Molecular Pathogenesis of Inflammatory Cytokines in Insulin Resistance Diabetes Mellitus

Abstract

Diabetes Mellitus Type 2 (T2DM) is a non-communicable and multifactorial disease. It is a leading cause of premature deaths worldwide. Inflammatory cytokines are reported that they have potential to enhance insulin resistance and hence T2DM. The current research was taken to investigate the possible role of inflammatory mediators: Tumor Necrosis Factor (TNF-α) and White blood cells (WBC’s) in mobilizing biological molecules mainly immunological nature. A total of 320 subjects were selected in this study among them 160 were T2DM cases and 160 were healthy controls. Serum concentration of Tumor Necrosis Factor-a (TNF-α) was quantified by ELISA method, WBC count was measured on Sysmax (Germany) hematology analyzer, biochemical and Immunoassay parameters were done on fully automatic analyzers. The expression of candidate pro-inflammatory cytokine (TNF-α), and (WBC’s) were elevated in T2DM. TNF-α shows association (p<0.001) with glycemic profile and insulin sensitivity in T2DM cases in comparison with healthy controls. Induction of inflammation and up regulation of pro-inflammatory cytokines has been purported to play a significant role in pathogenesis of T2DM and study confirms that the positive correlation of TNF-α with T2DM and hence to insulin sensitivity. These can act as early prediction biomarkers in diagnosis and prognosis of human disease i.e Diabetes Mellitus. Further studies are needed to help clinicians manage and treat T2DM effectively.

1. Introduction

The term Diabetes Mellitus describes a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia accompanied by distressed metabolism of carbohydrates, fats and proteins resulting from defects in insulin secretion, insulin action or both [1]. Diabetes Mellitus (T2DM), is a non-communicable, chronic disorder and progresses slowly because of multifactorial etiology and is a leading cause of premature deaths worldwide, also, its exceptional upsurge poses a severe threat on human society and imposes a huge economic burden worldwide [2]. As per recent reports of World Health Organization (WHO), 422 million people globally are affected from the diabetes mellitus with a prevalence rate of 8.5% and 46.3% still remains undiagnosed and number is projected to rise 552 million in 2030 [3]. Furthermore, highly effected population are living in developing countries and comprises of 40–60 age group. In 2017, studies reported that India alone has 72 million people affected with T2DM and is projected to rise 101.2 million in 2030 [3, 4]. The risk factors of T2DM are suggestively increased with changing lifestyle, blood pressure, central obesity, inadequate physical activity and unhealthy diet [5] Blood glucose fasting (FBG), Two-hour post prandial blood glucose (Two-hour-PP) and glycated hemoglobin (HbA1c) levels are most widely used as glycemic control markers which indicates progression of the disease and development of its complications. Studies reported diabetes mellitus are T2DM linked with lipid and lipoprotein irregularities, including reduced HDL cholesterol and raised triglycerides [6, 7, 8, 9, 10].

