Sonoma State University - AnimalPhysiology
Department of Biology - 1999

I. Introduction:

Glucose in the blood is a major source of energy for most cells. Some cells, as nerve cells, are dependent upon glucose for their energy. They don't have the enzyme systems to use fats or amino acids as an energy source. These are also not under the influence of insulin. In mammals, when the blood contains between 80-100 mg/dl and the animal is at rest, skeletal muscle does not take up glucose, but relies on fat as a major energy source. This conserves glucose for nerve and cardiac cells. Post prandial levels of glucose in the blood rise stimulating insulin secretion The insulin overcomes the barrier to glucose uptake and skeletal muscle cells take up excess glucose immediately phosphorylating it and storing it as glycogen or using the glucose directly. Since the body is composed of so much skeletal muscle, the glucose uptake by muscle accounts for nearly all of the buffering effect and preventing hyperglycemia. The liver also takes up glucose when in high concentration in the blood, however, insulin plays no role here. Fat cells take up more fat from the blood when insulin levels are high and the liver converts the sugar into fats. Insulin also promotes protein synthesis from amino acids partially by inhibiting the use of amino acids for catabolism.

Purpose:

The purpose of this experiment is to demonstrate the control system for blood glucose levels. We will compare the effects of injected insulin with a high glucose load with a high glucose load alone and sham filling of the stomach with water.

Procedure: (for each group; total 6 groups)

II. ANESTHESIA

In order to reduce the pain for the rat, we will use pentabarbital (Nembutal). We will use a light anesthetic dose of 35 mg/Kg. It is important that the calculations be accurate as an overdose will kill the rat and there is no way to remove the drug once it is injected. The solution contains 60 mg of pentabarbital/ml.

To pick up the rat, follow the instructors directions. While the rat is on the table, grasp the tail near its base and control the rat by the tail. Slip the other hand over the back and place the index and middle fingers on the sides of the neck so that they are under the jaw and you can control the head. You can now lay the rat back in your hand and support the whole animal. With the other hand control the hind feet. Inject the proper amount of penatbarbital into the lower quadrant away from the center line. Pop the needle in straight through the skin and muscle layer. Inject and remove the needle. Release the animal onto the table or in the cage.

III. ADMINISTRATION OF GLUCOSE PER OS (by mouth)

You will administer 5 ml of a 100g glucose/100ml. This solution is prepared for you. Again not following directions carefully can kill the rat by drowning. There are special, blunt needles made for this procedure. Select a needle size with the advice of the instructor. Hold the rat as above. Place the needle along the rat's head and body and determine how far the needle will penetrate to reach the bottom of the rib cage from the back of the throat. Place the tip of the needle in the back of the rat's throat. This should produce a swallow reflex and the rat will direct the needle into the esophagus and not the trachea. Gently advance the needle. If it stops abruptly short of the goal, you are in the trachea - do not push farther - but withdraw and try again. Remember that the esophagus is dorsal to the trachea. Once you are in the esophagus, and the needle is at the proper depth, slowly push the plunger allowing time for the stomach to expand. When the contents of the syringe are injected, slowly withdraw the needle.

IV. ADMINISTERING INSULIN

If you are in a group chosen to use insulin, inject .01 unit of insulin into a lower quadrant in the same manner as you injected the pentababarbital. You may use the same needle and syringe as you did with the pentabarbital.

V. DRAWING BLOOD

At the proper times (before glucose and at ½ hour intervals thereafter), Warm the rat's tail, swab with a small amount of alcohol and dry. Nick the tail with a sharp scalpel near the end where it is pink. You can squeeze out a large drop. Remove a test strip from the foil packet. Place the test strip into the meter and then, while the blood is still fresh and uncoagulated, touch the tip of a test strip to the blood so that it is drawn up by capillary action. Hold the test strip against the blood until after the meter beeps. The timer begins counting down from 59 seconds. After 59 seconds, the blood glucose levels appear in the display in mg glucose/100 ml blood. Using a tissue, remove the test strip and then within 2 seconds put it back in to delete the results.

	Before	½ hr	1 hr	1 ½ hr	2 hr
5 ml water
5 ml glucose
5 ml glucose; .01 unit insulin

After the lab is over be sure to put all needles in the "sharps container" with the cover on them, the syringes into the waste basket, and the other materials on the cart for return to the media kitchen.

Conclusions: (Insert a page if necessary)

Plot the blood glucose levels vs time.

Questions:

1) With insulin present, what should happen to blood amino acid levels?

2) What is the difference between the reactions of the brain and muscle to low blood glucose levels?

3) What is insulin shock? Diabetic coma?

Materials

6 X 1 ml syringe
6 X 5 ml syringe
6 X 27g needle
6 X intubation needles for per os administration
6 X scalpel
6 X lab tissues
24 Bayer glucose test strips
Bayer Glucose Meter
50 cotton swabs

Chemicals

100 ml of 1g glucose/ml
100 ml of 100 mg/100 ml pentabarbital (Nembutal)
10 ml of 0.1 unit/ml of insulin in 0.9% saline
6 X 50 ml 70% ethanol

ANIMALS

6 X Rats with cages

Permanent Materials in the lab.
6 label tapes
Box Pasteur Pipettes
Bulbs for pipettes
Waste glass container
Sharps Container
6 sharpie pens