Experts define diabetes mellitus as a group of metabolic diseases that are characterized by hyperglycemia. This condition arises from defects in insulin secretion, insulin action, or both. There are two types of presentation of diabetes which are Type 1 and Type 2 diabetes mellitus. ¹

The inability of the pancreas characterizes type 1 diabetes (known as insulin-dependent) to produce the insulin. It is most frequently observed in children and adolescents. Type 2 diabetes (known as non-insulin-dependent) is a chronic condition in which the body resists the effects of insulin or doesn't produce enough insulin to maintain normal glucose levels.²The global burden of diabetes is both substantial and escalating, with developing economies such as India experiencing a significant rise, primarily attributed to the increasing prevalence of overweight/obesity and unhealthy lifestyles. Type 2 is the most common form of diabetes and accounts for 90% of all diabetes cases across the world.³ And it most commonly occurs in adults (40-60 years), but a noted increase is seen in the adolescents as well. As of 2019, estimates revealed that a staggering 77 million individuals in India were grappling with diabetes, a number anticipated to surge to over 134 million by the year 2045. Alarmingly, approximately 57% of these cases go undiagnosed, underscoring the pressing need for enhanced awareness, early detection, and proactive management strategies to curb the escalating impact of diabetes on public health in India.⁴

When the condition is not effectively managed, diabetes complications can arise. These complications affect various parts of the body and can be both short-term and long-term. Short-term complications may include hypoglycemia (low blood sugar) or hyperglycemia (high blood sugar), leading to immediate health issues.⁵ Long-term complications, however, can be more severe and impact organs such as the heart, kidneys, eyes, and nerves. Cardiovascular diseases, kidney failure, vision impairment, and nerve damage are common long-term complications associated with diabetes.⁶

The diabetes death rate per 100,000 people increased from 10.0 in 1990 to 23.1 in 2016, with deaths accounting for 3.1% of total deaths, up from 0.98% in 1990. There was an almost threefold increase in diabetes-related deaths during this period.⁷ The rate of disability-adjusted life years (DALYs) from diabetes per 100,000 people rose from 440.1 in 1990 to 791.5 in 2016, an 80% increase. In absolute numbers, DALYs increased by 174% from 3.8 million to 10.4 million during the same period.⁸ DALYs because of diabetes accounted for 2.2% of the total DALYs in 2016, a 215% increase from 1990.^9-11

This is a prospective, case control and Randomized study with and without family history of type 2 diabetes was conducted in the Department of Biochemistry, Index Medical College.

Inclusion criteria

Age: Above 35 years.

Gender: Both male and female.

First degree relatives of Type 2 Diabetics.

Exclusion criteria:-Morbid obese,

Chronic alcoholism,

Chronic

Smoking,

Pregnant,

Lactating women,

Kyphosis,

Scoliosis.

Number of groups – two

• Group 1 (n=112): Apparently healthy individuals.
• Group 2 (n=112): First degree relatives of type 2 diabetes.

Methods for sampling: -

All patients will provide written informed consent when the institute ethics committee gives its approval. Every experiment was carried out in the research lab of the biochemistry department of Index Medical College in Indore.

• Before testing, the participant was required to void their urine and be made to sit comfortably in the lab to get used to the new surroundings.

Anthropometric and baseline measurements was made prior to the assessment of cardiopulmonary fitness.

Blood collection and storage: After a vein puncture, the blood was drawn, allowed to clot, and centrifuged at 3,000 RPM for 10 minutes at 4 °C (using a Remi refrigerated centrifuge). The serum will then be separated and frozen at -80 °C for analysis.

Measurement of IL6:- The microtiter plate provided in the kit was pre-coated with an antibody specific to IL6. Standards or samples were then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to IL 6. Next, Avidin conjugated to Horseradish Peroxidase (HRP) was added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain IL6, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically [model no:- 922 ] at a wavelength of 450nm ± 10nm. The concentration of IL 6 in the samples is then determined by comparing the O.D. of the samples

Measurement of hs CRP:-

This kit uses enzyme-linked immune sorbent assay (ELISA) [Model Read well touch, Robonik ] based on the Biotin double antibody sandwich technology to assay the Human High sensitivity C-Reactive Protein (hs-CRP). Add High sensitivity C-Reactive Protein(hs-CRP) to the wells, which are pre-coated with High sensitivity C-Reactive Protein(hs-CRP) monoclonal antibody and then incubate. After that, add anti hs-CRP antibodies labeled with biotin to unite with streptavidin-HRP, which forms immune complex. Remove unbound enzymes after incubation and washing. Add substrate A and B. Then the solution will turn blue and change into yellow with the effect of acid. The shades of solution and the concentration of Human High sensitivity C-Reactive Protein  (hs-CRP) are positively correlated.

