The effects due to diet variation changes plasma level of ghrelin, leptin and FFA in mice
Yu Chen, Lin
The University of Queensland, Australia
Abstract
The interaction of gastrointestinal regulated hormones plays in role in the maintenance of
energy expenditure with the influence of food deprivation. As we are interested to know the
hormonal regulation food dependent factor, mice with two different variances, normal food
supply and food deprivation were used for the study. The aim of this study was to find out if
fasting would stimulate or inhibit the release of ghrelin, leptin and FFA. It was speculated
that ghrelin and FFA will increase with food deprivation and the significance level in changes
of the ghrelin and leptin and their co-relationship. Food deprived mouse were subjected for
starvation for 18 hours while the control group was kept under normal food intake
circumstances, before collection of plasma samples. Plasma samples were assayed using
commercialized ELISA kit for presence of the hormones (ghrelin and leptin) and FFA under
both study groups. Absorbance reading at 450nm and 590nm was carried out for
quantification. Results returned showed remarkable high concentration of ghrelin in fasting
mice group. This is reflected on the level of FFA as well. Leptin showed a contrasting low
concentration in fasting mice. T test was done to show the high significance of the finding
with P value <0.0001 where the null hypothesis is rejected.
Background/Introduction
Under normal circumstances, glucose is the primary source of energy in the body. This is
often replenished with food. During food deprivation, glucose in the circulatory is first used
up and subsequently another source of energy fuel will be required to maintain normal
physiological activities within the body. Other sources includes glycogen, a stored form of
glucose in the liver will be converted to glucose. However, this source has small capacity. Fat
and protein are also alternative source of energy but protein is usually a preserved source,
thus the breakdown and release of free fatty acids (FFAs) that are stored in adipose tissues
can be used.
Ghrelin is a hormone produced mainly in P/D1 cell lining the fundus of human stomach and
epsilon cells in the pancreas. Concentration of ghrelin increase before meals and decrease
after meal is reflected with the desire to eat. Production of orexigenic signal during the
secretion of ghrelin in the stomach is one of its functions.
Counteracting to another hormone, leptin which is secreted by adipose tissue induces
satisfaction of food and thus stops food intake when present in high level usually observed
after food [1]. It is an anorexigenenic signal by contrast to ghrelin. Is it suggested that the
regulation of ghrelin and leptin is controlled by the presence of glucose in the circulatory [2].
Based on the orexigenic and anorexigenic actions of ghrelin and leptin, changes in peripheral
secretion of hormones would be observed during periods of fasting to promote food intake.
The aim of this study is to determine whether fasting in the mouse would stimulate or inhibit
the release of ghrelin, leptin and FFA. The impact of fasting on energy metabolism and
mechanisms that respond to physiological challenge of fasting in order to observe the
changes in levels of circulating ghrelin, leptin and FFAs. This is compared with a control
group where normal food availability is present.
The hypothesis of this study is mainly to prove that the level of ghrelin increment will be
observed in mice that are food deprived. Also, we are interested to see the significance level
in changes of the ghrelin and leptin and their co-relationship. FFA level was also
hypothesized to be increased during decrease level of leptin as it reflects the same action as of
ghrelin during food deprivation [2] [3].
The use of commercial ELISA kit was also used to quantify the concentration of ghrelin and
leptin. An understanding of “sandwich” Enzyme-Linked Immunosorbent Assay (ELISA)
theory would be applied as well.
Methods
Obtaining plasma samples from animal specimens
Mice of eight weeks old were used in obtaining plasma samples in this study. 10 mice were
used for the study were subjected to fasting, where the food was taken away at 10:30pm the
previous night and plasma samples were drawn at 3:30pm on average on 5th May 2010, with
a total fasting time of 18 hours. During the time of fasting, water was still given to the mice.
Control group of 10 mice were used to obtain plasma samples as well, but were not subjected
to any diet variance.
Mice were weighted at the start of the diet variance, and just before they were sacrificed. The
mice were anaesthetised with the injection of 32.5mg/kg of pentobarbital and plasma samples
were obtained with a syringe through the left ventricle only after the mice had no response
upon pedal withdrawal reflex. After blood had been collected, the mice were killed by
breaking the neck, to make sure they were dead before discarding. These were carried out by
trained personnel.
Ethical considerations and requirements were made so as to protect the use of animals in
research, where the stress on the animals was reduced as much as possible during the entire
course of the study. Negative impacts on the animals during extraction of blood samples
were minimized to where possible decreased the physiological impact on the mice in this
study.
