The present study was conducted in the Department of Biochemistry in association with Department of Medicine Government Medical College Amritsar, India. The subjects were recruited for the present study by conducting door to door survey of some villages of Amritsar and Tarn Taran district. Each diabetic individual was considered as one family unit and the offsprings of the diabetic individuals who were siblings amongst themselves in the age group of 10 to 55 years were included in the present study. Informed consent was taken from the diabetic individuals who were also the head of the family for inclusion of their families in the present study. The individuals were free to leave the study at any point. Blood samples were collected by conducting community camps of the registered individuals, all the individuals were advised to observe an overnight fast. To comply with these instructions the samples were collected early in the morning. Apart from various biochemical parameters i.e. Fasting plasma glucose [9], Glycated Hb [10], Insulin [11], C-peptide [12] and complete lipid profile [13–16], anthropometric measurements like waist hip ratio and BMI were also calculated. The status of beta cell function, %age sensitivity to insulin and insulin resistance were calculated using HOMA-IR 2 [17]. To avoid any instrumental error same procedure and measuring instruments were used to check the height, weight and waist hip measurements in all the individuals. The data collected was analyzed using SPSS software (version 16.0). The project was cleared by college ethical committee.

To collect the sample size a door to door survey of some of the villages of border belt of Amritsar and Tarn Taran district was conducted. A total of 400 families who had a history of type 2 diabetes gave their consent to join the present study. These families comprised of 1159 individuals (i.e. offsprings of diabetic individuals who were siblings amongst themselves). Individuals in the age group of 10 to 55 years were enrolled for the present study. Out of total 1159 individuals, 635 were males and 524 were females. These siblings were subdivided into four groups depending on their age i.e. group I ≤ 18 years, group II >18-35 years, group III >35-45 years and group IV >45 years [Table/Fig-1]. It was observed that maximum number of individuals belonged to the age group of >18-35 years.

All the individuals belonging to each group were divided into three categories depending on the levels of fasting plasma glucose as normal, IFG and diabetic [Table/Fig-2] as suggested by American Diabetes Association in its guidelines [18]. The % age of individuals with impaired fasting glucose (IFG) increased with advancing age both in males (from 15.2% to 30%) and females (from 22.9% to 60%), with prevalence being more in females than in males [Table/Fig-2].

Various biochemical investigations like glycosylated Hb, S. Insulin, C-peptide, lipid profile complete (Total cholesterol, Triglycerides, HDL-C, LDL-C and VLDL-C) along with anthropometric measurements used to calculate waist hip ratio and body mass index were estimated in all the individuals belonging to each age group.

A comparison of fasting plasma glucose levels of all the individuals included in each age group revealed that fasting plasma glucose levels were significantly more in individuals with IFG and diabetes when compared to that of normal individuals, also the diabetics had significantly raised fasting plasma glucose levels when compared to individuals with impaired fasting glucose. This trend was common both in males and females. Similar observations were revealed with glycosylated hemoglobin levels estimated in individuals with impaired fasting glucose and diabetes [Table/Fig-3]. Enzo Bonora and Jaakko Tuomilehto [18] states that the estimation of glycosylated Hb along with fasting glucose is important or the levels of glycosylated Hb clearly demarcated the individuals with IFG and diabetes from normal individuals.

Juvenile diabetes or type 1 diabetes was ruled out by estimating levels of S. Insulin and C-peptide in the age group of ≤ 18 years. Levels of S. Insulin were more in IFG and diabetics as compared to normal individuals thus clearly depicting synthesis and secretion of insulin thereby clearly indicating prevalence of pre diabetes or insulin resistance in these young individuals [Table/Fig-4]. Corresponding to insulin levels the c-peptide were more in individuals with IFG and diabetes as compared to normal and there was a positive correlation with insulin levels (r=+0.709, p<0.001).

As the levels of fasting plasma glucose progressed from normal and IFG to diabetes levels of insulin and c-peptide varied significantly but only in males. In females belonging to age group of ≤ 18 years and >18-35 years no significant variation in the levels of insulin was observed when IFG and diabetics were compared with each other thereby clearly indicating that hyperinsulinemia was prevalent in females belonging to these age groups [Table/Fig-4]. In adolescent group (≤ 18 years), this trend may be attributed to growth hormone levels that stimulate both synthesis and secretion of insulin [19].

