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Blood Sugar and Click

E X E R C I S E 4 Endocrine System Physiology O B J E C T I V E S 1. To define the following terms: metabolism, hormone replacement therapy, type 1 diabetes, type 2 diabetes, and glucose standard curve. 2. To explain the role of thyroxine in maintaining an animal’s metabolic rate. 3. To explain the effects of thyroid-stimulating hormone on an animal’s metabolic rate. 4. To understand how estrogen affects bone density. 5. To explain how hormone replacement therapy works. 6. To explain how fasting plasma glucose is used to diagnose diabetes. . To understand how levels of cortisol and ACTH can be used to diagnose endocrine diseases. T he endocrine system exerts many complex and interrelated effects on the body as a whole, as well as on specific tissues and organs. Studying the effects of hormones on the body is difficult to do in a wet lab because experiments often take days, weeks, or even months to complete and are expensive. In addition, live animals may need to be sacrificed, and technically difficult surgical procedures are sometimes necessary.

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This computer simulation allows you to study the effects of given hormones on the body by using “virtual” animals rather than live ones. You can carry out delicate surgical techniques with the click of a button and complete experiments in a fraction of the time that it would take in an actual wet lab environment. Hormones and Metabolism Metabolism is the broad term used for all biochemical reactions occurring in the body. Metabolism involves catabolism, a process by which complex materials are broken down into simpler substances, usually with the aid of enzymes found in the body cells.

Metabolism also involves anabolism, in which the smaller materials are built up by enzymes into larger, more complex molecules. When larger molecules are made, energy is stored in the various bonds formed. When bonds are broken in catabolism, energy that was stored in the bonds is released for use by the cell. Some of the energy liberated may go into the formation of ATP, the energyrich material used by the body to run itself. However, not all of the energy liberated goes into this pathway; some is given off as body heat. Humans are homeothermic animals, meaning they have a fixed body temperature.

Maintaining this temperature is important to maintaining the metabolic pathways found in the body. The most important hormone in maintaining metabolism and body heat is thyroxine, the hormone of the thyroid gland, which is found in the neck. The thyroid gland secretes thyroxine, but the production of thyroxine is really controlled by the pituitary gland, which secretes thyroid-stimulating hormone (TSH). TSH is carried by the blood to the thyroid gland (its target tissue) and causes the thyroid to produce more thyroxine. So in an indirect way, an animal’s metabolic rate is the result of pituitary hormones.

In the following experiments, you will investigate the effects of thyroxine and TSH on an animal’s metabolic rate (see Figure 4. 1b). To begin, select Exercise 4: Endocrine System Physiology from the drop-down menu and click GO. Before you perform the activities, watch the BMR Measurement video to see an 43 44 Exercise 4 (a) Hypothalamus TRH TSH Pituitary gland (hypophysis) Thyroid gland T3, T4 (b) F I G U R E 4 . 1 Metabolism and the thyroid gland. (a) Opening screen of the Metabolism experiment. (b) The regulation of thyroid secretion. ndicates stimulation of release, indicates inhibition of release, T3 triiodothyronine, T4 thyroxine, TRH = thyrotropin-releasing hormone, TSH thyroid-stimulating hormone. Endocrine System Physiology 45 experiment in which basal metabolic rate is measured. Then click Metabolism. The opening screen will appear in a few seconds (see Figure 4. 1a). Select Balloons On/Off from the Help menu for help identifying the equipment on-screen (you will see labels appear as you roll the mouse over each piece of equipment). Select Balloons On/Off to turn this feature off before you begin the experiments. Study the screen.

You will see a jar-shaped chamber to the left, connected to a respirometer-manometer apparatus (consisting of a U-shaped tube, a syringe, and associated tubing). You will be placing animals—in this case, rats—in the chamber in order to gather information about how thyroxine and TSH affect their metabolic rates. Note that the chamber also includes a weight scale, and under the timer is a weight display. Next to the chamber is a timer for setting and timing the length of a given experiment. Two tubes are connected to the top of the chamber. The left tube has a clamp on it that can be opened or closed.

Leaving the clamp open allows outside air into the chamber; closing the clamp creates a closed, airtight system. The other tube leads to a T-connector. One branch of the T leads to a fluid-containing U-shaped tube, called a manometer. As an animal uses up the air in the closed system, this fluid will rise in the left side of the U-shaped tube and fall in the right. The other branch of the T-connector leads to a syringe filled with air. Using the syringe to inject air into the tube, you will measure the amount of air that is needed to return the fluid columns to their original levels.

This measurement will be equal to the amount of oxygen used by the animal during the elapsed time of the experiment. Soda lime, found at the bottom of the chamber, absorbs the carbon dioxide given off by the animal so that the amount of oxygen used can easily be measured. The amount of oxygen used by the animal, along with its weight, will be used to calculate the animal’s metabolic rate. Also on the screen are three white rats in their individual cages. These are the specimens you will use in the following experiments.

