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Flaxseed Lignan Metabolites Modulate Hepatocellular Cholesterol Trafficking In HepaRG



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High blood cholesterol (HBC) is an important risk factor of cardiovascular disease (CVD), which is associated with high morbidity and mortality worldwide. Lifestyle changes and drugs (e.g. statins) are mainly advised by practitioners to manage cholesterol; however, safer alternatives such as natural products might be considered for mild to moderate hypercholesterolemia or in combination with statins in more severe conditions. The literature indicates the ability of flaxseed lignans to improve cholesterol blood profiles. However, the mechanism by which the metabolites of the non-bioavailable plant lignan, secoisolariciresinol diglucoside, modulate blood cholesterol levels is not yet known. This study examines a possible mechanism of cholesterol modulation in the liver that involves altered cholesterol trafficking based upon recent investigations in the Caco-2 cell line from my laboratory. In addition, the concomitant oral administration of statins and flaxseed lignans raises a possible role for a ‘drug-drug’ interaction at hepatic uptake transporters (OATP1B) which cannot be ignored as these transporters impact the pharmacokinetics and pharmacodynamics of statins. To address lignan mechanism of action, the effect of the mammalian lignan, enterolactone (ENL), and its metabolite, enterolactone glucuronide (ENL-Gluc), on hepatic trafficking of the fluorescent cholesterol probe NBD-cholesterol, was investigated using fluorescence microscopy in the HepaRG cell line as an in vitro liver model. Specific dye markers for endoplasmic reticulum was used to identify the intracellular location of cholesterol trafficking and accumulation. Furthermore, the INSIG-SREBP cholesterol regulation pathway was examined after treatment with ENL and ENL-Gluc by expression analysis of genes important in cholesterol metabolism and trafficking, namely, INSIG-1, SREBP-2, HMGCoA-reductase and LDL-receptor by qPCR and confirmed with western blot analysis. In addition, the possible inhibitory effect of ENL and ENL-Gluc on the uptake of the organic anion transporting polypeptide 1B1 and 1B3 substrate, fluorescein methotrexate (FMTX), was examined in HEK293 cells overexpressing human OATP1B1 & OATP1B3 transporters. Under high intracellular sterol conditions, both ENL and ENL-Gluc reduced the uptake of fluorescent cholesterol into HepaRG cells. In comparison to vehicle control (1% DMSO), treatment with 20 µM ENL and 20 µM ENL-Gluc reduced cholesterol uptake by 1.8 and 2.0-fold, respectively. This was confirmed by observing a surge in NBD-cholesterol accumulation in the endoplasmic reticulum (ER) following treatment with different concentrations of ENL and ENL-Gluc. These results suggest ENL and ENL-Gluc alter hepatocellular cholesterol homeostasis through increasing cholesterol retention within the endoplasmic reticulum. Furthermore, changes in the relative expression of multiple target genes that are responsible for activation of a membrane bound transcription factor, SREBP, as well as protein level measurements by western blot analysis suggest a transcriptional regulation of cholesterol biosynthesis via enhancement of HMGCoA-reductase degradation as well as inhibition of cholesterol uptake. Furthermore, the possible interaction between ENL or ENL-Gluc and hepatic uptake transporters OATP1B1 and OATP1B3 was observed by conducting an inhibitory uptake assay in HEK293 cells overexpressing human OATP1B1& OATP1B3 transporters. These investigations showed inhibition of probe substrate uptake by ENL and ENL-Gluc. In conclusion, we reported that flaxseed lignan ENL and its active glucuronidated form are suggested to be responsible for the modulation effect on cholesterol homeostasis and trafficking observed in HepaRG cells. Both ENL and ENL-Gluc showed a reduction in cholesterol uptake and an increase in cholesterol surge into the endoplasmic reticulum which involved downregulation of HMGCoA-R and LDL-R, and upregulation of SREBP-2, proteins of sterol sensing domain (SSD). This effect is apparently mediated via the active glucuronide form of ENL, which showed an inhibitory effect on OATP1B1 and OATP1B3 hepatic uptake-mediated statin transport. Further in vivo investigation is necessary to confirm the altered effect of lignans on cholesterol transport and metabolism as well as the inhibitory effect on OATP hepatic uptake transporters.



Cholesterol Trafficking, HepaRG, Flaxseed Lignan Metabolites, Enterolactone, Enterolactone Glucuronide, OATP hepatic uptake transporters,OATP1B1, OATP1B3.



Master of Science (M.Sc.)


Pharmacy and Nutrition




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