Lab 4: Pre-Lab

We have already learned a lot about the disease itself and the role glucose plays in diabetes. This lab will focus on the biochemistry behind some the symptoms of diabetes and necessitates further understanding of macromolecules (carbs, lipids and proteins) and of cells. You will explore diffusion and the insulin transduction pathway. You will conduct a series of tests to evaluate the effects of disruption of this pathway in diabetics. 

• Introduction
• Do you know enough?
• What will we do in lab?
• LABridge

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1) How does glucose get into our cells?

The phospholipid bi-layer around cells protects life from non-life. Comprised of two layers of amphipathic phospholipds (with hydrophilic phosphate heads facing outwards and hydrophobic lipid tails facing inward, the cell membrane regulates what gets into a cell and what gets out. Molecules can enter a cell in three primary ways:

• Simple diffusion- Solute moves down its concentration gradient, from areas of HIGH concentration to LOW concentration until an equilibrium is reach. This process requires no energy, it is passive and automatic. When water moves via this process, it is referred to as osmosis (more on this later).
• Facilitated diffusion- Solute requires help from proteins embedded in the membrane to enter the cell (ex: ion channels and carrier proteins). But because the solute is STILL moving down its concentration gradient (from areas of HIGH concentration to LOW concentration), this process also requires no energy. 
• Active transport- Moves substances against their gradient (from areas of LOW concentration to areas of HIGH concentration) and therefore requires an input of energy (often ATP).  Protein pumps perform active transport across the membrane. 

So what determines which process is required? That depends on the solute or molecule attempting to cross and the needs of the cell. We're interested in glucose and the role it plays in diabetes. 

​Click on the link below for a brief explanation!

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how glucose gets into our cells

Cell membrane - the phospholipid bi-layer

The role of cellular transport in digestion

Review the types of transport above. Make sure you understand which is which. Brainstorm some potential characteristics that may make it less likely for a substances to move via diffusion alone.

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2) Do you know what happens in individuals with Type 2 Diabetes?

What happens if glucose doesn't get into the cell? The effect is an increased concentration of glucose in the blood. This establishes a concentration gradient, with higher concentrations of glucose outside the cell than inside the cell, a condition referred to as hyperglycemia. As diabetics often use artificial insulin (which we will discuss later) to regulate their blood sugar, they can also experience extremely low blood sugar due to insulin dosing and diet. This condition is known as hypoglycemia.  In both conditions, osmosis will occur in an attempt to regulate the in-balance and maintain homeostasis. 

Osmosis is a particular type of diffusion. It is automatoc and passive (requires no energy). In osmosis, water moves from areas with low solute concentration to areas of high solute concentration until equilibrium is reached. You can think of water moving to another side to "water it down," like adding water to too sweet tea.

When predicting the flow of water during osmosis, you can think of differences between intracellular and extracellular environments in terms of tonicity. These terms refer to the solution of the extracellular environment in comparison to the intracellular environment:

• An Isotonic Solution: is equal in solute concentration to that of the intracellular environment.
• A Hypotonic Solution: is LOWER in solute concentration to that of the intracellular environment. This condition would cause red blood cells to swell and potentially burst as water rushes into the cell towards equilibrium.
• A Hypertonic Solution: is HIGHER in solute concentration to that of the intracellular environment.  This condition would cause red blood cells to shrivel as water rushes out of the cell towards equilibrium 

​Be sure you understand the difference between diffusion and osmosis and that you could diagram each process.

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​Please review the conditions of hypo (think LOW) and hyperglycemia including the associated symptoms of each. 

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Hypoglycemic and hyperglycemic blood glucose levels vs. normal. Click to enlarge.

Symptoms of hypo & hyperglycemia. Click to enlarge.

3) What will we do in lab and how will we do it?

Lab 4 will proceed in three parts. 

1) Predict the glucose concentration of two samples from 2 different patients: In the lab you will be provided with

• Dialysis tubing to make artificial cells
• Cellular solution matching "internal" cellular contents
• Serum solutions from three patients matching various "external" blood glucose levels

You will be asked to develop an osmosis-based protocol to determine which serum solution represents a hypoglycemic diabetes patient or a hyperglycemic diabetes patient. 

2) Determine the glucose level of the samples: You will accomplish this by using changes in mass, the Benedict's tests for simple sugars (from Lab 3), and Glucose Testing Strips. You will  compare the results to your predictions.

3) Review signal transduction pathways: If it's hard or impossible for glucose to move across the membrane of some cells, how does it get in? We will research this pathway and the vital role it plays in diabetes.

Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 4: Protocol

In today's lab you analyse variables that can affect the diffusion potential of a molecule. You will explore signal transduction pathways to understand the role they play in diabetes. Lastly, you will design an experiment which relies on the principles of osmoregulation to predict the condition of diabetic patients. 

