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Background and aims: Extensive research showed a diurnal rhythm of endogenous cholesterol synthesis, whereas recent research reported no diurnal rhythm of intestinal cholesterol absorption in males who consumed low-fat meals. Little is known about the acute effect of macronutrient consumption on cholesterol metabolism, and hence if meal composition may explain this absence of rhythmicity in cholesterol absorption. Therefore, we examined the effect of a high-fat, high-carbohydrate, and high-protein meal on postprandial intestinal cholesterol absorption and endogenous cholesterol synthesis in apparently healthy overweight and slightly obese males.Methods and results: Eighteen males consumed in random order an isoenergetic high-fat, high carbohydrate, and high-protein meal on three occasions. Serum total cholesterol concentrations, cholesterol absorption markers (campesterol, cholestanol, and sitosterol), and cholesterol synthesis intermediates (7-dehydrocholesterol, 7-dehydrodesmosterol, desmosterol, dihydrolanosterol, lanosterol, lathosterol, zymostenol, and zymosterol) were measured at baseline (T0) and 240 min postprandially (T240). Meal consumption did not significantly change total cholesterol concentrations and cholesterol absorption marker levels (all p > 0.05). Serum levels of 7dehydrocholesterol, lanosterol, lathosterol, zymostenol, and zymosterol decreased significantly between T0 and T240 (all p < 0.05). These decreases were not significantly different between the three meals (all p > 0.05), except for a larger decrease in dihydrolanosterol levels after the high-fat versus the high-carbohydrate meal (p Z 0.009).Conclusion: The high-fat, high-carbohydrate, and high-protein meal did not significantly influ-ence postprandial intestinal cholesterol absorption. Several cholesterol synthesis intermediates decreased postprandially, but the individual macronutrients did not differentially affect these intermediates, except for a possible effect on dihydrolanosterol.Trial registration: ClinicalTrials.gov, NCT03139890.(c) 2021 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Purpose Consumption of the algae spirulina (Arthrospira platensis or maxima) and wakame (Undaria pinnatifida) has been shown to lower LDL cholesterol concentrations in animals and humans, possibly due to the inhibition of intestinal cholesterol absorption. This mechanism, however, has never been investigated in humans. Therefore, we examined in non-hypercholesterolemic men and women the effects of spirulina and wakame consumption on serum markers for intestinal cholesterol absorption. Methods Thirty-five healthy men and women without hypercholesterolemia consumed in a random order daily 4.8 g spirulina, wakame or placebo for 17 days, separated by 14-day washouts. After 17 days, serum cholesterol-standardized campesterol, sitosterol and cholestanol, and lathosterol concentrations were measured as markers for intestinal cholesterol absorption and cholesterol synthesis, respectively. Concentrations of serum total cholesterol, LDL and HDL cholesterol, triacylglycerol, and plasma glucose, and blood pressure were measured as well. Results Compared with placebo, spirulina or wakame did not affect serum cholesterol-standardized campesterol (CI − 0.23 to 0.10 μmol/mmol, P = 0.435 and CI − 0.14 to 0.19 μmol/mmol, P = 0.729, respectively), sitosterol (P = 0.314 and P = 0.112), cholestanol (P = 0.610 and P = 0.809), or lathosterol (P = 0.388 and P = 0.102) concentrations. In addition, serum lipid and plasma glucose concentrations, and blood pressure were not changed. Conclusions Daily consumption of 4.8 g spirulina or wakame for 17 days did not affect plasma markers for intestinal cholesterol absorption or cholesterol synthesis in non-hypercholesterolemic men and women. Serum lipid and glucose concentrations, and blood pressure were also not altered. Electronic supplementary material The online version of this article (10.1007/s00394-019-02073-7) contains supplementary material, which is available to authorized users.