Measurement of TNF alpha:- The microtiter plate provided in the kit has been  pre-coated with an antibody specific to TNFalpha. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to TNFa. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain TNFα, biotin-conjugated antibody andenzyme-conjugated Avidin will exhibit a change in color. The enzyme- substrate reaction is terminated by the addition of sulphuric acid solution and the colour change is measured spectrophotometrically [model no:- 922 ] at a wavelength of 450nm ± 10nm. The concentration of TNFα in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Study Methods For Data Collection: -

After receiving approval from the ethical committee of the institute. Every patient will provide written informed consent. Every experiment was carried out in the biochemistry department research lab at Index Medical College in Indore. Prior to the measurements, the subject was advised to abstain from alcohol.

Prior to testing, the participants were required to empty their urine and was required to sit comfortably in the lab to get used to the unfamiliar surroundings. Anthropometric and baseline measurements were made prior to the assessment of cardiopulmonary fitness.

Data Analysis: -

Statistical Analysis plan:- R 3.2.3 for Windows were used to carry out the statistical analysis. SPSS software version 25 were used. The mean ± SD was used to express the data. Kolmogorov-Smirnov test was used to determine normality. To study the association of sympathovagal imbalance with other parameters, analysis was used depending on the normality of the data. 

Table 1:  Visceral fat (%) distribution among the study population

Mean Visceral Fat: Controls have a mean visceral fat percentage of 8.48%, while cases with a family history have a higher mean of 9.51%. P value: The p-value is 0.000, which is well below the 0.05 threshold for statistical significance. This indicates a statistically significant difference in visceral fat percentage between the two groups. The higher mean visceral fat percentage in individuals with a family history of type 2 diabetes suggests a notable association between having a family history and higher visceral fat.

Mean Visceral Fat: Controls have a mean of 8.38%, while cases have a mean of 11.35%.

P value: The p-value is 0.003, which is also below the 0.05 threshold. This indicates a statistically significant difference in visceral fat percentage, with cases showing a higher percentage compared to controls. The significant p-value reinforces the association between having a family history of type 2 diabetes and increased visceral fat.

Table 2:  Cardio respiratory fitness distribution among the study population

Mean Distance: Controls have a mean of 2773.0 meters, while cases have a mean of 2319.9 meters. The controls run significantly farther in 12 minutes compared to cases with a family history of type 2 diabetes. P value: The p-value is 0.006, which is below the 0.05 threshold for statistical significance. This indicates that the difference in the distance covered in the 12-minute run is statistically significant, suggesting that individuals with a family history of type 2 diabetes have lower endurance compared to those without such a history.

Mean Distance: Controls have a mean of 2940.4 meters, whereas cases have a mean of 2294.6 meters. Again, controls run significantly farther than cases.

P value: The p-value is 0.015, which is also below the 0.05 threshold, indicating that the difference in the 12-minute run distance between the two groups is statistically significant in this measurement as well.

Table 3:  Fasting Blood Sugar distribution among the study population

Mean FBS: Controls have a mean FBS level of 86.33 mg/dL, while cases with a family history have a mean of 90.5 mg/dL. Although the mean FBS level is higher in cases with a family history, the difference is not statistically significant.

P value: The p-value is 0.538, which is well above the 0.05 threshold for statistical significance. This indicates that there is no significant difference in FBS levels between the two groups in this measurement. Thus, having a family history of type 2 diabetes does not appear to be associated with a significantly different fasting blood sugar level in this sample.

Mean FBS: In this measurement, controls have a mean FBS of 85.95 mg/dL, and cases have a mean of 91.35 mg/dL. Similar to the first group, cases have higher FBS levels, but the difference is not statistically significant.

P value: The p-value is 0.750, also above 0.05, indicating no statistically significant difference in FBS levels between controls and cases in this measurement either.

