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The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina

View Article: PubMed Central - PubMed

ABSTRACT

The tumor form of pyruvate kinase M2 (PKM2) undergoes tyrosine phosphorylation and gives rise to the Warburg effect. The Warburg effect defines a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose, even in the presence of oxygen. Retinal photoreceptors are highly metabolic and their energy consumption is equivalent to that of a multiplying tumor cell. In the present study, we found that PKM2 is the predominant isoform in both rod- and cone-dominant retina, and that it undergoes a light-dependent tyrosine phosphorylation. We also discovered that PKM2 phosphorylation is signaled through photobleaching of rhodopsin. Our findings suggest that phosphoinositide 3-kinase activation promotes PKM2 phosphorylation. Light and tyrosine phosphorylation appear to regulate PKM2 to provide a metabolic advantage to photoreceptor cells, thereby promoting cell survival.

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Related in: MedlinePlus

Light-dependent PKM2 phosphorylation in rod- and cone-dominant retinas.Prefer-fixed sections of dark- (A,B) and light-adapted (C,D) mouse retinas were subjected to immunofluorescence with anti-p-PKM2 (Y105) (A,C), and anti-arrestin (B,D) antibodies. Panels (B,D) represent the merged images of p-PKM2 and arrestin, whereas panel (E) represents the omission of primary antibody. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Retinal lysates from dark- and light-adapted mice were subjected to immunoblot analysis with anti-pPKM2 (F) and anti-PKM2 (G) antibodies. Densitometric analysis of pPKM2 was performed in the linear range of detection, and absolute values were then normalized to PKM2 (H). Data are mean + SEM, n = 4. *p < 0.05. Pyruvate kinase activity was measured from dark- and light-adapted mouse retinas with an LDH-coupled enzyme assay. Data are mean ± SD, n = 3, *p < 0.05. Prefer-fixed sections of dark- (J) and light-adapted (K) Nrl−/− mouse retinas were subjected to immunofluorescence with anti-pPKM2 (J,K). Panel (L) represents the omission of primary antibody. POS, photoreceptor outer segments; OPL, outer plexiform; IPL, inner plexiform layer. Scale bar 50 μm. Full-length blots are presented in the Supplementary Information.
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f3: Light-dependent PKM2 phosphorylation in rod- and cone-dominant retinas.Prefer-fixed sections of dark- (A,B) and light-adapted (C,D) mouse retinas were subjected to immunofluorescence with anti-p-PKM2 (Y105) (A,C), and anti-arrestin (B,D) antibodies. Panels (B,D) represent the merged images of p-PKM2 and arrestin, whereas panel (E) represents the omission of primary antibody. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Retinal lysates from dark- and light-adapted mice were subjected to immunoblot analysis with anti-pPKM2 (F) and anti-PKM2 (G) antibodies. Densitometric analysis of pPKM2 was performed in the linear range of detection, and absolute values were then normalized to PKM2 (H). Data are mean + SEM, n = 4. *p < 0.05. Pyruvate kinase activity was measured from dark- and light-adapted mouse retinas with an LDH-coupled enzyme assay. Data are mean ± SD, n = 3, *p < 0.05. Prefer-fixed sections of dark- (J) and light-adapted (K) Nrl−/− mouse retinas were subjected to immunofluorescence with anti-pPKM2 (J,K). Panel (L) represents the omission of primary antibody. POS, photoreceptor outer segments; OPL, outer plexiform; IPL, inner plexiform layer. Scale bar 50 μm. Full-length blots are presented in the Supplementary Information.

Mentions: In tumor cells, PKM2 undergoes tyrosine phosphorylation (Y105), which results in the inhibition of PKM2 activity. This regulation of signaling diverts glucose metabolites from energy production to anabolic processes (protein, lipid, and nucleic acid synthesis), in addition to generating NADPH to support antioxidant metabolism27. To determine whether PKM2 undergoes tyrosine phosphorylation in the retina, retinal sections from dark- and light-adapted mice were subjected to immunohistochemistry with phospho-specific PKM2-Y105 and arrestin antibodies. The results revealed phospho-PMK2 immunoreactivity in dark-adapted retina (Fig. 3A,B): phosphorylation was enhanced under light-adapted conditions, especially in rod inner segments and the outer and inner plexiform layers (Fig. 3C,D). To quantify the PKM2-Y105 phosphorylation, retinal lysate was subjected to immunoblot analysis with anti-PKM2-Y105 and anti-PKM2 antibodies. These results suggest a significant phosphorylation of PKM2 in light-adapted retinas compared with dark-adapted retinas (Fig. 3F–H).


