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).