition to HbA1c could assist in reflecting glycemic control, and monitoring T2DM diabetic patients for a better understanding of the pathophysiology of this disease.

Material and Methods

The present study was performed on 30 patients (12 males/18 Females) with diagnosed type 2 diabetes mellitus (T2DM) who were on oral hypoglycemic drugs or injectable insulin from those attending the out-patient diabetes clinic of the National Institute of Diabetes and Endocrinology in the period from October to December 2018. The mean duration of diabetes for T2DM in cases was 12.3 ± 4.3 years, and their mean age was 47.6 ± 13.4. Exclusion criteria included patients with thyroid disease, current or previous intake of vitamin D or multivitamins, pregnant women, patients with chronic kidney disease, heart disease, fatty liver patients or patients with malabsorptive intestinal diseases or hepatitis B or C or HIV. The control group comprised 30 age and sex matched healthy subjects (14 males/16 females) whose mean age was 45.4 ± 15.6. They attended their routine appointment at the preventive health care service clinic on the same day of patients. They were healthy volunteers with normal fasting blood glucose, vitamin D, liver and kidney functions. All participants were subjected to full history taking and a detailed clinical examination.

Informed Consent Statement: All participants’ rights were protected and a written informed consent was obtained from each participant before the procedures were carried out according to the 1964 Helsinki Declaration.

Fasting blood samples were collected from all participants in suitable vacutainers. Sera and plasma were separated as the preferred schedule and kept at -200 C till assay time. Fasting blood sugar, urea, creatinine, total cholesterol, triglycerides, ALT, AST, were measured using routine methods on Hitachi 971 instrument (Roche Diagnostics GmbH, D, 68298 Mannheim). Glycosylated hemoglobin (HbA1c) was done by quantitative chromatographic spectrophotometric determination using a kit provided by Biosystem reagents and instruments, Barcelona (Spain) [13].

Thyroid stimulating hormone (TSH) was done by coat-A count TSH-IRMA provided by diagnostics products cooperation ISO13485:2003, catalog number IKTSI [14]. Serum vitamin D3 levels (1, 25 Dihydro- Cholecalciferol) was measured using a standard Radioimmunoassay Technique (RIA) by using 25(OH)D125 I RIA kit (DiaSource S.A., Belgium) [15]. Serum CXCL16 Levels were measured using a suitable ELISA kit specific for recombinant and natural Human C-X-C motif chemokine 16 N0: e0771h, provided by Wuhan EIAab Science Co., Ltd, China according to the manufacturer’s instructions on Infinite F50 TECAN ELISA instrument.

All data in this work was expressed as mean ± standard deviation. Correlations between Serum CXCL16, Vitamin D3, HbA1c as well as other variables were evaluated by Pearson’s Correlation test. Comparisons between the means of two groups were evaluated using Student’s two tailed t-test. All statistical analysis was carried out using Statplus version 2 for Macintosh statistical software.

Results

Serum CXCL16 levels and HbA1c % were shown to be significantly higher in type 2 diabetes mellitus cases joining in the study compared to the controls.

Discussion

Type 2 Diabetes Mellitus is regarded as one of the most important increasing community health concerns in Egypt. Its high prevalence remains to be on the rise as a result of multiple factors such as abdominal obesity, inactive life style, poor eating habits and the increased prevalence of hepatitis C [1]. Type 2 diabetes mellitus (T2DM) is an international health problem affecting people at diverse ages and walks of life with an increasing incidence leading to various complications. The longer duration of the disease, poor glycemic control in addition to an early age of onset comprise a group of risk factors leading the patient into microvascular and macrovascular diabetic complications such as kidney, eye and heart disease [16].

The role of vitamin D in human physiology has been investigated after the discovery of vitamin D receptors in the pancreas and immune cells. It has been reported frequently that subjects with vitamin D deficiency are at risk of developing type 2 diabetes mellitus [17]. Vitamin D has numerous effects on glucose metabolism pathways and several studies have emphasized the importance of the role that vitamin D deficiency plays in the pathogenesis of T2DM and its connected complications. In the current work, the level of vitamin D3 in T2DM patients was significantly lower compared to the healthy subjects. The results validate previous works ascertaining that vitamin D and its metabolites appear to have significant effects on insulin secretion by the pancreatic islets of Langerhans and subsequently the ability of beta cells to convert proinsulin to insulin [18].

