Abstract
Purpose:
Retinal photoreceptors are highly metabolic. Their energy consumption is equivalent to that of a proliferating cancer cell. Even though the retina is post-mitotic and thus non-dividing, photoreceptors shed 10% of their outer segments daily. Therefore, they must synthesize the membrane and protein equivalent of a proliferating cell each day. Most of this anabolic activity occurs early in the light phase of the diurnal cycle and requires ATP, ribose, and NADPH, among other co-factors. Ribose and NADPH are produced only by the hexose monophosphate shunt, also called the pentose phosphate pathway (PPP). Photoreceptors require NADPH, which is essential for membrane synthesis and promotes a reducing environment for proper structure and function maintenance. It has been shown that inhibition of glycolytic enzyme pyruvate kinase M2 (PKM2) activates the PPP in tumor cells. In this study, we characterized PKM2 in the retina.
Methods:
Localization and phosphorylation status of PKM2 was examined by immunohistochemistry and immunoblots performed on dark- and light-adapted wild-type retinal sections. Pyruvate kinase activity was measured by lactate dehydrogenase-coupled enzyme assay. Ex vivo retinal explant cultures were incubated in the presence and absence of phosphoinositide 3-kinase (PI3K) inhibitor. We then measured the PKM2 phosphorylation.<br />
Results:
Our immunohistochemical studies show that PKM2 is primarily localized to photoreceptor inner segments (RIS) and the inner (IPL) and outer plexiform layers (OPL) of the retina, irrespective of dark or light adaptation. We found that phospho-PKM2 immunoreactivity in dark-adapted retinas was enhanced under light-adapted conditions, especially in RIS, OPL, and IPL. The PKM2 enzyme activity was significantly lower in light-adapted retinas than in dark-adapted retinas. Our ex vivo experiments with PI3K-inhibitor suggest that PKM2 phosphorylation is PI3K-dependent.<br />
Conclusions:
Our data show that light-induced PKM2 regulates the hexose monophosphate shunt in the retina, and may provide NADPH for membrane synthesis and maintenance of a reducing environment to neutralize the toxic effect of free radicals. Our studies also suggest that PI3K regulates the PKM2 in the retina. <br />