Before     After

Betty Jean Jones   Cheryl Rankin  Eileen  Malloy Desormeaux   John Niemoth    Jim Sink   Jonathan Harris   If you have any questions or comments on this lab, please feel free to contact us by clicking on our names for our e-mail addresses.

NATIONAL SCIENCE EDUCATION Content Standards Focus
A. Student abilities are related to scientific inquiry
B. Understanding about scientific inquiry is evident
C. Understanding about science and technology is evident
D. Nature of scientific knowledge is evident
E. Change, constancy and measurement  are utilized
F. Form and function are utilized
G. Matter, energy and organization in living systems are evident

NATIONAL SCIENCE EDUCATION Non Content Standards Focus
A. Adequate time is allowed for learning to take place
B. Teachers are provided with current research
C. Teaching strategies allow for meaningful discourse among students
D. Outside resources are suggested
E. The curriculum coordinates with Mathematics
F. Clear goals and expectations are presented
G. Inquiry is emphasized as a way of learning

A Chi Square test determined that at a 95% confidence interval, there was not a significant difference between the control and the variables.  However, preliminary test results (using 150 mg glucose/100 ml water) did show an effect.  However,  these results are not reported because 150 mg glucose is above the upper limit for the glucose assay test used.  A glucose assay test with a upper limit range greater than 105 mg glucose should be used.

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        Teacher Objectives:
         This laboratory will allow teachers to
         *Emphasize inquiry as a way of learning
         *Coordinate curriculum with mathematics
         *Set clear goals and expectations
         *Use current research
         *Allow for meaningful discourse among students

• Biology students, Anatomy and Physiology students, AP Biology Students
• Grade Level (General to Advanced)

Notes to the Teacher: to top

1. Required of students:  Students should be given the lab ahead of time to read the introduction to obtain specific information on the interaction between glucose and insulin.
2. Students should already be familiar with the cell membrane, passive transport and facilitated transport, homeostasis and hormone regulation.
3. Because each group will test different combinations, class data will need to be collected and analyzed for meaningful results.
4. Preparation time needed:  Teachers should prepare the glucose and insulin solutions, set out all glassware, make sure the spectrophotometer is in good working order, and prepare all of the reagents specified in the glucose assay kit, one day prior to the lab.  The assay test requires a few transfers and dilutions that can be difficult for students if they are not explained ahead of time.
5. The rabbit psoas muscle should be cut by the teacher at the time the students need it.  It should not be cut ahead of time because it tends to shred.  As soon as students are given the muscle, they must add the glucose solution so that the rabbit muscle does not dry out.  The muscle can be cut into 12 equal sections.  The mass of each section will be approximately 0.25 g.
6. Insulin is measured in units.  The teacher will need to make up enough insulin to have 14 units/0.5 ml of water.
7. The glucose assay test will be done on the glucose remaining in solution (extracellular) not on the glucose that was taken into the cell (intracellular).  This avoids having to grind the muscle tissue to extract the glucose.
8. Hazards/Precautions:  To cut the rabbit psoas muscle, gloves should be worn and a sharp instrument should be used to prevent excessive shredding of the muscle.
9. Since students will be using glassware, teachers should review all laboratory safety rules.
10. The glucose assay kit used in this experiment had a glucose range from 70 - 105 mg/100 ml water.  The typical concentration of glucose in the blood is 90 mg glucose.  To better measure the results, a glucose assay kit which had a wider glucose range (especially on the upper end) would be more effective.  In a preliminary experiment, a 150 mg/100 ml glucose solution was used.  This test yielded more observable results.

    Each group of two students will need one of the following set ups.  Each set up will be used by four different  groups.

