Mursaleen Dar Muniza Mogri Mentors: Dr. P aresh Dandona,MD Dr. Husam Ghanim , PhD

1. Association Of Insulin Resistance And Testosterone Concentration In Young Pubertal And Post-pubertal Obese Males Mursaleen Dar MunizaMogri Mentors: Dr. PareshDandona,MD Dr. HusamGhanim, PhD Dr. Teresa Quattrin

2. Objective • To show whether obesity is associated with lower testosterone concentrations in pubertal (P) and post-pubertal (PP) obese males • Whether there is change in insulin receptor expression in obese (P) and (PP) males compared to lean population

3. Rationale • These young subjects could be treated for obesity to prevent the onset of diabetes and further complications and hypogonadotropic hypogonadism

4. Back Ground • Prevalence of obesity in the pediatric population has tripled from 1971–1974 to 2003–2004, and there has been a rise in the cases of type 2 diabetes(1,2) • Type 2 diabetes and obesity are associated with a high prevalence (25–33%) of hypogonadotropic hypogonadism in middle-aged and elderly men(3,4)

5. In all these studies, free testosterone (T) concentrations are negatively related to body mass index (BMI) • In addition, low T concentrations have been related to elevated HOMA-IR in obese men in all these studies

6. What is HOMA-IR • An index of insulin resistance • Homeostasis Model Assessment Of Insulin Resistance • Quantify insulin resistance and beta cell function • Fasting Glucose x Fasting Insulin/22.5

7. Back Ground Contd.. • This raises the question whether obesity is associated with lower testosterone concentrations even in younger males i.e; P and PP males and… • Whether there is change in insulin receptor expression in young obese males because of inverse co-relation of testosterone concentration with HOMA-IR

8. Hypothesis • On the basis of the above, it was hypothesized that: • 1. Obese boys and young obese men (14–20 years) have significantly lower total and free testosterone (TT and FT) and SHBG concentrations as compared to lean boys and young lean males and • 2. The insulin receptor expression is less in obese young males compared to the lean ones based on HOMA-IR

9. Materials & Methods • Cross-sectional observational study (Part 1) • Basic bench research (Western Blot) (Part 2)

10. Part 1 : Cross-sectional study • Observation of all members of a population or a representative subset at one specific point in time • “ A snapshot” of a population

11. Methodology • Study approved by Institutional Review Board of the Children’s Hospital of Buffalo and University at Buffalo • Informed consent taken • Parental consent was taken in addition to children’s consent for subjects less than 18 years of age

12. Steps in Methodology for Part 1 • 50 males between the ages of 14-20 years and tanner stage of >4 were consecutively recruited at the Endocrine and Diabetes Center of Women and Children’s Hospital of Buffalo • 25 obese subjects • BMI >95th percentile for age • 25 lean subjects • BMI < 85th percentile for age

13. Inclusion criteria • Males with age 14-20 years • Tanner stage 4-5 • Stable weight ( change in weight of less than 5% during last 6 months)

14. Exclusion Criteria • History of hypogonadism, panhypopituitarism, severe depression or psychiatric illness, diabetes, head trauma, renal failure, hemochromatosis, cirrhosis, hepatitis C, HIV • Treatment with testosterone or oral steroids were also excluded • Active infection or recent surgery or hospitalization in the prior 6 weeks

15. Maturity assessment • Tanner staging was assessed by one of the investigators (MM) trained by a board certified pediatric endocrinologist (TQ) using an orchidometer • Males with testicular volume between 12–15 ml were classified as Tanner stage 4 and those with testicular volume >15 ml were classified as Tanner stage 5

16. Methodology Contd… • Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer by trained personnel, and weight were measured to the nearest 0.1 kg using a digital scale • Blood pressure and heart rate were recorded. Subjects were healthy and without significant co-morbidities

17. Methodology contd... • One fasting blood sample was drawn between 8 and 10 am to measure total and free T and estradiol, SHBG, CRP, LH and FSH. • Total T and estradiol were measured by liquid chromatography and tandem mass spectrometry • SHBG, LH and FSH concentrations were measured by a solidphase, chemiluminescent immunometric assay • All these assays were performed by Quest Laboratories, Chantilly, VA, USA.

18. Methodology Contd... • Insulin concentrations were determined using an ELISA kit from Diagnostic Systems Laboratories Inc. (Webster, TX, USA) • Glucose concentrations were measured in plasma by YSI 2300 STAT Plus glucose analyzer

19. These assays were performed at the research laboratories of the division of Endocrinology and Metabolism, University at Buffalo • HOMA-IR was calculated from fasting insulin and glucose level using the formula [fasting insulin (mU/l) x fasting glucose (mmol/l)]/22.5.

