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Thomas Gardner, Mandy Losiewicz, Steven F Abcouwer, Patrice E Fort; Central role of glycolysis in the regulation of retinal protein synthesis through phosphorylation of the translational regulator, 4E-BP1. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1489.
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© ARVO (1962-2015); The Authors (2016-present)
The retina has a very high metabolic rate but the control mechanisms for biosynthetic processes have received little attention. The retina exhibits many features of the Warburg effect observed in tumors, in that the majority of retinal ATP is derived from aerobic glycolysis. Therefore, we hypothesized that retinal protein synthesis is highly dependent upon glycolytic flux.
Protein synthesis was assessed by 35S-methionine labeling in retinas from healthy adult male Sprague-Dawley rats. Glycolysis was inhibited with 2-deoxyglucose (2-DG) and 2-fluoro-deoxyglucose (2-FDG) and oxidative phosphorylation was inhibited by antimycin-A. Isoform-specific Akt activity and mTOR activity were analyzed by specific kinase activity assays using substrate-derived peptides. mTORC1 and mTORC2 complex activities were assayed by phosphorylation of 4E-BP1, 40S ribosomal protein S6 (RPS6) and PKC alpha. Phosphatase activity was determined from retinal lysates in the presence or absence of protein phosphatase inhibitors, okadaic acid and calyculin A. Analyses were performed using non-repeated measures ANOVA followed by the SNK test for multiple comparisons or t-test for a single comparison.
2-DG and 2-FDG caused dose-dependent reductions of retinal lactate production (p < 0.01), ATP content (p < 0.01) and protein synthesis (p < 0.001); D-mannose, but neither fructose nor L-mannose, prevented these effects. Glycolysis inhibition also reduced the kinase activities of Akt1/3 and mTORC2. Direct Akt inhibition also reduced protein synthesis. Glycolysis inhibition caused rapid dephosphorylation of 4E-BP1. Surprisingly, mTORC1 activity did not seem to be reduced, as evidenced by unaltered mTORC1-associated mTOR autophosphorylation. 2-DG, but not Akt inhibition, reduced 4E-BP1 phosphorylation by ≈ 50%, an effect that was greatly diminished by PP1/PP2A phosphatase inhibitors okadaic acid and calyculin A.
Collectively, these novel data support the hypothesis that normal retinal protein synthesis depends on glycolysis. Glycolysis inhibition impairs translation initiation associated with accelerated dephosphorylation of 4E-BP1, independent of the Akt-mTORC1 pathway. The retina utilizes glycolysis for essential anabolic processes so dysfunction of these pathways are likely to contribute to retinal degenerations.
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