Abstract
Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation. Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.
Highlights
In most organisms, energy usage is tightly regulated by tissue sensing of nutritional status and the coordination of cellular responses
HepG2 cells proliferated in media with reduced glucose or glutamine at a comparable rate to those in Dulbecco’s Modified Eagle’s medium (DMEM) and no adverse effects on cell viability were noted
Glucose and glutamine concentrations modulate transcript levels of selected Hypoxia-inducible factors (HIF)-target and dioxygenase genes Hypoxia and DNA hypermethylation are common in solid tumours, where reduced oxygen levels activate the HIF transcriptional complex[18,19], orchestrating the expression of a wide range of genes in an attempt to restore metabolic homeostasis[20,21]
Summary
Energy usage is tightly regulated by tissue sensing of nutritional status and the coordination of cellular responses. HIFs activate genes that are essential for cells to survive under low oxygen conditions which helps cells reduce energy demands by limiting oxygen consumption, for instance employing glycolysis in place of oxidative phosphorylation (Figure 1)[1]. Another gene family known as the alpha-ketoglutarate-dependent dioxygenases regulate metabolic homeostasis under low oxygen. CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression.
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