Download Effect of Ghrelin Antagonist on Serum Testosterone and Luteinizing

Journal of Rawalpindi Medical College (JRMC); 2014;18(1):32-36
Original Article
Effect of Ghrelin Antagonist on Serum Testosterone and
Luteinizing Hormone in Obese Type 2 Diabetic Mice
Lubna Siddique*, Umbreen Ahmed**, Tausif Ahmed Rajput***
*Department of Physiology, Margalla Institute of Health Sciences,Rawalpindi;** Department of Physiology, Army Medical College,
Rawalpindi;*** Department of Biochemistry and Pharmaceutical Chemistry, Margalla Institute of Health Sciences,Rawalpindi .
Abstract
resistance.More than 80 % cases of type 2 diabetes
mellitus are obese. 4 Obesity leads to a decrease in
expression and translocation of glucose transporter
type 4 (GLUT4) in adipocytes. In addition there is also
loss of tyrosine phosphatase activity leading to a loss
of 80-90% of its function. 5 Normalization of body
mass index leads to reversal of all these mechanisms
leading to an improvement in efficacy of blood glucose
control mechanisms. 6
It has been found that obesity directly and
indirectly affects the male reproductive functions,
leading to a decrease in the secretion of male sex
hormone i.e. testosterone. 7 A variety of cytokines,
adipokines and free fatty acids are secreted from
adipocytes which directly affects the plasma androgen
levels.8 The quantitative and qualitative efficacy of
leydig cells is also decreased. There is decrease in the
expression of glucose transporter type 3 (GLUT3) on
leydig cells. In addition there is also decrease in its
tyrosine phosphorylation activity leading to loss of its
function.9 Obesity may also affect male reproductive
system indirectly via insulin resistance. Insulin
resistance and plasma testosterone levels are found to
be in negative correlation with each other. 10 The
mentioned effects may also be exerted through ghrelin
hormone which is increased in obesity and type 2
diabetes mellitus. 11
Ghrelin a 28-amino acid peptide hormone was
discovered in 1999 from the stomach extracts of rats. 12
It is a hunger stimulating hormone secreted from
stomach and pancreas. Ghrelin receptors (G proteincoupled receptors), are expressed in a number of
tissues including pituitary, stomach, intestine,
pancreas, thymus, gonads, thyroid, and heart.13 The
wide expression of ghrelin receptors suggests that this
hormone has diverse functions in different tissues.
Ghrelin decreases the secretion of insulin from beta
cells of pancreas in response to hyperglycemia (by
decreasing the influx of calcium in the beta cells of
pancreas secondary to hyperglycemia), leading to an
increase in the plasma glucose levels. 14 D-Lys3 GHRP6
(His-D-Lys-Trp-D-Phe-Lys-NH2) acts as ghrelin
receptor antagonist. It acts on ghrelin receptor GHS-
Background: To determine the effect of ghrelin
antagonist (D-Lys3) on plasma glucose, insulin
resistance, serum testosterone and luteinizing
hormone levels, on obese and type 2 diabetic mice.
Methods: Fifty healthy BALB/c mice were divided
into five groups. Mice in group II to V were fed
high-fat diet for 4 weeks followed by intraperitoneal
(IP) injection of streptozotocin to group IV and V to
induce type 2 diabetes mellitus. Group I served as
control.Groups II and III were obese and group IV
and V were diabetic. Groups III and V were injected
ghrelin antagonist IP for 6 days. Terminal
intracardiac blood extraction was done and samples
were analyzed for fasting plasma glucose, serum
insulin, testosterone and luteinizing hormone.
Insulin resistance was calculated by homeostatic
model assessment for insulin resistance.
Results: Plasma glucose level and insulin resistance
decreased and serum testosterone and luteinizing
hormone levels increased after using ghrelin
antagonist in group III and V.
Conclusion: Glucose level and insulin resistance
were significantly decreased in obese and type 2
diabetic
mice
after
ghrelin
antagonist
administration. Whereas serum testosterone and
luteinizing hormone levels were increased
Key Words: Ghrelin antagonist ,testosterone,
luteinizing Hormone, obese Type 2 Diabetic Mice
Introduction
Obesity is considered as the epidemic of the
modern world. 1 The incidence of obesity has doubled
in the past twenty years, which may be due to altered
eating habits and sedentary life style. 2 It not only
affects the quality of life by decreasing the general
body performance, but also leads to the development
of many metabolic disorders including diabetes
mellitus, insulin resistance, fatty liver disease and
atherosclerosis. 3
Type 2 diabetes mellitus is characterized by
hyperglycemia,
hyperinsulinemia
and
insulin
32
Journal of Rawalpindi Medical College (JRMC); 2014;18(1):32-36
R1a and has been used in numerous studies.
