Antioxidant Trace Elements Depletion Induces Proinflammatory Cytokines Polarization in Anti Diabetics Drug Naive Non Obese Male Type 2 Diabetic Nigerians

Keywords

Cytokine responses • Type 2 diabetes mellitus • Cytokine balance • Antioxidant trace

Introduction

T2D is a substantial risk factor for cardiovascular disease and acute oxidative stress (OS) due to the increased generation of reactive oxygen species (ROS) associated with the lipotoxicity and glucotoxicity processes [1]. It is not entirely apparent how cytokines responses and the level of antioxidant trace elements (ATE) such nickel (Ni), zinc (Zn), magnesium (Mg), and chromium (Cr) affect immunological diseases [2]. However, the function of ATE as an important micronutrient has long been recognized as a possible option for treating metabolic diseases, such as glucose homeostasis in prediabetes [3]. Several studies have reported that the pathogenesis of type 2 diabetes (T2D) is related to an imbalance of some antioxidant trace elements (zinc, selenium, copper, manganese, chromium), which may adversely affect pancreatic islets and cause diabetes development, and is clearly associated with ROS production and insulin signaling [4]. T2D is related with elevated proinflammatory cytokines (TNF-, IL-6) that may contribute to the development of diabetes complications [5-7] and increased glycated haemoglobin production [4]. Several studies have shown that type 2 diabetes (T2D) is a low-grade inflammatory condition, and that lowering inflammation is a key result for T2D therapy because it causes a balance between pro-inflammatory and antiinflammatory cytokines [8-12]. The toxicological effects of unregulated reactive oxygen species production have been linked to various metabolic defects such as cardio-vascular attacks and auto-immune disorders, but there is still little known about the cytokine responses of anti-diabetics drug naive nonobese male type 2 diabetics in Nigeria. To the best of our knowledge, this is the first research to assess cytokine responses and antioxidant trace element status utilizing blood levels of interleukin-1 alpha, interleukin-4, zinc nickel, magnesium, and chromium circulating among anti-diabetics drug naive nonobese male type 2 diabetic Nigerians.

Materials and Methods

Before the commencement of the case-control study, ethical approvals were obtained from UBTH Ethical Review Committee. A total of 124 subjects (aged between 20 and 40 year) were randomly studied, among these were 64 known cases of type II diabetes mellitus with a poor glycemic control index and 60 apparently healthy individuals with a good glycemic index (see table 1). Informed consent was obtained from all subjects before the commencement of the study.

Collection of sample: For the heavy metals determination, about 3 ml of venous blood samples were collected aseptically into Potassium ethylene diamine tetra acetic acid (K-EDTA) anticoagulant tubes. The samples were labelled and immediately placed in ice pack at the site of collection and subsequently transferred into a refrigerator at 4ºC. For the full blood count (FBC) and Interleukin assays, 4 ml of venous blood were collected into K-EDTA tubes, mixed and FBC carried out immediately. The blood samples were centrifuged within 30 minutes of collection for 15 minutes at 2000 rounds per minute (RPM) and plasma separated from the cells into plain tubes and refrigerated at 2-8ºC.

Determination of blood zinc, magnesium, chromium and nickel and cytokines: A calibrated Buck 205 flame atomic absorption spectrophotometer (Perkin-Elmer, HGA-2100) was used to analyze the blood sample for Zinc, Magnesium, Chromium and Nickel as described by Iyengar GV, et al. [13] at Yitzhak Rabin Laboratory for Advanced Micropropagation and Biotechnology Research Centre, Nnamdi Azikiwe University Awka, Nigeria. This method is based on the principle that atoms of the element when aspirated into Atomic Absorption Spectrophotometer vaporizes and absorbed light of the same wavelength as that emitted by the element when in the excited state. The amount of light absorbed can be correlated in a linear fashion to the concentration of the analyte in the sample. The levels of IL-1β and IL-4 were determined using enzyme-linked immunosorbent assay (ELISA) method as described by Alfred EF, et al. [14].

Data analysis: Student’s t- test was used to compare independent variables. The probability values less than 0.05 were considered significant. The statistical analysis were done using SPSS version 20.0.

Results

(Table 1). Shows the mean ± (SD) of age and body mass index of the studied non obese diabetics group with the control group (Table 2). Shows the mean ± (SD) Levels of interleukin -1α, interleukin-4 and some haematological parameters of diabetics group and control group. The levels of interleukin -1α were significantly higher in the diabetics group as compared with the control group (p<0.002), whereas, The levels of interleukin-4, packed cell volume, total white blood count, red blood cells, mean cell haemoglobin and mean cell haemoglobin concentration were significantly lower as compared with the control group (P<0.005) (Table 3). Shows the mean ± (SD) levels of antioxidant trace elements in diabetics group as compared with the control group. The levels of Manganese, Chromium, Nickel, Zinc were significantly lower the diabetics group as compared with the control group (p<0.002).

Discussion

The observed significant increase in proinflammatory interleukin-1 levels in this study, coupled with significantly lower levels of interleukin-4, packed cell volume, total white blood count, red blood cells, mean cell haemoglobin, and mean cell haemoglobin concentration, could be associated with a depletion of antioxidant trace elements (Manganese, Chromium, Nickel, Zinc), which induces a state of low-grade inflammation (see table 2 & 3). Several researchers have also expressed their views in this regard, however they have emphasized that Zn is an important ATE component because: (i) it plays a vital function in the stability of insulin hexamers and hormone pancreatic storage [15], and (ii) it is an effective antioxidant [16]. Lower Zn plasma concentrations were reported in T2D is a risk factor for cardiovascular-metabolic syndrome [17,18] and lower Zn levels in diabetics appear to be associated to an increased risk for coronary artery disease [19]. It has been proven that ATE-induced immune cell activation contributes to the proinflammatory environment of the diabetic islet [20]. It has been found that higher levels of IL-6, IL-8, TNF-, and IL-1 beta are connected with elevated blood glucose levels seen in T2D patients [21-25] and ATE deficiency results in lower levels of the anti-inflammatory cytokine. The current investigation supports the findings of Jagannathan B, et al. [26], who found that blood from T2D patients with ATE depletion had higher levels of circulating Th17 cells, which are proinflammatory T cell subsets. Reduced inflammation has been proven in studies to be an essential result for T2D therapy because it causes a balance between pro-inflammatory and anti-inflammatory cytokines [27-30]. However, we hypothesize that ATE supplementation may decrease the overproduction of pro-inflammatory cytokines and reactive oxygen species caused by ATE deficiency.

Conclusion

Depletion of antioxidant trace elements in T2D is linked to increased proinflammatory cytokines and may contribute to the development of diabetic complications. ATE supplementation may be able to prevent the observed cytokine imbalance.

Competing Interests

The authors declare that they have no competing interests.

Authors Contributions

This work was carried out in collaboration between all authors. Author EFA and UO designed the study and performed the statistical analysis. Authors EFA, UO, UNE, DAK, EJI, EAO and URE conducted and managed the Laboratory analysis. All authors read and approved the final manuscript.

Acknowledgement

We acknowledge the cooperation of the members of staff of Iykenson Medicals and Research Centre, Awka, Nigeria.

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