One rat is normal; the second is thyroidectomized (abbreviated on the screen as Tx), meaning its thyroid has been removed; and the third is hypophysectomized (abbreviated on the screen as Hypox), meaning its pituitary gland has been removed. The pituitary gland is also known as the hypophysis, and removal of this organ is called a hypophysectomy. To the top left of the screen are three syringes with various chemicals inside: propylthiouracil, thyroid-stimulating hormone (TSH), and thyroxine. TSH and thyroxine have been previously mentioned; propylthiouracil is a drug that inhibits the production of thyroxine.

You will perform four experiments on each animal to: (1) determine its baseline metabolic rate, (2) determine its metabolic rate after it has been injected with thyroxine, (3) determine its metabolic rate after it has been injected with TSH, and (4) determine its metabolic rate after it has been injected with propylthiouracil. You will be recording all of your data on Chart 1. You may also record your data on-screen by using the equipment in the lower part of the screen, called the data collection unit. This equipment records and displays data you accumulate during the experiments.

Check that the data set for Normal is highlighted in the Data Sets window; you will be experimenting with the normal rat first. The Record Data button lets you record data after an experimental trial. Clicking the Delete Line or Clear Data Set button erases any data you want to delete. A C T I V I T Y 1 Determining Baseline Metabolic Rates First, you will determine the baseline metabolic rate for each rat. 1. Using the mouse, click and drag the normal rat into the chamber and place it on top of the scale. When the animal is in the chamber, release the mouse button. . Be sure the clamp on the left tube (on top of the chamber) is open, allowing air to enter the chamber. If the clamp is closed, click on it to open it. 3. Be sure the indicator next to the T-connector reads “Chamber and manometer connected. ” If not, click on the T-connector knob. 4. Click on the Weigh button in the box to the right of the chamber to weigh the rat. Record this weight in the Baseline section of Chart 1 in the row labeled “Weight. ” 5. Click the ( ) button on the Timer so that the Timer display reads 1 minute. 6. Click on the clamp to close it.

This will prevent any outside air from entering the chamber and ensure that the only oxygen the rat is breathing is the oxygen inside the closed system. 7. Click Start on the Timer display. You will see the elapsed time appear in the “Elapsed Time” display. Watch what happens to the water levels in the U-shaped tube. 8. At the end of the 1-minute period, the timer will automatically stop. When it stops, click on the T-connector knob so that the indicator reads “Manometer and syringe connected. ” 9. Click on the clamp to open it so that the rat can once again breathe outside air. 10.

Look at the difference between the levels in the left and right arms of the U-shaped tube. To estimate the volume of O2 that you need to inject to equalize the levels, count the divider lines on both sides. Then click the ( ) button next to the ml O2 display until the display shows your estimate. Click Inject and watch what happens to the fluid in the two arms. When the volume is equalized, the word “Level” will appear between the arms and stay on the screen. If too little was injected, click ( ) and then Inject until the arms are level. If too much was injected, the word “Level” will flash and then disappear.

You will then have to click the Reset button and try a lower volume. (The total amount injected to equalize the arm levels is equivalent to the amount of oxygen that the rat used up during 1 minute in the closed chamber. ) Record this measurement in the Baseline section of Chart 1 in the row labeled “ml O2 used in 1 minute. ” 11. Determine the oxygen consumption per hour for the rat. Use the following formula: ml O2 consumed 1 minute 60 minutes 1 hr ml O2/hr Record this data in the Baseline section of Chart 1 in the row labeled “ml O2 used per hour. ” 46 Exercise 4 CHART 1

