PHYSIOLOGY EXPERIMENTS

NOS. 19 & 20

SUBMITTED BY:
Section B - 6

FACILITATOR:
Dr. Cabansag
 Experiment 19
Epinephrine on blood sugar and liver glycogen

Introduction:

Epinephrine or adrenaline is a catecholamine, a sympathomimetic monoamine

derived from the amino acids phenylalanine and tyrosine. When secreted into the
bloodstream, it rapidly prepares the body for actions in emergency situations. It
increases supply of oxygen and glucose to the brain and the muscles, while suppressing
other non emergency bodily processes. It increases the heart rate and stroke volume,
dilates the pupils, and constricts arterioles in the skin and gut while dilating arterioles in
skeletal muscles. It elevates the blood sugar level by increasing the catalysis of
glycogen to glucose in the liver, and breakdown lipids into fat cells. Epinephrine acts
most readily to beta receptors.
Norepinephrine or noradrenaline is also a catecholamine. Its structure is almost
similar to that of epinephrine except for the absence of the methyl functional group at the
nitrogen atom of epinephrine. Along with epinephrine, it underlies the fight or flight
response, directly increasing heart rate, trigerring release of glucose from energy stores,
and increasing blood flow to the skeletal muscles, thus increasing its oxygenation as
well. Norepinephrine acts most readily to alpha receptors.
Both epinephrine and norepinephrine are synthesized from dopamine by
dopamine beta hydroxylase. They are released from the adrenal medulla.

General Objective:
• To determine the effects of epinephrine on blood sugar and liver glycogen levels.

Specific Objective:
• To measure blood sugar levels before and after the administration of epinephrine.
• To isolate and measure the amount of glycogen present in muscle and liver
tissues.

Procedure and Rationale:

1. Three well fed, adult rats, of almost the same weight, were labeled as rats A, B,
and C. Each rat was weighed before taking blood for sugar determination and
their respective weights were recorded.
2. 1 drop of blood was drawn from the tail of each rat and their blood sugar was
determined by an Optium Glucose Meter. Their blood sugars were recorded.
Only one student handled the Glucose meter so as not to cause inter-observer
 variability among the results. One student was also assigned per rat when blood
was being drawn.
3. Immediately after the first blood was drawn, the rats were injected
subcutaneously with 0.1 ml of 1:10,000 solution of epinephrine to rat A and 0.1 ml
of saline to rat B. Rat C had no injection administered. The time of injection was
recorded.
4. After 10 minutes from injection, a second blood sample was taken from each rat.
Blood glucose was once again determined.
5. A third sample was taken 20 minutes after injection and blood glucose levels
were analyzed. The different time intervals were done to determine levels of
blood glucose in effect to administration of the different drugs. Epinephrine is
metabolized quickly in the body, therefore possibly creating a variety in results
after a few minutes.
6. Test tubes (50 ml) and centrifuge tubes were then prepared. Without any content,
they were weighed individually and then 6 ml of 30% KOH were placed in each of
the 50 ml test tubes. They were again weighed and recorded.
7. After the last blood collection was done, the rats were sacrificed and the liver and
gastrocnemius muscles isolated. Each liver and muscle was weighed individually
and recorded.
8. The organs were then minced separately. The minced liver and muscle from the
epinephrine, saline and untreated rats were then placed in the test tubes
containing the 30% KOH for cellular digestion to take place. They were again
weighed and the difference of these values and the test tubes with KOH
determined the weight of the samples.
9. Glycogen isolation followed:
a. The test tubes were placed upright in a boiling water bath for 15-20
minutes. The solution was occasionally agitated to insure complete
disintegration.
b. 7 ml of 95% alcohol was added to each tube until boiling begins within the
tubes. The addition of alcohol facilitates the precipitation of glycogen.
c. As soon as the solution started to boil, it was removed and placed in ice
cold water to cool. Contents were then transferred to the centrifuge tubes
and centrifuged.
d. The precipitate was then drained and washed twice with 5 ml portions of
60% alcohol by centrifuge, decanting and draining as before. The last
traces of alcohol were expelled by immersing the tubes again in boiling
water and allowing the alcohol to evaporate.
e. The test tubes with precipitate were then weighed again and the amount
of glycogen derived was determined.
Results and Discussions:

Raw Data:
Weight Test Tube Weight
Weight Weight Test Tube Weight
Treatment of (with glycogen)
of Rats of Liver
Muscle For Liver For Muscle Liver Muscle
Epinephrine 133.1 g 2g 18 g 12 g 16.5 g 12.23 g 17.07 g
(Rat A)
Saline (Rat B) 124.1 g 5.2 g 1.1 g 16.5 g 15.50 g 17.08 g 15.94 g
None (Rat C) 139.9 g 2.7 g 1g 16.50 g 17.5 g 16.91 g 18.06 g

Results:
% Liver % Liver % Muscle % Muscle
Glycogen Body Weight Glycogen Body Weight
Epinephrine 11.5% 0.17% 3.17% 0.43%
Saline 11.16% 0.47% 4% 0.35
Control 15.19% 0.29% 56% 0.40%

Liver Glycogen= WeightTestTubew/glycogen - WeightTest Tube

Muscle Glycogen = WeightTestTubew/glycogen - WeightTest Tube

% Liver Glycogen = Liver Glycogen

WeightMuscle
% Muscle Glycogen = Muscle Glycogen
WeightLiver

Based on the groups’s result, the normal, which is represented by the control
group, obtained 15.19% of glycogen which is the largest value as compared with the
group that were injected with epinephrine and normal saline. This means that
epinephrine and normal saline have an effect on the liver glycogen mobilization to cause
a decrease in the amount, as compared with the normal rat’s liver glycogen.

