Association Of Insulin Resistance And Testosterone Concentration In Young Pubertal And Post-pubertal Obese Males

Mursaleen Dar

Muniza MogriMentors: Dr. Paresh Dandona,MD

Dr. Husam Ghanim, PhDDr. Teresa Quattrin

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

Rationale

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

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)

• 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

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

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

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

and2. The insulin receptor expression is less in obese

young males compared to the lean ones based on HOMA-IR

Materials & Methods

• Cross-sectional observational study (Part 1) • Basic bench research (Western Blot) (Part 2)

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

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

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

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)

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

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

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

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.

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

• 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.

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

StatisticsData LEAN (25) OBESE (25) P valueAGE 16.5+/-1.4 16.0+/-1.5 0.21

BMI 20.9+/-2.2 36.0+/-5.3 < 0.001Tanner Stage 4.7+/-0.5 4.7+/-0.5 0.9Total Testosterone(ng/dl)

610+/-238 310+/-149 <0.001

Free Testosterone calculated (ng/dl)

12.7+/-5.1 7.6+/-3.2 <0.001

SHBG (nmol/l) 37.2+/-17.2 21.7+/-11.6 0.001Glucose (mg/dl) 79+/-7 80+/-10 0.65Insulin (uU/mL) 5.3+/-2.4 18.1+/-15.7 <0.001

HOMA-IR 1.05+/-0.47 3.83+/-4.13 <0.001

Sys BP 120+/-11 130+/-10 0.001

Diastolic BP 68+/-9 74+/-11 0.06

Pulse 67+/-13 75+/-15 0.05

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

Part 2 : Western Blot

• Detects specific proteins

• 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)

Steps for Part 2 : Western Blot

• 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

Steps for Part 2 contd…

• The MNC band harvested repeatedly and washed with Hank’s balanced salt solution (HBSS)

• Stored at -80*

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

• Why 16 pairs instead of 25 pairs?

• Inadequate blood samples• Suboptimal protein samples• Lost samples• Gel membrane constraints

• 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

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

.

Rainbow marker

Lean 1

2 3 4 Obese 1

2 3 4 Control

• 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

Results of Part 2

INSR- p-INSR- p-INSR-INSR-

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100LEANOBESE

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

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

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• Thank you