MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF PHYSIOLOGY
NAME REG NUMBER COURSE
MWESIGE RONALD 19/U/0286 BMAM

THEME: HORMONAL EFFECT ON BLOOD SUGAR LEVELS.

ABSTRACT
This experiment was carried out to investigate the effect of two hormones; insulin and
adrenaline on blood glucose levels. Rabbit under anesthesia was used. The anesthesia was
made by mixing equal volume of 5% chloralose in 5% borax an 20% urethane in saline.
After injecting the animals with 20% glucose the first sample was collected ten minutes
after introducing glucose solution intravenously. Saline (0.9% sodium chloride solution),
insulin and adrenaline were introduced subcutaneously in that order and two blood samples each
collected at a 20 minutes interval. Saline was used as control.

The blood samples were heparinized to prevent coagulation and then centrifuged to
separate the cells from the plasma. The plasma was the treated with enzyme-dianisidine in
a series of various steps. The absorbances of this mixture, obtained using the two
hormones and saline were determined. Standard solutions of glucose were also made by
different dilutions. The results were tabulated and three graphs were plotted; the first was
a standard glucose curve, the second was of blood sugar levels against time for the various
treatments, and the third was the calculated percentage changes that occurred in the
samples against time.

It was established from the findings that insulin causes a reduction in the blood glucose
levels, while adrenaline causes an increase in blood glucose levels. Saline had no effect on
the blood glucose levels

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INTRODUCTION
It is essential that blood sugar level is maintained constant. In order to do this, there is
interplay of various hormonal factors including insulin, adrenaline, glucocorticoids
(cortisol), glucagon and somatotropin (human growth hormone).

AIM
To investigate the effects of insulin and adrenaline hormones on blood sugar level in a
rabbit.

OBJECTIVES
 To investigate the effect of insulin and adrenaline hormones on blood glucose levels.
 To explain the mechanism by which these hormones carry out their actions to bring
about the different effects on blood glucose levels.
 To state the relationship between insulin and the diabetic state.
 To plot a standard glucose curve
 To plot a curve of blood sugar levels against time for the various treatments.
 To calculate the percentage change occurring in the samples, and to plot these values
against time.

BACKGROUND
All metabolizing cells require a supply of glucose in order to continue functioning. glucose
is the key metabolite that generates energy in form of ATP. Brain cells which utilize glucose
D entirely are particularly sensitive to reduction in blood glucose levels which is 90mg per
100ml of blood. A rise in the blood glucose level can be equally dangerous. The supply of
carbohydrates in mammals fluctuates because they don’t eat continuously throughout the
day and the quantity of carbohydrate varies from meal to meal. Cells however metabolize
continuously and need a constant supply of glucose to sustain them. A system which
maintains a constant glucose level in blood, despite intermittent supplies from intestines, is
essential. The liver in conjunction with hormones such as insulin, glucagon, adrenaline,
thyroxin, growth hormone and Cortisol maintain the blood glucose levels constant.

In this experiment, the plasma was treated with a mixture of glucose oxidase and
peroxidase. These enzymes catalyze the reactions.
1. D-glucose + H2O + O2 = D-gluconic acid + H2O2
2. H2O2 + Donor – H2= 2 H2O2 + Donor

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‘Donor’ is a colored dye: the amount formed is a measure of the glucose oxidized. If o-
dianisidine is used as donor the optical density at about 450mu is related to the glucose
content

METHODOLOGY

APPARATUS

Three way tap cannula for iv infusion and for collection of blood samples

Calorimeter for estimation of blood glucose levels of the sample

Cuvettes for estimation of blood glucose levels

2ml or 5ml syringe for collection of the blood from the animal

Centrifuge for separation of the plasma and blood cells

Heparinized test tubes to prevent blood for clotting when taken out of the animal

Reagents used

a. Anesthetic
i. 5% Choralose in 5% sodium tetra hydroborate (borax)
ii. 20% Urethane in Saline
b. 20% glucose (2 ml/kg body weight)
c. Adrenaline 1 ml/ml (1 ml/kg body weight)
d. Insulin 2 U/ml (2 units/kg body weight)
e. Saline (1 ml/kg body weight)

PROCEDURE
The rabbit was weighed and, using an ear vein, 2.8 ml/kg of choralose – urathane was
injected. The anesthetic was not immediately effective but lasted for some hours and didn’t
need supplementing during the experiment. Induction time took 20 – 40 minutes.

RABBIT

This procedure of anesthetizing the rabbit up the end of cannulation was done for us.

