Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen

<ns4:p><ns4:bold>Background:</ns4:bold> 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. <ns4:italic>In vitro</ns4:italic> cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia.</ns4:p><ns4:p> <ns4:bold>Methods:</ns4:bold> 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.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> 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, <ns4:italic>CA9</ns4:italic> was the most upregulated gene in physiological glutamine media while <ns4:italic>TETs</ns4:italic> and <ns4:italic>FTO</ns4:italic> dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of <ns4:italic>TETs</ns4:italic> 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 <ns4:italic>TETs</ns4:italic> downregulation. Additionally, we observed that <ns4:italic>TET2</ns4:italic> expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate <ns4:italic>TET2</ns4:italic> expression resulting in global changes in DNA hydroxymethylation.  </ns4:p><ns4:p> <ns4:bold>Conclusion:</ns4:bold> Given the dramatic effects of nutrient availability on gene expression, future <ns4:italic>in vitro</ns4:italic> experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.</ns4:p>