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EFFECTS OF INSULIN AND GLUCAGON ON BLOOD GLUCOSE AND LIVER GLYCOGEN
BACKGROUND
The fate of glucose molecules in the blood is normally regulated by two hormones; insulin which
favors glucose storage and glucagon which favors glucose mobilization. In this experiment, you will
measure the effects of both hormones on plasma glucose levels. The glycogen stored in the liver is the
major short-term store of glucose that can be transferred to the blood. You will measure the amount of
glycogen remaining in the liver following treatment with either glucagon or insulin.
PROCEDURE
It is practicable for each group to test the effects of only one of the two hormones. Each group will be
assigned a number to help in keeping track of blood and liver samples. Results will be shared so that each
student will be able to prepare a report using all the results from his/her section. Delegate one member of
each group to be responsible for the glucose assay procedure.
1.Anesthetize the animal and insert tracheal and jugular cannulas. For this experiment, it is best to avoid
over anesthetizing the animal since urethane has a hyperglycemic effect in rabbits.
2.Fill a syringe filled with heparinized Locke's solution and attach it to the carotid cannula provided. Insert
the cannula into the carotid. This cannula should be flushed periodically to keep the blood from clotting.
3. Collect a control blood sample using the following procedure:
a. Get a heparinized microcentrifuge tube and label it with your group number and the sample time
using a marking pen. This is sample 0.
b. Clamp the cannula with hemostat and disconnect the syringe.
c. Unclamp the cannula and allow several drops of blood to drain into a waste beaker; this flushes
the cannula dead space.
d. Let enough fresh blood flow into the microcentrifuge tube to fill it at least half way up the
conical part of the tube.
e. Reattach the syringe and flush with heparinized Locke's to prevent a clot from forming.
4. Your TA will assign a drug for your group to use. Insulin group: Inject 1 ml (40 units/ml) insulin IP.
Glucagon group: Inject 1 ml (1mg/ml) glucagon IP.
NOTE THE TIME OF INJECTION! The reason for injecting the hormones IP is to spread out the
period of effectiveness over several tens of minutes. We can hope to follow the time course of the effect by
sampling at reasonable time intervals.
5. Take blood samples at 20 min intervals after the injection. The changes in blood glucose will probably
be maximal by the 40-60 minute samples. It would be preferable to continue to sample until the 80- minute
sample. Do not discard your samples until all the assays have been completed and the results inspected.
6. As soon as possible after sacrificing the animal at the end of the experiment, open the abdomen and
collect approximately 2 grams of liver. Place the tissue on a preweighed piece of plastic wrap and rapidly
determine its weight to the nearest 0.01 gm. Then put the wrapped tissue in a plastic bag labeled with your
section number, group number and the wet weight of the tissue. Freeze it as rapidly as possible with the
dry ice provided. You will carry out the glycogen assay in the next lab meeting.
ANALYSIS
Glucose Assay
The Glucometer Elite (Bayer) will be used to determine blood glucose levels. This automated method is
based on measurement of the electrical potential caused by the reaction of glucose with the reagents
(glucose oxidase and potassium ferrocyanide) on the electrode of the test strip. The blood sample is drawn
into the tip of the test strip through capillary action. In the reaction, electrons are generated and produce a
current that is proportional to the concentration of glucose in the sample. After a set reaction time, the
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glucose concentration is displayed. (A code strip is provided with each package of test strips. Each code
strip has a specific number. When the code strip is inserted, the glucometer is automatically calibrated.
When calibrated, the electronic system in the glucometer is automatically set to match to reactivity of that
specific package of test strips. The code strip is inserted into the test strip slot, and, when “F-#” is
displayed, calibration is complete.) It is important that the F-# set in the meter matches the code strip
number for the package of test strips in use. Refer to the operator’s manual for more information. Your
TA will demonstrate the use of the glucometer and test strips.
Liver Glycogen Assay
WEAR SAFETY GLASSES, DISPOSABLE GLOVES, AND A LAB COAT WHEN HANDLING
THESE CHEMICALS. TCA WILL EAT HOLES IN YOUR CLOTHES AND IN YOU, TOO.
Background
This assay is based on differential precipitation. First you will disrupt the tissue in trichloracetic acid
(TCA) which denatures, and so precipitates, the proteins and nucleic acids. Until this process is complete,
the sample must be kept cold to minimize loss of glycogen due to glycogen phosphorylase activity. Then
the glycogen-containing supernatant is treated with concentrated ethanol, which precipitates the glycogen.
Procedure
1. Recover your liver sample from the freezer. Place it in a precooled homogenizer sitting in an ice bath
containing a volume in milliliters of 10% TCA equal to the sample's weight in grams. Homogenize the
tissue, returning the homogenizer to an ice bath periodically to keep it cold. Add a pinch of sand to
facilitate the grinding.
2. Transfer the homogenate to a centrifuge tube labeled with your group number. Rinse the homogenizer
with a volume of TCA equal to the amount you used to homogenize the tissue, and add the rinsate to the
centrifuge tube. Centrifuge for 5 minutes at top speed in the tabletop centrifuge.
3. Decant off the opalescent supernatant into a 10 ml graduated cylinder. Add 2 volumes of 95% ethanol
and stir. A flocculent precipitate will form. Transfer the suspension to a centrifuge tube and centrifuge for
1-2 min. Decant off and discard the supernatant INTO THE WASTE CONTAINER INDICATED.
Remember, it contains TCA. The precipitate contains glycogen that must be cleaned up a bit by
reprecipitation.
4. Re-dissolve the precipitate in 5 ml H2O, and then re-precipitate the glycogen with 10 ml of 95% ethanol.
This step enhances the purity of the final product.
5. Place the suspension in a pre-weighed centrifuge tube (the weight is written on the side of the tube).
Centrifuge for 1-2 min. Discard the supernatant into an alcohol waste container. The precipitate is your
final product, but it must be dried before its weight can be determined.
6. Wash the precipitate three more times: once with 5 ml of 95% ethanol, then twice with 5 ml of 100%
ethanol. Each time, stir the precipitate thoroughly and centrifuge for 1-2 min. Discard the supernatant into
an alcohol waste container.
7. Perform the final wash in the fume hood. Using a glass pipette, add 5 ml of diethyl ether to the tube, stir
the precipitate thoroughly, and centrifuge for 1-2 minutes. Spin down the precipitate using the handcranked centrifuge, turning the handle slowly. Discard supernatant into the ether waste container.
8. After the last wash, spread the glycogen on the side of the centrifuge tube and allow it to dry, aided by
the flow of air in the fume hood. Then weigh the tube and its contents and subtract the weight of the tube.
In presenting your result, express the weight of glycogen as % wet weight of liver.
PREPARING YOUR REPORT
Some thought questions:
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1. How might the blood glucose results have differed if the samples were collected from the jugular rather
than the carotid?
2. How would the results have differed if the insulin animal had been adrenalectomized?
3. There is a negative correlation between normal blood glucose levels and body weight for different
mammalian species of different sizes. For example, the normal blood glucose concentration for an adult rat
is 130-140 mg%, for a rabbit it is 80-110 mg%, and for a human it is about 90 mg%. Can you think of
reasons why smaller animals might need higher blood glucose levels?
4. With regard to interpreting the liver glucogen results, the experiment could have been better designed. If
this were an original experiment, exactly what can we know from it, and what changes would you make in
the protocol to make it more informative, assuming more time and more animals were available?