JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 JPETThis Fast article Forward. has not been Published copyedited andon formatted. May 10, The 2007 final asversion DOI:10.1124/jpet.107.124040 may differ from this version. JPET #124040 Page 1 Flavonoid-glycosides are not transported by the human Na+/glucose transporter when expressed in Xenopus leavis oocytes, but effectively inhibit electrogenic glucose uptake Downloaded from jpet.aspetjournals.org Gabor Kottra and Hannelore Daniel Molecular Nutrition Unit, Department of Food and Nutrition TUM at ASPET Journals on October 2, 2021 Technische Universität München Am Forum 5, D-85350 Freising, Germany (G.K., H.D.) Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 This article has not been copyedited and formatted. The final version may differ from this version. JPET #124040 Page 2 Running title: Flavonoid-glycosides and hSGLT1 Corresponding author: Gabor Kottra Molecular Nutrition Unit, Department of Food and Nutrition Technische Universität München Am Forum 5, D-85350 Freising, Germany Telephone: +49 8161713405 Downloaded from Telefax: +49 8161713999 e-mail: [email protected] jpet.aspetjournals.org Number of text pages: 22 (including references) Number of tables: 1 Number of figures: 6 Number of references: 21 at ASPET Journals on October 2, 2021 Number of words in the Abstract: 234 Number of words in the Introduction: 749 Number of words in the Discussion: 1499 List of nonstandard abbreviations: hSGLT1 human sodium glucose cotransporter type 1 rPEPT1 rabbit intestinal proton-coupled peptide transporter α-MDG α-methyl-D-glucopyranoside GQ glycil-glutamine LPH lactase phlorizin hydrolase AUC area under the plasma concentration-time curve Q3G quercetin-3-O-glucoside Q4’G quercetin-4’-O-glucoside Recommended section assignment: Metabolism, Transport and Pharmacogenomics JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 This article has not been copyedited and formatted. The final version may differ from this version. JPET #124040 Page 3 Abstract There is a controversy on whether intestinal absorption of glycosylated flavonoids and particular of quercetin glycosides involves their uptake in intact form via the human sodium coupled glucose transporter hSGLT1. We here describe studies using Xenopus oocytes that express hSGLT1 and the two-electrode voltage clamp technique to determine the transport characteristics of a variety of flavonoids carrying glucose residues at different positions as well as of their aglycones (altogether 27 compounds). Neither quercetin, luteolin, apigenin, naringenin, pelarginidin, daidzein Downloaded from or genistein, nor any of their glycosylated derivatives generated significant transport currents. However, the inward current evoked by 1 mM of the hSGLT1 substrate α- jpet.aspetjournals.org MDG was potently reduced by the simultaneous application of various flavonoid- glycosides, but also by some aglycones. The inhibitory potency remained unchanged, when the attached glucose was replaced by galactose suggesting that at ASPET Journals on October 2, 2021 these residues may bind to SGLT1. Kinetic analysis by Dixon-plots revealed inhibition of competitive type with high affinities in particular when the glucose was attached to the position 4’ of the aromatic ring of the flavonoids. The affinities became lower when the glucose was attached to a different position. Unexpectedly, the aglycone form of luteolin also inhibited the transport competitively with high affinity. These data show that hSGLT1 does not transport any of the flavonoids and seems therefore not involved in their intestinal absorption. However, glycosylated but also a few non-glycosylated flavonoids show a structure-dependent capability for effective inhibition of SGLT1. JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 This article has not been copyedited and formatted. The final version may differ from this version. JPET #124040 Page 4 Introduction Flavonoids are a large group of polyphenolic compounds with a similar basic structure consisting of two phenolic benzene rings linked through a heterocyclic pyran or pyrone ring. Compounds are further divided into subclasses based on the link of the aromatic ring to the heterocyclic ring, their oxidation state and the functional groups attached to the heterocyclic ring. Within each subclass, individual compounds are characterized by specific hydroxylation and conjugation patterns. Flavonoids are widespread in higher plants and most flavonoids (with the exception Downloaded from of flavanols) occur naturally in conjugated form mainly with sugar residues. jpet.aspetjournals.org Epidemiological studies suggest health