Transcript Slide 1

Association Of Insulin Resistance
And Testosterone Concentration In
Young Pubertal And Post-pubertal
Obese Males
Mursaleen Dar
Muniza Mogri
Mentors: Dr. Paresh Dandona,MD
Dr. Husam Ghanim, PhD
Dr. 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 middleaged 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
and
 2. 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 ranksum 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
Statistics
Data
LEAN (25)
OBESE (25)
P value
AGE
16.5+/-1.4
16.0+/-1.5
0.21
BMI
Tanner Stage
Total
Testosterone(ng/dl)
Free Testosterone
calculated (ng/dl)
SHBG (nmol/l)
Glucose (mg/dl)
Insulin (uU/mL)
20.9+/-2.2
4.7+/-0.5
610+/-238
36.0+/-5.3
4.7+/-0.5
310+/-149
< 0.001
0.9
<0.001
12.7+/-5.1
7.6+/-3.2
<0.001
37.2+/-17.2
79+/-7
5.3+/-2.4
21.7+/-11.6
80+/-10
18.1+/-15.7
0.001
0.65
<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
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)
• 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
100
LEAN
OBESE
90
80
Arbitrary Units
70
60
50
40
30
20
10
0
INSR-
p-INSR-
p-INSR- INSR-
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
References
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• Thank you