Recent decade the diabetes mellitus, witnessed transformation from the epidemic to pandemic at global level. The global projections revealed that diabetes is affecting nearly 10% of the world’s population [11]. As per reports of World Health Organization (WHO), the prevalence of diabetes mellitus is likely to increase by 35% by the year 2030-45 [11]. It is the most common form of the disease, accounting for about 90 to 95% of all diagnosed cases of diabetes. T2DM is a group of genetically determined diseases which may be controlled by diet and/or hypoglycemic agents and/or exogenous insulin [12]. Although, it is mainly characterized by insulin resistance, but impairment in insulin secretion also occurs later in type 2 diabetes mellitus [13]. It occurs usually in individuals over 30 years of age and dramatically increases as a result of changes in human behavior and increased body mass index [14]. The global rise in diabetes mellitus is referred to population growth, aging, increasing trends towards an unhealthy diet, obesity and modern lifestyles [15]. Inflammation can be classified as acute, high-grade, or chronic low-grade inflammation [16]. Acute inflammation is essential for survival, because it initiates pathogen killing, initiates tissue repair processes, and helps to restore homeostasis after infection or tissue damage [16]. In general, acute inflammatory responses are short-term responses [16]. When clinical manifestations are minimal or absent, it is classified as low-grade inflammation [16]. Low-grade inflammation is characterized by slightly elevated blood concentrations of acute-phase proteins, cytokines, and mediators with endothelial activation capacity that are involved in acute inflammation as well [16]. It is likely that dysfunction of adipose tissue is a major contributor to chronic low-grade inflammation [16]. Adipose tissue dysfunction, is characterized by a reduced capacity to store dietary lipids and an impaired endogenous lipolysis, leading to lipid overflow and ectopic fat accumulation, which has been related to the development of insulin resistance. Adipose tissue has a dual function, in addition to acting as a storage repository of the body system has role in endocrine function system, secretes the inflammatory markers. Thus, any sort of imbalance in the secretion leads to low grade inflammation. The matured adipocytes, as observed in individuals with overweight, relate among others to an higher secretion of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) and a lower secretion of the anti-inflammatory cytokine, adipokine, adiponectin like IL-10. Besides secretion of cytokines by the adipocytes themselves, macrophages that infiltrate the obese adipose tissue can also secrete cytokines [17]. Being secreted, these pro-inflammatory cytokines can have autocrine and paracrine effects at the site of the adipose tissue [18]. Furthermore, these cytokines can be transported via the blood stream to act on distant targets, like the skeletal muscle and liver [18]. Besides adipose tissue, hyperglycemia itself can contribute to chronic-low grade inflammation. Hyperglycemia can stimulate the production of reactive oxygen species, which, in turn, stimulate production of pro-inflammatory cytokines, like TNF-α and IL-6 [19]. Insulin, however, could counterbalance the pro-inflammatory effect of glucose by suppressing the production of the pro-inflammatory cytokines and by activating the production of anti-inflammatory cytokines, like interleukin-4 and interleukin-10 [20]. Thus imbalance in cytokine expression can contribute to insulin resistance. TNF-α expression can affect the insulin signaling cascade by phosphorylation of the insulin receptor, insulin receptor substrate, and glucose transporter, can suppress expression of genes encoding for adiponectin, and can increase the expression of genes encoding for IL-6 [16, 20]. TNF-α and IL-6 also enhance oxidative stress by stimulation NF-kB or NADPH oxidase [19]. NF-kB causes a transcriptional response of genes involved in inflammatory processes. A high concentration of IL-6 stimulates the production of acute-phase protein C-reactive protein (CRP) in the liver [21]. CRP is a non-specific inflammation marker that may contribute to insulin resistance by increasing phosphorylation of IRS and by increasing the synthesis of cytokines like TNF-α and IL-6 [22]. In line with the proposed mechanisms, several prospective studies observed associations between slightly elevated concentrations of the inflammation markers CRP, TNF-α, and IL-6 and type 2 diabetes mellitus in different populations of world [23, 24, 25]. Weiyi et al. reported that circulating antibodies in plasma against inflammatory cytokines are associated with type 2 diabetes mellitus. Furthermore, some prospective cohort studies showed that participants with higher CRP, TNF-α, or IL-6 concentration had a higher risk of type 2 diabetes [26, 27].

Inclination of T2DM from metabolic disorder to inflammation is changed due to variations in pro and anti- inflammatory cytokines like tumor necrosis factor alpha-α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) [26]. It has been reported in insulin signaling pathways, cross linking and ultimately developing insulin resistance in β-cells of pancreas which further risks to T2DM [28, 29]. Steadiness among these pro and anti-inflammatory cytokines is compulsory to make β-cells immune to any infection which may lead to T2DM [30].

This chapter will focus on the studies about the role of, proinflammatory cytokine in diabetes mellitus.

