Glycolysis is a key metabolic pathway that breaks down glucose to support cellular energy production. When oxygen supply is limited (hypoxia), mammalian cells rely predominantly on glycolysis to compensate for reduced oxidative phosphorylation. Hypoxia is a common feature of the gastrointestinal epithelium, where the hypoxia-inducible factor (HIF) enhances expression of glycolytic genes. This metabolic shift is exacerbated in cancer, such as colorectal cancer, a hallmark known as the Warburg effect. Recent work suggests hypoxia promotes the assembly of glycolytic enzyme complexes. We hypothesize that hypoxia-induced glycolytic complex formation differs between normal and cancerous colon epithelial cells, and is regulated by post-translational modifications. This study aims to define how hypoxia promotes glycolytic enzyme complex formation in colon epithelial cells and whether this response is amplified in colorectal cancer. To first determine if glycolytic complexes form in vivo, colon tissues were collected from mice kept in hypoxia (9% O 2 ) or atmospheric conditions (21% O 2 ). Immunofluorescence staining revealed an increased colocalization of two key glycolytic enzymes, Phosphofructokinase (PFKP) and Hexokinase 2 (HK2). We quantified these interactions using a proximity ligation assay (PLA), which confirmed a significant hypoxia-dependent increase in HK2-PFKP complex formation. To define molecular drivers of this phenomenon, we next studied glycolytic activity in vitro using primary and cancerous intestinal epithelial cells. Extracellular lactate levels and glycolytic enzyme expression increased after 24 hours of hypoxia (1% O 2 ) in primary and cancer colon cells, but not in primary small intestinal cells, indicating colon-specific sensitivity to hypoxia. Immunoprecipitation and PLA further demonstrated a significant increase in HK2-PFKP interactions in hypoxic primary and cancer colon cells (p< 0.05), with cancer cells exhibiting a greater response than primary cells. In contrast, small intestinal epithelial cells did not show increased enzyme interactions. We next assessed lysine lactylation, a post-translational modification derived from the glycolytic end-product, lactate. Primary colon cells displayed a hypoxia-dependent increase in lactylation, whereas cancer colon cells maintained consistently high levels over time in both normoxia and hypoxia, suggesting lactylation is already maximized in cancer. Future studies will evaluate whether lactylation promotes glycolytic complex assembly in hypoxia. Overall, our findings show that colon epithelial cells exhibit a strong hypoxic glycolytic adaptation involving enzyme complex formation, which is further amplified in colorectal cancer cells. This enhanced metabolic response in the colon may contribute to the higher incidence of colorectal cancer compared to small intestinal malignancies, and highlights a cancer-specific metabolic adaptation with potential as a therapeutic target. Ongoing work will explore how post-translational modifications regulate hypoxia-driven glycolytic organization and metabolic remodelling. Funding agencies: Natural Science and Engineering Research Council of Canada, Science Foundation of Ireland. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
DeMichele et al. (Fri,) studied this question.