Association between subclinical inflammation & fasting insulin in urban young adult north Indian males

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1 Indian J Med Res 124, December 2006, pp Association between subclinical inflammation & fasting insulin in urban young adult north Indian males N.K. Vikram, Anoop Misra #, R.M. Pandey*, Manjari Dwivedi**, Kalpana Luthra**, Vibha Dhingra & K.K. Talwar + Departments of Medicine, *Biostatistics, **Biochemistry & + Cardiology, All India Institute of Medical Sciences, New Delhi, India Received September 1, 2005 Background & objectives: Elevated levels of c-reactive protein (CRP) are known to be associated with insulin resistance and metabolic syndrome in adults. A substantial prevalence of hyperinsulinaemia and elevated CRP levels have been shown in Indian young adults. We therefore studied the association of serum high-sensitivity C-reactive protein (hs-crp) with fasting insulin and insulin resistance in urban adolescent and young adult males in north India. Methods: In this cross-sectional study 324 healthy males, yr of age were selected randomly and their clinical and anthropometric profile [body mass index (BMI), waist and hip circumferences, waist-to-hip circumference ratio (W-HR), and skinfold thickness at four sites], percentage of body fat (%BF) and biochemical (fasting blood glucose, lipoprotein profile, fasting insulin and hs-crp) parameters were recorded. Insulin resistance was assessed by the homeostasis model of assessment (HOMA-IR). Results: Fasting insulin and hs-crp levels correlated significantly with BMI, waist circumference, and triceps and subscapular skinfold thickness. Fasting insulin also correlated with %BF, and hs- CRP correlated with W-HR. No correlation was observed between hs-crp and fasting insulin levels or insulin resistance. In multiple logistic regression analysis different independent risk factors for hyperinsulinaemia and elevated hs-crp levels were observed; hypercholesterolaemia, overweight and high subscapular skinfold thickness for the former, and high triceps skinfold thickness for the latter. Interpretation & conclusion: Lack of correlation between hs-crp and surrogate markers of insulin resistance and different risk factors for each, in young Indian males are unique observations of our study. Further studies on a larger sample of both genders need to be done to confirm these findings. Key words C-reactive protein - insulin resistance - young Indian males Present address: # Department of Diabetes & Metabolic Diseases, Ft. Lt. Rajan Dhall Hospital, Sector B, Pocket I, Aruna Asaf Ali Marg, Vasant Kunj, New Delhi , India Director, Post Graduate Institute of Medical Education & Research, Chandigarh , India 677

2 678 INDIAN J MED RES, DECEMBER 2006 C-reactive protein (CRP) correlates with generalized and abdominal adiposity 1, and robustly predicts future risk of coronary heart disease (CHD) and type 2 diabetes mellitus (T2DM) 2,3. Higher CRP levels in Asian Indians than white Caucasians, may contribute to a high prevalence of CHD and T2DM in this ethnic group 4. Subclinical inflammation and insulin resistance, forerunners to CHD and T2DM, may be pathophysiologically interlinked 5. High CRP concentrations significantly correlate with insulin resistance and the metabolic syndrome in adults 2,4,6,7 but these issues remain poorly investigated in children. CRP levels were shown to be associated with fasting insulin levels in healthy 2 to 3 yr old children 8, and young (18-22 yr) Japanese 9. The pathophysiological association of CRP and insulin resistance assumes great importance in Indian adolescents and young adults in whom substantial prevalence of hyperinsulinaemia 10 and elevated CRP levels 1 have been demonstrated by our group. We hypothesized that subclinical inflammation closely correlates with insulin resistance in young Asian Indians and studied the association of serum high sensitivity CRP (hs-crp) levels with fasting insulin and insulin resistance in a cross-sectional epidemiologic study involving urban adolescents and young adult males. Material & Methods Clinical and anthropometric profile: Male subjects yr of age (n=890), from the Epidemiological