Flavonoids can interact with multiple molecular targets to elicit their cellular effects, leading to changes in signal transduction, gene expression, and/or metabolism, which can, subsequently, ...affect the entire cell and organism. Immortalized cell lines, derived from tumors, are routinely employed as a surrogate for mechanistic studies, with the results extrapolated to tissues in vivo. Recent Advances: We review the activities of selected flavonoids on cultured tumor cells derived from various tissues in comparison to corresponding primary cells or tissues in vivo, mainly using quercetin and flavanols (epicatechin and (-)-epigallocatechin gallate) as exemplars. Several studies have indicated that flavonoids could retard cancer progression in vivo in animal models as well as in tumor cell models.
Extrapolation from in vitro and animal models to humans is not straightforward given both the extensive conjugation and complex microbiota-dependent metabolism of flavonoids after consumption, as well as the heterogeneous metabolism of different tumors.
Comparison of data from studies on primary cells or in vivo are essential not only to validate results obtained from cultured cell models, but also to highlight whether any differences may be further exploited in the clinical setting for chemoprevention. Tumor cell models can provide a useful mechanistic tool to study the effects of flavonoids, provided that the limitations of each model are understood and taken into account in interpretation of the data.
Acyl‐quinic acids (chlorogenic acids) are produced by many plants, including fruits, vegetables, and herbal remedies, with coffee and maté particularly rich dietary sources. Epidemiological and ...intervention studies suggest that they can reduce the risk of developing type 2 diabetes and cardiovascular disease. This review addresses their metabolic handling after oral consumption to provide a mechanistic basis to explain their possible effects on health. Intact acyl‐quinic acids are absorbed only to a small extent in the small intestine, but the cinnamic acids are efficiently absorbed after hydrolysis by either digestive or microbial enzymes in the colon. Metabolism results in phenolic conjugates in the blood and urine, but varying dependent on the acyl‐quinic acid, and subject to significant interperson variability. The balance between hydrogenation and complete β‐oxidation of the cinnamic acids, both by liver and gut microbiota, determines the profile of metabolites. Pharmacokinetic data suggest that some metabolites are bound to human serum albumin and/or sequestered in tissues, and some exhibit biological activity in vitro, consistent with proposed protective action in vivo. Significant gaps in the literature include lack of plasma and urinary data for free‐living individuals, and pharmacokinetic data for groups who consume coffee or maté at regular short intervals. Data are required for cis isomers. There is a critical need for precise urinary biomarkers of consumption of acyl‐quinic acids, accounting for variability in individual metabolism and in beverage composition, thus facilitating better translation of urinary metabolite measurements into accurate coffee consumption data to improve the outcomes of future epidemiological and intervention studies.
ABSTRACT
The gut microbiome supplies essential metabolites such as short‐chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by ...muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota‐derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN‐M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose—and insulin‐treated myotubes. One of the most effective was isovanillic acid 3‐O‐sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3‐O‐glucoside, a major compound in berry fruits. IVAS stimulated a dose‐dependent increase in glucose transport through glucose transporter GLUT4‐ and PI3K‐dependent mechanisms. IVAS also up‐regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.—Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism. FASEB J. 33, 1887–1898 (2019). www.fasebj.org
The origin of inter-individual variability in the action of bioactive small molecules from the diet is poorly understood and poses a substantial obstacle to harnessing their potential for attenuating ...disease risk. Epidemiological studies show that coffee lowers the risk of developing type 2 diabetes, independently of caffeine, but since coffee is a complex matrix, consumption gives rise to different classes of metabolites in vivo which in turn can affect multiple related pathways in disease development. We quantified key urinary coffee phenolic acid metabolites repeated three times in 36 volunteers, and observed the highest inter- and intra-individual variation for metabolites produced by the colonic microbiome. Notably, a urinary phenolic metabolite not requiring the action of the microbiota was positively correlated with fasting plasma insulin. These data highlight the role of the gut microbiota as the main driver of both intra- and inter-individual variation in metabolism of dietary bioactive small molecules.
Ferulic acid is released by microbial hydrolysis in the colon, where butyric acid, a major by-product of fermentation, constitutes the main energy source for colonic enterocytes. We investigated how ...varying concentrations of this short chain fatty acid may influence the absorption of the phenolic acid. Chronic treatment of Caco-2 cells with butyric acid resulted in increased mRNA and protein abundance of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4), previously proposed to facilitate ferulic acid absorption in addition to passive diffusion. Short term incubation with butyric acid only led to upregulation of MCT4 while both conditions increased transepithelial transport of ferulic acid in the apical to basolateral, but not basolateral to apical, direction. Chronic treatment also elevated intracellular concentrations of ferulic acid, which in turn gave rise to increased concentrations of ferulic acid metabolites. Immunofluorescence staining of cells revealed uniform distribution of MCT1 protein in the cell membrane, whereas MCT4 was only detected in the lateral plasma membrane sections of Caco-2 cells. We therefore propose that MCT1 may be acting as an uptake transporter and MCT4 as an efflux system across the basolateral membrane for ferulic acid, and that this process is stimulated by butyric acid.
