**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.

**Keywords:** inflammation, biomarkers, cytokines, mediators, type 2 diabetes mellitus

## **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 [6–10].

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 lowgrade 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 proinflammatory cytokines, like TNF-α and IL-6 [19]. Insulin, however, could counterbalance the pro-inflammatory effect of glucose by suppressing the production of *Molecular Pathogenesis of Inflammatory Cytokines in Insulin Resistance Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.100971*

the pro-inflammatory cytokines and by activating the production of antiinflammatory 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 NFkB 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 [23–25]. 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 (TNFα, 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. TNFα, 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 proinflammatory 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**).

#### *Molecular Pathogenesis of Inflammatory Cytokines in Insulin Resistance Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.100971*

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 carboxylend 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

#### **Figure 1.**

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

#### **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).*

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**).