2. Role of inflammatory mediators in T2DM

Numerous studies demonstrated that, the various inflammatory mediators in type 2 diabetes mellitus (T2DM), has been found abnormally high levels of cytokines, plasminogen activator inhibitor, chemokines, acute phase proteins (such as CRP) [24, 31]. The elevated concentrations of pro-inflammatory cytokines (TNF-α, IL-6 and CRP) initiates the activation of innate immune system in type 2 diabetic patients due to over-nutrition. Nutrients comprises of elements necessary for body functioning and development are minerals, vitamins, fats, carbohydrates, and proteins. Inflammatory mediators and CRPs, are considered to vary from individual to individual and tissue to tissue. In patients with T2DM, increased circulating levels of various proinflammatory cytokines and chemokines have been detected [32]. Consequently, one may not predict the degree and extent of inflammation in specific tissue by only observing the circulating levels of these pro-inflammatory mediators, which eradicates β-cells themselves leading to β-cell dysfunction.

2.1 Cytokines

The cytokines coined from two Greek words, “cyto” means cavity or cell“ and “kines” means movement. They are soluble proteins with low molecular weight proteins <30 kD, secreted by the cells of the bothinnate and adaptive immunity. These cytokines are chemically peptide molecules, and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have high affinity for receptors and are active in ‘picomole’ concentration. They function as autocrine, paracrine and endocrine signaling. Based on cellular sources there are three types of cytokines:- Monokines (mononuclear phagocyte), lymphokines (lymphocytes), interleukins (leukocytes) (TNF, IL-6, IL-10 etc.). In addition, a subfamily of cytokines called chemokines, which functions in directing migration of cells. Cytokines are produced by a wide series of immune cells, like macrophages, B lymphocytes, T lymphocytes and mast cells. They act through receptors, in the immune system. Cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Cytokines has been classed as lymphokines, interleukins, and chemokines, based on, cell of secretion, or target of action. Because cytokines have important characteristics of redundancy and pleiotropism. Cytokines are the key modulators of inflammation, participating in acute and chronic inflammation.

2.2 Tumor necrosis factor (TNF-α)

The term tumor-necrosis factor, which is abbreviated as TNF. TNF, is primarily produced as a 233-amino acid long type II transmembrane protein arranged in stable homotrimers. The TNF-α gene is present as a single copy gene on human chromosome 6 located on position (6p21.33). The gene consists of four exons and three introns. Interestingly, more than 80% of the mature TNF-α sequence is encoded in the fourth exon. Tumor necrosis factor (TNF-α) was initially identified in the 1970s as an endotoxin-induced serum factor responsible for the necrosis of certain tumours in vivo and in vitro. Subsequently, TNF-α was isolated and its gene was cloned. TNF-α, is an essential signaling protein in the innate and adaptive immune systems. It plays important role in tissue degeneration and repair. It stimulates the proliferation of normal cells, exerts cytolytic or cytostatic activity against tumor cells, and causes inflammatory, antiviral, and immunoregulatory effects.

TNF-α also performs in additional functions linked with lipid metabolism, coagulation, insulin resistance, and endothelial function. TNF-α is the prototypic member of the TNF superfamily of type II trans-membrane proteins that includes 30 receptors and 19 associated ligands with diverse functions in cell differentiation, inflammation, immunity and apoptosis. It is primarily secreted from activated macrophages, although it may also be secreted by other cell types including monocytes, T-cells, mast cells, NK-cells, keratinocytes, fibroblasts and neurons (Tracey et al., 2008). TNF-α is synthesized as a transmembrane precursor protein (m-TNF-α) with a molecular mass of 26 kDa, it is transported via the rough endoplasmic reticulum (RER), Golgi complex and the recycling endosome to the cell surface. The monomers of TNF-α associate at the plasma membrane as non-covalent trimmers prior to being cleaved by the metalloprotease, TNF-α converting enzyme(TACEorADAM17) (Black et al., 1997). Cleavage by TACE results in the production of 17 kDa soluble TNF-α (sTNFα) ectodomain and it is trimers of sTNFα that constitute the potent ligand that activates TNF receptors. Following TACE cleavage, the membrane proteolytically processed by the signal peptide peptidases (SPPLs) SPPL2a and SPPL2b. This cleavage produces an intra cellular domain (ICD) that translocates to the nucleus and induces pro-inflammatory cytokine signaling. Thus, the precursor TNF-α molecule is subjected to multiple cleavage events to release potent modulators of inflammation. TNF-α, is a pleiotropic cytokine which signals through two receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). The receptors are expressed on different cell types, with TNFR1 being widely expressed, while TNFR2 is expressed predominantly on leukocytes and endothelial cells. The two TNFRs have been reported to mediate distinct biological effects. Both TNFR1 and TNFR2 are single transmembrane glycoproteins with 28% homology in their extracellular domains (Figure 1).

