Association Of Insulin Resistance And Testosterone Concentration In Young Pubertal … · 2017. 12. 29. · gonadal dysfunction and insulin resistance during puberty. Journal of Pediatric

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

Obese Males Mursaleen Dar, MD

Muniza Mogri, MD

Mentors: Dr. Paresh Dandona, MD Dr. Husam Ghanim, PhD Dr. Teresa Quattrin, MD

Objective

• To show whether obesity is associated with lower testosterone concentrations in young 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 possibly future hypogonadotropic hypogonadism

Background • 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

Ogden et al. (2010) Prevalence of high body mass index in US children and adolescents. JAMA. Dabelea et al. (2007) Incidence of diabetes in youth in the United States. JAMA.

• Type 2 diabetes and obesity are associated with a high

prevalence (25–33%) of hypogonadotropic hypogonadism in middle-aged and elderly men

Dhindsa et al. (2004) Frequent occurrence of hypogonadotropic hypogonadism in type 2 diabetes. Journal of Clinical Endocrinology and Metabolism. Dhindsa et al. (2010) Testosterone concentrations in diabetic and nondiabetic obese men. Diabetes Care.

• In all these studies, free testosterone (FT) and total testosterone (TT) 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 (HOMA-IR) • Quantify insulin resistance and beta cell

function

22.5

Background...

• 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 suppression in insulin signaling activity in young obese males

Hypothesis

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 activity is less in obese

young males compared to the lean ones

Materials & Methods

• Cross-sectional observational study (Part 1) • Western Blot (Part 2) • Informed consent • Parental consent in addition to children’s

consent for less than 18 years of age • Institutional Review Board Approved –

Women & Children’s Hospital, University at Buffalo

Part 1 : Cross-sectional study

“…observation of a representative subset of a

population at one specific point in time…” “… a snapshot…”

Steps in Methodology for Part 1

• 50 males between the ages of 14-20 years and Tanner stage of 4-5 consecutively recruited at the Endocrine and Diabetes Center and Women and Children’s Hospital of Buffalo

• 25 obese subjects -BMI >95th percentile for age (CDC definition) • 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 less than 5%

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 • Active infection or recent surgery or

hospitalization prior 6 weeks

Methodology

• Healthy subjects, without significant co-morbidities

• Tanner staging assessed using orchidometer - testicular volume 12–15 ml Tanner stage 4 - testicular volume >15 ml Tanner stage 5 • 8 lean & 7 obese were Tanner 4, rest Tanner 5 • Height measured to nearest 0.1 cm • Weight measured to nearest 0.1 kg • Blood pressure, heart rate

Methodology...

• Fasting blood sample between 8 and 10 am – -Total and free testosterone (TT & FT) -Estradiol (E) -Sex Hormone Binding Globulin (SHBG) -Luteinizing Hormone (LH) -Follicle Stimulating Hormone (FSH)

Methodology...

• Insulin concentrations - ELISA kit • Glucose concentrations - glucose analyzer • HOMA-IR was calculated from above variables • These assays were performed at the research

laboratories of the division of Endocrinology and Metabolism, University at Buffalo and Quest Labs, Chantilly, VA

Statistical Analysis

• Group comparisons: 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 : ANCOVA and generalized linear model analysis

• P < 0.05 considered significant

Results

Data LEAN (25) OBESE (25) P value AGE 16.5±1.4 16.0±1.5 0.21

BMI 20.9±2.2 36.0±5.3 < 0.001

Tanner Stage 4.7±0.5 4.7±0.5 0.9

Sys BP 120±11 130±10 0.001

Diastolic BP 68±9 74±11 0.06

Pulse 67±13 75±15 0.05

Results

Data LEAN (25) OBESE (25) P value Total T (ng/dl) 610±238 310±149 <0.001

Free T (ng/dl) 12.7±5.1 7.6±3.2 <0.001

SHBG (nmol/l) 37.2±17.2 21.7±11.6 0.001

Total E (pg/ml) 18.0±11.1 20.6±9.9 0.33

Free E (pg/ml) 0.31±0.21 0.43±0.21 0.02

FSH (IU/l) 4.0 ± 3.9 3.2 ± 2.1 0.46

LH (IU/l) 3.5 ± 1.7 3.2 ± 1.4 0.49

Glucose (mg/dl) 79±7 80±10 0.65

Insulin (uU/mL) 5.3±2.4 18.1±15.7 <0.001

HOMA-IR 1.05±0.47 3.83±4.13 <0.001

Results Of Part 1

• Testosterone concentrations of young obese (P) and (PP) males are 40–50% lower than those with normal BMI (p<0.001)

