FIG. 4.

Hyperglycemic conditions mediate a shift from cap-dependent to cap-independent translation that is dependent on 4E-BP1. A: MEFs were incubated in medium containing high (25 mmol/L glucose) or low (5 mmol/L glucose, 20 mmol/L mannitol) glucose. Cells were transiently transfected with a bicistronic plasmid containing the IRES for either FGF-2 or VEGF. Whole-cell lysates were harvested, and the relative translation of LucR and LucF was evaluated. B: MEF cells were transiently transfected with bicistronic luciferase plasmid containing the VEGF IRES and were incubated in either 5 or 25 mmol/L glucose in the presence and absence of 3 mmol/L glucosamine. C: Wild-type and Eif4ebp1;Eif4ebp2 double knockout MEFs were incubated in medium containing either low glucose or high glucose with 3 mmol/L glucosamine. The content of 4E-BP1 relative to tubulin was assessed in whole-cell lysates by Western blot analysis. D: Whole-cell lysates were prepared, and the relative translation of LucR and LucF was evaluated. Values are means ± SE for two independent experiments (n = 4). E: Hepatocytes from Eif4ebp1;Eif4ebp2 knockout mice were transfected with a bicistronic plasmid containing the IRES for VEGF and maintained in medium containing either low glucose or high glucose with 3 mmol/L glucosamine. Values are means ± SE for two independent experiments (n = 3). Statistical significance was assessed by either Student t test or ANOVA followed by Holm-Sidak multiple-comparisons test to compare the mean of each group with the mean of every other group. Statistical significance is denoted by * (A and C) or the presence of different letters above the bars on the graphs (B, D, and E). Bars with different letters are statistically different; P < 0.05.