Effects of Hormones on Metabolic Rate Normal rat Thyroidectomized rat Hypophysectomized rat Baseline Weight ml O2 used in 1 minute ml O2 used per hour Metabolic rate 250. 2 grams ml ml ml O2/kg/hr 244. 8 grams 6. 1 ml 366ml 1. 49 7 420 1. 67 245. 8 grams 6 ml 360 ml ml O2/kg/hr 1. 46 ml O2/kg/hr With thyroxine Weight ml O2 used in 1 minute ml O2 used per hour Metabolic rate 250. 4 grams 7. 8 468 1. 86 ml ml ml O2/kg/hr 245 grams 244. 4 grams 7. 6 456 ml ml 7. 9 ml 474 ml 1. 93 ml O2/kg/hr 1. 86ml O2/kg/hr With TSH Weight ml O2 used in 1 minute ml O2 used per hour Metabolic rate 250. grams 8 ml 480 ml 1. 91 ml O2/kg/hr 245. 1 6 360 grams ml ml 2/kg/hr 244. 1 7. 4 444 1. 81 grams ml ml ml O2/kg/hr 1. 46 ml O With propylthiouracil Weight ml O2 used in 1 minute ml O2 used per hour Metabolic rate 249. 6 6. 2 372 grams ml ml 244. 3 grams 6. 1 ml 366 ml 245. 1 grams 6. 1 ml 366 ml 1. 49 ml O2/kg/hr 1. 49 ml O2/kg/hr 1. 49 ml O2/kg/hr 12. Now that you have the amount of oxygen used per hour, determine the metabolic rate per kilogram of body weight by using the following formula. (Note that you will need to convert the weight data from g to kg before you can use the formula. Metabolic rate ml O2/hr wt. in kg ml O2/kg/hr the Baseline section of Chart 1 in the appropriate column for each rat. Note that when you put the Tx vat in the chamber, the simulation highlights Tx under Data Sets (on the data collection box); likewise, it highlights Hypox when you move the hypophysectomized rat. Which rat had the fastest baseline metabolic rate? Record this data in the Baseline section of Chart 1 in the row labeled “Metabolic rate. ” 13. Click Record Data. 14. Click and drag the rat from the chamber back to its cage. 15. Click the Reset button in the box labeled Apparatus. 6. Now repeat steps 1–15 for the thyroidectomized (Tx) and hypophysectomized (Hypox) rats. Record your data in The normal rat with TSH had the highest metabolic rate of 1. 91 Endocrine System Physiology 47 Why did the metabolic rates differ? Why was this effect seen? ____________________________ the thyroxine increased The weight probably made it differ a bit. ¦ A C T I V I T Y 2 the metabolic rate of all the cells. What was the effect of thyroxine on the thyroidectomized rat’s metabolic rate? How does it compare to the thyroidectomized rat’s baseline metabolic rate? hyroxine injection is faster then thyroidectomized rat. Determining the Effect of Thyroxine on Metabolic Rate Next, you will investigate the effects of thyroxine injections on the metabolic rates of all three rats. Note that in a wet lab environment, you would normally need to inject thyroxine (or any other hormone) into a rat daily for a minimum of 1–2 weeks in order for any response to be seen. However, in the following simulations, you will inject the rat only once and be able to witness the same results as if you had administered multiple injections over the course of several weeks.

In addition, by clicking the Clean button while a rat is inside its cage, you can magically remove all residue of any previously injected hormone from the rat and perform a new experiment on the same rat. In a real wet lab environment, you would need to either wait weeks for hormonal residue to leave the rat’s system or use a different rat. 1. Choose and click on a rat to test. You will eventually test all three, and it doesn’t matter in what order you test them. Do not drag the rat to the chamber yet. Under Data Sets, the simulation will highlight Normal, Tx, or Hypox depending on which rat you use. . Click the Reset button in the box labeled Apparatus. 3. Click on the syringe labeled thyroxine and drag it over to the rat. Release the mouse button. This will cause thyroxine to be injected into the rat. 4. Click and drag the rat into the chamber. Perform steps 1–12 of Activity 1 again, except this time record your data in the With Thyroxine section of Chart 1. 5. Click Record Data. 6. Click and drag the rat from the chamber back to its cage, and click Clean to cleanse it of all traces of thyroxine. 7. Now repeat steps 1–6 for the remaining rats.

Record your data in the With Thyroxine section of Chart 1 in the appropriate column for each rat. What was the effect of thyroxine on the normal rat’s metabolic rate? How does it compare to the normal rat’s baseline metabolic rate? It made up for the Why was this effect seen? ____________________________ thyroxine that was supposed to be there but that was removed. What was the effect of thyroxine on the hypophysectomized rat’s metabolic rate? How does it compare to the hypophysectomized rat’s baseline metabolic rate? The metabolic rate after thyroxine is faster after thyroxin injection.

The gland did not make Why was this effect seen? ____________________________ TSH so it could not produce thyroxine ¦ so it compensated for it. A C T I V I T Y 3 Determining the Effect of TSH on Metabolic Rate Now you will investigate the effects of TSH injections on the metabolic rates of the three rats. Select a rat to experiment on first, and then proceed. 1. Under Data Sets, highlight Normal, Tx, or Hypox, depending on which rat you are using. 2. Click the Reset button in the box labeled Apparatus. 3. Click and drag the syringe labeled TSH over to the rat and release the mouse button, injecting the rat. . Click and drag the rat into the chamber. Perform steps 1–12 of Activity 1 again. Record your data in the With TSH section of Chart 1. 5. Click Record Data. 6. Click and drag the rat from the chamber back to its cage, and click Clean to cleanse it of all traces of TSH. 7. Now repeat this activity for the remaining rats. Record your data in the With TSH section of the chart in the appropriate column for each rat. On the normal rat, the metabolic rate after thyroxine is faster then baseline. 48 Exercise 4 What was the effect of TSH on the normal rat’s metabolic rate?

How does it compare to the normal rat’s baseline metabolic rate? 2. Click the Reset button in the box labeled Apparatus. 3. Click and drag the syringe labeled propylthiouracil over to the rat and release the mouse button, injecting the rat. 4. Click and drag the rat into the chamber. Perform steps 1–12 of Activity 1 again, except this time record your data in the With Propylthiouracil section of Chart 1. 5. Click Record Data. 6. Click and drag the rat from the chamber back to its cage, and click Clean to cleanse the rat of all traces of propylthiouracil. 7. Now repeat this activity for the remaining rats.