20. Statistical Analysis • Group comparisons were performed by one-way ANOVA, two tailed t-tests, Mann–Whitney rank-sum tests and chi-squared tests as appropriate • Adjustment for variables such as age, BMI, SHBG and Tanner stage in group comparisons was carried out with ANCOVA and generalized linear model analysis • Data are presented as means ± SD for normally distributed data and median [25th, 75th percentile] for non-normal data • P < 0.05 was considered significant

21. Statistics

22. Results Of Part 1 • Testosterone concentrations of young obese (P) and (PP) males are 40–50% lower than those with normal BMI • And low testosterone levels in obese population had inverse correlation with HOMAIR

23. Part 2 : Western Blot • Detects specific proteins

24. Steps for Part 2 : Western Blot • Blood samples were collected in Na-EDTA as an anticoagulant • 4.5mL of anticoagulated blood sample were carefully layered over 3.5 mL of PMN isolation media ( Robbins Scientific Corp., Sunnyvale, CA)

25. Peripheral Mononuclear cells (MNCs) were isolated by Ficoll-Hypaque method • Samples were centrifuged • 2 bands separate out at the top of the RBC pellet • Top band consists of MNC

26. Steps for Part 2 contd… • The MNC band harvested repeatedly and washed with Hank’s balanced salt solution (HBSS) • Stored at -80*

27. Immunoblotting • MNC total cell lysates of 32 samples (16 obese and 16 lean) were prepared by lysing the cells in 150uL of lysis buffer, and protease and phosphatase inhibitors

28. Why 16 pairs instead of 25 pairs? • Inadequate blood samples • Suboptimal protein samples • Lost samples • Gel membrane constraints

29. After 30 min incubation on ice, samples were centrifuged at 12000rcf for 10 mins, supernatants collected and total protein concentrations determined • 60 ug of total cell lysate were boiled in 2X SDS buffer

31. . • Gels loaded and proteins separated by SDS PAGE electrophoresis and then transferred to PVDF membrane

34. Polyclonal and monoclonal antibodies against insulin receptor (phosphotyrosine kinase) and actin were used and the membranes developed using super signal, chemiluminescence reagent • Mixed-control was made by mixing equal amounts of all the samples

36. Results of Part 2

38. Discussion • Based on the results we found out that young obese (P) and (PP) males have lower total and free T concentrations as well as high HOMA-IR compared to their lean counterparts. However the expression of insulin receptors on the MNCs is the same in both the groups. Showing that there is no change in IR expression or signaling does not mean that our hypothesis is wrong. MNC might not reflect the truth or HOMA-IR may not reflect the truth. For future research and to get all answers we need to do biopsy of adipose tissue or muscles and check IR expression or do clamp method to check insulin resistance

39. Conclusion • Young obese (P) and (PP) males have significantly lower total and free (T) concentrations compared to their lean counterparts • High HOMA-IR • Had inverse co-relation of testosterone concentration to HOMA-IR • There was no difference in the insulin receptor expression between the two groups

40. References • 1 Ogden, C.L., Carroll, M.D., Curtin, L.R. et al. (2010) Prevalence • of high body mass index in US children and adolescents, 2007– • 2008. JAMA, 303, 242–249. • 2 Dabelea, D., Bell, R.A., D’ Agostino, R.B. Jr, et al. (2007) Incidence of diabetes in youth in the United States. JAMA, 297, • 2716–2724. • 3 Dhindsa, S., Prabhakar, S., Sethi, M. et al. (2004) Frequent occurrence of hypogonadotropichypogonadism in type 2 diabetes. • Journal of Clinical Endocrinology and Metabolism, 89, 5462–5468. • 4 Dhindsa, S., Miller, M.G., McWhirter, C.L., et al. (2010) Testosterone concentrations in diabetic and nondiabetic obese men. • Diabetes Care, 33, 1186–1192. • 5 Dandona, P. & Dhindsa, S. (2011) Update: hypogonadotropic • hypogonadism in type 2 diabetes and obesity. Journal of Clinical • Endocrinology and Metabolism, 96, 2643–2651. • 6 Chandel, A., Dhindsa, S., Topiwala, S. et al. (2008) Testosterone • concentration in young patients with diabetes. Diabetes Care, 31, • 2013–2017. • 7 Bhatia, V., Chaudhuri, A., Tomar, R. et al. (2006) Low testosterone and high C-reactive protein concentrations predict low • hematocrit in type 2 diabetes. Diabetes Care, 29, 2289–2294. • , dehydroepiandrosterone, and testosterone with pediatric • and adult reference intervals. Clinical Chemistry, 56, 1138–1147

41. 9 Moriarty-Kelsey, M., Harwood, J.E., Travers, S.H. et al. (2010) • Testosterone, obesity and insulin resistance in young males: evidence for an association between gonadal dysfunction and insulin resistance during puberty. Journal of Pediatric Endocrinology • and Metabolism, 23, 1281–1287. • 10 Salameh, W.A., Redor- Goldman, M.M., Clarke, N.J. et al. • (2010) Validation of a total testosterone assay using highturbulence liquid chromatography tandem mass spectrometry: • total and free testosterone reference ranges. Steroids, 75, 169–175. • 11 Beal, S.L. (2001) Ways to fit a PK model with some data below • the quantification limit. Journal of Pharmacokinetics and Pharmacodynamics, 28, 481–504. • 12 Sodergard, R., Backstrom, T., Shanbhag, V. et al. (1982) Calculation of free and bound fractions of testosterone and estradiol-17 • beta to human plasma proteins at body temperature. Journal of • Steroid Biochemistry, 16, 801–810. • 13 Vermeulen, A., Verdonck, L. & Kaufman, J.M. (1999) A critical • evaluation of simple methods for the estimation of free testosterone in serum. Journal of Clinical Endocrinology and Metabolism, • 84, 3666–3672. • 14 Hofstra, J., Loves, S., van Wageningen, B. et al. (2008) High prevalence of hypogonadotropichypogonadism in men referred for • obesity treatment. Netherlands Journal of Medicine, 66, 103–109. • 15 Kushnir, M.M., Blamires, T., Rockwood, A.L., et al. (2010) Liquid • chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric • and adult reference intervals. Clinical Chemistry, 56, 1138–1147

42. Thank you