Study of Adolescents and Young Adults (ESAY) study 1 were included in this study. Multistage cluster sampling methodology according to the modified World Health Organization Expanded Program of Immunization Sampling Plan was adapted 11 for the ESAY study. A random selection of 40 clusters, defined as a school or a college in southwest New Delhi was made based on the proportional allocation of socio-economic strata. A section was considered as the primary sampling unit and all the students in that section were recruited. A written informed consent was obtained from the subjects >18 yr of age, and from the parents of those <18 yr of age. The study was initiated in August 2000 after getting an approval from the Director of Education, Ministry of Education, Government of New Delhi, and the institutional ethics committee. After excluding subjects who smoked (current or within the past 6 months), had any acute infective illness (current or within the past 6 months), or had any chronic illness, a total of 750 subjects remained. Of these, 324 subjects were randomly selected for the present analysis due to limited availability of ELISA kits for hs-crp assay. However, there were no significant differences between the selected sample and those not selected for age and body mass index (BMI). The study was conducted from 2001 to 2003 in southwest New Delhi. Clinical and demographic data were obtained for all the subjects 1. Anthropometric parameters [BMI, waist circumference (WC), waist-tohip circumference ratio (W-HR) and skinfold thickness (triceps and subscapular) were measured according to methods described earlier 12. Percentage of body fat (%BF) was measured by bioelectrical impedance 1. Waist and hip circumferences and skinfold thickness measurements were obtained in all the males by the same research personnel who was initially trained in the technique of various measurements. The intraobserver variation was less than 10 per cent in the measurement of skinfold thickness and still lower in the measurement of waist and hip circumferences. Laboratory methods: Subjects were instructed to fast from 2200 h the evening prior to blood sampling. Blood samples were obtained between 0800 and 0900 h and were transported within one hour to the central laboratory in an insulated container. Samples were centrifuged and the sera were divided into separate aliquots and stored at C. Estimation of fasting blood glucose (FBG), total cholesterol (TC), serum triacylglycerol (TG), and high-density lipoprotein cholesterol (HDL-c) was done on the same day as described previously 1. The value of LDL-c was computed according to the Friedewald s equation 13.

3 VIKRAM et al: CRP & INSULIN RESISTANCE IN YOUNG INDIAN MALES 679 Estimation of fasting serum insulin was done using a commercially available radioimmunoassay kit (Medicorp, Canada). The assay for fasting insulin was performed in batches. The samples for each batch were randomly selected from the total number of the subjects included in this study. Few samples were assayed in duplicate to determine the intra- and interassay variation. The intra-assay and inter-assay percentage coefficient variables were 2.6 and 3 per cent, respectively. Measurement of hs-crp was done by using a kit based on the ELISA method (Biocheck, Inc. CA, USA). The intra-assay and inter-assay variations were 1.7 and 3.3 per cent, respectively. Values >85th percentile from the 1800 subjects recruited in ESAY study were used to define high values of BMI (overweight, >23 kg/m 2 ), %BF (>28.5%), WC (>79 cm), W-HR (>0.86), triceps skinfold thickness (>19.7 mm) and subscapular skinfold thickness (>21.9 mm). Values >95th percentile were used to define hypercholesterolaemia (>4.37 mmol/l), high TG (>1.34 mmol/l) and high levels of low-density lipoprotein cholesterol (LDL-c) (>2.79 mmol/l) values <5th percentile (<0.98 mmol/l) for defining low level of HDL-c 1,14,15. The criteria defined by the American Diabetic Association were used for the diagnosis of diabetes 16. Elevated serum level of hs-crp was defined as >2.1 mg/l 17. Hyperinsulinaemia was defined as values in the highest quartile (>126.6 pmol/l) 18. Insulin resistance was also measured by homeostasis