A positive correlation of fasting plasma glucose with insulin (r= +0.425, p<0.001) indicated secretion of insulin in response to hyperglycemia. C-peptide is a byproduct of insulin which is cleaved off for production of insulin was found to be more in IFG and diabetics as compared to normal individuals [Table/Fig-4]. In females belonging to the age group of ≤ 18 years no significant variation in the levels of C-peptide was observed when IFG and diabetics were compared amongst each other. In all the other individuals belonging to various age groups levels of c-peptide were more in diabetics as compared to normal and IFG both in males and females.

Levels of total cholesterol were more in individuals with hyperinsulinemia and hyperglycemia [Table/Fig-5] As stated by Manisha C [20], Angelo, [21], Wilckon DE, [22], Enas A, [23] and OMP Ganda, [24] levels of cholesterol are more in individuals with IFG due to biosynthesis of cholesterol via HMG Co A pathway and preventing its uptake by the tissues for utilization. The IFG male individuals belonging to the age group of >45 years had more levels of cholesterol as compared to diabetics and this variation was statistically significant in males [Table/Fig-5], whereas in females the variation amongst IFG and diabetics was statistically not significant as these individuals were on treatment hence no definite conclusion could be drawn.

Mean LDL-C levels (in males and females) in all the age groups i.e. ≤ 18 years, >18-35 years, >35-45 years and >45 years although were more in IFG and diabetic individuals yet the values were within normal limits [Table/Fig-6].

Hyperglycemia leading to hypercholesterolemia and hypertriglyceridemia may be responsible for slight variation in LDL-C levels (LDL-C with fasting plasma glucose r= +0.950, p<0.001, LDL-C with Total Cholesterol r= +0.950, p<0.001). Raised levels of triglycerides in response to hyperglycemia (r=+1.0, p<0.001) leads to raised levels of VLDL-C in individuals with IFG . No significant variation was observed when IFG and diabetics were compared amongst each other [Table/Fig-6]. As stated by Coppack et al., [25], unrestricted lipolysis in insulin resistance leads to increased fatty acid flux in liver and increased synthesis of triglycerides in liver thereby affecting the levels of VLDL-C. Levels of HDL-C are affected by physical inactivity.

Hyperinsulinemia associated with hypertriglyceridemia and low levels of HDL are atherogenic, moreover glycosylation of HDL-C and LDL-C takes place leading to decrease in the half life of glycosylated HDL-C, receptors do not recognize to scavenge it, whereas glycosylated LDL stays longer in circulation. These glycosylated lipoproteins are immunogenic and can damage the arterial endothelium, the situation is aggravated by increased circulating C-peptide which induces local inflammation [26].

HOMA-IR calculations in all the individuals belonging to various age groups indicated loss of β-cell function in diabetes and IFG as compared to normal individuals both in males and females [Table/Fig-7].

There are five stages in progression to diabetes from IFG which are marked by changes in β-cell function [27]. As the levels of fasting glucose rises from normal to IFG and diabetes the function of β-cell decreases.

All the individuals (both males and females) were less sensitive hence more resistant to insulin. Maximum resistance to insulin was seen in individuals belonging to the age group of >18-35 years and >35-45 years [Table/Fig-7].

Anthropometric measurements i.e. height, waist and waist hip ratio were recorded and BMI of all the individuals was calculated. The waist hip ratio did not vary significantly when young IFG and diabetic individuals (belonging to the age group of ≤ 18 years and >18-35 years were compared amongst each other). In the age group of >35-45 years the females having IFG had considerably more waist hip ratio as compared to IFG males whereas in the age group of >45 years the ratio was reverse [Table/Fig-8].

[Table/Fig-9]:

Comparison of Waist hip ratio and BMI of individuals belonging to various age groups

It can be concluded from the present study that pre diabetes or insulin resistance, hyperinsulinemia along with hypertriglyceridemia is present in adolescents and young individuals i.e. belonging to the age group of ≤ 18 years and >18-35 years, thus predisposing these individuals to coronary events. Females in the reproductive years are more prone to pre diabetes or insulin resistance. Impaired fasting glucose along with deranged waist hip ratio and BMI play a significant role in predicting potential diabetics.