Table 4: Tumor necrosis factor alpha (TNF-α) distribution among the study population

Mean TNF-alpha: Controls have a mean TNF-alpha level of 122.00 pg/ml, while cases have a much higher mean of 277.00 pg/ml. This indicates a significant increase in TNF-alpha levels in individuals with a family history of type 2 diabetes. P value: The p-value is 0.001, which is significantly below the 0.05 threshold for statistical significance. This indicates a highly significant difference between the two groups, suggesting that individuals with a family history of type 2 diabetes have elevated TNF-alpha levels compared to controls.

Mean TNF-alpha: Controls have a mean of 147.55 pg/ml, whereas cases have a mean of 298.71 pg/ml. The difference in means is considerable, showing a clear increase in TNF-alpha levels in those with a family history. P value: The p-value is 0.081, which is above the 0.05 threshold but relatively close. This suggests that while there is a trend towards a significant difference, it does not reach conventional levels of statistical significance in this subgroup.

Table 5: Interleukin 6 levels based distribution among the study population

Mean IL-6: Controls have a mean IL-6 level of 6.99 pg/ml, while cases have a mean of 15.33 pg/ml. This indicates that individuals with a family history of type 2 diabetes have higher IL-6 levels compared to controls. P value: The p-value is 0.544, which is much higher than the 0.05 threshold for statistical significance. This indicates that the difference in IL-6 levels between the two groups is not statistically significant, suggesting no strong evidence of an association between family history of type 2 diabetes and IL-6 levels in this sample.

Mean IL-6: Controls have a mean of 6.55 pg/ml, and cases have a mean of 15.89 pg/ml. Again, cases have significantly higher IL-6 levels compared to controls.

P value: The p-value is 0.560, which is also above the 0.05 threshold, indicating that this difference is not statistically significant in this subgroup as well.

Table 6: High-sensitivity C-reactive protein (mg/l)  levels distribution among the study population

Mean hsCRP: Controls have a mean hsCRP level of 4.86 mg/L, while cases have a significantly higher mean of 7.25 mg/L. This indicates that individuals with a family history of type 2 diabetes have higher levels of systemic inflammation as measured by hsCRP.

P value: The p-value is 0.000, which is well below the 0.05 threshold for statistical significance. This indicates a highly significant difference between the two groups, suggesting that a family history of type 2 diabetes is strongly associated with elevated hsCRP levels.

Mean hsCRP: Controls have a mean hsCRP level of 4.45 mg/L, while cases have a mean of 6.42 mg/L. The difference is substantial, with cases showing elevated hsCRP levels compared to controls. P value: The p-value is 0.000, indicating a statistically significant difference between the groups. This further supports the finding that individuals with a family history of type 2 diabetes have higher hsCRP levels, reflecting increased inflammation.

Table 7: Lipid profile based on distribution among the study population

       FBS Levels: Controls: The mean FBS level for controls is 86.33 mg/dl, and the sub-group mean is slightly lower at 85.95 mg/dl. Cases: The mean FBS level for cases with a family history of type 2 diabetes is 90.5 mg/dl, with the sub-group mean being 91.35 mg/dl.

      According to Pappachan JM et al stated that increase in blood glucose level in case control study.¹² Similary, a study also reported by Laffel LM et al there is change in FBG between case and control group.¹³ Differing, a study also reported by Zuckerman Levin N et al there is no change of FBG between case and control group.¹⁴

    Genetic predisposition to β-cell failure

A hereditary propensity to β-cell failure is highly supported by the data. Early-onset type 2 diabetes is commonly caused by a genetic variant of the illness that is characterised by heterozygous mutations in β-cell transcription factors, β-cell malfunction, an autosomal dominant mode of inheritance, and diagnosis at less than 25 years of age. However, environmental variables prevail and it is hard to establish a genetic defect clinically in the majority of patients in clinical practice.¹⁵

        Mechanisms responsible for decline in insulin secretion

Increased insulin production from individual β-cells and/or an increase in β-cell mass can be observed in normal β-cell adaptation to insulin resistance. Certain individuals exhibit normal glucose levels despite having lower insulin secretion or β-cell mass; their insulin sensitivity is sufficient to guarantee appropriate insulin secretion.¹⁶