    Student Groups (4) testing the control (Glucose only):
    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)   4 ml of  105 mg glucose/100 ml of water solution
    (4)  19 ml distilled water

    Student Groups (4) testing the variable of insulin (Glucose + Insulin):
    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)  4 ml of 105 mg glucose/100 ml of water solution
    (2)  0.5 ml solution containing 14 units of insulin
    (4)  19 ml distilled water

    Student Groups (4) testing the variable of insulin and chromium picolinate (Glucose + Insulin +
    Chromium Picolinate):

    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)  4 ml of 105 mg glucose/100 ml of water solution
    (2)  0.5 ml solution containing 14 units of insulin
    (2)  0.1 g of chromium picolinate
    (4)  19 ml distilled water
    (1)   mortar and pestle

    All Student Groups will need the following:

     (6) test tubes
     (6) 50 ml beakers
     (1) 25 ml graduated cylinder
     (2) 1 ml pipettes
     (6) spectrophotometer cuvettes or properly sized test tubes
     (1) test tube rack
     (1)  pipette bulb

    The Class needs the following:

    Spectrophotometer 20
    Glucose Assay Kit (Sigma Diagnostics 510-A or one that shows a greater testing range)
 
    Representative Results:
 
 
 

A Chi Square test determined that at a 95% confidence interval, there was not a significant difference between the control and the variables.  However, preliminary test results (using 150 mg glucose/100 ml water) did show an effect.  However,  these results are not reported because 150 mg glucose is above the upper limit for the glucose assay test used.  A glucose assay test with a upper limit range greater than 105 mg glucose should be used.
 
 

 
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Carbohydrates are made of small molecules called sugars.  Glucose is an example of a group of carbohydrates known as monosaccharides.  Monosaccharides are organic compounds composed of one sugar molecule.  The molecular structure of glucose is C₆H₁₂O₆.  Glucose contains two functional groups: an aldehyde (C=O) and an hydroxyl (OH) group.  The functional groups make glucose soluble in aqueous solutions.  When a glucose molecule is in solution, the aldehyde group on the first carbon combines with a hydroxyl group on the fifth carbon to form a ring structure.

Glucose plays  major roles in the energy processes in living systems.  Glucose  can be converted into fatty acids via glycolysis and acetyl CoA.  Glucose also produces NADPH required for fatty acid synthesis upon oxidation via the pentose shunt pathway.  This simple sugar is essential for triglyceride synthesis as well..

 The hormone insulin has been discovered in many simple organisms including some protozoan, fungi, and prokaryotes including E. coli. . Because of its similarity to human insulin, the insulin of E. coli is able to promote glucose oxidization in human fat cells.  Because of this, it is assumed that insulin originated as a non glandular messenger.  As the vertebrate pancreas evolved, the insulin production and mechanism also evolved and adapted.

Specific cells in the pancreas called the beta cells of the islets of Langerhans produce insulin.  It is a protein containing two amino acid chains connected by disulfide bridges.  Proinsulin, a single chain of 86 amino acids is first formed, which allows for the correct alignment of disulfide bonds.  This molecule is then cleaved, resulting in insulin, which consists of an A chain made of 21 amino acids and a B chain with 30 amino acids.  The two chains form a highly ordered structure with several alpha helical regions in both the A and B chains.  It is the carboxyl-terminal portion of the B chain and the amino and carboxyl-terminal residues of the A  chain that form the surface of the molecule which interacts with the receptor sites.  In isolation, the A and B chains of insulin are inactive.