model of assessment (HOMA-IR) 19. Statistical analysis: Anthropometric and biochemical parameters were confirmed for the approximate normality and, subsequently, mean and standard deviations summarized the variables. Since the values of hs-crp and insulin were not normally distributed, transformed values of hs-crp (log) and fasting insulin (square root) were used to compute the Pearsons correlation coefficient (r) with various anthropometric and biochemical parameters. Independent risk factors associated with hyperinsulinaemia and elevated hs-crp levels were determined using step-wise logistic regression analysis. This model was further evaluated by using another statistical method; Bootstrap technique with 100 replications 20. In Bootstrap methodology rather than making an assumption about an underlying population distribution to estimate the standard error it is estimated on the basis of repeated random samples (with replacements) from the sample itself. Usually 100 subsamples are taken. While huge computing resources are required, the underlying mathematics is fairly uncomplicated compared with most advanced methods. STATA 8.0 Intercooled version (STATA Corporation, College Station, TX, USA) was used for the statistical analysis. Results None of the subjects had diabetes. The BMI was 20.1 ± 3.5 kg/m 2 (Table I). The prevalence of hyperinsulinaemia was higher in subjects with overweight (50%), with high %BF (41.5%) and with high WC (50%) as compared to those with normal values of these variables (P<0.001 for all). The mean values of hs-crp were higher though not significantly in subjects with overweight, with high %BF, and significantly higher in those with high W-HR (P=0.01) as compared to subjects with normal values of these parameters. The prevalence of elevated hs-crp was 13.1 per cent (overall); 21.3 per cent (overweight subjects), 23.8 per cent (with high %BF), 32.4 per cent (with high triceps skinfold thickness) and 23.1 per cent (with high subscapular skinfold thickness). Serum levels of fasting insulin and hs-crp correlated significantly with BMI (r=0.19, P<0.01 and 0.14, P<0.01), WC (r=0.17 in both, P<0.01), triceps skinfold thickness (r=0.25 and 0.18, P<0.001) and subscapular skinfold thickness (r=0.22, P<0.001 and 0.12, P<0.05), respectively (Table II). In addition, fasting insulin levels showed significant correlation with %BF (r=0.31, P<0.001), TC (r=0.11, P<0.05), TG (r=0.14, P<0.05) and hs- CRP levels correlated with W-HR (r=0.16, P<0.01). Serum hs-crp levels did not show any significant correlation with fasting insulin, HOMA-IR and lipid parameters. The correlations of HOMA-IR were similar to those for fasting insulin.

4 680 INDIAN J MED RES, DECEMBER 2006 On step-wise multivariate logistic regression analysis, overweight [OR (95% CI): 2.4 ( )], high subscapular skinfold thickness [OR (95% CI): 3.0 ( )] and hypercholesterolaemia [OR (95% CI): 2.6 ( )] were observed to be independent risk factors associated with hyperinsulinaemia. In the Bootstrap analysis the frequencies of selection of overweight, high subscapular skinfold thickness and hypercholesterolaemia as independent risk factors of hyperinsulinaemia were 63, 67 and 53 per cent, respectively (Fig.). Similarly, high triceps skinfold thickness [OR (95% CI): 4.3 ( )] was observed to be the independent risk factor associated with elevated hs-crp levels and in the Bootstrap analysis it was selected with a frequency of 75 per cent (Fig.). Discussion This is perhaps the first study showing significant but dichotomous correlations of CRP and fasting insulin Table I. Anthropometric and biochemical profiles of subjects (n=234) Parameters Mean (SD) Population mean (95% CI) Anthropometry: Body mass index (kg/m 2 ) 20.1 (3.5) 19.9 ( ) Percentage of body fat 21.7 (6.5) 22.3 ( ) Waist circumference (cm) 70.7 (7.8) 71.1 ( ) Waist-hip circumference 0.82 (0.04) 0.82 ( ) ratio Triceps skinfold 11.8 (5.4) 13.1 ( ) thickness (mm) Subscapular skinfold 13.3 (6.9) 14.5 ( ) thickness (mm) Biochemical parameters: Fasting blood glucose 4.91 (0.52) 89.7 ( ) (mmol/l) Total cholesterol (mmol/l) 3.45 (0.63) ( ) Serum triacylglycerols 0.96 (0.36) 88.8 ( ) (mmol/l) High-density lipoprotein 1.22 (0.16) 