beneficial effects of dietary flavonoids by reducing the risk of carcinogenesis, hypertension and cardiovascular diseases (for reviews, see (Ren et al., 2003; Arts and Hollman, 2005; Scalbert et al., 2005)). at ASPET Journals on October 2, 2021 Flavonoids not only have antioxidant activity but also possess more specific effects on a variety of cellular and molecular processes. In view of the effects of flavonoids on human health, not only on the amount consumed via the diet but also their bioavailability is important. A recent review has summarized the data on bioavailability of polyphenols, including flavonoids as derived from 97 publications (Manach et al., 2005). The comparison of pharmacokinetic data such as maximal plasma concentration, time to reach this concentration and the area under the plasma concentration-time (AUC) curve revealed huge differences between the polyphenols and suggest different mechanisms of absorption and different sites in the gastrointestinal tract where absorption takes place. Uptake of flavonoids can occur at the gastric level (only JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 This article has not been copyedited and formatted. The final version may differ from this version. JPET #124040 Page 5 observed for aglycones), in the small intestine (aglycones and glucosides) and after interaction with the microflora in the colon (predominantly for glycosides with complex sugar moiety like rutinoside) (Manach et al., 2004). Regardless of the mechanism of absorption, intact glycosides of flavonoids are usually not found in the plasma and urine (for references, see (Manach et al., 2004)). Since most flavonoids in foods are present in glycosylated form, a considerably number of publications were devoted to the characterization of transport mainly by Downloaded from using quercetin glucosides for which uptake via the intestinal sodium-coupled glucose transporter (SGLT1) was proposed. jpet.aspetjournals.org To test whether quercetin-4’-O-glucoside (Q4’G) is a substrate for SGLT1, its cellular uptake was examined with Caco-2 cells and chinese hamster ovary cells stably at ASPET Journals on October 2, 2021 transfected with SGLT1. Fluorescent microscopy as well as HPLC analysis revealed a sodium-dependent cellular accumulation of Q4’G after apical loading. Uptake was inhibited by the addition of glucose or phlorizin as a high affinity inhibitor of SGLT1. These findings suggested that Q4’G was taken up into cells by SGLT1 (Walgren et al., 2000). In contrast, studies conducted in isolated segments of the small intestine of rats showed that the absorption of genistein-7-glucoside (genistin) was increased 2.5-fold when phlorizin was simultaneously perfused with genistin (Andlauer et al., 2004). Employing Ussing-chamber with rat jejunum, Wolffram and coworkers observed the disappearance of quercetin-3-O-glucoside (Q3G) from the luminal, but not from the serosal compartment with the aglycone quercetin appearing in the serosal JPET Fast Forward. Published on May 10, 2007 as DOI: 10.1124/jpet.107.124040 This article has not been copyedited and formatted. The final version may differ from this version. JPET #124040 Page 6 compartment when Q3G was applied on the mucosal side. The addition of glucose or of phlorizin largely reduced the disappearance of Q3G from the luminal side whereas fructose had no effect. The transport of quercetin was observed only in jejunum, but not in the proximal colon. These findings led the authors conclude that SGLT1 played a role in cellular uptake of Q3G (Wolffram et al., 2002). These conclusions were disputed in a letter to the editor (Arts et al., 2002) with the main objection that the inhibitors chosen are not specific for SGLT1, but could also Downloaded from have inhibited lactase phlorizin hydrolase (LPH), an enzyme present in the brush border membrane of the small intestine capable of hydrolyzing Q3G on the cell jpet.aspetjournals.org surface with uptake of the aglycone quercetin via passive processes. This hypothesis was finally followed experimentally to assess transport of Q3G and Q4’G in the presence and absence of phlorizin and of a specific inhibitor of LPH (5). The results at ASPET Journals on October 2, 2021 suggested that Q3G was transported only as aglycone via an SGLT1-independent mechanism, whereas in the transport of Q4G may have involved both pathways (Day et al., 2003). The rather contradictory findings from these studies on transport of quercetin glycosides and other compounds via SGLT1 prompted us to test the transport by directly measuring transport currents in Xenopus oocytes expressing the human SGLT1. By selecting flavonoids belonging to different