   Glucose is transported by facilitated diffusion through the cell membrane by the attachment of insulin on the cell surface.  After entering the cell, glucose is utilized in the cell to produce ATP, stored as glycogen or converted to fat in adipose tissue.  If  insulin levels are low, glucose transporters, GLUT4, are held in cytoplasmic vesicles within the cell.  When insulin is present, the hormone acts on these target cells to move the transporters to the cell surface.  The transporters become imbedded in the membrane where they can remove glucose from the blood.  Insulin is released from the pancreas and is taken up by cells containing an insulin receptor. The insulin receptor is composed of two alpha and two beta  polypeptide chains.  The alpha chains reside on the outside surface of the cell and the alpha chain transmits a signal to the interior of the cell to release two phosphate groups.  The phosphate binds to form insulin receptor transporters (IRS).  The activation of the receptor sites initiates signals within the cell to stimulate DNA synthesis and cell division.  Another signal will stimulate the movement of glucose transporters to the cell membrane.  With the lowering of blood sugar and the resultant decrease in insulin, the transporters are returned to the cell interior by endocytosis.  Insulin is responsible for initiating a cascade of activities by various cells:
       1.  Insulin stimulates the absorption of additional glucose from the blood by  muscle and adipose cells, and indirectly causes the liver to take up glucose  and convert it into fat and glycogen.
        2. It promotes both oxidation of glucose and the conversion of glucose  into glycogen in muscle as well as liver cells.
        3. It inhibits metabolic breakdown of stored glycogen in liver and muscle cells.
        4. It promotes the synthesis of fats from glucose by adipose cells and also   inhibits the metabolic breakdown of fat.
        5. It promotes the uptake of amino acids by liver and muscle cells and favors protein synthesis while inhibiting protein breakdown (Gould p. 944).
 
 Chromium is an essential mineral that is required for the maintenance of human health and is a vital component of GTF, or glucose tolerance factor.  Glucose tolerance factor works in concert with insulin to stabilize blood sugar levels and to aid in the uptake and metabolism of glucose, or blood sugar, by the cells of the body.  Adequate levels of chromium are required for the synthesis of enzymes, proteins, fats and cholesterol.  A key indication of coronary artery disease is dangerously low levels of chromium in blood plasma.  Inadequate intake of chromium can improve the production of GTF, limiting insulin activity.  This results in high blood sugar levels or glucose intolerance, and can lead to symptoms similar to adult-onset diabetes.

 Good dietary sources of chromium include:  Brewer's yeast, whole grain breads and cereals, molasses, brown rice, cheese, and lean meat.  Studies indicate that for optimal benefits, adults should take between 200 to 400 micrograms per day, and up to 600 micrograms if engaged in strenuous exercise programs.  Chromium supplements are available, usually in 100 to 200 microgram capsules either as niacin-bound chromium or chromium picolinate.   Nine confirming scientific studies with humans and animals claimed significant reduction in the body fat when chromium picolinate is added to the diet.  These  studies also show a consistent trend toward increased lean muscle.  Muscle burns calories; fat merely stores  calories. As a further note, there has also been a study linking high amounts of chromium to cancer and chromosome damage.

Introduction:  
     Modify the Introduction and Background material to produce a student introduction that meets your needs.
Purpose:
   To determine the uptake of glucose into a muscle cell.
   To understand the role of insulin and chromium picolinate in the uptake of glucose into a cell.
Equipment:
  The Materials and Equipment listed below are needed for the following groupings:
                Class of 24 students
                Class divided into 12 groups of two students each
                Each group doing one standard and two trials of either the control or one of the variables
 
    The Materials and Equipment listed below are needed for each group of two students:

    Student Groups (4) testing the control (Glucose only):
    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)   4 ml of  105 mg glucose/100 ml of water solution
    (4)  19 ml distilled water

    Student Groups (4) testing the variable of insulin (Glucose + Insulin):
    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)  4 ml of 105 mg glucose/100 ml of water solution
    (2)  0.5 ml solution containing 14 units of insulin
    (4)  19 ml distilled water

    Student Groups (4) testing the variable of insulin and chromium picolinate (Glucose + Insulin +
    Chromium Picolinate):

    (2)  0.25 g sections of Rabbit Psoas Muscle
    (3)  4 ml of 105 mg glucose/100 ml of water solution
    (2)  0.5 ml solution containing 14 units of insulin
    (2)  0.1 g of chromium picolinate
    (4)  19 ml distilled water
    (1)   mortar and pestle

    All Student Groups will need the following:

     (6) test tubes
     (6) 50 ml beakers
     (1) 25 ml graduated cylinder
     (2) 1 ml pipettes
     (6) spectrophotometer cuvettes or properly sized test tubes
     (1) test tube rack

    The Class needs the following:

    Spectrophotometer 20
    Glucose Assay Kit (Sigma Diagnostics 510-A)
 
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Procedure A: This is to be done by all groups.
To establish baseline data for the spectrophotometer and construct a standard curve.