           According to Miyazaki et al., there was an increase in TNF-α prior to the start of diabetes, and this rise was not linked to an increase in insulin resistance. However, Bluher et al. found no evidence linking TNF-α to the development of early insulin resistance. More specifically, they linked non-esterified fatty acids to the development of insulin resistance. No relationships were discovered between insulin resistance and TNF-α. Demirbas et al. demonstrated that in individuals with prediabetes, serum TNF-α concentration rose along with increases in concentrations of insulin and HOMA IR.¹⁷

      Mechanisms behind the decline in TNF-α

       In our study, Mean TNF-alpha: Controls have a mean TNF-alpha level of 122.00 pg/ml, while cases have a much higher mean of 277.00 pg/ml. This indicates a significant increase in TNF-alpha levels in individuals with a family history of type 2 diabetes. P value: The p-value is 0.001, which is significantly below the 0.05 threshold for statistical significance. This indicates a highly significant difference between the two groups, suggesting that individuals with a family history of type 2 diabetes have elevated TNF-alpha levels compared to controls.

      Tumour necrosis factor-a (TNF-α) is an inflammatory cytokine that has drawn a lot of interest. It has been shown that TNF-a can either positively or negatively affect the development of diabetes in NOD mice; however, it is unclear how TNF-α causes these varying effects.¹⁸

     Paradies Y et al., have reported increased TNF alpha levels in diabetics among a Southern Karnataka population.¹⁹ Similary, a study also reported by Suglia SF et al there is change TNF alpha between case and control group.²⁰

     (IL-6) INTERLEUKIN 6

      In this study, Mean IL-6: Controls have a mean IL-6 level of 6.99 pg/ml, while cases have a mean of 15.33 pg/ml. This indicates that individuals with a family history of type 2 diabetes have higher IL-6 levels compared to controls. P value: The p-value is 0.544, which is much higher than the 0.05 threshold for statistical significance. This indicates that the difference in IL-6 levels between the two groups is not statistically significant, suggesting no strong evidence of an association between family history of type 2 diabetes and IL-6 levels in this sample.

        Mean IL-6: Controls have a mean of 6.55 pg/ml, and cases have a mean of 15.89 pg/ml. Again, cases have significantly higher IL-6 levels compared to controls. P value: The p-value is 0.560, which is also above the 0.05 threshold, indicating that this difference is not statistically significant in this subgroup as well.

Similary, a study also reported by Carnethon MR et al there is change of IL6 between case and control group.²¹ Contrary, a study also reported by Butler AM et al there is no change of IL6 between case and control group.²²

       The mechanisms behind the rise in IL-6

       In conclusion, research on the relationship between type 2 diabetes and variations in the IL-6 gene has produced conflicting results. It is unclear if this link may be explained by increased IL6 promoter activity combined with other type 2 diabetes-predisposing genes to cause insulin resistance/β-cell failure. In glucose-tolerant individuals, −174G was linked to metabolic syndrome characteristics. It is uncertain if the reduced IL6 promoter activity in IL6−/− mice, which acquire maturity-onset obesity presumably because of CNS effects on hunger and energy expenditure control, may account for this connection.²³
         Furthermore, it is well acknowledged that IL-6 has an anti-inflammatory and immunosuppressive effect in both systemic and local inflammatory responses (Yang P, 2023). TNF-α and IL-1β expression is believed to be inhibited by muscle-derived IL-6 during exercise.²⁴

        C-REACTIVE PROTEIN (CRP)

Mean hsCRP: Controls have a mean hsCRP level of 4.86 mg/L, while cases have a significantly higher mean of 7.25 mg/L. This indicates that individuals with a family history of type 2 diabetes have higher levels of systemic inflammation as measured by hsCRP. P value: The p-value is 0.000, which is well below the 0.05 threshold for statistical significance. This indicates a highly significant difference between the two groups, suggesting that a family history of type 2 diabetes is strongly associated with elevated hsCRP levels.

Controls have a mean hsCRP level of 4.45 mg/L, while cases have a mean of 6.42 mg/L. The difference is substantial, with cases showing elevated hsCRP levels compared to controls. P value: The p-value is 0.000, indicating a statistically significant difference between the groups. This further supports the finding that individuals with a family history of type 2 diabetes have higher hsCRP levels, reflecting increased inflammation.

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