Exercise I. Predict the glucose concentration of two samples.

Exercise II. Compare predictions with testing strips 

​Exercise III. Review signal transduction pathways

Lab Objectives: Following today's lab, you should be able to...

• Exercise I
• Exercise II
• Exercise III

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Exercise I. Predict the condition of diabetic patients: Hyper or Hypoglycemic?

As you read in the Pre-Lab, insulin enters our cells in several ways. The most important pathway is the insulin transduction pathway (which you will learn more about in Exercise III).  This pathway is disrupted in diabetics either because insulin doesn't bind to the receptor or the receptor isn't activated upon binding. Regardless of the cause, the effect is an increased concentration of glucose (solute) in the blood. When this happens water moves into and out of cells attempting to reach an equilibrium, a process called osmoregulation.

Materials: Your Tool Kit

At Your Stations

• Serum samples from Patient 1 and 2 (in beakers labeled "Solution 1" and "Solution 2"). These are extracellular solutions from Patient 1 and Patient 2.
• Cell samples for two cells (labeled "Cell 1" and Cell 2"). These are intracellular solutions from Patient 1 and Patient 2.
• Benedict's Reagent
• Waterbath
• Gloves, goggles
• Labeling tape, markers
• 1 mL and 5mL pipette & tips,
• Dialysis tubing (soaking in a beaker labeled "DI H2O") 
• clips
• a small funnel
• Digital balance & weigh boats
• Droppers
• Glucose Monitor and test strips

​In this experiment you are going to use the water loss or water gain of an artificial "cell" to predict the status of the extracellular environment (e.g., hypertonic or hypotonic) and the condition of each patient (hyperglycemic or hypoglycemic).

Procedure

1. Create 2 cells: Today we will use dialysis tubing as a substitute for a cellular membrane as both are semi-permeable. The tubing has been pre-cut into 15cm segments and is soaking in a beaker of DI water at your station.
2. You’ve likely heard of kidney dialysis before, in which a cellulose membrane tube is used to filter a patient’s blood removing waste and excess solutes, in place of failing kidneys. Dialysis tubing is semi-permeable, glucose is too large to quickly pass through, so it is a good stand-in for a cell membrane.
3. Get your tubing from the beaker labeled "DI Water." Gradually roll the tubing between your fingers and thumb to open it up.
4. Fold over one end of the tubing and clamp with clip to create leak-proof seal.
5. Using a funnel and graduated cylinder, fill your cell with 20 mL of the intracellular solution from the beaker labeled "Cell 1." Use a funnel and go slow.
6. Reduce the amount of air in the "cell" as much as possible.
7. Fold over the other end and clamp it.
8. Place your cell on a clean dry paper towel and gently dry off the exterior surface. Allow the rest to air dry completely.
9. Then hold the cell from each end to check for leaks. Re-fold and re-dry if leaks appear.
10. Repeat for "Cell 2."
11. Next, weigh each cell and record the mass. Use these instructions for the digital balance at your station.
12. Your lab has been provided simulated serum samples from two different diabetic patients in beakers labeled "Solution 1 and 2."
13. Each beaker is your testing environment for one of the cells you have made.
14. Place each cell into its corresponding solution.
15. Allow osmosis to occur: Record the time and insert your cell into your test beaker. Allow osmosis to occur for 30 minutes. 
16. As osmosis is occurring think about which direction the water may or may not be moving based on the principles of osmoregulation and the extracellular environment.
17. You will use the changes in the mass of each cell to predict the condition (i.e, hyper or hypoglycemic) of each diabetic patient based on their corresponding samples.
18. While the experiment is running, you can begin work on  on your notebook entry for Exercise III. Be sure to consult the blood glucose (BGL) chart in the side bar. 
19. Once 30 minutes have elapsed, remove and dry the "cell." 
20. Once the cells are completely dry, re-weigh each and record the results in your notebook.​
21. Also record your results in the class data sheet (front of lab).​
22. Make your predictions! Based on the change in mass, which patients may be struggling diabetes? 

Tonicity of the extracellular environment.

Related conditions in diabetics.

Lab 4 Notebook Guide. Click to download.

Blood Glucose Level (BGL) Chart. Click to enlarge.

Exercise II. Test your predictions

Procedure: Test 1

1. After you have made your predictions, you must check-in with your instructor before proceeding.
2. Locate the Benedict's solution to test the concentration of "blood glucose level" in each of the the two serum samples (Solution 1 and 2).
3. Use the same protocol we used in Lab 3 to conduct the test. It's in the sidebar as well.
4. Compare your samples to each other (no need for + or - controls).
5. Record the results in your Lab Notebook Guide.
6. Do they match your original predictions based on the change in mass?

