A comprehensive evaluation of omega-3 fatty acid supplementation in cystic fibrosis patients using lipidomics.

The evaluation of the benefits of omega-3 fatty acid supplementation in humans requires the identification and characterization of suitable biomarkers of its incorporation in the body. The reference method for the evaluation of omega-3, gas chromatography, is difficult to apply in clinical practice because of its low throughput and does not provide information about the incorporation of specific fatty acids in lipid species and the potential effects of supplementation on lipid classes. We used a quantitative lipidomic approach to follow the incorporation of omega-3 fatty acids into plasma lipids in cystic fibrosis patients (n=50) from a randomized controlled clinical trial after the supplementation of seaweed oil enriched with docosahexaenoic acid (DHA). Lipidomic analysis accurately showed the distribution of fatty acids in different lipid classes after omega-3 supplementation, and the performance in determining the compliance to supplementation was similar to that of gas chromatography coupled to mass spectrometry. Twelve months after fatty acid supplementation, DHA was predominantly incorporated into highly unsaturated cholesteryl esters (110.9±16.2 vs. 278.6±32.6 µM, mean±S.E.M.) and phosphatidylcholine (142.4±11.9 vs. 272.9±21.4 µM) and, to a lesser extent, into phosphatidylethanolamine (9.4±0.8 vs. 15.5±1.5 µM) and triglycerides (0.4±0.04 vs. 1.1±0.12 µM). In addition, a technique was developed for the fast measurement of the DHA/arachidonic acid ratio to simplify the follow-up of nutritional intervention with DHA-enriched foods. We conclude that lipidomics is a suitable approach for monitoring the incorporation of omega-3 fatty acids in nutritional studies.

Hydroxytyrosol supplementation modulates the expression of miRNAs in rodents and in humans.

Dietary microRNAs (miRNAs) modulation could be important for health and wellbeing. Part of the healthful activities of polyphenols might be due to a modulation of miRNAs' expression. Among the most biologically active polyphenols, hydroxytyrosol (HT) has never been studied for its actions on miRNAs. We investigated whether HT could modulate the expression of miRNAs in vivo. We performed an unbiased intestinal miRNA screening in mice supplemented (for 8 weeks) with nutritionally relevant amounts of HT. HT modulated the expression of several miRNAs. Analysis of other tissues revealed consistent HT-induced modulation of only few miRNAs. Also, HT administration increased triglycerides levels. Acute treatment with HT and in vitro experiments provided mechanistic insights. The HT-induced expression of one miRNA was confirmed in healthy volunteers supplemented with HT in a randomized, double-blind and placebo-controlled trial. HT consumption affects specific miRNAs' expression in rodents and humans. Our findings suggest that the modulation of miRNAs' action through HT consumption might partially explain its healthful activities and might be pharmanutritionally exploited in current therapies targeting endogenous miRNAs. However, the effects of HT on triglycerides warrant further investigations.

Docosahexaenoic acid modulates the enterocyte Caco-2 cell expression of microRNAs involved in lipid metabolism.

Consumption of the long-chain ω-3 (n-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) is associated with a reduced risk of cardiovascular disease and greater chemoprevention. However, the mechanisms underlying the biologic effects of DHA remain unknown. It is well known that microRNAs (miRNAs) are versatile regulators of gene expression. Therefore, we aimed to determine if the beneficial effects of DHA may be modulated in part through miRNAs. Loss of dicer 1 ribonuclease type III (DICER) in enterocyte Caco-2 cells supplemented with DHA suggested that several lipid metabolism genes are modulated by miRNAs. Analysis of miRNAs predicted to target these genes revealed several miRNA candidates that are differentially modulated by fatty acids. Among the miRNAs modulated by DHA were miR-192 and miR-30c. Overexpression of either miR-192 or miR-30c in enterocyte and hepatocyte cells suggested an effect on the expression of genes related to lipid metabolism, some of which were confirmed by endogenous inhibition of these miRNAs. Our results show in enterocytes that DHA exerts its biologic effect in part by regulating genes involved in lipid metabolism and cancer. Moreover, this response is mediated through miRNA activity. We validate novel targets of miR-30c and miR-192 related to lipid metabolism and cancer including nuclear receptor corepressor 2, isocitrate dehydrogenase 1, DICER, caveolin 1, ATP-binding cassette subfamily G (white) member 4, retinoic acid receptor ß, and others. We also present evidence that in enterocytes DHA modulates the expression of regulatory factor X6 through these miRNAs. Alteration of miRNA levels by dietary components in support of their pharmacologic modulation might be valuable in adjunct therapy for dyslipidemia and other related diseases.

Comparative effects of dietary supplementation with red grape juice and vitamin E on production of superoxide by circulating neutrophil NADPH oxidase in hemodialysis patients.