• And low testosterone levels in obese population had inverse correlation with HOMA-IR (p<0.001)

Part 2: Basic Bench Research

Clinical & Translational Research Center

© buffaloctrc.org

Part 2: Basic Bench Research: Western Blot

• Detection of Insulin Receptor by Western Blot • Insulin receptor is a protein • Sample Source : Blood • PMN Cells – peripheral mononuclear cells or

MNCs

Image source

Insulin Receptor

Western Blot overview

Image source

• Blood samples were collected in Na-EDTA anticoagulant

• 4.5mL of anticoagulated blood sample were carefully layered over 3.5 mL of PMN isolation media

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 • The MNC band were harvested repeatedly and washed with Hank’s balanced salt solution (HBSS) • Stored at -80*

Sample preparation

Preparing the lysate

• Out of 50 samples, 32 were found suitable for testing

• MNC cell lysates of 32 samples (16 obese and 16 lean) were prepared

Image source

• After 30 min incubation on ice with lysis buffer and protease and phosphatase inhibitors, samples were vortexed and centrifuged for 10 mins

• Supernatants collected and total protein concentrations determined

• 60 ug of total cell lysate were boiled in 2X SDS buffer followed by Gel Electrophoresis

Preparing the lysate

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Wells

Lean 1

Lean 2

Lean 3

Lean 4

Obese 1

Obese 2

Obese 3

Obese 4

Sample Sequence

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Gel Electrophoresis

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Transfer of Proteins

Detection of insulin receptor

• Polyclonal and monoclonal antibodies against insulin receptor-β subunit (IR-β) and phosphotyrosine IR-β (p-IR-β) were used and the membranes developed using super signal, chemiluminescence reagent

Image source

Results of Part 2

IR-β

p-IR-β

INSR-β p-INSR-β p-INSR-β/INSR-β

Arbi

trary

Uni

ts

0

10

20

30

40

50

60

70

80

90

100LEANOBESE

Colorimetric Analysis

Discussion • Based on the results of part 1 & 2, we found out that young obese

(P) and (PP) males have lower total and free T concentrations • Consistent with the studies in adults, the concentrations of LH and

FSH were not elevated and hence were inappropriately low indicating central suppression of the hypothalamo-hypophyseal-gonadal axis

• Obese have high HOMA-IR compared to their lean counterparts • However the expression and phosphorylation of insulin receptor on

the MNCs is not different between the two groups • Does it mean there is properly functioning insulin receptor in obese

individuals? • If so, why is HOMA-IR high? • Do we have the right body tissue?

Discussion • Right body tissue? – If Yes: why high insulin

burden • Insulin receptor not modulating GLUT4? • Intermediary pathway problems..?

Discussion • Right body tissue? – If Yes: why high insulin

burden

©openi.nlm.nih.gov

Discussion • Right body tissue? If NO… • Biopsy adipose tissue or muscles and check

Insulin Receptor expression • Use clamp method to check insulin resistance

instead of HOMA-IR

Take home points… • Young obese (P) and (PP) males have

significantly lower total and free (T) concentrations compared to their lean counterparts

• High HOMA-IR • Inverse co-relation of testosterone

concentration to HOMA-IR • No difference in the insulin receptor

expression between obese and lean

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Acknowledgements

• Sanaa Abuaysheh (CTRC) • Dr. Khalid J. Qazi, MD • Dr. Henri Woodman, MD • Dr. Zubair Butt, MD

© buffaloctrc.org