Figure 1.

Mechanism of TNF-α receptors and association with other inflammatory cytokines (source: Sujuan et al., 2018. Front. Immunol; 9:784).

It comprises of four cysteine-rich domains (CRDs), each of which comprises three cysteine-cysteine disulphide bonds, and a pre- ligand binding assembly domain (PLAD) involved in trimerisation of the receptor. Importantly, the receptors differ by the presence of an intracellular death domain (DD) at the carboxyl-end of TNFR1, that is able to drive either apoptosis or inflammation through interaction with associated adaptor molecules (Figure 1). Recruitment of TRADD to TNFR1 is required for both signaling pathways. Subsequently, one of two complexes is formed, either at the cell surface (complex-I) or following internalization (complex-II). The formation of complex-I requires TNFR-associated factor 2 (TRAF2) and receptor- interacting protein (RIP), leading to kinase cascades that trigger pro- inflammatory gene expression. Alternatively, should the first complex fail to signal, Complex II is formed to induce apoptosis. In Complex II, proteolysis and internalization of the receptor results in the recruitment of FADD and pro-caspase-8 to form the death-inducing signaling complex. The distinct cytoplasmic domains could account for the differential signaling of the receptors by sTNFα and mTNFα. It was found that mTNFα was a more potent activator of TNFR2 than sTNFα and induced distinct biological outcomes. Further, activation of TNFR1 was found to stimulate NF-κB expression to a significantly greater extent than TNFR2. Finally, Scatchard analysis of ligand binding to TNFR1 and TNFR2 found that the former had a higher affinity for TNF-α. Thus, TNFR1 is considered to be the more important of the two receptors for the activation of pro- inflammatory signaling pathways (Figure 2).

Figure 2.

Mechanism of TNF receptor1 and 2, activating signaling pathways of pro-inflammatory cytokines (source: Ana Falvia et al., 2019. World J Gastrointest Oncol. Apr 15, 2019; 11(4): 281–294).

3. Materials and methods

3.1 Subjects and study design

We included in our study 340 T2DM cases and 160 healthy controls, 30–60 years old as per American Diabetes Association (ADA) criteria 2016 (Table 1).

FBG ≥126 mg/dl.; Fasting means no food ingestion for ≥ 8 hours

2-hr BG ≥200 mg/dl

HbA1C ≥6.5%.

Random BG ≥200 mg/dl.

Table 1.

American Diabetes Association (ADA) 2016 criteria for diagnosing T2DM.

Inclusion criteria: Confirmed T2DM patients.

Exclusion criteria: Pregnant women, patients suffering from (thyroiditis, rheumatoid arthritis, inflammatory bowel syndrome, skin diseases, any cancer).

3.2 Anthropometric measurement

Height (cm) was noted by a scale on wall and Weight (kg) was measured by digital weighing machine. The body mass index (BMI) of subjects was calculated by formulae = weight (Kg) / height (m²). Participants with a BMI ≥30.0 kg/m² were considered obese as per NCEP ATPIII criteria. “Waist circumference” (WC) was evaluated in the middle, between the lower rib margin and the iliac crest with subjects in upright position.

3.3 Biochemical and immunoassay analysis

Glycated hemoglobin (HbA1c) levels and clinical chemistry was evaluated for all cases and healthy controls. The Insulin resistance (IR) of subjects was accepted by calculating the index of HOMA-IR (homeostatic model assessment – insulin resistance) which is as under: “HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mg/dl)/405” (24). Following HOMA-IR score was used as reference range for classification of IR.