Experimental Approach
Blood samples collected were spun down with “Tomy” bench top microcentrifuge at 5000g
for 5 minutes. Plasma was separated from the pellets containing other blood components and
aliquot into one eppendorf tube labelled “PMSF” of 100µL, and three labelled “Plasma” of
50µL each. 6µL of PMSF (phenylmethanesulfonylfluoride) was added to the “PMSF” tube
and resuspended, which is required for ghrelin assay. The other plasma containing tubes were
reserved for leptin and FFA assay. All eppendorf tubes were then stored in dry ice until
assayed.
Hormones and FFA Analysis
Quantification of ghrelin, leptin and FFA in the plama samples collected was done with
various commercial assay kit. The quantitation of mice ghrelin (active) in the collected
plasma samples were analyzed by the Linco® Rat/Mouse Ghrelin (active) ELISA kit (cat #
EZRGA-90K). The quanitification of leptin found in the blood plasma samples collected,
Millipore® Mouse Leptin ELISA kit (cat # EZML-82K). FFA was determined using Wako
NEFA-C assay. Plasma samples of various mice were analyzed and the plate had the
absorbance read using Tecan at 450nm and 590nm.
Statistic
Raw data of absorbance of various standard concentrations and plasma samples in ghrelin,
leptin and FFA assay were analysed where the background produced by the reagents were
accounted for, to produce an accurate concentration of ghrelin, leptin and FFA in different
mice variance. The concentrations were calculated through plotting of the standard regression
graph. T test was carried out to show the 5% significance in the hypothesized approach to
this study in each assays ran.
Results
Weight of Control Mice
27.0
Weight (g)
26.0
25.0
Start
24.0
Finish
23.0
22.0
21.0
20.0
1
2
3
4
5
6
7
8
9
10
Mice Specimens
Figure 1: The difference in weight (g) of the control mice group (no diet variant) at the start of the experiment
(blue) and just before they were scarified for collection of blood sample (red). The average weight of the 10
mice at the start of this study is 24.3g (range: 21.6 - 26.0g) and the average weight just before the extraction of
plasma samples is 23.7g (range: 20.8 - 25.4g). It was observed an average decrease of 0.6g during the period of
no diet variance in control mice.
Weight (g)
Weight of Fasting Mice
29.0
28.0
27.0
26.0
25.0
24.0
23.0
22.0
21.0
20.0
Start
Finish
1
2
3
4
5
6
7
8
9
10
Mice Specimens
Figure 2: The difference in weight (g) of the fasting mice group (diet variance) at the start of the experiment and
just before they were scarified for collection of blood samples (red). The average weight of the 10 mice at the
start of this study is 25.3g (range: 24.0 - 26.6g) and the average weight just before the extraction of plasma
samples is 23.8g (range: 22.4 – 26.3g). It was observed an average decrease of 1.5g during the period of diet
variance in fasting mice group.
Abs against Concentration (pg/mL)
0.8000
y = 0.0004x - 0.0182
R² = 0.9879
0.7000
0.6000
OD Reading
0.5000
0.4000
0.3000
0.2000
0.1000
0.0000
-0.1000 0
500
1,000
1,500
2,000
[Ghrelin] (pg/mL)
Figure 3: Known standard concentrations of ghrelin plotted against absorbance reading (OD Reading) in
regression curve, obtaining the R2 value of 0.9879. Concentration of ghrelin in each mice sample of control and
fasting mice group was then derived with the standard curve equation plotted.
Figure 4: Higher level of ghrelin concentration (pg/mL) in fasting mice group’s plasma sample was observed as
compared to control mice group which has no diet deprivation. T test was done to generate P<0.0001, showing
the 5% confidence interval and thus proving the significance of increase in ghrelin level during low food input
in mice, with the null hypothesis rejected. The level of ghrelin in fasting mice group shows 4.33 times higher
than that of control mice group on average. Error bars showed the standard deviation of ghrelin concentration
range and the column itself shows the mean of data.
Figure 5: Higher level of leptin concentration (ng/mL) was observed in control mice group’s plasma samples, as
compared to fasting mice group. This is shown as a counteracting effect as to the level of ghrelin in the plasma
samples, had higher level in fasting mice group. R2 value produced from the standard concentration curve of
leptin was 0.9913. P value of 8.82322E-08 was obtained with paired T test. Leptin level in control mice shows a
6.3 times higher than that in fasting mice on average. Error bars showed the standard deviation of leptin
concentration range in both mice group with the column bar itself shows the mean of data.
Figure 6: Higher level of FFA concentration (uEq/L) was observed in fasting mice group’s plasma samples, as
compared to control mice group. FFA was more prevalent in fasting mice group due to the low level of glucose,
resulting in breaking down of stored energy sources to FFA. FFA concentration in fasting mice group shows a
6.3 times higher than in control mice group on average. Error bars showed the standard deviation of leptin
concentration range in both mice group with the column bar itself shows the mean of data. FFA and ghrelin
concentration in plasma samples both showed similarity in fasting mice group as higher than that of in control
mice group.
