Investigating the Warburg effect and the role of pyruvate kinase M2 in retinal Müller glial cells

Abstract

Surprisingly, similar to various cancer cells, Müller glial cells and light-sensing photoreceptors of the mammalian retina display the Warburg effect (aerobic glycolysis), an unusual metabolism whereby the cells tend to convert glucose into lactate via glycolysis regardless of oxygen availability. In cancer, the glycolytic enzyme pyruvate kinase M2 (PKM2) promotes lactate production and acts as a coactivator for the transcription factor hypoxia-inducible factor-1 (HIF-1), stimulating glycolysis; thus, PKM2 is implicated in driving the Warburg effect. PKM2 has also been shown to be expressed in the retina and cultured Müller cells (MCs). This thesis elucidates MC glucose metabolism and tests the hypothesis that PKM2 drives the Warburg effect in cultured MCs. Primary rat MCs, the SV40-immortalised rMC-1 MC line and a novel spontaneously immortalised rat SIRMu-1 MC line were used as experimental models. The SIRMu-1 is a novel spontaneously immortalised cell line that was derived from primary MCs during the course of this thesis. It retains similar cellular morphology to cultured primary rat MCs. Immunofluorescence, western blotting and RNA sequencing show that the SIRMu-1 cells closely resemble primary MCs in regard to overall transcriptome and expression of the MC markers cellular retinaldehyde-binding protein, glutamine synthetase, S100, vimentin and glial fibrillary acidic protein at both the mRNA and the protein levels. SIRMu-1 cells, however, proliferate rapidly, do not senesce and have a high transfection efficiency. RNA sequencing also shows that the SIRMu-1 cells were derived from a male rat. The cell line is a valuable experimental tool to study MCs. Glucose metabolism of primary MCs, rMC-1 and SIRMu-1 cells was investigated using lactate assays and a Seahorse XFe96 Analyser system. Primary MCs and rMC-1 cells display the Warburg effect, while SIRMu-1 cells depend predominantly on oxidative phosphorylation. This shows that the Warburg effect does not always exist in highly-proliferative cells as commonly postulated. As the rMC-1 cells retain the Warburg effect observed in primary MCs, they were used to investigate the role of PKM2. Short hairpin RNA-mediated PKM2 knockdown and CRISPR/Cas9-mediated PKM2 knockout did not significantly alter the high glycolytic activity of rMC-1 cells, indicating that PKM2 is not a main driver of the Warburg effect in cultured MCs. This demonstrates that the role of PKM2 in the Warburg effect is cell type-dependent. This project improves our understanding of MC metabolism and may contribute to research on retinal diseases associated with MC abnormality.