Background Apolipoprotein-I (ApoA-I), the major component of high-density lipoprotein (HDL) particles, mediates cholesterol efflux by which it facilitates the removal of excess cholesterol from peripheral tissues. Therefore, elevating ApoA-I production leading to the production of new pre-beta-HDL particles is thought to be beneficial in the prevention of cardiovascular diseases. Recently, we observed that amoxicillin treatment led to decreased HDL concentrations in healthy human volunteers. We questioned whether this antibiotic effect was directly or indirectly, via changed short-chain fatty acids (SCFA) concentrations through an altered gut microflora. Therefore, we here evaluated the effects of amoxicillin and various SCFA on hepatic ApoA-I expression, secretion, and the putative underlying pathways. Methods and Results Human hepatocytes (HepG2) were exposed to increasing dose of amoxicillin or SCFA for 48 hours. ApoA-I messenger RNA (mRNA) transcription and secreted protein were analyzed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To study underlying mechanisms, changes in mRNA expression of KEAP1, CPT1, and PPAR alpha, as well as a PPAR alpha transactivation assay, were analyzed. Amoxicillin dose-dependently decreased ApoA-I mRNA transcription as well as ApoA-I protein secretion. SCFA treatment resulted in a dose-dependent stimulation of ApoA-I mRNA transcription, however, the ApoA-I protein secretion was decreased. Furthermore, SCFA treatment increased PPAR alpha transactivation, PPAR alpha and CPT1 mRNA transcription, whereas KEAP1 mRNA transcription was decreased. Conclusion Direct treatment of HepG2 cells with amoxicillin has either direct effects on lowering ApoA-I transcription and secretion or indirect effects via modified SCFA concentrations because SCFA were found to stimulate hepatic ApoA-I expression. Furthermore, BET inhibition and PPAR alpha activation were identified as possible mechanisms behind the observed effects on ApoA-I transcription.

PURPOSE: Lowering of LDL cholesterol levels by plant sterols and stanols is associated with decreased risk of cardiovascular disease in humans. Plant sterols and stanols also lower triacylglycerol (TG). However, it is not fully understood how reduction in TG is achieved and what the full potential of plant sterols and stanols is on whole-body metabolism. We here hypothesize that high levels of plant sterols and stanols stimulate whole-body energy expenditure, which can be attributed to changes in mitochondrial function of brown adipose tissue (BAT), skeletal muscle and liver.METHODS: Phytosterolemic mice were fed chow diets for 32 weeks to examine whole-body weight gain. In vitro, 24-h incubation were performed in adipocytes derived from human BAT, human myotubes or HepG2 human hepatocytes using sitosterol or sitostanol. Following mitochondrial function was assessed using seahorse bioanalyzer.RESULTS: Chow feeding in phytosterolemic mice resulted in diminished increase in body weight compared to control mice. In vitro, sitosterol or sitostanol did not change mitochondrial function in adipocytes derived from human BAT or in cultured human myotubes. Interestingly, maximal mitochondrial function in HepG2 human hepatocytes was decreased following sitosterol or sitostanol incubation, however, only when mitochondrial function was assessed in low glucose-containing medium.CONCLUSIONS: Beneficial in vivo effects of plant sterols and stanols on lipid and lipoprotein metabolism are well recognized. Our results indicate that alterations in human mitochondrial function are apparently not involved to explain these beneficial effects.

