The anti-inflammatory properties of COL-3 derives from its ability to restrain glucose uptake and consequent glucose-dependent NADPH production
Our data showed that the treatment with inflammogens LPS or αSa substantially elevated glucose consumption by microglial cells, as it seems to be a prerequisite to the shift into an activated profile [43], estimated through the capture of [3H]-2-DG, a synthetic analog of glucose labeled with tritium. This corroborates previous results from our group showing LPS increases glucose uptake as a substrate to NADPH-dependent ROS production, which leads to an inflammatory state of microglial cells [32]. This is the first time it is shown that the aggregated inflammatory form of αS can increase glucose consumption in microglial cells, which points to a common mechanism of these aggregated form of the protein with LPS to induce the inflammatory state in microglial cells. Of interest, COL-3 strongly inhibited [3H]-2-DG uptake in LPS- and αSa-stimulated cells, indicating the non-antibiotic tetracycline curtailed inflammatory-type responses by reducing glucose uptake. Again, a higher concentration of the reference antibiotic tetracycline DOX reproduced these findings; however, the microglial uptake of [3H]-2-DG was unresponsive to DEX, indicating that the mechanisms of action of the glucocorticoid and the tetracyclines were most probably partly different.
It should be emphasized that the uptake of [3H]-2-DG was also strongly reduced by inhibiting NADPH oxidase activity with APO, which indicates that COL-3 prevented the uptake of glucose through inhibition of ROS dependent-signaling events [44]. Noticeably, COL-3 anti-inflammatory action was also mirrored by unlabeled 2-DG used at concentrations that competitively inhibit hexokinase, the glycolytic enzyme that converts glucose into glucose-6-P [45], and saturating concentrations of GLUT-dependent glucose uptake [46]. Our current data is one more evidence that the control of glucose availability by anti-inflammatory drugs largely contributes to the reduction of microglial cell activation [32,47].
NADPH oxidase (NOX2), a multimeric complex, utilizes NADPH as substrate and reduces molecular oxygen to produce superoxide, that is then further dismutated into stable and diffusible hydrogen peroxide pro-oxidant [48]. The first metabolite product of glucose, glucose-6-phosphate, can be translocated into the pentose phosphate shunt [49], which serves to produce nucleotide precursors and helps regenerate the reducing agent NADPH, which is also a requisite substrate for NOX2 [45,49]. Consistent with previous findings from our group and others, the pentose-phosphate shunt pathway is essential for ROS response in immune cells like macrophages and microglial cells [32,48]. Therefore, we tested the possibility that the increase in glucose uptake observed in primary microglial cells may serve to stimulate an increase in NADPH levels and that COL-3 may possibly resolve inflammatory processes by preventing this effect. Here we observed a robust elevation of NADPH synthesis in LPS- and αSa-challenged microglial cells.
In a recent study from our group, the phytocannabinoid anti-inflammatory cannabidiol prevented the rise in NADPH synthesis elicited by LPS treatment, indicating that the anti-inflammatory action of cannabidiol may be also dependent of this suppressive effect [32]. Our current results sustain our previous data, as the tetracyclines COL-3 and DOX reduced NADPH levels produced after LPS and αSa stimulation in microglial cells. Like cannabidiol, COL-3 operated by interfering with an alternative glucose-dependent mechanism. Therefore, it is possible to imply that the inhibitory effects that COL-3 exerts on glucose consumption and NADPH synthesis may strengthen the intrinsic antioxidant potential of this compound in a self-reinforcing process.
Reinforcing our hypothesis, we found that compounds capable of restricting LPS and αSa-induced TNF-α release and Iba-1 expression were also effective in preventing not only [3H]-2-DG uptake, but also the consequent production of intracellular NADPH derived from the preferential activation of the pentose-phosphate shunt pathway. In this way, the NOX2 inhibitor APO and the saturating concentration of unlabeled 2DG prevented the production of NADPH, confirming the resulting inflammatory process caused by these inflammogens results from a signaling event that requires an increase in glucose uptake and consequent NADPH production, and the anti-inflammatory effect of COL-3 comes from blocking the signaling cascade of the pentose-phosphate shunt pathway.
Our results also show that DEX, despite reducing inflammatory factors, was ineffective in avoiding LPS- and αSa-dependent [3H]-2AG uptake increase. Although there are reports showing DEX inhibits glucose uptake in contracting myotubes [50], glucocorticoids classically suppress the transcription of several genes that encode pro-inflammatory cytokines and chemokines, therefore suggesting it may not have a direct action over glucose uptake and consumption and oxidative stress response.