•Dietary fibre gives both short chain fatty acids and phenolic acids in the colon.•Ferulic acid is partially taken into cells by the transporter MCT1.•Ferulic acid is effluxed basolaterally by the transporter MCT4.•Butyrate upregulates transporters of phenolic acids in a colonic model.•A short chain fatty acid can regulate phenolic acid absorption.
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Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no ...information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-14C(U)-glucose or D-14C(U)-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting.
In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (−)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings.
Although polyphenols inhibit glucose absorption and transport in vitro, it is uncertain whether this activity is sufficient to attenuate glycaemic response in vivo. We examined this using orange ...juice, which contains high levels of hesperidin. We first used a combination of in vitro assays to evaluate the potential effect of hesperidin and other orange juice components on intestinal sugar absorption and then tested whether this translated to an effect in healthy volunteers. Hesperidin attenuated transfer of 14C-labelled glucose across differentiated Caco-2/TC7 cell monolayers. The involvement of the sugar transporter GLUT2 was demonstrated by experiments carried out in the absence of Na to exclude the contribution of sodium-glucose linked transporter 1 and further explored by the use of Xenopus laevis oocytes expressing human GLUT2 or GLUT5. Fructose transport was also affected by hesperidin partly by inhibition of GLUT5, while hesperidin, even at high concentration, did not inhibit rat intestinal sucrase activity. We conducted three separate crossover interventions, each on ten healthy volunteers using orange juice with different amounts of added hesperidin and water. The biggest difference in postprandial blood glucose between orange juice and control, containing equivalent amounts of glucose, fructose, sucrose, citric acid and ascorbate, was when the juice was diluted (ΔC max=-0·5 mm, P=0·0146). The effect was less pronounced when the juice was given at regular strength. Our data indicate that hesperidin can modulate postprandial glycaemic response of orange juice by partial inhibition of intestinal GLUT, but this depends on sugar and hesperidin concentrations.
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Commonly used drugs for treating many conditions are either natural products or derivatives. In silico modelling has identified several natural products including quercetin as ...potential highly effective disruptors of the initial infection process involving binding to the interface between the SARS-CoV-2 (Covid-19) Viral Spike Protein and the epithelial cell Angiotensin Converting Enzyme-2 (ACE2) protein. Here we argue that the oral route of administration of quercetin is unlikely to be effective in clinical trials owing to biotransformation during digestion, absorption and metabolism, but suggest that agents could be administered directly by alternative routes such as a nasal or throat spray.
Consumption of foods rich in ferulic acid (FA) such as wholegrain cereals, or FA precursors such as chlorogenic acids in coffee, is inversely correlated with risk of cardiovascular disease and type 2 ...diabetes. As a result of digestion and phase II metabolism in the gut and liver, FA is converted predominantly into ferulic acid-4-O-sulfate (FA-sul), an abundant plasma metabolite. Although FA-sul is the main metabolite, very little has been reported regarding its bioactivities. We have compared the ex vivo vasorelaxing effect of FA and FA-sul (10−7–3.10−5M) on isolated mouse arteries mounted in tissue myographs. FA-sul, but not FA, elicited a concentration-dependent vasorelaxation of saphenous and femoral arteries and aortae. The FA-sul-mediated vasorelaxation was blunted by 1H-1,2,4oxadiazolo4,3-aquinoxalin-1-one, a soluble guanylate cyclase (sGC) inhibitor. The role of sGC was confirmed in femoral arteries isolated from sGCα1(−/−) knockout mice. Furthermore, 4-aminopyridine, a specific inhibitor of voltage-dependent potassium channels, significantly decreased FA-sul-mediated effects. In anesthetized mice, intravenous injection of FA-sul decreased mean arterial pressure, whereas FA had no effect, confirming the results obtained ex vivo. FA-sul is probably one of the major metabolites accounting for the blood pressure-lowering effects associated with FA consumption.
Consumption of dietary bioactives is an avenue to enhancing the effective healthiness of diets by attenuating the glycaemic response. The intestinal brush border enzyme sucrase-isomaltase (SI) is the ...sole enzyme hydrolysing consumed sucrose, and we previously showed the acute effects of olive leaf extract (OLE) on sucrase activity when given together with sugars both in vitro and in vivo. Here we tested whether OLE could affect sucrase expression when pre-incubated chronically, a "priming" effect not dependent on competitive interaction with SI, in both a cell model and a human intervention. Using differentiated Caco-2/TC7 cells, long-term pre-treatment with oleuropein-rich olive leaf extract (OLE) lowered SI mRNA, surface protein and activity, and attenuated subsequent sucrose hydrolysis. Based on these results, a randomised, double-blinded, placebo-controlled, crossover pilot study was conducted. OLE (50 mg oleuropein) was consumed in capsule form 3 times a day for 1 week by 11 healthy young women followed by an oral sucrose tolerance test in the absence of OLE. However this treatment, compared to placebo, did not induce a change in post-prandial blood glucose maximum concentration (Glc
), time to reach Glc
and incremental area under the curve. These results indicate that changes in SI mRNA, protein and activity in an intestinal cell model by OLE are not sufficient under these conditions to induce a functional effect in vivo in healthy volunteers.
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