Concentrations of apolipoprotein A-I (ApoA-I) decrease during inflammation, which may lead to dysfunctional ApoA-I-poor high-density lipoprotein (HDL) particles, and as such, elevate cardiovascular risk. Therefore, rescuing ApoA-I concentrations, especially during inflammation, seems beneficial. Recently, short-chain fatty acids (SCFAs) have received more attention as a strategy in reversing atherosclerosis. We here evaluated the effects of SCFAs on inflammatory pathways in relation to ApoA-I transcription. SCFAs dose&ndash, response studies were performed in the presence and absence of inflammatory cytokines. ApoA-I and interleukin 8 (IL-8) mRNA expression were analyzed using qPCR and ELISA, respectively. To study underlying mechanisms, nuclear factor kappa B (NF-&kappa, B) transactivation and changes in mRNA expressions of the genes targets of bromodomain and extra-terminal (BET) inhibition, peroxisome proliferator-activated receptor-alpha (PPAR&alpha, ) transactivation and activator protein 1 (AP-1) pathway were analyzed. SCFAs (except hexanoic acid) increased ApoA-I mRNA transcription in both normal and inflammatory conditions and lowered IL-8 mRNA expression. This anti-inflammatory effect of SCFAs was confirmed by inhibition of NF-&kappa, B transactivation. Moreover, butyric acid increased carnitine palmitoyltransferase 1 (CPT1), PPAR&alpha, target gene, mRNA transcription in both conditions, and there was a negative correlation between CPT1 and NF-&kappa, B. Therefore, PPAR&alpha, transactivation is probably involved in the anti-inflammatory effects of SCFAs, which rescues ApoA-I transcription. In conclusion, propionate, butyrate and valerate elicit anti-inflammatory effects which might rescue ApoA-I transcription in inflammatory conditions via PPAR&alpha, transactivation mediated NF-&kappa, B inhibition.

Although phytosterols, plant-derived sterol-like components, are well known for their cholesterol-lowering properties, their atherogenic potential is still under debate. Although they are known to share structural similarities with cholesterol, it is unclear whether their oxidized forms (oxyphytosterols) have the capacity to mediate proinflammatory responses in macrophages. In the present study, bone marrow-derived macrophages were treated with oxidized low-density lipoproteins, oxyphytosterols (7keto-sito/campesterol [7keto-sit/camp] or 7-beta-hydroxy-sito/campesterol [7OH-sit/camp]), nonoxidized phytosterol (-sitosterol), or carrier-control (cyclodextrin) in a dose- and time-dependent manner. Inflammatory cytokine release, activity, and the corresponding mRNA expression levels were analyzed. 7OH-sit/camp, rather than 7keto-sit/camp, induced a modest proinflammatory response in wild-type cells derived from C57Bl/6 mice. The observed mild inflammatory effects are independent of the low-density lipoprotein receptor and Cluster of differentiation 36/Scavenger receptor-a. These data suggest that exogenously added oxyphytosterols do not affect macrophage-mediated inflammatory responses, at least in vitro.

Background: Changes in the microbiota composition have been implicated in the development of obesity and type 2 diabetes. However, not much is known on the involvement of gut microbiota in lipid and cholesterol metabolism. In addition, the gut microbiota might also be a potential source of plasma oxyphytosterol and oxycholesterol concentrations (oxidation products of plant sterols and cholesterol). Therefore, the aim of this study was to modulate the gut microbiota by antibiotic therapy to investigate effects on parameters reflecting cholesterol metabolism and oxyphytosterol concentrations.Design: A randomized, double blind, placebo-controlled trial was performed in which 55 obese, prediabetic men received oral amoxicillin (broad-spectrum antibiotic), vancomycin (antibiotic directed against Gram-positive bacteria) or placebo (microcrystalline cellulose) capsules for 7 days (1500 mg/day). Plasma lipid and lipoprotein, non-cholesterol sterol, bile acid and oxy(phyto)sterol concentrations were determined at baseline and after 1-week intervention.Results: Plasma secondary bile acids correlated negatively with cholestanol (marker for cholesterol absorption, r = -0.367; P Conclusion: Despite strong correlations between plasma bile acid concentrations and cholesterol metabolism (synthesis and absorption), amoxicillin and vancomycin treatment for 7 days did not affect plasma lipid and lipoprotein, plasma non-cholesterol sterol and oxy(phyto)sterol concentrations in obese, pre-diabetic men. (C) 2017 Elsevier B.V. All rights reserved.

