Question

6) In unstimulated muscle cells, the glucose transporter Glute 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. In order for insulin to elicit this change in Glute 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 Glutt 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. Glut on the cell surface was detected by an anti-Glute antibody, which fluoresces green; the insulin receptor on the cel 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 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 (12)

Answer 1

The GLUT4 on surface of the cell is detected by green fluorescence linked to ant-GLUT4 antibody. The Insulin receptor (IR) is detected by red fluorescence linked to ant-insulin receptor antibody.

The cells expressing either IR or GLUT4 or both are shown in the image.

Quadrant 1 (Q1): Cells expressing GLUT4

Quadrant 2 (Q2): Cells expressing GLUT4 and IR

Quadrant 3 (Q3): Cells expressing neither GLUT4 nor IR

Quadrant 4 (Q4): cells expressing IR

a. Not all cells express IR on their cell surface. The cells in Q1 and Q3 do not express IR. Only cells in Q2 and Q4 express IR.

The expression of IR is not dependent on insulin. However, more cells express both IR and GLUT4 when insulin is present. This is because in absence of insulin, most cells only express IR (-Insulin Q4). There are no cells expressing IR along with GLUT4 (- Insulin Q2). When cells are exposed to insulin, most cells that express IR also start expressing GLUT4 (+ Insulin Q2) and cells that express only IR reduce in numbers (+insulin-Q4). However, insulin does not affect the expression of IR as number of cells that do not express IR and GLUT4 is not altered in presence and absence of insulin (+ insulin, -Insulin Q3).

b. Not all cells express GLUT4 on their surface. Cells in Q3 and Q4 do not express GLUT4 on their surfaces. Only cells in Q1 and Q2 express GLUT4 on their surfaces.

GLUT4 expression is not dependent on presence of IR. This is because in absence of insulin, GLUT4 is not expressed by any cells. Even cells that have IR on their surfaces, do not express GLUT4 (-Insulin, Q4).

GLUT4 expression depends on presence of insulin. In absence of insulin, there are no cells that express GLUT4 (- Insulin, Q1 and Q2). However, when insulin is present, it binds to IR and initiates signaling events via PI1K-AKT-mTOR to induce GLUT4 expression on cells. Hence, majority of cells that were originally expressing IR (-insulin, Q4) will now start expressing GLUT4 as well (+insulin, Q2). This will cause decrease in number of cell that only express IR (-Insulin, + Insulin, Q4). Thus, GLUT4 expression is dependent on presence of insulin.

(a) - Insulin (b) + Insulin Q1 Q1 GLUT4 Q2 GLUT4 Q2 GLUT4 IR GLUT4 GLUT4 Geuren IR Green fluorescence Green fluorescence IR 100 10° 104 107 10° 104 Red fluorescence - 107 IR Red fluorescence ->IR