Record your data in the With Propylthiouracil section of Chart 1 in the appropriate column for each rat. What was the effect of propylthiouracil on the normal rat’s metabolic rate? How does it compare to the normal rat’s baseline metabolic rate? The metabolic rate is faster after the injection. Why was this effect seen? ____________________________ it increased the production of thyroxine What was the effect of TSH on the thyroidectomized rat’s metabolic rate? How does it compare to the thyroidectomized rat’s baseline metabolic rate? It is the same as the baseline.

It is slower then the metabolic rate. It had no thyroid Why was this effect seen? ____________________________ gland so it had nothing to act on. They react together in Why was this effect seen? ___________________________ a way that makes the effects decrease. What was the effect of propylthiouracil on the thyroidectomized rat’s metabolic rate? How does it compare to the thyroidectomized rat’s baseline metabolic rate? What was the effect of TSH on the hypophysectomized rat’s metabolic rate? How does it compare to the hypophysectomized rat’s baseline metabolic rate? It was faster then the baseline.

It is the same as the baseline. Why was this effect seen? ____________________________ The pituitary gland It has no effect on it Why was this effect seen? was removed and it produced thyrocine ¦ A C T I V I T Y 4 since the rat cannot produce thyroxine. What was the effect of propylthiouracil on the hypophysectomized rat’s metabolic rate? How does it compare to the hypophysectomized rat’s baseline metabolic rate? Determining the Effect of Propylthiouracil on Metabolic Rate Next, you will investigate the effects of propylthiouracil injections on the metabolic rates of the three rats.

Keep in mind that propylthiouracil is a drug that inhibits the production of thyroxine by blocking the attachment of iodine to the amino acid tyrosine and interfering with the conversion of thyroxine to triiodothyronine. Select a rat to experiment on first, and then proceed. 1. Under Data Sets, the simulation will highlight Normal, Tx, or Hypox, depending on which rat you are using. It is the same as the baseline. They plan on eachother Why was this effect seen? ___________________________ and compensate for what the other is missing. 8. If you wish, click Tools > Print Data to print all of your recorded data for this experiment. Endocrine System Physiology 49 F I G U R E 4 . 2 Opening screen of the Hormone Replacement Therapy experiment. Hormone Replacement Therapy Follicle-stimulating hormone (FSH) stimulates ovarian follicle growth. While the follicles are developing, they produce the hormone estrogen. As the female enters menopause, the ovaries stop producing estrogen. One of the symptoms of menopause is loss of bone density, which can result in osteoporosis and bone fractures. Postmenopausal treatments to prevent osteoporosis include the administration of estrogen to increase bone density.

Calcitonin is a hormone that inhibits osteoclast activity and stimulates calcium uptake for deposit in bone. In this experiment we will use three ovariectomized rats because they are no longer producing estrogen due to the removal of their ovaries. The three rats were chosen because each has a baseline T score of 2. 6, indicating osteoporosis. T scores are interpreted as follows: normal 1 to 0. 99; osteopenia (bone thinning) 1. 0 to 2. 49; osteoporosis 2. 5 and below. You will administer either estrogen therapy or calcitonin therapy, two types of hormone replacement therapy.

The third rat will serve as an untreated control and receive daily injections of saline. The vertebral bone density (VBD) of each rat will be measured with dual X-ray absorptiometry (DXA) to obtain the T score. Start by selecting Hormone Replacement Therapy from the Experiment menu. A new screen will appear (Figure 4. 2) with three ovariectomized rats in cages. (Note that if this were a wet lab, the ovariectomies would have been performed on the rats a month prior to the rest of the experiment in order to ensure that no residual hormones remained in the rats’ systems. Also on screen are a bottle of saline, a bottle of estrogen, a bottle of calcitonin, a clock, and a dual X-ray absorptiometry bone density scanner. A C T I V I T Y 5 Hormone Replacement Therapy 1. Click on the syringe, drag it to the bottle of saline, and release the mouse button. The syringe will automatically fill with 1 ml of saline. 2. Click and hold the syringe, drag it to the control rat, and place the tip of the needle in the rat’s lower abdominal area. Injections into this area are considered intraperitoneal and will quickly be picked up by the abdominal blood vessels. 50

Exercise 4 Release the mouse button—the syringe will empty into the rat and automatically return to its holder. Click Clean on the syringe holder to clean the syringe of all residue. 3. Click on the syringe again, this time dragging it to the bottle of estrogen, and release the mouse button. The syringe will automatically fill with 1 ml of estrogen. 4. Click and hold the syringe, drag it to the estrogentreated rat, and place the tip of the needle in the rat’s lower abdominal area. Release the mouse button—the syringe will empty into the rat and automatically return to its holder.

