Hyperglycemia upregulates DC glycolysis activity
We then examined the expression of genes involved in glycolysis under different glucose concentrations in BMDC. All gene expressions were adjusted relative to the inactivated cells cultured in 11 mM glucose. A glucose dose-dependent increase was observed in mRNA of all three analyzed genes in inactivated and activated DC (Figure 3. A – C). Cells cultured in 11 mM glucose were significant elevation in HK2 and LDHA but not GLUT1 upon activation by AGE and LPS, while cell activation significantly upregulated LDHA in 25 mM glucose culture, but not a statistical increase for HK2 and GLUT1 levels, (Figure 3 A – C). Similar increase in the expression of genes associated with glycolysis in response to increasing glucose levels was observed in monocyte-differentiated DC. Furthermore, these glycolysis genes were significantly up in the presence of AGE and LPS (Figure 3 D – F).
To further corroborate the effect on DC glycolysis, we utilized Seahorse Glycolysis Street Test to measure the glycolysis activity under 5.5 mM, 11 mM, and 25 mM glucose with or without LPS and AGE stimulations. In the absence of AGE and LPS, the inactivated mDC previously cultured in higher glucose concentrations exhibited high extracellular acidification rate (ECAR) that was proportional to the relative glycolysis activity when glucose was supplemented (Figure 3G). This increase in ECAR value was observed to be directly dependent on glucose concentrations. Activation of mDC by AGE and LPS further upregulated the ECAR and the cell glycolysis, such that higher ECAR values was observed with the similar glucose dose-dependent increasing trend, except for the activated mDC cultured in 25 mM glucose (Figure 3H). This alteration in cell metabolic flux was also reflected on the glycolysis rate and maximal glycolytic capacity (Figure 3I – J).
Extracellular L-Lactate secreted from the cells also demonstrated a dose-dependent increase with the increasing glucose concentration in both inactivated and activated DC, and the cell activations resulted in a higher L-Lactate release under all glucose conditions (Figure 3K). On the other hand, the DC OXPHOS activity, measured by the relative intensity of the cell membrane potential, gradually decreased with the increasing glucose concentrations, with or without the stimulation of AGE and LPS (Figure 3L).