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Diabetic nephropathy (kidney disease) is characterized in part by a significant decrease in the glomerular filtration rate. Podocytes are differentiated cells that surround the glomerular capillaries and counteract the pressure of blood filtration. Hyperglycemia (elevated blood glucose) and capillary hypertension (high blood pressure) , both of which are common in diabetic patients, can subject podocytes to mechanical stress. This mechanical stress has been shown to regulate glucose uptake by podocytes, possibly by altering the production of glucose transport (GLUT) proteins.Researchers investigating GLUT2 and GLUT4 expression in rat podocytes measured the impact of mechanical stress on glucose uptake. Cells were grown for 4 hours on membranes exposed to atmospheric (atm) or high air pressure (HP) to mimic mechanical stress. Antibodies specific to GLUT2 and GLUT4 were used to visualize the expression of each under normal glucose (NG) and hyperglycemic (HG) conditions, and to assess the effect of mechanical stress on expression. The uptake of a radiolabeled glucose analogue, [³H]-2-deoxy-D-glucose (3H-2DG) , was then determined. The quantity of 3H-2DG transported by each GLUT protein was directly proportional to the cell surface expression.
Figure 1 Cell surface expression of GLUT transporters in response to mechanical stress and varying glucose concentration.In vivo studies have shown that transport via GLUT2 is noninducible and depends only on extracellular glucose concentrations. However, GLUT4-mediated uptake is induced by insulin binding and occurs under high glucose conditions when insulin is released. The standard K_m values for GLUT2 and GLUT4 are 19.4 and 4.3 mM, respectively, at normal blood glucose levels after a meal (~7.8 mM) . The data shown in Figure 2 represent the combined kinetic properties of both transporters.
Figure 2 Plot of glucose uptake in podocytes with and without mechanical stress.
Adapted from Lewko, B., Bryl, E., Witkowski, J. M., Latawiec, E., Angielski, S., & Stepinski, J. (2005) . Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes. Nephrology Dialysis Transplantation, 20(2) , 306-311.
-What stress-induced change in Figure 2 provides evidence that mechanical stress alters the binding properties of the GLUT transporters?

Question

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Diabetic nephropathy (kidney disease) is characterized in part by a significant decrease in the glomerular filtration rate. Podocytes are differentiated cells that surround the glomerular capillaries and counteract the pressure of blood filtration. Hyperglycemia (elevated blood glucose) and capillary hypertension (high blood pressure) , both of which are common in diabetic patients, can subject podocytes to mechanical stress. This mechanical stress has been shown to regulate glucose uptake by podocytes, possibly by altering the production of glucose transport (GLUT) proteins.Researchers investigating GLUT2 and GLUT4 expression in rat podocytes measured the impact of mechanical stress on glucose uptake. Cells were grown for 4 hours on membranes exposed to atmospheric (atm) or high air pressure (HP) to mimic mechanical stress. Antibodies specific to GLUT2 and GLUT4 were used to visualize the expression of each under normal glucose (NG) and hyperglycemic (HG) conditions, and to assess the effect of mechanical stress on expression. The uptake of a radiolabeled glucose analogue, [³H]-2-deoxy-D-glucose (3H-2DG) , was then determined. The quantity of 3H-2DG transported by each GLUT protein was directly proportional to the cell surface expression.

Figure 1 Cell surface expression of GLUT transporters in response to mechanical stress and varying glucose concentration.In vivo studies have shown that transport via GLUT2 is noninducible and depends only on extracellular glucose concentrations. However, GLUT4-mediated uptake is induced by insulin binding and occurs under high glucose conditions when insulin is released. The standard K_m values for GLUT2 and GLUT4 are 19.4 and 4.3 mM, respectively, at normal blood glucose levels after a meal (~7.8 mM) . The data shown in Figure 2 represent the combined kinetic properties of both transporters.

Figure 2 Plot of glucose uptake in podocytes with and without mechanical stress.
Adapted from Lewko, B., Bryl, E., Witkowski, J. M., Latawiec, E., Angielski, S., & Stepinski, J. (2005) . Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes. Nephrology Dialysis Transplantation, 20(2) , 306-311.
-What stress-induced change in Figure 2 provides evidence that mechanical stress alters the binding properties of the GLUT transporters?

Quizplus · Accepted Answer

11ecba2d_0e7d_c220_a3bf_0330d06abe16_MD0008_00 Lineweaver-Burk plots can be used to compare the effects of different experimental conditions on kinetic parameters. The linear graphs are derived from the Michaelis-Menten equation and display the inverse reaction velocity (1/V_o) versus the inverse substrate concentration (1/[S]). Consequently, K_m is the reciprocal of the negative x-intercept, and V_max is the reciprocal of the y-intercept. Figure 2 shows a Lineweaver-Burk plot of the inverse rate of glucose uptake as a function of inverse 3H-2DG concentration under both atmospheric pressure (atm) and high pressure (HP). The question states that mechanical stress alters the binding properties of the GLUT transporters. The binding affinity of a protein for its substrate is exhibited by the K_m value, for which lower values indicate higher binding affinities. Therefore, changes in the x-intercept should be evident in podocytes exposed to HP. In comparison to atm pressure, there is a right-shift in the x-intercept under HP conditions. A right-shift shows that the K_m value has increased, which suggests a lower binding affinity for the substrate. (Choice A) A left-shift in the x-intercept on a Lineweaver-Burk plot would indicate that the K_m value has decreased. Stress generated by HP in Figure 2 resulted in a right-shift of K_m, indicating an increased K_m value. (Choice B) Changes in the binding affinity of a protein do not alter the value of V_max. A downward-shift in a Lineweaver-Burk plot corresponds to an increase in V_max, which suggests that glucose uptake is fastest under mechanical stress. (Choice C) Catalytic efficiency is the k_cat/K_m ratio of an enzyme and is inversely proportional to the slope of a Lineweaver-Burk plot. In Figure 2, the slope of the plot decreases, indicating an increase in catalytic efficiency. Educational objective: Changes in the binding properties of a protein can alter the K_m value. On a Lineweaver-Burk plot, K_m and V_max are the reciprocals of the x- and y-intercepts, respectively. A right-shift of the x-intercept corresponds to an increase in K_m whereas a left-shift indicates a decrease.

Passage Diabetic Nephropathy (Kidney Disease) Is Characterized in Part by a a Significant