Intersection between Glucose Metabolism and the HIF-1α Pathway
The next series of experiments were aimed at confirming the effect on HIF-1α signaling by determining the changes in PHD2 enzymatic activity, HIF-1α protein expression, and the expression of selected HIF-1α target genes. At, and beyond, the transition from anaerobic to mixed aerobic-anaerobic metabolism (≥ 4 mL), intracellular pyruvate, PHD2 activity, and HIF‑1α protein expression were significantly affected (Figure 2a). Similar to the tissue formation and glucose metabolism studies, maximal changes were also observed under intermediate volumes of media (4 mL) (PHD2 activity : -45%, HIF-1α : 8-fold increase) (Figure 2a). Corresponding HIF-1α gene targets (GLUT1, PDK1 and SOX9) were also maximally upregulated under these conditions (2.0- to 2.5-fold increases) (Figure 2b). Loss-of-function experiments utilizing YC-1 to degrade HIF-1α paralleled these results, with maximal inhibition of ECM synthesis occurring at 4 mL (~70% reduction in collagen and proteoglycan synthesis) (Figure 3). Lastly, HIF-1α expression (by Western blot) after treatment with YC-1 was not detectable (data not shown).
A targeted metabolomic approach was undertaken to determine whether intracellular metabolites of glucose metabolic pathways (glycolysis, fermentation, TCA, and pentose phosphate pathways) were correlated with PHD2 activity. Of the 14 intracellular metabolites investigated, only lactate and succinate appeared to be significantly correlated with PHD2 activity (p<0.02; Table S3). Both intracellular lactate and succinate had strong negative correlations with PHD2 activity (ρ = – 0.999 and – 0.986, respectively) with maximal changes also observed under intermediate media volumes (4 mL) (lactate : 2-fold increase, succinate : 1.6-fold increase) (Figure 4). However, the relative concentrations of these metabolites were different, with lactate present in substantially higher amounts compared to succinate (order of 100 vs 0.1 nmol/µg DNA).