In a recent human study, we observed that amoxicillin treatment decreased HDL-C concentration. We hypothesize that antibiotics lower the transcription and secretion of ApoA-I, the responsible protein for HDL production. HepG2 and Caco-2 cells were exposed to increasing dose of amoxicillin, penicillin, and streptomycin. Secreted ApoA-I protein and mRNA transcripts were analyzed using ELISA and qPCR, respectively. To unravel underlying mechanisms, KEAP1, CPT1, and CHOP mRNA expressions were determined as well as PPAR&alpha, transactivation. In HepG2 and Caco-2, amoxicillin decreased ApoA-I transcription and secretion. Effects on ApoA-I expression were clearly there for amoxicillin while no effects were observed for penicillin or streptomycin. KEAP1, CPT1, and CHOP mRNA expressions were reduced by amoxicillin treatments. Moreover, a significant correlation between ApoA-I and CPT1 mRNA expressions was found. Furthermore, amoxicillin lowered PPAR&alpha, transactivation. All together, these data suggest that inhibited PPAR&alpha, transactivation is involved in the effects of amoxicillin on ApoA-I. In conclusion, the direct effect of amoxicillin in treated HepG2 and Caco-2 cells was a lower ApoA-I secretion and transcription. Based on evaluating alterations in KEAP1, CPT1, and CHOP mRNA expressions plus PPAR&alpha, transactivation, we suggest that a reduced PPAR&alpha, activation is a potential mechanism behind the observed amoxicillin effects on ApoA-I expression.

Increasing apolipoproteinA-I (apoA-I) production may be anti-atherogenic. Thus, there is a need to identify regulatory factors involved. Transcription of apoA-I involves peroxisome-proliferator-activated-receptor-alpha (PPARα) activation, but endoplasmic reticulum (ER) -stress and inflammation also influence apoA-I production. To unravel why PPARα agonist GW7647 increased apoA-I production compared to PPARα agonist fenofibric acid (FeAc) in human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (CaCo-2) cells, gene expression profiles were compared. Microarray analyses suggested CCAAT/enhancer-binding-protein-beta (C/EBP-β) involvement in the FeAc condition. Therefore, C/EBP-β silencing and isoform-specific overexpression experiments were performed under ER-stressed, inflammatory and non-inflammatory conditions. mRNA expression of C/EBP-β, ATF3, NF-IL3 and GDF15 were upregulated by FeAc compared to GW7647 in both cell lines, while DDIT3 and DDIT4 mRNA were only upregulated in HepG2 cells. This ER-stress related signature was associated with decreased apoA-I secretion. After ER-stress induction by thapsigargin or FeAc addition, intracellular apoA-I concentrations decreased, while ER-stress marker expression (CHOP, XBP1s, C/EBP-β) increased. Cytokine addition increased intracellular C/EBP-β levels and lowered apoA-I concentrations. Although a C/EBP binding place is present in the apoA-I promoter, C/EBP-β silencing or isoform-specific overexpression did not affect apoA-I production in inflammatory, non-inflammatory and ER-stressed conditions. Therefore, C/EBP-β is not a target to influence hepatic apoA-I production. J. Cell. Biochem. 118: 754-763, 2017. © 2016 Wiley Periodicals, Inc.

Background: Non-cholesterol sterols are validated markers for fractional intestinal cholesterol absorption (cholestanol) and endogenous cholesterol synthesis (lathosterol). This study’s objective was to evaluate markers for cholesterol synthesis and absorption in children exposed to two different intravenous lipid emulsions that rapidly change serum plant sterol concentrations as part of their parenteral nutrition (PN). Methods: Serum samples from two different studies were used: (1) nine PN-dependent children with intestinal failure associated liver disease (IFALD) whose soy-based, plant sterol-rich lipid (SO) was replaced with a fish-based, plant sterol-poor (FO) lipid, and (2) five neonates prescribed SO after birth. In the first study, samples were collected at baseline (prior to FO initiation) and after 3 and 6 months of FO. In study 2, samples were collected at 1 and 3 weeks of age. Results: In study 1, a 7-fold reduction in campesterol, a 12-fold reduction in sitosterol, and a 15-fold reduction in stigmasterol was observed 6 months after switching to FO. Serum cholesterol concentrations did not change, but cholesterol-standardized lathosterol increased (3-fold) and cholesterol-standardized cholestanol decreased (2-fold). In study 2, after 3 weeks of SO, sitosterol and campesterol concentrations increased 4-5 fold. At the same time, cholesterol-standardized lathosterol increased 69% and cholesterol-standardized cholestanol decreased by 29%. Conclusion: Based on these finding we conclude that changes in serum plant sterol concentrations might have direct effects on endogenous cholesterol synthesis, although this needs to be confirmed in future studies. Moreover, we speculate that this changed synthesis subsequently affects intestinal cholesterol absorption.