1.  Follow spectrophotometer directions for warming up the machine.
2.  Follow spectrophotometer directions for establishing a baseline.
3.  Make glucose concentration standard using the directions in the Sigma Diagnostics Glucose Assay
   Kit (Procedure No. 510).

Procedure B:  This is to be done by groups doing Glucose only.
To determine the uptake of glucose alone into a cell.

1. Place rabbit muscle in distilled water overnight before using.
2. Place  0.25g of rabbit muscle into a test tube.
3. Add 4 ml of  glucose solution to the test tube..
4. Wait 45 minutes (at room temperature) to allow glucose to enter the cells.
5. Carefully, read and follow the directions given in the glucose assay kit to determine the concentration of the glucose.
6.  Record results.
7.  Repeat steps 1 - 6 one more time.

Procedure C: This is to be done by groups doing Glucose + Insulin
To determine the uptake of glucose into a cell using insulin.

1.  Repeat Steps 1-3 in Procedure B.
2.  Add 0.5 ml of insulin into the test tube.
3.  Repeat Steps 4-7 in Procedure B.

Procedure D:  This is to be done by groups doing Glucose + Insulin + Chromium Picolinate
To determine the uptake of glucose into a cell using chromium picolinate.

1.  Repeat Steps 1 - 3 in Procedure B.
2.  Add 0.1 g of chromium picolinate into the test tube
3.  Repeat Steps 4-7 in Procedure B.
 
 
 Observation:
A Class Data Table must be established: (Sample Table below):
 

Group	Standard	Glucose Only	Glucose + Insulin	Glucose + Insulin + Chromium
1	.	.	.	.
2	.	.	.	.
3	.	.	.	.
4	.	.	.	.
5	.	.	.	.
6	.	.	.	.
7	.	.	.	.
8	.	.	.	.
9	.	.	.	.
10	.	.	.	.
11	.	.	.	.
12	.	.	.	.
Total	.	.	.	.
Average	.	.	.	.

Data Analysis:
Use the class data to determine if insulin or insulin and chromium picolinate affect glucose uptake into a cell.
Students should graph the data from the Class Data Table.
Students should do a Chi Square test to determine if variables had an effect on glucose uptake.

Conclusions:
 

 
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      Biology, 5th edition. Curtis, H., Barnes, N.S.,Worth  Publishers, Inc., NY, NY. 1989.

      Goodman and Gilman, The Pharmacological Basis of Therapeutics 9th edition /J.G. Hardman,
      A. Goodman Gilman, L.E. Limbird, McGraw-Hill Co., 1996.
 
      Insulin Receptors/C.R. Kahn, L.C. Harrrison. Liss, NY, NY. 1988

      Cell and Molecular Biology. Karp. G., John Wiley & Sons Inc.: NY, NY.1996
 
      Molecular Cell Biology. Lodish, H., W.H. Freeman and Company: NY, NY. 1995
 
      Biochemistry. second edition. Voet, D. John Wiley & Sons Inc.: NY, NY. 1995

      Insulin Biosynthesis and its Hormonal Functions
      http://www.chem.uwec.edu/Chem406/Webpages/Ying/overview.htm

      http://www.smartbasic.com/glos.minerals/chromium.glos.html

      http://www.chromium.edu/intro.htm

      http://www.joslin.harvard.edu/research/cmp/bc.html
 
      http://www.chromium.edu/intro.htm

      http://w3.ouhsc.edu/biochem/olson.htm

      http://www.mmeade.com/cheat/chromium.html

      http://www.prn.usm.my/bulletin/sun/1996/sun4.htm

      http://www.anndeweesallen.com/dal_ra01.htm

  National Science Education Standards
        Center for Science, Mathematics, and Engineering Education
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        Washington, D. C. 20418