Low oxygen availability induces a metabolic shift towards enhanced glucose utilization in non-lipid-loaded and lipid-loaded HepG2 cells

Liver function is impaired in metabolic dysfunction-associated fatty liver disease. Previous studies demonstrated that oxygen availability in the tissue microenvironment affects adipose tissue and skeletal muscle function, but its hepatic effects remain unclear. This study aimed to investigate the impact of oxygen levels on metabolic pathways in HepG2 cells. Non-lipid-loaded and lipid-loaded HepG2 cells, were exposed to different physiological O 2 levels (5% and 10%) or standard laboratory conditions (21% O 2 ) for 24h. Thereafter, we determined lipid content, gene expression of metabolic markers, glycogen content and glucose release. Furthermore, mitochondrial respiration and glycolytic activity were assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Exposure to 5% O₂ increased the expression of the gluconeogenic gene G6PC1 in both non-lipid-loaded and steatotic HepG2 cells compared to 21% O 2 (p<0.001). Furthermore, 5% O 2 decreased the expression of lipogenic genes ( SREBP1C, ACC2 and FASN) in non-lipid-loaded and/or steatotic cells (all p<0.05), while genes involved in fatty acid oxidation ( PPARGC1A, p<0.001; PPARA, p=0.038) were downregulated in steatotic cells. Low oxygen exposure increased glycogen content in non-lipid-loaded and steatotic cells (both p<0.001) and reduced glucose release (p<0.05). Finally, low oxygen exposure reduced OCR (p<0.05) and increased glycolysis (p<0.001) in both non-lipid-loaded and steatotic cells compared to 21%. In conclusion, our findings demonstrate that reduced oxygen availability in the microenvironment has marked effects on metabolic pathways in non-lipid-loaded and steatotic hepatocytes, inducing a metabolic shift to enhanced reliance on glucose as energy source.