Analyze the Data Chapter 4: FACS of Glucose Transport In unstimulated muscle cel
ID: 196185 • Letter: A
Question
Analyze the Data Chapter 4: FACS of Glucose Transport
In unstimulated muscle cells, the glucose transporter Glut4 is present in the membrane of endosomes. Upon stimulation of these cells with insulin, the endosomes fuse with the plasma membrane, thereby depositing Glut4 in the plasma membrane, where it serves to transport glucose into the cells (see Figure 15-34 on page 659). In order for insulin to elicit this change in Glut4 localization, insulin must bind to its receptor, present in the plasma membrane of some cells. To determine if a stem cell line can be induced in culture to differentiate into muscle cells in which Glut4 is expressed and exhibits this insulin-dependent change in behavior, the following studies were undertaken.
A stem cell line was incubated under culture conditions in which stem cells can differentiate into various specific cell types. The cells were then incubated in the presence and absence of insulin. To determine if either Glut4 or the insulin receptor were present on the cell surface of cells within this population, antibodies that specifically recognize each protein were used to label the cells. Glut4 on the cell surface was detected by an anti-Glut4 antibody, which fluoresces green; the insulin receptor on the cell surface was detected by an anti-insulin receptor antibody, which fluoresces red. These cells were then placed in a fluorescence activated cell sorter FACS machine (see Figure 9-29 in the text) and sorted based on both red and green fluorescence. The following data were obtained. Each • on the graphs represents 20 cells. An increased value of fluorescence intensity indicates that more antibody has bound to those cells.
A. Do all cells in the population express insulin receptor on their surface? Is this expression
dependent on the presence of insulin?Explain.
(a) Insulin (b) Insulin 10 10 0 104 100 10 104 Red fluorescence 10 10 Red fluorescenceExplanation / Answer
Do all cells in the population express insulin receptor on their surface? Is this expression dependent on the presence of insulin? Explain.
Yes, all cells in the population express insulin receptor. In the absence of insulin, as shown in figure (a), the number of cells expressing insulin receptor was high. Dots representing cells was found to be higher in the fluorescence intensity range of 104 to 107. In the presence of insulin, as shown in figure (b), the number of cells expressing insulin receptor drastically reduced. However, the expression of Glut4 is highly dependent on the presence of insulin receptors. Thus cells showing increased green fluorescnce must express insulin receptor. So, all cells in the population would have expressed insulin receptor.
The expression of the insulin receptor in stem cells is not dependent on the presence of insulin.
Explanation:
1. In the present study, a stem cell line was incubated to obtain different specific cell types
2. During this process, some cells in this cell line would have differentiated into muscle cells.
3. The differentiated cells were incubated in presence and absence of insulin.
4. In the absence of insulin, the endosomes of muscle cells did not fuse with the plasma membrane. Thus Glut4 was not expressed and as a result, lower green fluorescence was observed. In contrast to this observation, the expression of insulin receptor was found to be higher and this expression was not dependent on the presence of insulin. Insulin is a key regulatory molecule needed for glucose metabolism in all cells. Hence, the expression of the insulin receptor is not dependent on the presence of insulin.
5. In the presence of insulin, endosomes present in differentiated muscle cells fused with the plasma membrane. This lead to the expression of Glut4. The activation of Glut4 is dependent on the presence of insulin and the binding of insulin to the insulin receptor is inevitable. Thus in presence of insulin, the Glut4 gets expressed and thus there is an increased green fluorescence.