Administration of ghrelin receptor antagonist (D-Lys3 )
in normal and obese mice, leads to an increase in the
secretion of insulin accompanied with decrease in
blood glucose levels.15, 16
Ghrelin acts as a negative modulator of male
reproductive system. It causes a decrease in number of
leydig and sertoli cells in rats. 17 The high level of
ghrelin is associated with a decrease in plasma
testosterone and gonadotropin hormones (leutinizing
hormone and follicle stimulating hormone.18 Ghrelin
also acts upstream in hypothalamo- hypophyseal
gonadal axis and reduces the pulse frequency of
gonadotropin-releasing hormone (GnRH). 19 Ghrelin
antagonist improves testosterone production in obese
mice. 20 The mechanism involved is not clear. Ghrelin
antagonist may act via luteinizing (LH) which in turn
increases the secretion of testosterone. The purpose of
this study was to identify the site of action of ghrelin
antagonist in the hypophysial-gonadal axis, which is of
clinical significance in obese and diabetic human male
reproductive health.
induces rapid and extensive degradation of cellular
deoxyribonucleic acid (DNA) of beta cells of
pancreas.21, 22 All the other groups were injected
intraperitoneally with normal saline. Tail vein blood
samples were used to measure (fasting) blood glucose
levels and to confirm diabetes. The blood glucose
level >252 mg/dl was taken as the cut off value for
confirming diabetes mellitus. 22 This was followed by
intraperitoneal injection of ghrelin antagonist 10
μmol/kg for 6 days in 9th week 23 to group III and V.
On 62nd day , terminal blood sample was collected by
intracardiac puncture. 24 The samples were stored at 20o C for further analysis. The statistical significance of
differences across the groups was determined by
applying analysis of variance Post-hoc (ANOVA)
followed by Tukey’s HSD to find the difference in
various pairs of groups. P value < 0.05 was considered
significant.
Results
There was no significant difference in body weight
of mice at the start of study (Table 1). The average
weight of the mice was 28.07g ± 0.10. There was
continuous weight gain by the mice throughout the
experiment. Weight of the mice in obese group
treated with ghrelin antagonist (group III) were
significantly reduced compared with obese group II (p
=0). Ghrelin antagonist treated diabetic group (group
V) mice showed significant reduction in weight as
compared with diabetic group IV (p = 0).
Materials and Methods
This study was conducted at Department of
Physiology, Army Medical College, Rawalpindi, in
collaboration with National Institute of Health (NIH),
Islamabad, Pakistan. The study was approved by
animal ethics committee of Army Medical College,
NUST
and
all
animals
received
humane
care.Streptozotocin (STZ) and ghrelin antagonist were
purchased from Bioplus Fine Research Chemicals and
Sigma Chemicals, USA respectively. Glucose was
measured by GOD-POD method.Enzyme linked
immunosorbent assay (ELISA) was used to measure
serum insulin, serum testosterone and serum LH
levels.
Five weeks old, 50 healthy BALB/c male mice were
purchased from National Institute of Health,
Islamabad. An average weight of mouse was 28.07 g ±
0.1. Mice were divided into five equal groups. They
were given free access to food and water. The room
was well ventilated with controlled temperature range
of 20-22°C and 12 hours light and dark cycles were
maintained. After acclimatization the animals were
randomly divided into five groups. Group 1 (control)
was fed normal pellet diet, while group II-V were
given high fat diet ad libitum (to induce obesity and
insulin resistance) for 4 weeks. After 4 weeks, group
IV and V were given intraperitoneal injections of
streptozotocin 50mg/kg body weight for 5 days, as
previously described to induce type 2 diabetes. STZ
Table1. Weight of BALB/c mice at start and end of
experiment
Weight
of
mice in grams
Group I
Control
Group
II
Obese
Group III
Obese with
ghrelin
antagonist
Group
IV
Diabet
-ic
Group V
Diabetic
with
ghrelin
antagonist
Start
of
experiment
28.1±
0.27
28.09±
0.45
28.18± 0.21
27.91±
0.38
28.07± 0.38
End
of
Experiment
32.81±
1.56
39 ±
0.68
36.7 ± 0.62
41.2 ±
1.48
37.21± 0.37
There was no significant difference in the blood
glucose level of different groups at the start of the
experiment (Table 2). After 8 weeks of study i.e. on
day 55, fasting blood glucose levels were significantly
high in group IV and group V compared with the
control (group I) (p = 0). There was no significant
difference between other groups.At the end of the
experiment blood glucose level in terminal blood
samples showed that group II was significantly raised
compared with the control and group III (p = 0) while
33
Journal of Rawalpindi Medical College (JRMC); 2014;18(1):32-36
it was significantly low compared with group IV and
group V ( p = 0 ). Group IV was significantly high
compared with Group I and V (p = 0). Blood glucose
level at the end of the experiment was significantly
higher in Group V compared with the control (p = 0).