Vitamin D directly stimulates insulin receptors increasing insulin response to glucose by binding its active circulating form to its receptor in beta cells to inhibit insulin resistance. Indirectly, vitamin D acts to decrease insulin resistance by regulating intracellular and extracellular calcium ion content. Calcium is well known to be an essential element for the activity of cellular glucose transporters [19]. Reduced serum vitamin D levels documented in the cases participating in the present study are in agreement with the findings of previous studies and can be considered as a risk factor for diabetes mellitus progress to complications including retinopathy [20], microvascular complications [21], metabolic syndrome [17], cardiovascular diseases [22], inflammatory bowel diseases [23] and multiple sclerosis [24].

In the current study, the level of vitamin D3 in T2DM patients correlated negatively with HbA1c. The results are in accordance with previous findings [25] and active vitamin D can be regarded as being a metabolic biomarker that may reflect the inflammatory and immunologic conditions of the body.

CXCL16 is a documented chemokine that is usually expressed in both dendritic cells and macrophages. It plays a pivotal role in the recruitment of T cells and NK cells [26]. The inflammatory cytokines TNF-alpha and IFN-gamma stimulate the expression of CXCL16 [27]. Although much is known from animal-based studies, little is known about CXCL16 in human-based studies particularly in patients with type 2 diabetes mellitus. Alterations in the levels of serum CXCL16 have not yet been fully examined in type 2 diabetes mellitus patients without complications such as heart disease and chronic kidney disease. Therefore, the present study was dedicated to investigating changes in serum concentrations of CXCL16 and its diagnostic value in type 2 diabetes mellitus patients. Moreover, the present study was a trial to find an interrelationship between vitamin D3 and CXCL16 levels in diabetic patients for a better understanding of the pathophysiology of type 2 diabetes mellitus.

Data of the present study revealed that serum CXCL16 levels were significantly higher in T2DM patients compared to the controls. These results are in harmony with a similar study [28] that investigated CXCL16 levels in patients suffering from T2DM with or without coronary artery disease and found that CXCL16 levels were significantly higher than the controls in both diabetic patient groups. Similar results were obtained by a study carried out on cases with gestational diabetes mellitus. Significantly higher serum CXCL16 levels were found in these cases compared to the controls [29]. On the other hand, another study [11] did not find any significant differences in serum CXCL16 levels between healthy controls and T2DM Caucasian diabetic patients.

Also, a study carried out on Chinese diabetic patients, serum CXCL16 levels didn’t show significant changes in diabetic patients when compared to the controls [30]. However, the mechanism responsible for the elevation of CXCL16 concentration and its role in the pathophysiology of diabetes is not fully understood. We speculate that the elevation of CXCL16 levels may be related to abnormalities in cholesterol metabolism which is supported by the fact that the long duration of CXCL16 level elevation associated with high levels of ox-LDL was found in streptozotocin induced diabetes in mice [31]. In the current study serum cholesterol levels were significantly higher in diabetic patients compared to the controls.

Numerous studies have highlighted the fact that chronic low-grade inflammation plays an important role in the development and progression of diabetes mellitus through a multitude of immunologic mechanisms [32][33]. Results of the present study found a significant negative correlation between CXCL16 and active vitamin D. A similar correlation was documented by a study carried out on diabetic kidney disease patients [34]. This indicates that vitamin D controls inflammation and is closely related to chronic diseases mainly T2DM and its deficiency acts as a risk factor that increases inflammatory markers and cytokines.

Conclusion

CXCL16 plays a fundamental role in inflammation and could partly contribute to the development and progression of type 2 diabetes mellitus. CXCL16 can be used in monitoring the inflammatory status and glycemic control of patients together with vitamin D and HbA1c and may help to predict diabetic complications. Additional studies are necessary to clarify the precise mechanism.

Ethical approval: All procedures performed in the current study were in agreement with the ethical criteria of the National Center for Radiation Research and Technology (NCRRT) research ethics committee (reference number 6H/ 19, dated 30/02/2019) and with the 1964 Helsinki declaration and its later amendments in 2013.

Funding: The authors received no specific funding for this study.

Conflict of Interests Statement: The authors state that there is no conflict of interests concerning the publication of this work.