A small area along the ventral midline of the neck was clipped, the skin was incised and one
carotid artery was isolated. The vessel lies lateral (2 – 5 mm) to the trachea and could be
identified by its thick walled appearance and its pulsation. The accompanying vagus nerve
was separated off and two loose ligatures put round the artery 15 – 25 mm apart.

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A polythene cannula, fitted with a 3 – way tap was filled with heparinized saline (500 U/ml
in 0.9% NaCl solution) and was positioned close to the exposed artery.

The head ward ligature was tied and a small clip was used to occlude the artery between
the second ligature and the heart. A loose loop was made in this second ligature ready for
tying in the cannula.

A small cut was made in the artery, close to the tied ligature and the cannula was
introduced as far as possible with the tip pointing towards the heart. The ligature was now
tied round the cannula and the head ward ligature also tied in order to make the cannula
more secure. After making sure that the tap was closed to the artery, the clip was removed
and the skin edges brought together with a few stitches.

When the cannulation had been completed, the animals were left undisturbed for 15 – 20
minutes. During this time, the injection solutions in saline were prepared as follows:

Working in three different groups;

a) 20% glucose (2 ml/kg body weight)

b) Adrenaline 1 ml/ml (1ml/kg body weight)
c) Insulin 2 U/ml (2 units/kg body weight)
d) Saline (1 ml/kg body weight) (control)

20% glucose was injected through the catheter and we waited for 10 minutes before taking
sample (1) at zero time. Immediately after taking sample (1), adrenaline was injected
subcutaneously. Samples were thereafter taken at intervals of 20, 40, 80 and 100 minutes,
making a total of six (6) samples.
This procedure was done depending on the group but using insulin or saline (as the
control), and a total of six samples collected for each.

BLOOD SAMPLING

ESTIMATION OF BLOOD SUGAR USING THE SPECIFIC METHOD FOR GLUCOSE

Reagents used.

1. Fresh, dilute perchloric acid; 1.8 ml 60% made to 50 ml with water.

2. O – dianisidine solution; 5 mg/ml in 95% ethyl alcohol, stored in dark bottle and
cold.
3. Enzyme; 30 mg glucose oxidase powder and 2 mg peroxidase, dissolved in 100 ml of
0.3M sodium phosphate buffer pH 6.5. (Kept cold for 2 – 4 weeks).
4. Enzyme dianisidine reagent. Before use, 0.25 ml dianisidine solution was added to
10 ml enzyme solution. (2.0 ml was necessary for each sample, including blanks and

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 standards). This solution is carcinogenic and extra care had to be taken while
handling it. For our experiment, this mixture had been made for us.
5. Glucose stock standard 2.0 mg/ml.

METHOD

Blood was collected from the carotid artery that was already cannulated and fitted to a 3-
way tap and a 2ml syringe with heparinized saline.

A. COLLECTION OF PLASMA SAMPLE

1. Using the attached syringe (1), 0.5 ml of blood were withdrawn.
2. The 3-way tap was turned to close off syringe (1), and another 2ml syringe was
inserted into the 3-way tap and 2ml sample of blood was with withdrawn.
3. The 3-way tap was closed and the syringe was also removed.
4. The blood in syringe (1) was returned into the animal followed by some heparinized
saline from the same syringe that was to clear the cannula.
5. The sample was immediately delivered into a centrifuge to separate the cell from
the plasma at 300 rpm for 15 minutes.
6. Using a Pasteur pipette, the plasma was taken off into a clean dry tube.
7. 0.4 ml of the plasma was pipetted into a clean dry centrifuge tube and 3.6ml of
dilute perchloric acid was added.
8. The solution was mixed thoroughly and left for 10 minutes.
9. The mixture was centrifuged.
10. The supernatant was poured off into a clean dry test tube.
11. 2.0ml of supernatant was pipetted into a clean test tube; 3.0 ml of water was added
and then mixed.

B. PREPARATION OF STANDARD GLUCOSE

Using the stock glucose, the following dilutions were made:
1. 1.0 ml stock + 9.0ml water =200 µg/ml
2. 0.7 ml stock + 9.3ml water = 140 µg/ml
3. 0.4 ml stock + 9.6ml water = 80 µg/ml

C. ASSAY
i. 10 test tubes were used.
ii. Into tube 1, 1.0 ml water was pipette. This was the blank.
iii. Into tube 2 to 4, 1.0 ml standard glucose solutions that were prepared by
dilution (1 to 3) above were put.
iv. Into tube 5 to 10, 1.0 ml plasma supernatant solution from blood samples 1 to 6
was put.