BACKGROUND: Atherosclerotic cardiovascular disease is the most common cause of death among hemodialysis patients; it has been attributed to increased oxidative stress, dyslipidemia, malnutrition, and chronic inflammation. Activation of neutrophils is a well-recognized feature in dialysis patients, and superoxide-anion production by neutrophil NADPH oxidase may contribute significantly to oxidative stress. OBJECTIVE: The aim of the study was to compare the effects of dietary supplementation with concentrated red grape juice (RGJ), a source of polyphenols, and vitamin E on neutrophil NADPH oxidase activity and other cardiovascular risk factors in hemodialysis patients. DESIGN: Thirty-two patients undergoing hemodialysis were recruited and randomly assigned to groups to receive dietary supplementation with RGJ, vitamin E, or both or a control condition without supplementation or placebo. Blood was obtained at baseline and on days 7 and 14 of treatment. RESULTS: RGJ consumption but not vitamin E consumption reduced plasma concentrations of total cholesterol and apolipoprotein B and increased those of HDL cholesterol. Both RGJ and vitamin E reduced plasma concentrations of oxidized LDL and ex vivo neutrophil NADPH oxidase activity. These effects were intensified when the supplements were used in combination; in that case, reductions in the inflammatory biomarkers intercellular adhesion molecule 1 and monocyte chemoattractant protein 1 also were observed. CONCLUSIONS: Regular ingestion of concentrated RGJ by hemodialysis patients reduces neutrophil NADPH-oxidase activity and plasma concentrations of oxidized LDL and inflammatory biomarkers to a greater extent than does that of vitamin E. This effect of RGJ consumption may favor a reduction in cardiovascular risk.

Concentrated red grape juice exerts antioxidant, hypolipidemic, and antiinflammatory effects in both hemodialysis patients and healthy subjects.

BACKGROUND: Patients treated with hemodialysis frequently experience cardiovascular complications attributed, among other causes, to dyslipidemia, increased oxidative stress, and inflammation. OBJECTIVE: The aim of the study was to study the effects of dietary supplementation with concentrated red grape juice (RGJ), a source of polyphenols, on lipoprotein profile, antioxidant capacity, LDL oxidation, and inflammatory biomarkers. DESIGN: Twenty-six patients receiving hemodialysis and 15 healthy subjects were instructed to drink 100 mL RGJ/d for 14 d. Blood was drawn at baseline, twice during RGJ supplementation, and twice during the 6-mo follow-up period. As a control, 12 other randomly recruited hemodialysis patients not receiving RGJ were studied. Lipids, apolipoproteins, oxidized LDL, and antioxidant vitamins were measured in plasma. The bioavailability of RGJ polyphenols was assessed in healthy subjects. RESULTS: The maximum plasma concentration of quercetin was achieved 3 h after RGJ ingestion, which indicates that supplement-derived polyphenols are rapidly absorbed. In both healthy subjects and hemodialysis patients, RGJ consumption increased the antioxidant capacity of plasma without affecting concentrations of uric acid or ascorbic acid; reduced the concentration of oxidized LDL; and increased the concentration of cholesterol-standardized alpha-tocopherol. RGJ supplementation also caused a significant decrease in LDL-cholesterol and apolipoprotein B-100 concentrations, while increasing the concentrations of HDL cholesterol and apolipoprotein A-I. In a further study in hemodialysis patients, RGJ supplementation for 3 wk significantly reduced plasma monocyte chemoattractant protein 1, an inflammatory biomarker associated with cardiovascular disease risk. CONCLUSION: Dietary supplementation with concentrated RGJ improves the lipoprotein profile, reduces plasma concentrations of inflammatory biomarkers and oxidized LDL, and may favor a reduction in cardiovascular disease risk.

Red grape juice polyphenols alter cholesterol homeostasis and increase LDL-receptor activity in human cells in vitro.

Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryl-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, DiI-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis.

Differential effects of ergosterol and cholesterol on Cdk1 activation and SRE-driven transcription.

Cholesterol is essential for cell growth and division, but whether this is just a consequence of its use in membrane formation or whether it also elicits regulatory actions in cell cycle machinery remains to be established. Here, we report on the specificity of this action of cholesterol in human cells by comparing its effects with those of ergosterol, a yeast sterol structurally similar to cholesterol. Inhibition of cholesterol synthesis by means of SKF 104976 in cells incubated in a cholesterol-free medium resulted in cell proliferation inhibition and cell cycle arrest at G2/M phase. These effects were abrogated by cholesterol added to the medium but not by ergosterol, despite that the latter was used by human cells and exerted similar homeostatic actions, as the regulation of the transcription of an SRE-driven gene construct. In contrast to cholesterol, ergosterol was unable to induce cyclin B1 expression, to activate Cdk1 and to resume cell cycle in cells previously arrested at G2. This lack of effect was not due to cytotoxicity, as cells exposed to ergosterol remained viable and, upon supplementing with UCN-01, an activator of Cdk1, they progressed through mitosis. However, in the presence of suboptimal concentrations of cholesterol, ergosterol exerted synergistic effects on cell proliferation. This is interpreted on the basis of the differential action of these sterols, ergosterol contributing to cell membrane formation and cholesterol being required for Cdk1 activation. In summary, the action of cholesterol on G2 traversal is highly specific and exerted through a mechanism different to that used for cholesterol homeostasis, reinforcing the concept that cholesterol is a specific regulator of cell cycle progression in human cells.