The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina
Light-dependent PKM2 phosphorylation in rod- and cone-dominant retinas.Prefer-fixed sections of dark- (A,B) and light-adapted (C,D) mouse retinas were subjected to immunofluorescence with anti-p-PKM2 (Y105) (A,C), and anti-arrestin (B,D) antibodies. Panels (B,D) represent the merged images of p-PKM2 and arrestin, whereas panel (E) represents the omission of primary antibody. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Retinal lysates from dark- and light-adapted mice were subjected to immunoblot analysis with anti-pPKM2 (F) and anti-PKM2 (G) antibodies. Densitometric analysis of pPKM2 was performed in the linear range of detection, and absolute values were then normalized to PKM2 (H). Data are mean + SEM, n = 4. *p < 0.05. Pyruvate kinase activity was measured from dark- and light-adapted mouse retinas with an LDH-coupled enzyme assay. Data are mean ± SD, n = 3, *p < 0.05. Prefer-fixed sections of dark- (J) and light-adapted (K) Nrl−/− mouse retinas were subjected to immunofluorescence with anti-pPKM2 (J,K). Panel (L) represents the omission of primary antibody. POS, photoreceptor outer segments; OPL, outer plexiform; IPL, inner plexiform layer. Scale bar 50 μm. Full-length blots are presented in the Supplementary Information.
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f3: Light-dependent PKM2 phosphorylation in rod- and cone-dominant retinas.Prefer-fixed sections of dark- (A,B) and light-adapted (C,D) mouse retinas were subjected to immunofluorescence with anti-p-PKM2 (Y105) (A,C), and anti-arrestin (B,D) antibodies. Panels (B,D) represent the merged images of p-PKM2 and arrestin, whereas panel (E) represents the omission of primary antibody. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Retinal lysates from dark- and light-adapted mice were subjected to immunoblot analysis with anti-pPKM2 (F) and anti-PKM2 (G) antibodies. Densitometric analysis of pPKM2 was performed in the linear range of detection, and absolute values were then normalized to PKM2 (H). Data are mean + SEM, n = 4. *p < 0.05. Pyruvate kinase activity was measured from dark- and light-adapted mouse retinas with an LDH-coupled enzyme assay. Data are mean ± SD, n = 3, *p < 0.05. Prefer-fixed sections of dark- (J) and light-adapted (K) Nrl−/− mouse retinas were subjected to immunofluorescence with anti-pPKM2 (J,K). Panel (L) represents the omission of primary antibody. POS, photoreceptor outer segments; OPL, outer plexiform; IPL, inner plexiform layer. Scale bar 50 μm. Full-length blots are presented in the Supplementary Information.
Mentions: In tumor cells, PKM2 undergoes tyrosine phosphorylation (Y105), which results in the inhibition of PKM2 activity. This regulation of signaling diverts glucose metabolites from energy production to anabolic processes (protein, lipid, and nucleic acid synthesis), in addition to generating NADPH to support antioxidant metabolism27. To determine whether PKM2 undergoes tyrosine phosphorylation in the retina, retinal sections from dark- and light-adapted mice were subjected to immunohistochemistry with phospho-specific PKM2-Y105 and arrestin antibodies. The results revealed phospho-PMK2 immunoreactivity in dark-adapted retina (Fig. 3A,B): phosphorylation was enhanced under light-adapted conditions, especially in rod inner segments and the outer and inner plexiform layers (Fig. 3C,D). To quantify the PKM2-Y105 phosphorylation, retinal lysate was subjected to immunoblot analysis with anti-PKM2-Y105 and anti-PKM2 antibodies. These results suggest a significant phosphorylation of PKM2 in light-adapted retinas compared with dark-adapted retinas (Fig. 3F–H).

View Article: PubMed Central - PubMed

ABSTRACT

The tumor form of pyruvate kinase M2 (PKM2) undergoes tyrosine phosphorylation and gives rise to the Warburg effect. The Warburg effect defines a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose, even in the presence of oxygen. Retinal photoreceptors are highly metabolic and their energy consumption is equivalent to that of a multiplying tumor cell. In the present study, we found that PKM2 is the predominant isoform in both rod- and cone-dominant retina, and that it undergoes a light-dependent tyrosine phosphorylation. We also discovered that PKM2 phosphorylation is signaled through photobleaching of rhodopsin. Our findings suggest that phosphoinositide 3-kinase activation promotes PKM2 phosphorylation. Light and tyrosine phosphorylation appear to regulate PKM2 to provide a metabolic advantage to photoreceptor cells, thereby promoting cell survival.

No MeSH data available.


Related in: MedlinePlus