• < 3 = Normal IR b) Between 3 and 5 = Moderate IR c) >5 = Severe IR

White Blood Cell count analysis: whole Blood samples were taken in EDTA vials were analyzed for WBC count on Sysmax hematology analyzer (Germany).

Estimation of Pro-inflammatory cytokine (TNF-α) by Enzyme linked Immunoassay (ELISA) Analysis.

TNF-α assay: Quantitative measurement of TNF- α was done by ‘Diaclone Human TNF-α ELISA kit’ (France).

Statistical analysis: Data was compiled on Microsoft excel 2011 spread sheet. All the data were expressed as a mean ± standard deviation and significance value (p) were calculated. Data analysis were performed by using statistical ‘software SPSS 16.1’ (Chicago, IL). Students T-test was done on biochemical, immunoassay and inflammatory mediators. Chi-square test was done on socio-demographic characters. Correlation analysis was performed for determining the association between serum TNF-α, and other parameters, Pearson correlation coefficient (r) was obtained. P < 0.05 were considered statistically significant.

4. Result and discussion

Total 320 subjects were included for the study among 160 were cases and 160 were controls (Table 2). The mean ± SD age of cases were (49.9 ± 9.4) Years and that of healthy controls were (46.9 ± 9.9) years which is statistically significant (p = 0.003). In this study, It was observed that BMI was (42.2 ± 8.1) kg/m² in T2DM cases and in healthy controls was (21.2 ± 2.2) kg/m² which is statistically significant (p = 0.003). Among 160 cases 81 were males and 79 females and in healthy controls 80 were males and 80 were females, on gender wise comparison difference in patients and controls are significant (p = 0.005).

Variables	T2DM Cases (n = 160)	Controls (n = 160)	p value
Age (Years)	49.9 ± 9.4	46.9 ± 9.9	0.003
Gender (M/F)	81/79	80/80	0.005
BMI (kg/m^2)	42.2 ± 8.1	21.2 ± 2.2	0.002

Table 2.

Anthropometric analysis in study subjects.

In Table 3, biochemical profile of T2DM cases and healthy controls were summarized and it was found that there were increase trend in parameters of lipid profile like serum Triglycerides (TG), total cholesterol (TC), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL) among T2DM cases as compared to healthy controls and the trend were significantly high (p < 0.05). The glycemic profile (Glucose Fasting and HbA1c) in T2DM cases was higher as compared to healthy controls and are found to be statistically significant (p < 0.05).

Variables	Diabetes mellitus (n = 160)	Controls (n = 160)	p value
Fasting Glucose (mg/dl)	168.4 ± 32.7	81.9 ± 7.7	0.119
Post-parandial Glucose (mg/dl)	316.2 ± 51.6	122.1 ± 9.1	0.001
Total Cholesterol (mg/dl)	298.5 ± 54.1	109.1 ± 27.9	0.002
Triglycerides (mg/dl)	319.5 ± 57.1	146.1 ± 29.6	0.003
HDL (mg/dl)	92.4 ± 22.6	52.9 ± 10.1	0.024
LDL (mg/dl)	148.3 ± 9.1	69.8 ± 29.8	0.002
HBA1c (%)	9.9 ± 2.8	4.9 ± 0.8	0.014

Table 3.

Levels of clinical chemistry parameters in study group.

Figure 3, Histogram representing graphical analysis of Insulin and HOMA-IR of study group where there was elevation in the Insulin (μU/ml) levels among T2DM case (32.6 ± 7.5) as compared to healthy controls (7.8 ± 2.1) and it was found that the elevation level among the T2DM cases was significantly (p = 0.001) higher than healthy controls. The HOMA-IR index for insulin sensitivity was calculated by a standard formula in both T2DM cases and healthy controls and was found significantly (p < 0.05) higher in T2DM cases. Table 4, describes the levels of serum inflammatory mediators (TNF-α, and WBC) in T2DM cases and healthy controls; the mean ± SD value of inflammatory markers in T2DM cases was as WBC = 8495 ± 1943, TNF-α = 36.5 ± 7.8 while in healthy controls it was WBC =7389 ± 1504, TNF-α =13.7 ± 4.4 and it was found that in T2DM patients the levels of inflammatory mediators were highly significant (p < 0.05) in comparison with healthy controls.