Ghrelin antagonist significantly reduced the blood
glucose levels in group III (p = 0) and Group V (p = 0)
compared with group II and group IV respectively.
in group III compared with group II (p = 0.05).
Testosterone levels in group V (72.5% increase) were
significantly high (p = 0.006) compared with group IV.
The levels of testosterone were significantly lower in
all groups compared with the control group I (p <
0.05).The levels of LH were significantly high in group
III compared with group II (p = 0) and in group V
compared with group IV (p = 0). The levels of
luteinizing hormone was significantly lower in all
other groups compared with the control group 1 (p =
0).)Table 3)
Table 3. Serum testosterone and luteinizing hormone
levels in BALB/c mice at the end of the experiment
Table 2. Fasting blood glucose, insulin and HOMA
IR levels in BALB/c mice
Blood
parameters
Initial Blood
glucose level
(mg/dl)
Blood
glucose level
at 8 weeks
(mg/dl)
Blood
glucose level
in
terminal
samples
(mg/dl)
Serum
Insulin levels
(µIU/ml)
(terminal
sample)
Insulin
Resistance
HOMA-IR
(terminal
sample)
Group
I
n = 10
97.2 ±
11.87
Group
II
n = 10
101.89
± 16.07
Group
III
n = 10
107.70 ±
13.74
Group
IV
n = 10
105.90
± 0.93
Group
V
n = 10
109.67 ±
17.25
100.2 ±
9.59
116 ±
11.05
118 ±
10.91
383.6 ±
17.06
395.67 ±
20.73
92.6 ±
10.17
169.9 ±
9.8
103 ±
7.3
488.8 ±
17.19
257.56 ±
20.09
2.17 ±
0.10
3.11 ±
0.09
3.43 ±
0.14
4.77 ±
0.13
5.07 ±
0.13
0.49 ±
0.05
1.29 ±
0.09
0.87 ±
0.08
5.75 ±
0.22
3.24 ±
0.29
Hormonal
Profile
Serum
Testosterone
pg/ml
Serum LH
(mIU/ml)
Group I
791.11
±
107.02
8.3±
0.45
Group
II
434.35
±
82.80
5.16 ±
0.55
Group
III
585.07
±
124.88
7.68 ±
0.24
Group
IV
268.19
±
132.19
2.59 ±
0.45
Group
V
462.75
±
138.75
6.34 ±
0.42
Discussion
This study was performed to find the link between
obesity, type II diabetes and infertility in males.
Previous
studies
showed
that
ghrelin,
a
gastrointestinal hormone, may be involved in this
cascade. Ghrelin increases the level of plasma glucose
and decreases insulin and testosterone levels. 15, 25 This
study was performed to see the reversal of these
effects by ghrelin antagonist. We used mice model in
our study as has previously been used. 15 High fat diet
with 60% fat was given to induce obesity. Diabetes
type II and insulin resistance was confirmed in group
IV and V by fasting blood glucose levels as previously
described.22
Ghrelin is a recently identified potential regulator
of both energy homeostasis and reproductive function.
Previous studies show that it has a diabetic like effect,
increasing blood glucose levels and decreasing plasma
insulin levels. 15 It interacts with the reproductive axis,
decreasing plasma testosterone levels. 25 On the other
hand, Ghrelin antagonist causes a reversal of these
effects, causing an increase in plasma insulin levels
and normalizing blood glucose. 26 Our results also
support the previous studies in animal models where
ghrelin antagonist decreased blood glucose levels in
normal and obese mice. 15,16,27 This decrease in blood
glucose level may be due to an increased secretion of
insulin promoted by ghrelin antagonist. Our results
showed that insulin levels were significantly increased
in both obese and diabetic groups treated with ghrelin
antagonist (III and V). The insulin levels were 9.3 %
more in obese group III compared with obese group II.
