Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

Abstract

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

“…Interestingly, knockdown of UCP2 in macrophages increases lactate production. The increased lactate production indicates increased energy production through glycolysis, a common indicator of electron transport chain dysfunction (50). Additionally, this may also explain UCP2 dependency of the lower basal respiration of FABP4/aP2-deficient macrophages.…”

The influence of phase II conjugation on the biological activity of flavonoids

“…After the consumption of foods rich in quercetin glycosides, the main metabolites in plasma are reported to be quercetin-3-O-glucuronide and quercetin-3'-O-sulphate; further glucuronidated, sulfated and/or methylated conjugates can be found at lower concentrations. Most conjugates detected in plasma are also reported to be present in urine [36,37,[103][104][105]. After the consumption of endive, kaempferol-3-O-glucuronide is the only kaempferol conjugate reported to be present in plasma, in addition to small amounts of the aglycone.…”

“…In human brain, Q-3G was detected by an anti-Q-3G antibody in the epithelial cells of the choroid plexus; in fresh infarcts, Q-3G was localized in the cytoplasm of the cortical neurons, and in recent infarcts Q-3G appeared to be localized in foamy macrophages in the necrotic core [102]. Upon Q-3G exposure, murine RAW264 macrophages and MG6 microglia are reported to accumulate Q-3G and quercetin, as well as further conjugate quercetin to methylated quercetin [101][102][103]. When RAW264 macrophages were stimulated with LPS, intracellular levels of Q-3G and quercetin were higher than in cells not stimulated, and no methylated conjugate was detected [101][102][103].…”

“…Upon Q-3G exposure, murine RAW264 macrophages and MG6 microglia are reported to accumulate Q-3G and quercetin, as well as further conjugate quercetin to methylated quercetin [101][102][103]. When RAW264 macrophages were stimulated with LPS, intracellular levels of Q-3G and quercetin were higher than in cells not stimulated, and no methylated conjugate was detected [101][102][103]. Ishisaka et al proposed that Q-3G is deconjugated extracellularly before diffusing into the cells [103].…”

“…When RAW264 macrophages were stimulated with LPS, intracellular levels of Q-3G and quercetin were higher than in cells not stimulated, and no methylated conjugate was detected [101][102][103]. Ishisaka et al proposed that Q-3G is deconjugated extracellularly before diffusing into the cells [103]. Human neutrophils, which are another type of phagocytes, are reported to be able to deconjugate quercetin and luteolin glucuronides and thereby release the aglycone, especially in sites of inflammation [104,105].…”