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

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.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia

The FIH (Factor Inhibiting HIF) asparaginyl hydroxyls regulates oxidative metabolism and accelerates adaptation to hypoxia

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The Factor Inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants, and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/VHL pathway to accelerate HIF-mediated metabolic responses to hypoxia