Effect of short-term hyperglycemia on protein kinase C alpha activation in human erythrocytes

Abstract

Background: Diabetes mellitus, characterized by chronic hyperglycemia, is known to have a deleterious effect on erythrocyte structure and hemodynamic characteristics, which eventually contribute to diabetes-associated vascular complications. Protein kinase C alpha (PKCα) is a major regulator of many metabolic processes and structural changes in erythrocytes, and may play a significant role in the development of hyperglycemia-mediated cellular abnormalities.

Aim: We hypothesized that acute hyperglycemic stress may affect erythrocyte structure and metabolic properties through its effect on PKCα membrane content and activity.

Results: Erythrocytes, from healthy individuals acutely exposed to a glucose enriched media, showed a significant decrease in the membranous fraction of PKCα and its phosphorylation (p = 0.005 and p = 0.0004, respectively). These alterations correlated with decreased affinity of PKCα to its membrane substrates (4.1R and GLUT1) and reduced RBC deformability (p = 0.017). Pre-activation of erythrocytes with PKC activator, PMA, minimized the effect of glucose on the membrane PKCα fraction and RBC deformability (p > 0.05).

Conclusions: Acute glycemia-induced inhibition of PKCα membranous translocation and activation is associated with reduced erythrocyte membrane deformability.

Figure 1. Total and membrane fractions of PKCα in healthy erythrocytes

Representative Western blots (A) illustrate a comparison between the total and membranous fractions of total and activated (phosphorylated in Threonine 638) fractions of PKCα for randomly chosen three participants. The results of the Western blots were quantified by Adobe Photoshop densitometry, and presented as the percentage of the membrane fraction from the total cellular fraction of the protein kinase (B). The results represent the mean for seven individuals ± SE. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control to verify equivalent amounts of protein throughout the lanes. Molecular mass markers are indicated in kDa.

Figure 2. Changes in the membrane fraction of PKCα in erythrocytes acutely exposed to various concentrations of D-glucose

The erythrocytes were incubated in PBS supplemented with 0 mM (white bars), 5 mM (grey bars), or 20 mM D-glucose (black bars) for 5 minutes at 37ºC. When indicated, the cells were pretreated with 25 µM PMA. A representative Western blot shows the membrane fractions of PKCα and phosphorylated PKCα (PKCα-T638) (A). Molecular mass markers are indicated in kDa. Averaged results of Western blots of the membrane fractions of PKCα (B) or PKCα-T638 (C) were quantified using Adobe Photoshop, and normalized to "0 mM D-glucose"-treated cells. Data are means of 4 to 13 independent experiments ± SE. * p < 0.05; ** p < 0.01 vs. correspondent data.

Figure 3. Changes in cellular ATP in erythrocytes acutely exposed to various concentrations of D-glucose

Erythrocytes, either pretreated with 25 µM PMA or untreated, were acutely incubated in PBS with 0 mM (white bars), 5 mM (grey bars), or 20 mM D-glucose (black bars). Cellular ATP concentrations were quantified by luciferin-luciferase assay, and normalized to “0 mM D-glucose”-treated cells. Data are means of 8 to 13 independent experiments ± SE. * p < 0.05; ** p < 0.01 vs. correspondent data.

Figure 4. Co-immunoprecipitations of PKCα with GLUT1 and 4.1R

RBC lysates were exposed to protein A-Sepharose coupled to anti-GLUT1 or anti-4.1R. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blot, as described under "Materials and methods". A representative result is presented in the figure. Similar results were obtained in two additional experiments. Data of 4.1R, GLUT1, and light chain of correspondent antibodies were used as controls to verify equivalent amounts of protein throughout the lanes. In the control 'no lysate' study, protein A-Sepharose coupled to an appropriate antibody was exposed to a RBC-free sample. Molecular mass markers are indicated in kDa.

Figure 5. Acute glucose effect on RBC deformability

The figure shows the elongation ratio (ER) or the percentage from the total RBC population in accordance to ER of erythrocytes incubated in PBS supplemented with with 0 mM (white bars), 5 mM (grey bars), or 20 mM D-glucose (black bars) for 5 min at 37ºC. When indicated, cells were pretreated with 25 µM PMA. A. Median elongation ratio. B. Percentages of undeformable (ER ≤ 1.1) erythrocytes. C. Percentage of erythrocytes with decreased 1.1-1.5 ER deformability. D. Percentage of erythrocytes with median 1.5-2 ER deformability. E. Percentage of erythrocytes with increased 2-2.5 deformability. F. Percentage of erythrocytes with increased 2.5-3.0 deformability. Data are means of 4 to 13 independent experiments ± SE. * p < 0.05; ** p < 0.01 vs. correspondent data.