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 v. To each of the 10 tubes, 2.0 ml of enzyme-dianisidine reagent were added and
then mixed.
vi. The tubes were then left for 40’ at room temperature.
vii. 2.0 ml 25% H2SO4 from a burette were added, then mixed.
viii. After 5 minutes, the absorbance was read using a calorimeter with a blue filter
(622nm) against blank (tube1) which was 0.

RESULTS
TEST TUBE ABSORBANCE
1 Blank 0.00
2 Standard glucose 1 3.60
3 Standard glucose 2 2.60
4 Standard glucose 3 1.50
5 Blood sample at 0 minutes 1.66
6 Blood sample at 10 minutes 1.69
7 Blood sample at 20 minutes 1.79
8 Blood sample at 40 minutes 1.50
9 Blood sample at 60 minutes 1.40
10 Blood sample at 80 minutes 1.80
11 Blood sample at 100 minutes 2.50

A standard glucose curve showing the absorbance of the standard concentrations

against time

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Blood glucose concentration of different the samples in the test tubes were read off
from the graph

The blood glucose concentrations were found out considering the 25-dilution factor
that was done during the experiment

Blood glucose concentration=glucoseconcentration∈the test tube x 25

Using a value of 100 µg/ml at 0 minutes, the percentage change of blood sugar
occurring in the other samples was calculated. These values were plotted against
time.

glucose concentration−100
percentage change= x 100
100

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 BLOOD [GLUCOSE]
TEST (mg/ 100ml) (25 PERCENTAGE
TUBE ABSORBANCE [GLUCOSE](µg/ml) dilution) CHANGE (%)
TIME
0 5 1.66 87.5 218.75 0

10 6 1.69 88.75 221.875 -11.25

20 7 1.79 93.75 234.375 -6.25

40 8 1.5 78.75 196.875 -21.25

60 9 1.4 72.5 181.25 -27.5

80 10 1.8 93.75 234.375 -6.25

100 11 2.5 131.25 328.125 31.25

A graph of blood glucose concentration against time

350
blood sugar levels (mg/100ml of blood)

300

250

200

150

100

50

0
0 20 40 60 80 100 120
time in minutes

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 A gragh of percentage change against time
40
30
percentage change (%)

20

10
0
0 20 40 60 80 100 120
-10
-20
-30

-40
time in minutes

From the results it is observed that initially blood sugar increases then decreases with
injection of insulin

With injection of epinephrine, blood sugar increases.

A standard glucose curve was drawn. This was a straight-line graph through the origin.
This was used to determine the glucose concentration of the unknown samples

A graph was plotted for blood sugar levels against time for the various treatments.

DISCUSSION

INSULIN
The blood glucose levels decrease with subcutaneous infusion of the rabbit with insulin.
This is because insulin diffused in to blood and it was transported by blood to its target
organs especially the liver, muscles, and the adipose tissues. Insulin increases glucose
uptake from the blood by all the tissues by activating the glucose transporter 4 (GLUT 4)

In the liver, insulin increases glucose increases the rate of glucose conversion to glycogen
for storage by activating glycogen synthase enzyme. Insulin also increases the glycolysis
pathway and thus glucose conversion into ATP, by activating the phosphofructokinase 1
enzyme and the pyruvate kinase enzyme.

In the muscles the insulin increases the rate of glycogenesis, and the rate of glucose
conversion into energy in form of ATP

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In the adipose tissues the glucose that is taken up is converted to glycerol – 3 phosphate
and used for lipogenesis

Insulin also inhibits gluconeogenesis mainly by decreasing the quantities and activities of
the liver enzymes required for gluconeogenesis. However, part of the effect is caused by an
action of insulin that decreases release of amino acids from muscle and other extrahepatic
tissues and in turn the availability of these

ADRENALINE

The blood glucose levels increased when the rabbit was subcutaneously infused with
adrenaline hormone.

This is because adrenaline diffused in to blood and it was transported to the liver, while in
the liver, adrenaline acts by second messenger mechanism and it causes activation of many
cystolic enzymes and it also caused transcription of mRNA and translation of the mRNAs to
proteins that increase the blood glucose levels

Adrenaline activates, and causes synthesis of gluconeogenic enzymes which all increases
the amount of glucose formed by the liver and released into blood. Adrenaline also
activates the glycogenolytic enzymes i.e glycogen phosphorylase enzyme thus increased
glycogen breakdown to release glucose into blood

Adrenaline also inactivates the glycolytic enzyme like the pyruvate kinase and the PFK 1
thus reducing the glucose catabolism for energy such that all glucose is released into blood

Mechanism of action of insulin hormone.

