Physio Lab Exp 19

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

Weight

of Rats

of Liver

Epinephrine

133.1 g

(Rat A) Saline (Rat B) None (Rat C)

124.1 g 139.9 g

Treatment

Weight

Test Tube Weight

of

Test Tube Weight

2g

Muscle 18 g

For Liver 12 g

For Muscle 16.5 g

(with glycogen) Liver Muscle 12.23 g 17.07 g

5.2 g 2.7 g

1.1 g 1g

16.5 g 16.50 g

15.50 g 17.5 g

17.08 g 16.91 g

15.94 g 18.06 g

Results: % Epinephrine Saline Control

Liver % Liver

Glycogen 11.5% 11.16% 15.19%

Body Weight 0.17% 0.47% 0.29%

%

Muscle % Muscle

Glycogen 3.17% 4% 56%

Body Weight 0.43% 0.35 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 Glucagon

Stimulus Decrease in plasma

Inhibitor High levels of FFA

Effect on CHO Promotes

(Pancreas)

glucose

GLP – 1

glycogenolysis and

(fasting/starvation),

gluconeogenesis;

increase in plasma

inhibits glycolysis

AA (Arg, Ala), PNS Growth hormone

stimulation 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

Cathecholamine –

ghrelin Stress,

Epinephrine

hypoglycemia,

glycogenolysis,

(Adrenal Medulla)

exercise, pure

Reduced glucose

Cortisol (adrenal

protein feeding Stress,

cortex)

hypoglycemia,

gluconeogenesis,

exercise, pure

reduced glucose

protein feeding Moment-to-moment

A2 adrenergic

uptake Decreased plasma

fluctuations in blood

receptor agonist

blood glucose

Increased plasma

Increased glucose

Insulin (pancreas)

Degraded by MAO

Via feedback control

Increased

uptake Increased

glucose concentration,

Leucine,

levels of FFA

Vagal stimulation

uptake, reduced 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.