Click Clean on the syringe holder to clean the syringe of all residue. 5. Click on the syringe again, this time dragging it to the bottle of calcitonin, and release the mouse button. The syringe will automatically fill with 1 ml of calcitonin. 6. Click and hold the syringe, drag it to the calcitonintreated rat, and place the tip of the needle in the rat’s lower abdominal area. Release the mouse button—the syringe will empty into the rat and automatically return to its holder. Click Clean on the syringe holder to clean the syringe of all residue. 7. Click on the clock.

You will notice the hands sweep the clock face twice, indicating that 24 hours have passed. 8. Repeat steps 1–7 until each rat has received a total of 7 injections over the course of 7 days (1 injection per day). Note that the # of injections displayed below each rat cage records how many injections the rat has received. The control rat should receive 7 injections of saline, the estrogen-treated rat should receive 7 injections of estrogen, and the calcitonintreated rat should receive 7 injections of calcitonin. 9. You are now ready to measure the effect of each of the solutions.

First, predict the effect that each solution will have on the rat’s vertebral bone density. Saline injections 13. Click Record Data. 14. Click and drag the rat to return it to its cage. 15. Repeat steps 10–14 for the estrogen-treated rat. T score (estrogen): -2. 0 + . 15 16. Repeat steps 10–14 for the calcitonin-treated rat. T score (calcitonin): -2. 6 + . 15 17. Click Tools > Print Data to print your recorded data for this experiment. Recall that the baseline value for all three rats was 2. 6. T scores are interpreted as follows: normal 1 to 0. 99; osteopenia (bone thinning) 1. to 2. 49; osteoporosis 2. 5 and below. What effect did the administration of estrogen injections have on the estrogen-treated rat? Estrogen changed the rates T score from the osteoporosis range to the osteopenia range. What effect did the administration of calcitonin injections have on the calcitonin-treated rat? It has little to no effect on the rats. How did these results compare with your predictions? They were just what I thought. Brittle or the same ¦ Make them weaker, or have Estrogen injections no effect. Calcitonin injections Insulin and Diabetes Still be the same.

Insulin is produced by the beta cells of the endocrine portion of the pancreas. It is vital to the regulation of blood glucose levels because it enables the body’s cells to absorb glucose from the bloodstream (see Figure 4. 4b). When insulin is not produced by the pancreas, type 1 diabetes mellitus results. When insulin is produced by the pancreas but the body fails to respond to it, type 2 diabetes mellitus results. In either case, glucose remains in the bloodstream, unable to be taken up by the body’s cells to serve as the primary fuel for metabolism. The following experiment is divided into two parts.

In Part I, you will obtain a glucose standard curve, which will be explained shortly. In Part II, you will use the standard 10. A gaseous anesthetic will be applied to immobilize the rats for imaging. Click on the Anesthesia button for the control rat to immobilize the rat. 11. Click on the control rat and drag it to the exam table. Release the mouse to release the rat. 12. Click the Scan button to activate the scanner. Record the T score: T score (control): -2. 7 + . 15 Endocrine System Physiology 51 F I G U R E 4 . 3 Opening screen of the Insulin and Diabetes experiment, Part I. urve to measure fasting plasma glucose levels in patients to diagnose diabetes mellitus. Part I A C T I V I T Y 6 Obtaining a Glucose Standard Curve To begin, select Insulin and Diabetes-Part 1 from the Experiment menu (see Figure 4. 3). Select Balloons On/Off from the Help menu for help identifying the equipment on-screen. (You will see labels appear as you roll the mouse over each piece of equipment. ) Select Balloons On/Off to turn this feature off before you begin the experiments. On the right side of the opening screen is a special spectrophotometer.

The spectrophotometer is one of the most widely used research instruments in biology. It is used to measure the amounts of light of different wavelengths absorbed and transmitted by a pigmented solution. Inside the spectrophotometer is a source for white light, which is separated into various wavelengths (or colors) by a prism. The user selects a wave- length (color), and light of this color is passed through a special tube, or cuvette, containing the sample being tested. (For this experiment, the spectrophotometer light source will be preset for a wavelength of 450 nanometers, or nm. The light transmitted by the sample then passes onto a photoelectric tube, which converts the light energy into an electrical current. This current is measured by a meter. Alternatively, the light may be measured before the sample is put into the light path, and the amount of light absorbed—called optical density—is measured. Using either method, the change in light transmitted or light absorbed can be used to measure the amount of a given substance in the sample being tested. In Part II, you will use the spectrophotometer to determine how much glucose is present in blood samples taken from two rats.

Before using the spectrophotometer, you must obtain a glucose standard curve so that you have a point of reference for converting optical density readings into glucose readings, which will be measured in mg/deciliter (mg/dl). To do this, you will prepare five test tubes that contain known amounts of glucose: 30 mg/dl, 60 mg/dl, 90 mg/dl, 120 mg/dl, and 150 mg/dl, respectively. You will then use the spectrophotometer to determine the corresponding optical density 52 Exercise 4 readings for each of these known amounts of glucose. Information obtained in Part I will be used to perform Part II.

