GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs.

Energy Metabolism Regulates Stem Cell Pluripotency

“…Indeed, the bivalent metabolic phenotype of naïve PSCs may reflect the metabolic preference of the morula and blastocyst, which utilizes a combination of pyruvate oxidation and glycolysis to meet their metabolic demands, while primed PSCs become almost exclusively dependent on glycolysis, reflecting implantation into the hypoxic uterine wall. Indeed, the importance of reduced oxygen tension has been examined across a number of stem cell populations (Mohyeldin et al, 2010), including a role in improving the acquisition (Yoshida et al, 2009) and maintenance of pluripotency (Ezashi et al, 2005;Forsyth et al, 2006;Prasad et al, 2009;Lengner et al, 2010;Mathieu et al, 2013;Christensen et al, 2015). In part these beneficial effects of physiologically relevant oxygen levels (2-5%) may be due to a reduction in mitochondrial function and oxygen utilization associated with elevated utilization of glucose via glycolysis and amino acid turnover (Forristal et al, 2013;Christensen et al, 2014;Turner et al, 2014;Lees et al, 2015Lees et al, , 2019Harvey et al, 2016b), although these metabolic changes can occur in the absence of changes in self-renewal (Harvey et al, 2016b).…”

Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans

“… No large change in amino acid utilisation or production  ↑Glycine consumption  ↓ serine uptake (82) hESC and hiPSC 2 >5  Genomic screen suggested culture at 2 kPa shifted cells towards neuronal lineages (expression of SOX1, HES5 and FOXG1), specifically towards glial differentiation vs neuronal  Effect was abolished by HIF-2 siRNA and HIF-a inhibition  HIF regulates MYC and thus LIN28/let-7, a key neuronal lineage regulator (666) Blastocystderived…”

Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery

“…The other is the interaction of PKM2 with Oct4, a major regulator of self-renewal and differentiation in stem cells, decreases the transcriptional activity of Oct4 for ‘stemness’ maintaining of glioma stem cells and thus induces differentiation [37]. Nevertheless, some contradictory results need attention, with one study demonstrating the high PKM2 expression in poorly differentiated esophageal squamous cell cancer tissues [38], another showing that PKM2 promotes OCT4 expression in hypoxic hESCs and also enhances its transcriptional activity by binding to OCT4 [42]. Given that PKM2 can promote cell proliferation by interaction with a lot of molecules (e.g.…”