Differential Impact of Flavonoids on Redox Modulation, Bioenergetics, and Cell Signaling in Normal and Tumor Cells: A Comprehensive Review

Significance: 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. Critical Issues: 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. Future Directions: 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

Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography–tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography

Fatty acyl-Coenzyme A species (acyl-CoAs) are key biomarkers in studies focusing on cellular energy metabolism. Existing analytical approaches are unable to simultaneously detect the full range of short-, medium-, and long-chain acyl-CoAs, while chromatographic limitations encountered in the analysis of limited amounts of biological samples are an often overlooked problem. We report the systematic development of a UHPLC–ESI-MS/MS method which incorporates reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations in series, in an automated mode. The protocol outlined encompasses quantification of acyl-CoAs of varying hydrophobicity from C2 to C20 with recoveries in the range of 90–111 % and limit of detection (LOD) 1–5 fmol, which is substantially lower than previously published methods. We demonstrate that the poor chromatographic performance and signal losses in MS detection, typically observed for phosphorylated organic molecules, can be avoided by the incorporation of a 0.1% phosphoric acid wash step between injections. The methodological approach presented here permits a highly reliable, sensitive and precise analysis of small amounts of tissues and cell samples as demonstrated in mouse liver, human hepatic (HepG2) and skeletal muscle (LHCNM2) cells. The considerable improvements discussed pave the way for acyl-CoAs to be incorporated in routine targeted lipid biomarker profile studies

Quercetin preserves redox status and stimulates mitochondrial function in metabolically-stressed HepG2 cells

Hyperglycemia augments formation of intracellular reactive oxygen species (ROS) with associated mitochondrial damage and increased risk of insulin resistance in type 2 diabetes. We examined whether quercetin could reverse chronic high glucose-induced oxidative stress and mitochondrial dysfunction. Following long-term high glucose treatment, complex I activity was significantly decreased in isolated mitochondria from HepG2 cells. Quercetin dose-dependently recovered complex I activity and lowered cellular ROS generation under both high and normal glucose conditions. Respirometry studies showed that quercetin could counteract the detrimental increase in inner mitochondrial membrane proton leakage resulting from high glucose while it increased oxidative respiration, despite a decrease in electron transfer system (ETS) capacity, and lower non-ETS oxygen consumption. A quercetin-stimulated increase in cellular NAD+/NADH was evident within 2 h and a two-fold increase in PGC-1α mRNA within 6 h, in both normal and high glucose conditions. A similar pattern was also found for the mRNA expression of the repulsive guidance molecule b (RGMB) and its long non-coding RNA (lncRNA) RGMB-AS1 with quercetin, indicating a potential change of the glycolytic phenotype and suppression of aberrant cellular growth which is characteristic of the HepG2 cells. Direct effects of quercetin on PGC-1α activity were minimal, as quercetin only weakly enhanced PGC-1α binding to PPARα in vitro at higher concentrations. Our results suggest that quercetin may protect mitochondrial function from high glucose-induced stress by increasing cellular NAD+/NADH and activation of PGC-1α-mediated pathways. Lower ROS in combination with improved complex I activity and ETS coupling efficiency under conditions of amplified oxidative stress could reinforce mitochondrial integrity and improve redox status, beneficial in certain metabolic diseases

Pomegranate juice, but not an extract, confers a lower glycemic response on a high–glycemic index food: randomized, crossover, controlled trials in healthy subjects

Background: Low–glycemic index diets have demonstrated health benefits associated with a reduced risk of developing type 2 diabetes. Objectives: We tested whether pomegranate polyphenols could lower the glycemic response of a high–glycemic index food when consumed together and the mechanism by which this might occur. Design: We compared the acute effect of a pomegranate juice and a polyphenol-rich extract from pomegranate (supplement) on the bread-derived postprandial blood glucose concentration in 2 randomized, crossover, controlled studies (double-blinded for the supplements), each on 16 healthy volunteers. An additional randomized, crossover, controlled study on 16 volunteers consuming constituent fruit acids in a pH-balanced solution (same pH as pomegranate) and bread was conducted to determine any contributions to postprandial responses caused by acidic beverages. Results: As primary outcome, the incremental area under the curve for bread-derived blood glucose (−33.1% ± 18.1%, P = 0.000005) and peak blood glucose (25.4% ± 19.3%, P = 0.0004) were attenuated by pomegranate juice, compared with a control solution containing the equivalent amount of sugars. In contrast, the pomegranate supplement, or a solution containing the malic and citric acid components of the juice, was ineffective. The pomegranate polyphenol punicalagin was a very effective inhibitor of human α-amylase in vitro, comparable to the drug acarbose. Neither the pomegranate extract nor the individual component polyphenols inhibited 14C-D-glucose transport across differentiated Caco-2/TC7 cell monolayers, but they inhibited uptake of 14C-glucose into Xenopus oocytes expressing the human glucose transporter type 2. Further, some of the predicted pomegranate gut microbiota metabolites modulated 14C-D-glucose and 14C-deoxy-D-glucose uptake into hepatic HepG2 cells. Conclusions: These data indicate that pomegranate polyphenols, when present in a beverage but not in a supplement, can reduce the postprandial glycemic response of bread, whereas microbial metabolites from pomegranate polyphenols exhibit the potential to further modulate sugar metabolism much later in the postprandial period. This trial was registered at clinicaltrials.gov as NCT02486978, NCT02624609, and NCT03242876