Also on the screen are three dropper bottles, a test tube washer, a test tube dispenser (on top of the washer), and a test tube incubation unit with numbered cradles that you will need to prepare the samples for analysis. 1. Click and drag the test tube (on top of the test tube washer) into slot 1 of the incubation unit. You will see another test tube pop up from the dispenser. Click and drag this second test tube into slot 2 of the incubation unit. Repeat until you have dragged a total of five test tubes into the five slots in the incubation unit. 2. Click and hold the mouse button on the dropper cap of the glucose standard bottle.

Drag the dropper cap over to tube 1. Release the mouse button to dispense the glucose. You will see that one drop of glucose solution is dropped into the tube. 3. The dropper will automatically slide over to each of the remaining samples. Notice that each subsequent tube will automatically receive one additional drop of glucose standard (i. e. , tube 2 will receive two drops, tube 3 will receive three drops, tube 4 will receive four drops, and tube 5 will receive five drops). 4. Click and hold the mouse button on the dropper cap of the deionized water bottle. Drag the dropper cap over to tube 1.

Release the mouse button to dispense the water. Notice that four drops of water are automatically added to the first tube. 5. The dropper will automatically slide over to each of the remaining samples. Notice that each subsequent tube will receive one less drop of water than the previous tube (i. e. , tube 2 will receive three drops, tube 3 will receive two drops, tube 4 will receive one drop, and tube 5 will receive no drops of water). 6. Click on the Mix button of the incubator to mix the contents of the tubes. 7. Click on the Centrifuge button. The tubes will descend into the incubator and be centrifuged. 8.

When the tubes resurface, click on the Remove Pellet button. Any pellets from the centrifuging process will be removed from the test tubes. 9. Click and hold the mouse button on the dropper cap of the enzyme color reagent bottle. Still holding the mouse button down, drag the dropper cap over to tube 1. When you release the mouse, you will note that five drops of reagent are added to the tube. 10. The dropper will automatically slide over to each of the remaining samples. 11. Now click Incubate. The tubes will descend into the incubator, incubate, and then resurface. 12. Using the mouse, click on Set Up on the spectrophotometer.

This will warm up the instrument and get it ready for your readings. 13. Click and drag tube 1 into the spectrophotometer (above the Set Up button) and release the mouse button. The tube will lock into place. 14. Click Analyze. You will see a spot appear on the screen and values appear in the Optical Density and Glucose displays. 15. Click Record Data on the data collection unit. 16. Click and drag the tube into the test tube washer. 17. Repeat steps 13–16 for the remaining test tubes. 18. When all five tubes have been analyzed, click on the Graph button. This is the glucose standard graph, which you will use in Part II of the experiment.

Click Tools > Print Data to print your recorded data. ¦ Part II A C T I V I T Y 7 Measuring Fasting Plasma Glucose Select Insulin and Diabetes-Part 2 from the Experiment menu. A new screen will appear (Figure 4. 4a). Four reagents and five patient samples are present. To undergo the fasting plasma glucose (FPG) test, patients must fast for a minimum of 8 hours prior to the blood draw. Plasma samples will be measured in the spectrophotometer, and the glucose standard curve generated in Part I will be used to determine fasting plasma glucose levels in the five patient samples.

A patient with two separate FPG tests greater than or equal to 126 mg/dl is diagnosed with diabetes. FPG values between 110 and 126 mg/dl are indicative of impairment or borderline impairment of glucose uptake by cells. FPG values less than 110 mg/dl are normal. If the FPG is borderline, another test, the oral glucose tolerance test (OGTT), is performed. In this test, the patient also fasts for 8 hours. The patient then ingests a concentrated glucose solution, and blood is drawn and tested at periodic intervals. Glucose and sometimes insulin levels are measured.

The 2-hour glucose level should be below 140 mg/dl. A 2-hour OGTT level between 140 and 200 mg/dl indicates impaired glucose tolerance, and a level above 200 mg/dl confirms the diabetes diagnosis. Individuals with impaired fasting glucose values and impaired glucose tolerance are at a higher risk of developing type 2 diabetes. If a patient is pregnant, an FPG value greater than 110 mg/dl could indicate gestational diabetes and a strict diet should be followed for the remainder of the pregnancy. 1. Click and drag a test tube (on top of the test tube washer) into slot 1 of the incubation unit.

You will see another test tube pop up from the dispenser. Click and drag the second test tube into slot 2 of the incubation unit. Repeat until you have dragged a total of five test tubes into the five slots in the incubation unit. 2. Click and hold the mouse button on the dropper cap of Sample 1 and then drag the dropper to the first test tube. The dropper will automatically dispense 3 drops of blood into the test tube and automatically return to the vial. 3. Repeat step 2 for the remaining patient samples. 4. Click and hold the mouse button on the dropper cap of the deionized water bottle.