Figure 3.

Histogram representing immunoassay analysis of study group.

Variables	Diabetes mellitus (n = 160)	Controls (n = 160)	p value (<0.05)
TLC (thousands)	8495 ± 1943	7389 ± 1504	0.002
TNF-α (pg/ml)	36.5 ± 7.8	13.7 ± 4.4	0.002

Table 4.

Levels of inflammatory mediators in the study group.

Table 5 shows the comparison of inflammatory mediators within gender groups and it was found that in female cases levels of inflammatory mediators was highly significant (p < 0.05) as compared to male cases while WBC was not statistically significant, which provides us the information that females may be at higher risk to T2DM.

Inflammatory mediators	Male T2DM (n = 81)	Female T2DM (n = 79)	Male controls (n = 80)	Female controls (n = 80)	p-value <0.05
TNF-α (pg/dl)	8.8 ± 0.8	8.7 ± 1.0	3.6 ± 0.5	3.7 ± 0.4	0.001
WBC (thousands)	1974 ± 206	1784 ± 184	1459 ± 169	1385 ± 165	0.082

Table 5.

Comparison of inflammatory mediators in T2DM male and female patients versus control subjects.

Figures 4–7 shows the correlation of inflammatory mediators in T2DM cases and controls with glycemic profile and insulin sensitivity and was studied by Pearson’s correlation analysis. TNF- α shows positive correlation with glycemic profile (Glucose fasting, HbA1c) and insulin sensitivity (Insulin assay, HOMA-IR) in T2DM cases and were statistically significant (p < 0.05).

Figure 4.

Correlation of TNF-α with glycemic profile in controls.

Figure 5.

Correlation of TNF-α with glycemic profile in cases.

Figure 6.

Correlation of TNF-α with insulin sensitivity in controls.

Figure 7.

Correlation of TNF-α with insulin sensitivity in cases.

Table 6, describes the relationship of inflammatory mediators with glycemic profile and the Table 7, depicts the relationship of insulin sensitivity as per gender wise in cases and controls. We observed in Males and Female T2DM cases there was a positive correlation (p = 0.001) of TNF-α with glycemic profile and Insulin sensitivity and other inflammatory mediators show negative and weak correlation. Worldwide people are suffering from T2DM and it is projected to increase from present 415 million people to 642 million by 2040. In all developing countries it was seen that number of T2DM patients is increasing and 75% of people with T2DM are living in these developing countries [33]. In this study, we observed that Socio-Demographic factors like Education, Lifestyle and Smoking has significant association with T2DM except Residence (Urban and rural of same geographical area) which had no substantial influence on the levels of inflammatory mediators of study like, TNF-α, and WBC (32). From the results we infer that there were increased expression of inflammatory markers (TNF-α, and WBC) between cases and controls which supports the findings of Phosat, et al. [34] as they found in their study that there were greater risk of T2DM with higher levels of inflammatory mediators [34]. On comparison between sex wise within case group it was observed that there was an elevation in levels of TNF-α in Female T2DM cases as compared to Male T2DM Cases which are in agreement with the findings of Insha et al., [9, 10, 33, 35]. There are many research studies on this subject which demonstrated that levels of markers of inflammatory reactions increased with the decrease in insulin sensitivity depending on the severity of T2DM [36, 37]. In this study both Male and Female sexes have confirmed the importance of inflammatory mediators in the pathogenesis of T2DM. The levels of TNF-α rise significantly in both sexes compared to control group showing correlation with glycemic profile and Insulin sensitivity thus, being considered an independent predictor of risk of developing T2DM [34].