Dietary plant sterols, such as campesterol and sitosterol, reduce plasma cholesterol concentrations, but any relationship to plaque development and CVD remains unclear. Some epidemiologic studies have suggested that elevated plasma plant sterol concentrations are atherogenic, including the Framingham Offspring Study that identified a positive association between plant sterol concentrations and CVD status. We hypothesized that this suggested atherogenicity relates to the oxidation status of plant sterols (i.e., concentrations of plasma oxyphytosterols). Therefore, in the Framingham Offspring Study cohort, we measured plasma oxyphytosterol concentrations in 144 patients with documented CVD and/or more than 50% carotid stenosis and 383 matched controls. We analyzed plasma oxyphytosterol concentrations by GC/MS/MS and performed conditional logistic regression analysis to determine associations between plasma plant sterol or oxyphytosterol concentrations and CVD status. We found that higher total cholesterol (TC)-standardized campesterol concentrations [odds ratio (OR): 2.36; 95% CI: 1.60, 3.50] and higher sitosterol concentrations (OR: 1.47; 95% CI: 1.09, 1.97) were significantly associated with increased CVD risk, as in the earlier study. However, the sum of absolute oxyphytosterol concentrations (OR: 0.99; 95% CI: 0.81, 1.21) and the sum of TC-standardized oxyphytosterol concentrations (OR: 0.98; 95% CI: 0.80, 1.19) were not associated with an increased CVD risk. Results were comparable for individual absolute and TC-standardized oxycampesterol and oxysitosterol concentrations. Plasma nonoxidized TC-standardized sitosterol and campesterol concentrations showed weak or no correlations with oxyphytosterol concentrations, while all individual plasma concentrations of oxyphytosterol correlated with each other. In conclusion, circulating plasma oxyphytosterols are not associated with CVD risk in the Framingham Offspring Study.

The kinetics of plant stanol uptake and routing in 8-week-old C57BL/6J mice were determined after a plant stanol ester gavage. In addition, acute changes in intestinal and hepatic gene expression were investigated. Mice were fed a plant sterol/stanol poor diet from weaning. At the age of 8 weeks, they received an oral gavage consisting of 0.25 mg cholesterol + 50 mg plant stanol esters dissolved in olive oil. Animals were euthanized at different time points. In a second comparable set-up, mesenteric lymph-cannulated versus sham-operated mice received the same oral gavage, which was now deuterium labeled. Intestinal and hepatic sitostanol concentrations increased within 15 min post-gavage. This rapid hepatic appearance was absent in lymph-cannulated mice, suggesting a very fast lymph-mediated uptake. Hepatic mRNA expression of SREBP2 and its target genes rapidly decreased, whereas expression of LXR target genes increased. The intestinal SREBP2 pathway was increased, whereas the expression of LXR target genes hardly changed. The fivefold and sixfold increased expression of intestinal LDLr and PCSK9 is suggestive of TICE activation. We conclude that in C57BL/6J mice plant stanol kinetics are fast, and affect intestinal and hepatic gene expression within 15 min postprandial after lymph-mediated uptake. Electronic supplementary material The online version of this article (doi:10.1007/s11745-015-4020-1) contains supplementary material, which is available to authorized users.

PLoS one 9(10), e110758 (2014). doi:10.1371/journal.pone.0110758, Published by PLoS, Lawrence, Kan.