Drag the dropper cap over to test tube 1. Release the mouse to dispense the water. Five drops of water will be dispensed into the tube. 5. The dropper will automatically slide over to the remaining tubes and will add five drops to each tube. The dropper Endocrine System Physiology 53 F I G U R E 4 . 4 Insulin and diabetes. (a) Opening screen of the Insulin and Diabetes experiment, Part II. will automatically return to the vial when the dispensing is complete. 6. Click and hold the mouse button on the dropper of barium hydroxide. Drag the dropper cap over to test tube 1.

Release the mouse button to dispense the barium hydroxide. Five drops of the solution will be dispensed. 7. The dropper will automatically slide over to the remaining tubes and will add five drops to each tube. The dropper will automatically return to the vial when the dispensing is complete. (Barium hydroxide is used for clearing proteins and cells so that clear glucose readings may be obtained. ) 8. Click and hold the mouse button on the dropper of the heparin bottle. Drag the dropper cap over to tube 1. Release the mouse button to dispense the heparin. 9.

The dropper will automatically slide over to the remaining tubes and will add one drop to each tube. The dropper will automatically return to the vial when the dispensing is complete. (Heparin is an anticoagulant that prevents blood clotting. ) 10. Click on the Mix button of the incubator to mix the contents of the tubes. 11. Click on the Centrifuge button. The tubes will descend into the incubator, be centrifuged, and then resurface. 12. Click on the Remove Pellet button to remove any pellets from the centrifugation process. 13. Click and hold the mouse button on the dropper of the enzyme color reagent bottle.

Drag the dropper cap to test tube 1. Release the mouse to dispense the reagent. 14. The dropper will automatically slide over to the remaining tubes and will add five drops to each tube. The dropper will automatically return to the vial when the dispensing is complete. 15. Click Incubate. The tubes will descend into the incubator, incubate, and then resurface. 16. Click Set Up on the spectrophotometer to warm up the instrument and get it ready for your readings. 17. Click Graph Glucose Standard. The graph from Part I of the experiment will appear on the monitor. 54 Exercise 4

Stimulates glucose uptake by cells Insulin Tissue cells Stimulates glycogen formation Glucose Glycogen Blood glucose falls to normal range Pancreas Liver Stimulus: Rising blood glucose level Imb ala n ce Homeostasis: Normal blood glucose level (about 90 mg/100 ml) Imb ala n ce Stimulus: Declining blood glucose level Blood glucose rises to normal range Pancreas Glycogen Glucose Stimulates glycogen breakdown Glucagon Liver (b) F I G U R E 4 . 4 (continued) Insulin and diabetes. (b) Regulation of blood sugar levels by insulin and glucagon. 18. Click and drag tube 1 to the spectrophotometer and release the mouse button.

The tube will lock into place. 19. Click Analyze. You will see a horizontal line appear on the screen and a value appear in the Optical Density display. 20. Drag the movable ruler (the vertical line on the far right of the spectrophotometer monitor) over to where the horizontal line (from step 19) crosses the glucose standard line. Watch what happens to the Glucose display as you move the movable ruler to the left. What is the glucose reading where the horizontal line crosses the glucose standard line? Sample 1: glucose concentration of 21. Click Record Data on the data collection unit. 22.

Click and drag the test tube from the spectrophotometer into the test tube washer, then click Clear under the display. 23. Repeat steps 17–22 for the remaining test tubes. Record your glucose readings for each test tube here: 111 Sample 2: glucose concentration of Sample 3: glucose concentration of Sample 4: glucose concentration of Sample 5: glucose concentration of mg/deciliter 128 mg/deciliter 117 mg/deciliter 137 mg/deciliter 95 mg/deciliter This is your glucose reading for the patient being tested. For which patient(s) were the glucose reading(s) in the normal Endocrine System Physiology 55 range? patient 1 TA B L E 4 . Cortisol and ACTH Disorders Cortisol level ACTH level For which patient(s) were the fasting plasma glucose reading(s) in the diabetic range? 3 and 5 For which patient(s) were the fasting plasma glucose reading(s) in the impaired range? 2 and 4 What recommendations would you make to a patient with an impaired FPG value who also tested in the impaired range with the oral glucose tolerance test? Cushing’s syndrome (primary hypercortisolism) Cushing’s disease (secondary hypercortisolism) Iatrogenic Cushing’s syndrome Addison’s disease (primary adrenal insufficiency) Secondary adrenal insufficiency (hypopituitarism)