Nutritional implications of olives and sugar: attenuation of post-prandial glucose spikes in healthy volunteers by inhibition of sucrose hydrolysis and glucose transport by oleuropein

Purpose: The secoiridoid oleuropein, as found in olives and olive leaves, modulates some biomarkers of diabetes risk in vivo. A possible mechanism may be to attenuate sugar digestion and absorption. Methods: We explored the potential of oleuropein, prepared from olive leaves in a water soluble form (OLE), to inhibit digestive enzymes (α-amylase, maltase, sucrase), and lower [¹⁴C(U)]-glucose uptake in Xenopus oocytes expressing human GLUT2 and [¹⁴C(U)]-glucose transport across differentiated Caco-2 cell monolayers. We conducted 7 separate crossover, controlled, randomised intervention studies on healthy volunteers (double-blinded and placebo-controlled for the OLE supplement) to assess the effect of OLE on post-prandial blood glucose after consumption of bread, glucose or sucrose. Results: OLE inhibited intestinal maltase, human sucrase, glucose transport across Caco-2 monolayers, and uptake of glucose by GLUT2 in Xenopus oocytes, but was a weak inhibitor of human α-amylase. OLE, in capsules, in solution or as naturally present in olives, did not affect post-prandial glucose derived from bread, while OLE in solution attenuated post-prandial blood glucose after consumption of 25 g sucrose, but had no effect when consumed with 50 g of sucrose or glucose. Conclusion: The combined inhibition of sucrase activity and of glucose transport observed in vitro was sufficient to modify digestion of low doses of sucrose in healthy volunteers. In comparison, the weak inhibition of α-amylase by OLE was not enough to modify blood sugar when consumed with a starch-rich food, suggesting that a threshold potency is required for inhibition of digestive enzymes in order to translate into in vivo effects

Ferulic acid-4-O-sulfate rather than ferulic acid relaxes arteries and lowers blood pressure in mice

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 may be the main metabolite, very little has been reported regarding its bioactivities. We have therefore compared the ex vivo vasorelaxing effect of FA and FA-sul (10−7 - 3.10−5 M) 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, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 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

The effects of dictatorship on health: the case of Turkmenistan

BACKGROUND: There is a health crisis in Turkmenistan similar to, but more severe than, in other Central Asian countries. This paper asks whether the health crisis in Turkmenistan is attributable to the consequences of the dictatorship under president Niyazov, who died in 2006. METHODS: The basis for this paper was a series of semi-structured in-depth interviews with key informants complemented by an iterative search of internet sites, initially published as a report in April 2005, and subsequently updated with feedback on the report as well as a comprehensive search of secondary information sources and databases. RESULTS: This paper describes in depth three areas in which the dictatorship in Turkmenistan had a negative impact on population health: the regime's policy of secrecy and denial, which sees the "solution" to health care problems in concealment rather than prevention; its complicity in the trafficking of drugs from Afghanistan; and the neglect of its health care system. CONCLUSION: The paper concludes that dictatorship has contributed to the health crisis facing Turkmenistan. One of the first tests of the new regime will be whether it can address this crisis

Long term treatment with quercetin in contrast to the sulfate and glucuronide conjugates affects HIF1α stability and Nrf2 signaling in endothelial cells and leads to changes in glucose metabolism

Endothelial functionality profoundly contributes to cardiovascular health. The effects of flavonoids shown to improve endothelial performance include regulating blood pressure by modulating endothelial nitric oxide synthase and NADPH oxidases, but their impact on glucose uptake and metabolism has not been explored. We treated human umbilical vein endothelial cells (HUVEC) with the flavonoid quercetin and its circulating metabolites acutely and chronically, then assessed glucose uptake, glucose metabolism, gene transcription and protein expression. Acute treatment had no effect on glucose uptake, ruling out any direct interaction with sugar transporters. Long term treatment with quercetin, but not quercetin 3-O-glucuronide or 3'-O-sulfate, significantly increased glucose uptake. Heme oxygenase-1 (HO-1) was induced by quercetin but not its conjugates, but was not implicated in the glucose uptake stimulation since hemin, a classical inducer of HO-1, did not affect glucose metabolism. Quercetin increased stability of the transcription factor hypoxia induced factor 1α (HIF1α), a powerful stimulant of glucose metabolism, which was also paralleled by treatment with a prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which regulates the rate of glycolysis, was upregulated by both quercetin and DMOG. Pyruvate dehydrogenase kinase (PDK) isoforms regulate pyruvate dehydrogenase; PDK2 and PDK4 were down-regulated by both effectors, but only DMOG also upregulated PDK1 and PDK3. Quercetin, but not DMOG, increased glucose-6-phosphate dehydrogenase. Chronic quercetin treatment also stimulated glucose transport across the HUVEC monolyer in a 3D culture model. Gene expression of several flavonoid transporters was repressed by quercetin, but this was either abolished (Organic anion transporter polypeptide 4C1) or reversed (Multidrug resistance gene 1) by both conjugates. We conclude that quercetin and its circulating metabolites differentially modulate glucose uptake/metabolism in endothelial cells, through effects on HIF1α and transcriptional regulation of energy metabolism

Butyric acid increases transepithelial transport of ferulic acid through upregulation of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4)

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

Indirect Chronic Effects of an Oleuropein-Rich Olive Leaf Extract on Sucrase-Isomaltase In Vitro and In Vivo

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 (Glcmax), time to reach Glcmax 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