At the end of experiment fasting serum insulin
levels were significantly high in group II, IV and V
compared with control. There was no significant
difference between group III and control. In addition,
insulin levels were significantly raised in ghrelin
antagonist treated groups compared with their
respective untreated groups.
The levels were
significantly raised in obese group III and diabetic
group V compared with obese group II (p = 0.001) and
diabetic group IV (p = 0) respectively (Table 2). Insulin
resistance (HOMA-IR) was decreased in ghrelin
antagonist treated groups compared with untreated
groups. HOMA-IR in group III was significantly low
compared with group II (p = 0). Group V was
significantly low compared with diabetic group IV (p
= 0).
Levels of testosterone were increased in ghrelin
antagonist treated groups. Although not reaching
significant difference, the levels were 34.7% increased
34
Journal of Rawalpindi Medical College (JRMC); 2014;18(1):32-36
Similarly blood insulin levels were 5.7 % more in
diabetic group V compared with group IV. 28 It is
suggested that ghrelin antagonist enhances the insulin
secretion by blocking ghrelin receptor thus preventing
ghrelin inhibitory action on beta cells of pancreas.28
Increase in ghrelin is associated with insulin
resistance. Ghrelin depresses the expression and
translocation of glucose transporter type 4 (GLUT 4) in
muscles causing insulin resistance and ghrelin
antagonist leads to its improvement as confirmed by
HOMA-IR measurement. 23 Insulin resistance in
antagonist treated groups was significantly low
compared with their respective untreated groups
(obese group III vs obese group II and diabetic group
V vs diabetic group IV).Serum ghrelin levels in obese
subjects are positively related to insulin resistance.29
The significant reduction in HOMA-IR in our study
suggests a positive role of ghrelin antagonist in
normalizing the plasma glucose levels.
Previous studies suggest a close association
between obesity, diabetes and male reproductive
dysfunctions leading to a decrease production of
testosterone by the leydig cells. 7, 30 We also showed
that the levels of testosterone in obese group II, was
markedly reduced compared with the control.
Although we did not measure the enzyme activity in
our study, previous studies show that there is an
increased activity of enzyme aromatase (involved in
conversion of testosterone to estrogen) in the adipose
tissue.31 Enhanced activity of aromatase in obesity
causes a feedback inhibition of the gonadotrophinmediated testosterone secretion.32 Both these effects
lead to a decrease in testosterone production.
Metabolic disturbances like diabetes mellitus also
lead to impaired reproductive function by disturbing
the pituitary hypothalamic reproductive axis leading
to decreased levels of luteinizing hormone, follicular
stimulating hormone and testosterone.28 In rat model,
ghrelin significantly decreased the testosterone
secretion and produced degenerative changes in the
testis by decreasing the stem cell factor, which
regulates the development of leydig cells.26 Ghrelin
has anti-proliferative effects on different testicular cell
types and is a negative modulator of male
reproductive system, leading to a decreased
testosterone production by altering the cellular
morphology and affecting different cell organelles. 33
These negative effects of ghrelin on testosterone,
luteinizing hormone and follicular stimulating
hormone levels are also seen in humans.18 It is
suggested that ghrelin negatively modulates the
hypothalamic-pituitary axis and reduces the pulse
frequency of gonadotropin-releasing hormone which
ultimately
suppresses
levels
of
luteinizing
hormone.19,34,35 There is a down regulation of Kiss1
expression in hypothalamus that in turn decreases the
pulsatile secretion of LH. 36 Few researchers have
shown that ghrelin decreases the testosterone
synthesis by affecting the enzymes involved in
movement of cholesterol from outer to inner
mitochondrial membrane or P450cc that is mainly
involved in conversion of pregnenolone to
testosterone. 19
Conclusions
1.Glucoes level and insulin resistance decrease
significantly in obese and type 2 diabetic mice after
ghrelin antagonist (D-Lys3) administration
2.Administration of ghrelin antagonist to obese and
diabetic groups significantly improves the levels of
testosterone and luteinizing hormone
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