47.6 ( ) cholesterol (mmol/l) Low-density lipoprotein 1.80 (0.60) 78.1 ( ) cholesterol (mmol/l) Serum insulin (pmol/l) (33.4) - HOMA-IR 3.24 (1.04) - C-reactive protein (mg/ml)* 0.63 ( ) - *Geometric mean (95 % CI) concentrations with measures of generalized and regional adiposity, and the absence of correlation between CRP and surrogate markers of insulin resistance in a large number of young Indian males. The risk factors for elevated CRP also have been described in this study. In Bootstrap analysis, overweight and high subscapular skinfold thickness emerged as independent risk factors for hyperinsulinaemia in two out of every three studies, implying a strong association. However, the association of hypercholesterolaemia with hyperinsulinaemia was observed to be weaker. These data contrast with those from studies on adult migrant Asian Indians 4,6,21 and adult white Caucasians 21. The association of CRP with insulin resistance has been shown to be stronger in women as compared to men 22,23, suggesting a potential gender difference. Obesity may also influence the association of CRP with insulin sensitivity, being stronger in non-obese individuals 7. Persistent and mild elevations in CRP levels in Asian Indians residing in India could be due to increased exposure to repeated infections. Thus, a single value of an elevated CRP level may not have Table II. Correlations of fasting insulin and hs-crp with various anthropometric and biochemical parameters Variable Square root of LogCRP fasting insulin Square root of 0.02 fasting insulin LogCRP 0.02 Body mass index 0.19*** 0.14** Percentage body fat 0.31*** 0.07 Waist circumference 0.17** 0.17** Waist-hip ratio ** Triceps skinfold thickness 0.25*** 0.18*** Subscapular skinfold 0.22*** 0.12* thickness Total cholesterol 0.11* Serum triglycerides 0.14* 0.07 High-density lipoprotein cholesterol Low-density lipoprotein cholesterol *P<0.05; **<0.01; ***<0.001

5 VIKRAM et al: CRP & INSULIN RESISTANCE IN YOUNG INDIAN MALES 681 Fig. Validation of independent risk factors of hyperinsulinaemia and elevated C-reactive protein (CRP) in 100 replications using the Bootstrap method. adequate predictive importance for T2DM and CHD, particularly in children, and may not correlate with measures of insulin resistance as has been shown for other ethnic groups. to our preliminary analysis on diet and lifestyle, some factors (e.g., saturated fat, polyunsaturated fatty acids, and physical inactivity) may influence CRP levels and insulin resistance (unpublished data). In vivo release of interleukin-6, linked closely to CRP pathway, but not tumour necrosis factor-a, which is related to insulin resistance, has been reported in human subcutaneous adipose tissue (SAT) 24. We speculate that the relatively large SAT mass (truncal and peripheral SAT) in Asian Indians, may generate relatively more CRP and preferentially drive this pathway rather than the insulin resistance pathway, although both appear to be interlinked. This is in keeping with our data wherein triceps skinfold thickness is an independent risk factor associated with elevated CRP. Importantly, when compared to white Caucasian and blacks, significantly triceps skinfold thickness was significantly more in Asian Indian children 10. Diet and physical activity may influence insulin resistance and subclinical inflammation. According The findings of this investigation need to be confirmed in a larger number of Asian Indians of both genders, and the clinical significance of the correlations observed in this study need to be evaluated in prospective studies. Effect of other factors like non-esterified fatty acids, insulin-like growth factor system and adipocytokines on the relationship between CRP and insulin sensitivity needs to be investigated in Asian Indians. Finally, there is a need to carry out prospective studies in which markers of insulin resistance and CRP levels could be correlated to the future risk of development of T2DM and CHD in Asian Indians. Acknowledgment The study was supported by a financial grant from the Department of Science and Technology, Ministry of Science and Technology, Government of India.

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