Discussion
Weights of mice that are sacrificed for this study were recorded at the start of any diet
variance between 10 fasting mice and 10 control mice. In fasting mice group, food was
removed at 10:30pm, 18 hours before blood samples were drawn. Both control and fasting
mice has the constant water availability during the period of 18 hours, until they were brought
to the laboratory for blood sample collection. The average starting weight of the control mice
is 24.3g (range: 21.6-26.0g) and the average weight before the start of blood sample drawing
is 23.7g (range: 20.8-25.4). It shows an average weight loss of 2.4691%. The average weight
of the fasting mice is 25.3g (range: 24.0-28.0g) and the average weight before the start of
blood sample drawing is 23.8g (range: 22.4-26.3g), showing an average weight loss of
5.9289%. A higher decrease of weight is observed in fasting mice due to the exhaustion of
glucose and subsequently glycogen and fats during the period of no external source of fuel
given, as well as normal water excretion. The observed decrease in weight of control mice
can be presumed for the loss of normal water excretion.
Mice have been used in this study instead of human specimens as it is of academic purpose,
and also limited to regulations and time containment. Mice show a much exaggerated impact
of starvation as compared to the human body due to the difference in body size as well as
metabolic rates. Thus, consideration of fasting period was taken into account to, to promote a
similar simulation in a human specimen.
In all the quantification assays of ghrelin, leptin and FFA, changes were observed in the level
of each hormones and FFA that we are interested to find out during an impact of diet
deprived condition, as compared to a normal control, where diet is not compromised.
In ghrelin, there is a significant increase in concentration observed in fasting mice. The
increment is calculated to an average of 62.48%. This observation is supported in
publications showing that the secretion of ghrelin enhances the need and for normal drive of
food when the body is low in primary source of fuel [2]. T test was done to show the
confidence interval at 5% significance to prove that our hypothesis was supported. P value of
<0.0001 was generated, which shows the high significance that the production of ghrelin
increases during food deprivation was supported.
In leptin, a drastic difference averaged to 72.62%, which is much lower, observed in fasting
mice. It is suggested that the regulation of leptin is dependent on the amount of glucose in the
circulatory, as during low plasma glucose level, it is seen that level of leptin decreased [3] [4].
This is also observed in our study showing the drop of leptin level in fasting mice as to
control mice.
In FFA, it is observed that there is an increment average of 54.58% was seen when the mice
were fasted. This is due to the breakdown and release of energy from FFAs that are stored in
adipose tissue. Persistent fasting increases the level of FFA in the circulation due to the
depletion of glucose or glycogen.
In the raw data of leptin assay, standard concentration of 20ng/mL did not fit as well to the
consistency of the other concentration, thus affecting the R2 value overall, and generating
negative values for subsequent concentration calculation of leptin in control and fasting mice.
This was due to human error of insufficient addition of detection antibody or the enzyme
substrate as there was no colour observed unlike other wells.
Also, quality control (QC) of the test kit was done as well to ensure the kit is working and the
results generated from the kit are of accuracy. QC1 and QC2 samples were added before the
additional of control and fasting mice plasma samples. This also ensures that during the
course of the assay, no contamination of wells or any unexpected results showing at the end.
The expected ranges of QC1 and QC2 in ghrelin assay are 31-65pg/mL and 256-533pg/mL
respectively. However, the actual result of QC1 and QC2 were 81.556pg/mL and
282.625pg/mL. For leptin assay, the expected range for QC1 and QC2 are 1.03-2.13ng/mL
and 3.1-6.4ng/mL, and the results produced were 0.0744ng/mL and 2.347ng/mL respectively.
This indicates that the concentration of ghrelin and leptin in control and fed mice has a
possibility of inaccuracy within.
In conclusion of the data and concentration generated for ghrelin, leptin and FFA, there is
clear relationship between these variable, with the diet variance of control and fasting mice.
In control mice group, lower level of ghrelin is observed as compared to the concentration of
leptin. Contrastingly, it is observed a higher level of ghrelin than leptin in fasting mice. This
clearly shows an inversely proportional distribution on the concentration of ghrelin and leptin
during control and fasting circumstances. The reason being both has functions that is
opposition to each other, ghrelin produces orexigenic signal, stimulating the desire to eat,
while leptin produces anorexigenic signal, stimulating the control of stopping food intake.
To improve the experiment design, transportation of mice can be decreased by having smaller
practical class and doing the extraction of plasma samples can be carried out in the
comfortable environment to that of mice. By this, stress level due to environment changes in
the mice can be decreased. Consistency of one handler should be observed in doing assays,
which is able to variations in handling techniques and thus producing a more reliable result.
However, inconsistency is inevitable due to nature of this study is carried out which is for
academic demonstration, which should be accounted for.
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