Inflammatory mediators	Cases				Controls
	HBA1c		Fasting glucose		HBA1c		Fasting glucose
	Males n = 81	Females n = 79	Males n = 81	Females n = 79	Males n = 80	Females n = 80	Males n = 80	Females n = 80
TNF-α	*p = 0.02; *r = 0.89	p = 0.003; r = 0.883	p = 0.004; r = 0.459	p = 0.005; r = 0.546	*p = 0.035; *r = 0.388	p = 0.011; r = 0.368	p = 0.013; r = 0.260	p = 0.063; r = 0.211

Table 6.

Pearson correlation coefficients of inflammatory mediators with glycemic profile, sex-wise.

Inflammatory mediators	Insulin		HOMA-IR		Insulin		HOMA-IR
	Males n = 81	Females n = 79	Males n = 81	Females n = 79	Males n = 80	Females n = 80	Males n = 80	Females n = 80
TNF-α	*p = 0.008; *r = 0.478	p = 0.009; r = 0.368	p = 0.008; r = 0.374	p = 0.004; r = 0.388	*p = 0.012; *r = 0.016	p = 0.011; r = 0.019	p = 0.111; r = 0.099	p = 0.008; r = 0.319

Table 7.

Pearson correlation coefficients of inflammatory mediators with insulin sensitivity (sex-wise).

This study experimentally determined that only pro-inflammatory cytokine TNF-α can leads to pathogenesis of T2DM while other inflammatory cytokines shows negative and weak correlation with T2DM. This research study showed vibrant changes in concentrations of pro-inflammatory cytokines, in T2DM. Our findings are in concurrence with the results of [32], which showed serum expression of candidate mediators (TNF-α) are elevated in T2DM cases which are independent of physical activity and other risk factors [38]. It is suggest that TNF-α is an important predictor for the development of T2DM for Male and female, in both rural and urban populations.

Interestingly, results of our study showed a high degree of correlation between these promising cytokines (TNF, WBC) in T2DM in comparison to healthy controls. The results are statically significant. In this case–control study, we found in our T2DM cases there were significantly higher concentration of TNF-α as compared to those of controls which may be the possible cause of low grade inflammation and predisposes subjects to the T2DM or towards its complications. These assertions aggress with the findings of AL-Shukaili, et al. [39]. Furthermore our experimental finding provides evidence that the pattern and variation of these cytokines (TNF-α, and WBC) are important in the pathogenesis of T2DM [32]. Significant correlation of TNF-α inflammatory mediator in T2DM cases with glycemic profile and insulin sensitivity leads to pathogenesis of diseases in this ethnic population [32]. These findings are in agreement with the fact that inflammatory reactions depends on group of cytokines rather than a single one. The reports of inflammation has a role in pathogenesis of T2DM has been elucidated in several studies in different populations.

5. Conclusion

The study findings confirms that TNF-α, plays a positive role in the pathogenesis of T2DM and can act as early prediction biomarkers which can prevent T2DM in this population. Further studies on the wider range of inflammatory mediators in association with other biochemical, immunoassay and hematological parameters are needed to establish role of inflammatory markers as early prediction biomarkers which can prevent T2DM.

6. Highlights of chapter

1. Inflammation is initiated by trauma or injury, infection, and hence effects cascades of numerous cytokines and white blood cells. The low grade inflammation triggers inflammatory cells like neutrophils, macrophages and monocytes in blood stream and also expresses the pro-inflammatory cytokines like Tumor necrosis factor-alpha, and interleukin-6.

2. The liver cells synthesize acute-phase proteins under the stimulus of some cytokines, which flow through the bloodstream, reach the site of inflammation, and eradicate the pathogens through opsonization and eliciting the complement pathways.

3. The variations in the serum concentrations of TNF-α leads to pathogenesis of T2DM.

4. Diagnostic routine tests are sometimes invasive. To augment the modern diagnostics in patient care, the employment of noninvasive biomarkers are needed.

5. Molecular biological tools have modernized the field of the biomarkers. For the development of biomarkers, genomics and proteomics, pathophysiology of a disease are needed to understand, the most available technique is correlating serologic markers with clinical parameters.