​​ Procedure: Test 2​

1. You will use the glucose monitor and test strips to confirm or refute the predictions you just made.
2. Because the blood sugar levels of diabetics are artificially kept in the normal range via insulin, consistent monitoring is required so the timing and dosage of insulin treatments can match the individual needs of each patient.  
3. View this video to see what glucose monitoring entails for diabetics. 
4. There is a testing station located at the back bench which includes sterile serum solutions for patients 1 and 2.
5. Watch the demo in the side bar on how to use the monitor.
6. The Quick Reference Guide may also be helpful.
7. Complete Exercise II in your lab notebook guide.

Benedict's test protocol. Click to enlarge.

Blood Glucose Level (BGL) Chart. Click to enlarge.

1. Before you move on to Exercise III, please ensure your station looks exactly as pictured at the start of lab. Replace your paper towels and be sure the monitor goes back into the box. Dump your extracellular solutions down the sink, rinse the beakers (no soap) and dry them. All your leftover Benedict's test solutions go in hazardous waste. Clean the test tubes and put them back in your rack, upside down so they can drain onto a paper towel.. 

Exercise III. Explore the insulin transduction pathway

What does the evidence from our Pre-Lab, Exercise I and your research tell you about glucose? For some cells, an alternate pathway to simple diffusion is required. Specifically, some cells need to employ a signal trandsuction pathway to actively transport glucose when there are high concentrations in the blood.

Signal transduction is imperative for cell-to-cell signaling. These biochemical pathways convert an extracellular signal to an intracellular signal, thus passing messages across our bodies. Transduction pathways occur when a signaling molecule binds to the surface of a cell, and its signal is then transferred and converted through a series of events to reach a target inside the cell, and cause the intended response.

​​Insulin is a signaling molecule secreted by the pancreas when blood glucose levels are high. Insulin is a peptide hormone, in-soluble in lipids, and therefore cannot cross the cell membrane.  Instead, a transduction pathway is required. In the pathway, insulin binds to a receptor in the cell membrane which triggers glucose uptake into the cell by a glucose transporter (typically GLUT-4). This uptake eventually decreases blood glucose levels. A transduction pathway is required for this to occur because:

1. Insulin cannot cross the phospholipid bi-layer. It is lipid in-soluble.
2. Glucose is too large to easily cross (diffuse) across the cell membrane in some types of cells.

Insulin is not required for all types of glucose uptake, but is required for muscle and fat tissues. Insulin-mediated glucose uptake is the pathway most responsible for blood sugar regulation.

Insulin Pathway Video

Procedure

1. Move to Exercise III in your Notebook Guide.
2. Please review the general steps of signal transduction in the figure at the top of the sidebar.
3. Review the specific steps of the insulin transduction pathway in the figure in the sidebar.
4. Watch the video. It lasts just 4 minutes. The protein that acts as the glucose transporter is commonly referred to as GLUT-4. Other types of GLUT work in different types of cells.
5. ​Complete your concept map in the Notebook Guide.​ A concept map or flowchart use shapes to represent different types of actions or steps in a process. Lines and arrows show the sequence of the steps, and the relationships among them. See the example in the sidebar.

If you finish early, work on your report.
LAB 2 Exercise III. has all the details.

General transduction pathway. Click to enlarge.

Insulin transduction pathway. Click to enlarge.

Another representation of the insulin pathway. Click to enlarge.

Flow chart/ Concept Map example of G-protein pathways. Click to enlarge.

Lab 4 BIOL 120 CONNECTIONS
Section 6.2: Phospholipid Bilayers
Section 6.3: How Substances Move Across Lipid Bilayers: Diffiusion & Osmosis 
Section 6.4: Proteins Alter Membrane Structure & Function
​Section 11.3: How do Distant Cells Communicate?
Section 41.4: Nutritional Homeostasis Glucose as a Case Study

Faculty Spotlight: Dr. Ajay Srivastava

Email: ajay.srivastava@wku.edu

The Srivastava lab uses fruit flies (Drosophila) to better understand cell development and disease. Dr. Srivastava is particularly interested in the connection between the Extracellular Matrix (ECM) and the production and formation of tumors. The ECM is a dense network that connects the phospholipid bi-layer to the external cellular environment and is vital in cell structural support, growth and signaling. Just as our understanding of the cell membrane helps in the treatment of diabetes, Dr. Srivastava hopes that expanding our knowledge of the ECM can augment our understanding of cancer (Lab Web Page). He is always looking for talented and interested undergrads to help with his research! Check here for available positions.
Research Key Words: The ECM and the basement membrane, microscopy, genetics, gene expression, cell growth and repair, cancer, tumors
​Recent Publication:  Powers, N, Srivastava, A. JAK/STAT signaling is involved in air sac primordium development of Drosophila melanogaster. FEBS Lett. 2019 Mar 10. doi: 10.1002/1873-3468.13355. [Epub ahead of print] PMID: 30854626 (Selected for "Editor's Choice" and on the front cover of the journal)