An investigation into pre-diabetes-associated changes in immune cells, red blood cell indices and long noncoding ribonucleic acids in patients with pre-diabetes from Durban, South Africa.

Abstract

Background: Pre-diabetes is a metabolic condition that often precedes the onset of type 2 diabetes (T2D). This asymptomatic condition is characterised by moderate hyperglycaemia that is below the threshold for a diagnosis of T2D. Risk factors that are implicated in the development of pre-diabetes includes chronic consumption of unhealthy diets, as well as sedentary lifestyles. The asymptomatic nature of this condition has made it difficult to diagnose and study the condition in humans. Studies using an animal model of pre-diabetes have reported that there are abnormalities such as immune activation, upregulation of inflammatory markers and haematological changes during this condition. The findings indicated changes in immune cells such as neutrophils, lymphocytes, monocytes, basophils and eosinophils. The studies also showed upregulation of inflammatory markers such as CRP, IL-6, TNF-α, fibrinogen, sCD40L and P-selectin during pre-diabetes. Additionally, these studies reported on changes on red blood cell indices such as MCH, MCHC, RBCs, HCT, HGB, MCV and RDW. These findings from animal studies raised the question if these also occur in humans with pre-diabetes. Furthermore, studies have shown that long noncoding ribonucleic acids (lncRNAs) expressed during inflammatory conditions such as T2D contribute to abnormalities such as atherosclerosis. These include lncRNAs such as noncoding RNA expressed in dendric cells (LncRNA-DC), noncoding transcript in T- cells (lncRNA-NTT) and noncoding Repressor of nuclear factor activated T-cells (lncRNA-NRON), however, there have been no studies to investigate if these are expressed during pre-diabetes. Recent studies have reported on increasing prevalence of pre-diabetes among adults in Durban, South Africa with the highest prevalence found in those between 25-45 years of age. This made this area and population ideal to study pre-diabetes to investigate pre-diabetes-associated changes in immune cells, red blood cell indices and long noncoding ribonucleic acids in patients with pre-diabetes. Methods: Upon ethics approval, the blood samples (n=292) were collected from King Edward Hospital. They were divided into 3 experimental groups; non-diabetic (ND, n = 30 which consist of samples from 20 females and 10 males), pre-diabetic (PD, n = 90 which consists of samples from 56 females and 34 males) and type 2 diabetic (T2D, n = 172 which consists of samples from 113 females and 59 males). This was done using the American Diabetes Association criteria. In each sample, the concentration of immune cells and red blood cell indices were determined using haemocytometer which were analysed and reported on study 1 and study 2 respectively. Additionally, ELISA and Multiplex assay were used to measure concentration of select inflammatory markers for both study 1 and study 2. For study 3, samples were divided into 3 experimental groups; non-diabetic (ND, n = 9 which consist of samples from 6 females and 3 males), pre-diabetic (PD, n = 15 which consists of samples from 11 females and 4 males) and type 2 diabetic (T2D, n = 22 which consist of samples from 16 females and 6 males). Real time polymerase chain reaction was used to measure the relative expression of lncRNAs. Results: Results showed abnormal ranges of neutrophils (below normal range = 40-60%) and basophils (above normal range = 0.5-1%) in all 3 groups. Lymphocytes, monocytes and eosinophils were within the normal range. Results showed an increase in basophils, eosinophils, and fibrinogen on PD group by comparison with ND group. There was also a statistically significant (p< 0.05) increase in CD40L and TNF-α on PD group by comparison with the ND group. The results showed a decrease in neutrophils, lymphocyte, monocytes, IL-6, CRP and P-selectin on PD group by comparison with ND group. The results also showed a decrease in RBC, HGB, HCT, MCV, MCH, and MCHC for all females per group by comparison to males and an increase in RDW in females by comparison to males per group. Results showed an increase in all RBC indices in the PD group by comparison with ND group. Findings also showed a statistically significant (p< 0.05) increase in expression of lncRNA-NRON in the PD group by comparison with the ND group. There was an increase in the expression lncRNA-DC in the PD group by comparison with the ND group while there was a decrease in expression of lncRNA-NTT in the PD group by comparison with the ND group. Conclusion: The findings of this study indicated that there is immune activation, sub-clinical inflammation, hematological changes, and the expression of various lncRNAs during the pre-diabetic state in humans. While these findings warrant further investigations, they will form a foundation for further investigation of pre-diabetes-associated changes in immune and haematological indices.