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).