**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**). **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


#### **Table 1.**

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

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

formulae = weight (Kg) / height (m<sup>2</sup> ). Participants with a BMI ≥30.0 kg/m<sup>2</sup> 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/m<sup>2</sup> 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).

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


**Table 2.** *Anthropometric analysis in study subjects.*


#### **Table 3.**

*Levels of clinical chemistry parameters in study group.*

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).

**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.*

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


**Table 4.**

*Levels of inflammatory mediators in the study group.*


**Table 5.**

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

**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.

**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).

**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

**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.*

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

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

**Figure 7.** *Correlation of TNF-α with insulin sensitivity in cases.*

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].

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.


**Table 7.**

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

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