High High High Low Low Low High Low High Low due to damage to the pituitary gland. Levels of ACTH are also low in secondary adrenal insufficiency. A variety of endocrine disorders are related to both high and low levels of cortisol and adrenocorticotropic hormone. Sugars are restricted Table 4. 1 summarizes these endocrine disorders. Start by selecting Measuring Cortisol and Adrenocorticotropic Hormone from the Experiment menu. A new screen will appear (Figure 4. 5a) with five patient plasma samples and an HPLC (high-performance liquid chromatograPatient 3 is pregnant; how might this change the diagnosis? hy) column that will be used to simulate the measurement of What recommendations would you make to this patient? cortisol and adrenocorticotropic hormone (ACTH). There is a I would say she has gestational syringe that will be used to inject the samples into the HPLC injector for analysis. The Cortisol and ACTH buttons are used to diabetes. A special diet would be recommended where she is to restrict prepare the column with solvents used to separate the two different hormones. The detector will measure the amount of the sugars. hormone and convert it into a concentration value. ¦ 1.

Start the experiment by clicking on the Cortisol button. This will prepare the column for the separation and measureA C T I V I T Y 8 ment of cortisol. Measuring Cortisol and Adrenocorticotropic Hormone Cortisol, a hormone secreted by the adrenal cortex, is key to the long-term regulation of stress. Cortisol release is stimulated by adrenocorticotropic hormone (ACTH), a hormone released by the anterior pituitary. ACTH release is stimulated by a hypothalamic hormone, corticotropin-releasing hormone (CRH). Increased levels of cortisol negatively feed back to inhibit the release of both ACTH and CRH.

See Figure 4. 5b for the regulation of cortisol secretion. Increased cortisol in the blood, or hypercortisolism, is referred to as Cushing’s syndrome if it is due to an adrenal tumor. Hypercortisolism caused by a pituitary tumor also causes levels of ACTH to increase and is referred to as Cushing’s disease. Cushing’s syndrome can also be iatrogenic; that is, physician induced. This occurs when glucocorticoid hormones such as prednisone are administered for the treatment of rheumatoid arthritis, asthma, or lupus and is often referred to as “steroid diabetes” because it results in hyperglycemia.

Hypocortisolism can occur due to adrenal insufficiency. In primary adrenal insufficiency, also known as Addison’s disease, the low cortisol is directly due to gradual destruction of the adrenal cortex, and ACTH levels are typically elevated as a compensatory effect. Secondary adrenal insufficiency also results in low levels of cortisol, usually 2. Click and hold the syringe and drag it over to the first patient sample. Then release the mouse. The syringe will fill with plasma. 3. Click and hold the syringe again and drag it over to the HPLC injector. Then release the mouse.

The sample will enter the tubing and flow through the column. The detector will display the concentration of cortisol in the first patient sample. 4. Click Record Data. 5. Click the Clean button under the syringe to prepare it for the next sample. 6. Click the Clean Column button near the top of the screen to remove residual cortisol from the column. 7. Repeat steps 2–6 for the remaining four patient samples. 8. Next, prepare the column for ACTH separation and measurement by clicking on the ACTH button. 9. Click and hold the syringe and drag it over to the first patient sample.

Then release the mouse. The syringe will fill with plasma. 10. Click and hold the syringe again and drag it over to the HPLC injector. Then release the mouse. The sample will enter the tubing and flow through the column. The detector will display the concentration of ACTH in the first patient sample. 56 Exercise 4 Stress CRH Hypothalamus Anterior pituitary ACTH Adrenal cortex Cortisol (b) F I G U R E 4 . 5 Following the cortisol release pathway. (a) Opening screen of the Measuring Cortisol and ACTH experiment. (b) The regulation of cortisol secretion. ndicates stimulation of release, indicates inhibition of release, CRH corticotropin-releasing hormone, ACTH = adrenocorticotropic hormone. Endocrine System Physiology 57 TA B L E 4 . 2 Abnormal Morning Cortisol, ACTH Levels High Low 5 mcg/dl 20 pg/ml 16. Click Tools > Print Data to print your recorded data for this experiment. 17. Record your results for each patient here and circle High or Low: Patient 1: Cortisol ACTH Cortisol ACTH Note: 1 mcg 1 g 23 mcg/dl 80 pg/ml 1 microgram 3+1 mcg/dl High/Low 18 +2 pg/ml High/Low mcg/dl High/Low Patient 2: Cortisol 35+5 ACTH 13+2 pg/ml 11. Click Record Data. 2. Click the Clean button under the syringe to prepare it for the next sample. 13. Click the Clean Column button near the top of the screen. 14. Repeat steps 9–13 for the remaining four patient samples. 15. Select a row in the Data Set and choose High or Low based on the breakpoints shown in Table 4. 2 for cortisol and ACTH in plasma from a morning blood draw. Patient 3: Cortisol ACTH Patient 4: Cortisol High/Low High/Low 45+5 mcg/dl 86+5 pg/ml High/Low 3+1 100+5 mcg/dl High/Low ACTH _______ pg/ml Patient 5: Cortisol 50+5 High/Low High/Low ¦ mcg/dl ACTH _______ pg/ml 18+2 High/Low

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