The effects of insulin hormone were produced in the following ways

Once insulin reached its target organs, it attaches on its specific plasma receptors forming a
hormone receptor complex. Some of the short time effects is increasing the target organ’s
permeability to glucose by activating the GLUT 4 transporters into the cells from blood

Insulin activates a phospholipid second messenger mechanism by after activating its

specific receptor on the plasma membrane. It is done by activation of phospholipase c
enzyme that breaks down phosphotidyl inositol diphosphate to diacyl glyceride (DAG) and
inositol triphosphate (IP3). DAG and IP3 act as second messengers which increase
transcription of mRNAs and their translation into proteins most of which are enzyme for
glycogenesis and glycolysis, and for inactivation of gluconeogenetic enzymes such that
much of the glucose is taken out of blood and by conversion into energy and glycogen and
almost no glucose is synthesized

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Mechanism of action of adrenaline hormone.

Excitation or Inhibition of the Effector Cell by Changing Its Membrane Permeability.

Because the receptor protein is an integral part of the cell membrane, a conformational
change in structure of the receptor protein often opens or closes an ion channel through
the interstices of the protein molecule, thus altering the permeability of the cell membrane
to various ions. For instance, sodium and/or calcium ion channels frequently become
opened and allow rapid influx of the respective ions into the cell, usually depolarizing the
cell membrane and exciting the cell.

Receptor Action by Altering Intracellular “Second Messenger” Enzymes.

Another way a receptor often functions is to activate or inactivate an enzyme (or other
intracellular chemical) inside the cell. The enzyme often is attached to the receptor protein
where the receptor protrudes into the interior of the cell.

For instance, binding of epinephrine (adrenaline) with its receptor on the outside of many
cells increases the activity of the enzyme adenylate cyclase on the inside of the cell, which
causes formation of cyclic adenosine monophosphate (cAMP). The cAMP in turn can
initiate any one of many different intracellular actions, with the exact effect depending on
the specific effector cell and its chemical machinery.

Actions of adrenaline (and noradrenaline) are executed by binding with receptors called
adrenergic receptors, which are present in the target organs.

Adrenergic receptors are of two types:

1. Alpha-adrenergic receptors, which are subdivided into alpha – 1 and alpha – 2

receptors

2. Beta-adrenergic receptors, which are subdivided into beta – 1 and beta – 2 receptors.

RELATIONSHIP BETWEEN INSULIN AND THE DIABETIC STATE

Diabetes mellitus is a syndrome of impaired carbohydrate, fat, and protein metabolism

caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin. It
results in to very high blood glucose levels especially after a meal

There are two general types of diabetes mellitus:

 Type 1 diabetes, also called insulin – dependent diabetes mellitus, is caused by lack of
insulin secretion.

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 Type 2 diabetes, also called non – insulin-dependent diabetes mellitus, is initially
caused by decreased sensitivity of target tissues to the metabolic effect of insulin. This
reduced sensitivity to insulin is often called insulin resistance.

In both types of diabetes mellitus, metabolism of all the main food stuffs is altered. The
basic effect of insulin deficiency or insulin resistance on glucose metabolism is to prevent
the efficient uptake and utilization of glucose by most cells of the body, except those of the
brain. As a result, blood glucose concentration increases, cell utilization of glucose falls
increasingly lower, and utilization of fats and proteins increases.

Various manifestations of diabetes mellitus develop because of three major setbacks of

insulin deficiency, which include:

i. Increased blood glucose level (300 to 400 mg/dl) due to reduced utilization by
tissues.
ii. Mobilization of fats from adipose tissue for energy purpose, leading to elevated fatty
acid content in blood. This causes deposition of fat on the wall of arteries and
development of atherosclerosis
iii. Depletion of proteins from the tissues.

CONCLUSION

Hormones mediate the body cells biochemical activities. Deficiency or complete absence of
hormones would lead to malfunction and death. Over secretion brings about hyper activity
or over production of certain metabolites. This would also lead to malfunction and at times
death.

Insulin hormone decreases blood sugar levels.

Adrenaline/epinephrine increases blood sugar levels.

REFERENCES
 Dinesh Puri. Textbook of Medical Biochemistry 3rd edition; Elsevier inc.
Ganong’s Review of Medical Physiology 24TH edition by Kim E. Barret, Susan M.
Barman, Scott Boitano, Heddwen L. Brooks.

 Guyton Arthur Clifton, & Hall John E. (2016). Textbook of medical physiology
13th edition. Philadelphia: Elsevier,inc.
 K Sembulingam & Prema Sembulingam . (2012). Essentials of Medical
Physiology 6TH edition. New Delhi: Jaypee Brothers Medical Publishers (P) LTD

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 

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