Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Abstract

Objective: In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs), dimeric catechins, inhibited the transport of di-peptides across Caco-2 monolayers by suppressing the expression of PEPT1 through AMP-activated protein kinase (AMPK) activation. In this study, we investigated the structural requirement of theaflavins for the effect, and the mechanism(s) underling theaflavin-induced AMPK activation.Methods: Theaflavin-3’-O-gallate (TF3’G) was used for this study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 μM sample (TF3’G or its related compounds) in an Ussing Chamber. The amount of Gly-Sar (a model of PEPT1-transporing peptide) transport at fixed time-points to 60 min was determined by fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay (Ex/Em: 405 nm/460 nm). The apparent permeability coefficient (Papp) was used to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 μM TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μM STO-609 as CaMKK inhibitor) was evaluated by Western blot.Results: The Papp value of Gly-Sar significantly (P < 0.05) decreased in 20 μM purprogallin-treated Caco-2 cells as well as in TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Papp reduction. In TF3'G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C, but not STO-609.Conclusion: The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport, and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway, but not CaMKK/AMPK pathway.Keywords: Theaflavin-3’-Ο-gallate, Peptide transport, PEPT1, Benzotropolone, AMP-activated protein kinase, Calmodulin-dependent protein kinase kinase

Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1

“…We found that TFs inhibited intestinal peptide transport via the downregulation of the peptide transporter PEPT1 by activated AMPK in Caco-2 cells. 4,5) In the present study, we further showed that TFs activated AMPK resulting in enhanced TJ-related protein expression and closure of the TJ route (Figs. 5-7).…”

“…4,5) For Caco-2 transport experiments, cells were seeded into BD Falcon™ cell culture insert (PET membrane, 0.9 cm 2 , 1.0 μm pore size; BD Biosciences) coated with type I collagen (collagen gel culturing kit, Cellmatrix type I-A, Nitta Gelatin, Osaka, Japan) at a density of 4.0 × 10 5 cell/mL. Seeded cells were cultured in a seeding basal medium containing MITO+™ serum extender for 48 h. The medium was replaced with an enterocyte differentiation medium containing MITO+™ serum extender every day for three days to allow the cells to form monolayers.…”

“…4) TFs suppressed intestinal transport of small peptides via the downregulation of AMP-activated protein kinase (AMPK)-mediated peptide transporter (PEPT1) in Caco-2 cell monolayers. 4,5) We previously found that TFs induced AMPK activation in Caco-2 cells. Thus, TFs may influence other AMPK-related intestinal transport systems, since Zheng and Cantley have found that epithelial AMPK activation was closely associated with enhanced tight junction (TJ) integrity in MDCK cells.…”

Theaflavin-3, 3′-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways

“…TF3 possesses a benzotropolone skeleton that is formed from co-oxidation of EGCG and ECG, one with a vic-trihydroxy moiety and the other with an ortho-dihydroxy structure ( 9 ). The benzotropolone moiety has been proven to be important in the antioxidant activity ( 55 ), anti-inflammatory activity ( 56 ), anti-peptide transport activity and activating AMP-activated protein kinase ( 57 ). Thus, we speculated that benzotropolone moiety might also be vital for its anticancer activity.…”

Condensed catechins and their potential health-benefits

“…In theaflavin-treated AMPK-activated Caco-2 cells, theaflavins induced the significant (Po 0.05) suppression of peptide transporter 1 (PepT1) expression (Takeda et al, 2013;Park et al, 2013) and the significant (Po 0.05) promotion of the expression of TJ-related proteins including occludin, claudin-1, and zonula occluden (ZO)-1, in which the candidate structure required for such actions was a benzotropolone moiety of theaflavins (Park et al, 2015). In turn, non-absorbable theaflavins are an active intestinal regulator for the absorption of compounds transported through both PepT1 and TJ pathways (Fig.…”