Glycogen synthesis and degradation occurs in the liver cells. It is here that the
hormone insulin (the primary hormone responsible for converting glucose to glycogen)
acts to lower blood glucose concentration. Insulin stimulates glycogen synthesis;
thereby, inhibiting glycogen degradation.

Epinephrine, on the other hand, is one of the two primary hormones (the other
being glucagon) that breakdown glycogen. Epinephrine will bind to the receptor on the
outside of a liver cell allowing a conformational change to occur. This receptor shape
change allows G protein to bind, and become active. The activation G protein causes a
conformational change on the molecule causing adenylate cyclase to bind. Once
adenylate cyclase has been activated ATP binds to the complex. Adenylate Cyclase
breaks down ATP into Cyclic AMP, which becomes the second messenger protein in this
process. Cyclic AMP activates protein kinase, which activates phosphorylase catalyzing
the breakdown of glycogen to glucose. Also, the breakdown of glycogen has caused
blood glucose level to rise.

On the result the control group obtained 56% muscle glycogen. And accordingly
the results generated from the epinephrine and saline injected group is lower than it. So
it then again imparts that epinephrine and normal saline have an effect on the
mobilization of muscle glycogen stores.

A second major source of stored glucose is the glycogen of the skeletal muscle.
However, muscle glycogen is not generally available to other tissues, because muscle
lacks the enzyme glucose-6-phosphatase. Epinephrine binds to the receptors on the
surface of muscle cells. It induces conversion of muscle glycogen into glucose.

On the results obtained from the saline injected group, it was surprising to have
results almost similar to that obtained from the epinephrine injected group. It can be said
that the stress induced by the needle from the injection itself, could have caused
sympathetic stimulation on the rat, causing the acetylcholine release from preganglionic
sympathetic fibers innervating the medulla, that have been the cause of the secretion of
epinephrine. Thus, the saline injected group had almost the same liver and muscle
glycogen.

Guide Questions:

A. What are the consequences if starved rats were used in this experiment?

During conditions of starvation, the hepatic glycogen stores will be eventually

depleted as a mechanism (glycogenolysis) in order to elevate blood glucose which is
used by the body as fuel for its cells. When epinephrine is infused to the rats, it will
increase blood glucose by means of increasing the breakdown of lipids in adipocytes
(increased lipolysis) and further utilize the glycogen in the liver. Hence, this will increase
the free fatty acids in circulation which can be used as a substitute fuel source for most
tissues in the body in the absence of glucose, and increase blood sugar per se.
Epinephrine may also stimulate gluconeogenesis from other types of substrate found in
the body.

Therefore, measuring the liver glycogen amount will be useless since it may be depleted
or exhibit a very low amount in starved rats even before any substance was injected to
them.
 B. What other hormones (aside from epinephrine and insulin) influence the blood
glucose level? Discuss their effects on blood glucose level.

The regulatory effects of hormones are one of the mechanisms used by the body
to maintain homeostatic levels of blood glucose.

There are actually two groups of metabolic hormones which act antagonistically
to one another in regulating blood glucose:

1. Catabolic Hormones – increases blood glucose includes glucagons, growth

hormone, and the cathecholamines
2. Anabolic Hormones – decreases blood glucose, one example is insulin

It can also be categorized as regulatory and counter-regulatory hormones. With insulin

being the main regulatory hormone while glucagons, cathecholamine, cortisol, and
growth hormones as counter-regulatory.

Hormone Stimulus Inhibitor Effect on CHO

Glucagon Decrease in plasma High levels of FFA Promotes
(Pancreas) glucose GLP – 1 glycogenolysis and
(fasting/starvation), gluconeogenesis;
increase in plasma inhibits glycolysis
AA (Arg, Ala), PNS
stimulation
Growth hormone Insulin-induced Somatostatin Reduces liver
(Anterior Pituitary) hypoglycemia, Dietary uptake of Glucose,
exercise, stress, carbohydrate, Promotes
increased plasma glucocorticoids gluconeogenesis in
AA, estradiol, the Liver
ghrelin
Cathecholamine – Stress, Degraded by MAO Increased
Epinephrine hypoglycemia, glycogenolysis,
(Adrenal Medulla) exercise, pure Reduced glucose
protein feeding uptake
Cortisol (adrenal Stress, Via feedback control Increased
cortex) hypoglycemia, gluconeogenesis,
exercise, pure reduced glucose
protein feeding uptake
Insulin (pancreas) Moment-to-moment A2 adrenergic Decreased plasma
fluctuations in blood receptor agonist blood glucose
glucose
concentration, Increased plasma Increased glucose
 Leucine, levels of FFA uptake, reduced
Vagal stimulation glycogenesis,
TNF-alpha reduced
gluconeogenesis

C. Are effects of epinephrine in muscle and liver similar? What will be expected
findings if the blood and liver glycogen determination was done 4 hours later after
epinephrine was injected?

Generally, the effects of epinephrine in muscle and liver is similar in that it

promotes the restoration of normal plasma glucose levels in order to prevent
hypoglycemia. However, in the liver, it promotes gluconeogenesis, while in the muscle, it
promotes glycolysis. It also promotes lipolysis and inhibits insulin secretion.

If the blood and liver glycogen determination was done 4 hours later, the results
of the experiment would have been confounded. Epinephrine is metabolized as quickly
as 2 minutes in the body, and the blood glucose level would have already normalized
after 4 hours of exogenous infusion.