Chronic exposure to short chain fatty acids modulates transport and metabolism of microbiome-derived phenolics in human intestinal cells

Dietary fibre-derived short chain fatty acids (SCFA) and phenolics produced by the gut microbiome have multiple effects on health. We have tested the hypothesis that long term exposure to physiological concentrations of SCFA can affect the transport and metabolism of (poly)phenols by the intestinal epithelium using the Caco-2 cell model. Metabolites and conjugates of hesperetin (HT) and ferulic acid (FA), gut-derived from dietary hesperidin and chlorogenic acid respectively, were quantified by LC–MS with authentic standards following transport across differentiated cell monolayers. Changes in metabolite levels were correlated with effects on mRNA and protein expression of key enzymes and transporters. Propionate and butyrate increased both FA transport and rate of appearance of FA-glucuronide apically and basolaterally, linked to an induction of MCT1. Propionate was the only SCFA that augmented the rate of formation of basolateral FA-sulfate conjugates, possibly via basolateral transporter upregulation. In addition, propionate enhanced the formation of HT-glucuronide conjugates and increased HT-sulfate efflux towards the basolateral compartment. Acetate treatment amplified transepithelial transport of FA in the apical to basolateral direction, associated with lower levels of MCT1 protein expression. Metabolism and transport of both HT and FA were curtailed by the organic acid lactate owing to a reduction of UGT1A1 protein levels. Our data indicate a direct interaction between microbiota-derived metabolites of (poly)phenols and SCFA through modulation of transporters and conjugating enzymes, and increase our understanding of how dietary fibre, via the microbiome, may affect and enhance uptake of bioactive molecules

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia

Endothelial cells are routinely exposed to elevated glucose concentrations post-prandially in healthy individuals and permanently in patients with metabolic syndrome and diabetes, and so we assessed their sugar transport capabilities in response to high glucose. In human umbilical vein (HUVEC), saphenous vein, microdermal vessels and aorta, GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT6 (SLC2A6), and in microdermal vessels also GLUT12 (SLC2A12), were the main glucose transporters as assessed by mRNA, with no fructose transporters nor SGLT1 (SLC5A1). Uptake of 14C-fructose was negligible. GLUT1 and GLUT3 proteins were detected in all cell types and were responsible for ~ 60% glucose uptake in HUVEC, where both GLUT1 and GLUT3, but not GLUT6 siRNA knock-down, reduced the transport. Under shear conditions, GLUT1 protein decreased, GLUT3 increased, and 14C-deoxy-glucose uptake was attenuated. In high glucose, lipid storage was increased, cell numbers were lower, 14C-deoxy-glucose uptake decreased owing to attenuated GLUT3 protein and less surface GLUT1, and trans-endothelial transport of glucose increased due to cell layer permeability changes. We conclude that glucose transport by endothelial cells is relatively resistant to effects of elevated glucose. Cells would continue to supply it to the underlying tissues at a rate proportional to the blood glucose concentration, independent of insulin or fructose

Exploring mechanisms of action of flavonoids and phenolic acids on pathways of lipid metabolism

Epidemiological studies- indicate an association between the consumption of a 'diet rich in polyphenols and several health benefits. As diabetes and other lipid associated diseases are now reaching menacing proportions worldwide, the need for the development of cost-efficient prevention strategies effective at the population level becomes more essential than ever. Studies in the biological effects of polyphenols are now starting to include their hypoglycaemic and hypolipidemic properties and to investigate potential mechanisms through which they may exert their actions. The focus of the present study was to examine the action of various flavonoids and phenolic acids in pathways of energy metabolism and how they may affect the gene expression of critical enzymes involved in lipid and glucose metabolism. Studied flavonols (quercetin, kaempferol, galangin), and coffee phenolic acids and their metabolites (caffeic acid, dihydroferulic acid, protocatechuic acid) were identified as novel regulators of CPTIA, while quercetin was found to importantly upregulate CPTIA & PDK4 gene expression, to restrict glucose uptake and citrate synthase activity, and therefore was shown to potentially modulate energy metabolism in HepG2 cells. Dihydroferulic acid, the main microbial metabolite of chlorogenic acids from coffee was found to significantly increase PDK4 mRNA levels and enhance citrate synthase activity while it increased glucose uptake. On the other hand, a mixture of flavonols at in vivo relevant concentrations was able to produce a synergistic upregulating effect on CPTIA & PDK4, similar to that observed with quercetin, but an increase in glucose uptake. Further studies showed that the induction of CPTIA and PDK4 by quercetin was mediated by more than one transcription factors, with AMPK to have a pivotal role but Nrf2 driven antioxidant mechanisms not to be involved. From our data we hypothesised for quercetin that it sets the cell under a similar with mild starvation state as in between meals. Phenolic acids were further studied for their role in p-oxidation processes and it was demonstrated for the first time that they may form intermediates of p-oxidation as part of their metabolism in HepG2 cells. Our data suggest a novel but distinct role between flavonols and phenolic acids in lipid metabolism. Their action is defined both by direct interactions with enzymes involved in p-oxidation as well as by indirect actions on the transcriptional level. The biological importance of these findings requires further studies with additional models, which will expand our knowledge with regards to the role of polyphenols in lipid metabolism and any possible beneficial in vivo effects. IIIEThOS - Electronic Theses Online ServiceGBUnited Kingdo

The cardiovascular benefits of dark chocolate

Dark chocolate contains many biologically active components, such as catechins, procyanidins and theobromine from cocoa, together with added sucrose and lipids. All of these can directly or indirectly affect the cardiovascular system by multiple mechanisms. Intervention studies on healthy and metabolically-dysfunctional volunteers have suggested that cocoa improves blood pressure, platelet aggregation and endothelial function. The effect of chocolate is more convoluted since the sucrose and lipid may transiently and negatively impact on endothelial function, partly through insulin signalling and nitric oxide bioavailability. However, few studies have attempted to dissect out the role of the individual components and have not explored their possible interactions. For intervention studies, the situation is complex since suitable placebos are often not available, and some benefits may only be observed in individuals showing mild metabolic dysfunction. For chocolate, the effects of some of the components, such as sugar and epicatechin on FMD, may oppose each other, or alternatively in some cases may act together, such as theobromine and epicatechin. Although clearly cocoa provides some cardiovascular benefits according to many human intervention studies, the exact components, their interactions and molecular mechanisms are still under debate

Cellular asymmetric catalysis by UDP-glucuronosyltransferase 1A8 shows functional localization to the basolateral plasma membrane.

UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (-)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pre-treatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells and (-)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and western blotting showed the presence of UGT1A in basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that in HT29-MTX goblet cells at least some of the UGT1A8 enzyme is not endoplasmic reticulum resident but plasma membrane spanning, resulting in much more efficient conjugation of substrate from the basal than from the luminal side, coupled with rapid efflux by functionally-associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell