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Our previous study showed that lycopene reduced the absorption of cholesterol in Caco-2 cells through inhibiting Niemann-Pick C1-Like 1 (NPC1L1) expression. Herein, we aimed to explore whether lycopene supplementation can decrease cholesterol absorption in the intestine and prevent atherosclerosis progression in high-fat diet (HFD)-fed apolipoprotein E knockout (ApoE-/-) mice. Male ApoE-/- mice were fed a high-fat diet with or without lycopene for 19 weeks. Supplementation of lycopene markedly lowered serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. Additionally, serum high-density lipoprotein cholesterol (HDL-C) levels were increased after lycopene administration. Lycopene also downregulated the expression of NPC1L1 and hepatocyte nuclear factor-1α (HNF-1α) in the small intestine. Furthermore, the Oil Red O staining of the aorta and aortic sinus showed that lycopene supplementation remarkably reduced atherosclerotic lesions. These results indicated that lycopene inhibited intestinal cholesterol absorption and protected against HFD-induced atherosclerosis through inhibiting HNF-1α and NPC1L1 expression. Lycopene exhibits a potential antiatherosclerotic effect through suppressing intestinal cholesterol absorption.

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/).

Cholesterol gallstone disease is a common global condition. This study investigated the role of plant sterols (PS) in the prevention of gallstone formation and the underlying mechanisms. Adult male mice were fed a lithogenic diet (LD) alone or supplemented with PS (LD-ps), phospholipids (LD-pl) or both PS and phospholipids (LD-ps/pl) for 8 weeks. Incidences of gallstone formation were compared among the groups. Lipids in the bile, liver and serum were analyzed. The expression of genes involved in cholesterol absorption, transport and metabolism in the liver and small intestine was determined. The incidences of gallstone formation were 100% (10/10), 20% (2/10), 100% (10/10) and 40% (4/10) in the LD, LD-ps, LD-pl and LD-ps/pl groups, respectively. Serum cholesterol and intestinal cholesterol absorption were decreased in PS-supplemented mice. The expression of genes related to cholesterol transport and metabolism in the liver was down-regulated by dietary PS. PS supplementation decreased Niemann-Pick C1-like 1 expression in the small intestine and reduced intestinal cholesterol absorption. Our results demonstrated that PS could inhibit intestinal cholesterol absorption and thus prevent cholesterol gallstone formation.

Plasma concentrations of phytosterols and non-cholesterol sterol precursors of cholesterol synthesis have been used as markers of intestinal cholesterol absorption and synthesis in inherited and secondary dyslipidemias and in population-based investigations to evaluate the risk for cardiovascular disease, respectively. The method aims at replacing initial research procedures such as the use of stable isotopes associated with fecal steroid balance, which are limited by the high cost and tedious procedures. However, we show in this review that numerous results obtained with serum sterol measurements are contradictory. In this regard, the following points are discussed: 1) how phytosterols relate to atherosclerosis considering that defects in biliary output or in the transport of phytosterols from the intestinal mucosa back into the intestinal lumen provide increased content of phytosterols and other sterols in plasma and tissues, thus not allowing to conclude that their presence in arteries and atheromas represents the etiology of atherosclerosis; 2) serum non-cholesterol sterols as markers of cholesterol synthesis and absorption, such as cholestanol, present discrepant results, rendering them often inadequate to identify cases of coronary artery disease as well as alterations in the whole body cholesterol metabolism; 3) such methods of measurement of cholesterol metabolism are confounded by factors like diabetes mellitus, body weight and other pathologies including considerable hereditary hyperlipidemias biological variabilities that influence the efficiency of synthesis and intestinal absorption of cholesterol.

Background and aims Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme catalyzing the final step of triglyceride synthesis by esterifying a diglyceride with a fatty acid. We have previously shown that apolipoprotein E-knockout (ApoE−/−) mice lacking Dgat1 have reduced intestinal cholesterol absorption and potentiated macrophage cholesterol efflux, and consequently, exhibit attenuated atherogenesis. However, hematopoietic Dgat1 deficiency lacked beneficial effects on atherosclerosis. Due to our recent results on the critical role of intestinal Dgat1 in murine cholesterol homeostasis, we delineated whether intestinal Dgat1 deficiency regulates atherogenesis in mice. Methods We generated intestine-specific Dgat1−/− mice on the ApoE−/− background (iDgat1−/−ApoE−/−) and determined cholesterol homeostasis and atherosclerosis development. Results When fed a Western-type diet, iDgat1−/−ApoE−/− mice exhibited a substantial decrease in fasting plasma cholesterol content in ApoB-containing lipoproteins. Although lipid absorption was delayed, iDgat1−/−ApoE−/− mice had reduced acute and fractional cholesterol absorption coupled with an elevated fecal caloric loss. In line, increased appearance of i.v. administered [³H]cholesterol in duodena and stool of iDgat1−/−ApoE−/− animals suggested potentiated cholesterol elimination. Atherosclerotic lesions were markedly smaller with beneficial alterations in plaque composition as evidenced by reduced macrophage infiltration and necrotic core size despite unaltered collagen content, indicating improved plaque stability. Conclusions Disruption of Dgat1 activity solely in the small intestine of ApoE−/− mice strongly decreased plasma cholesterol levels by abrogating the assimilation of dietary cholesterol, partly by reduced absorption and increased excretion. Consequently, the reduced cholesterol burden significantly attenuated atherogenesis and improved the lesion phenotype in iDgat1−/−ApoE−/− mice.

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.

Reverse cholesterol transport (RCT) and transintestinal cholesterol efflux (TICE) are two important pathways for body cholesterol elimination. We studied these pathways in an animal model of diabetes and obesity (ob/ob) where HDL function is compromised as a result of hyperglycemia, low-grade inflammation and oxidative stress. Co-treatment of ob/ob mice with PPAR-α (fenofibrate) and LXR (T0901317) agonists increased fecal cholesterol by 12-fold; PPAR-α and LXR agonists individually showed 2.6- and 4.0-fold fecal cholesterol excretion, respectively. We investigated the mechanism of synergistic efficacy of PPAR-α and LXR agonists in fecal cholesterol excretion. LXR agonist and the combination of PPAR-α and LXR agonists had greater HDL-C elevation. Ex vivo cholesterol efflux showed correlation with the fecal cholesterol excretion but was not sufficient to explain 12-fold increases in the fecal cholesterol in the co-treated mice. Therefore, we examined TICE to explain the 12-fold increases in the fecal cholesterol. A strong positive correlation of fecal cholesterol with ATP binding cassette transporter G5 (ABCG5) and G8 and anegative correlation with NPC1L1 was observed. ABCG5, G8 and NPC1L1 are involved in intestinal cholesterol absorption. The extent of influence of PPAR-α and LXR agonists on RCT and TICE was distinctly different. PPAR-α agonist increased fecal cholesterol primarily by influencing TICE, while LXR agonist influenced fecal cholesterol excretion via both RCT and TICE mechanisms. Synergistic efficacy on fecal cholesterol excretion following co-treatment with PPAR-α and LXR agonists occurred through a combination of RCT, TICE, and the key enzyme in bile synthesis, cholesterol 7-α hydroxylase (cyp7a1). These results suggest that cholesterol efflux, biliary cholesterol excretion, and TICE collectively contributed to the 12-fold increases in the fecal cholesterol excretion in ob/ob mice co-treated with PPAR-α and LXR agonists.

Background: Patients with metabolic syndrome (MetS) have increased endogenous synthesis of cholesterol, together with lower level of intestinal cholesterol absorption. However, less is known about ...

Aims Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. Methods and results Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E−/− (Apoe −/−) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe −/− mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [−15.9 mg/dL (−8.1%) vs. −1.6 mg/dL (−0.5%), P = 0.016], total [−19.6 mg/dL (−7.3%) vs. −5.3 mg/dL (−1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: −18.9 mg/dL (−9.1%) vs. −0.6 mg/dL (−0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. Conclusion Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.

Phytosterol serum concentrations are under tight genetic control. The relationship between phytosterols and coronary artery disease (CAD) is controversially discussed. We perform a genome-wide meta-analysis of 32 phytosterol traits reflecting resorption, cholesterol synthesis and esterification in six studies with up to 9758 subjects and detect ten independent genome-wide significant SNPs at seven genomic loci. We confirm previously established associations at ABCG5/8 and ABO and demonstrate an extended locus heterogeneity at ABCG5/8 with different functional mechanisms. New loci comprise HMGCR, NPC1L1, PNLIPRP2, SCARB1 and APOE. Based on these results, we perform Mendelian Randomization analyses (MR) revealing a risk-increasing causal relationship of sitosterol serum concentrations and CAD, which is partly mediated by cholesterol. Here we report that phytosterols are polygenic traits. MR add evidence of both, direct and indirect causal effects of sitosterol on CAD., Physterols are cholesterol homologs derived from plants, which are found in humans through consumption of plant products. Here the authors have performed a genome-wide meta-analysis of 32 serum phytosterol traits, with evidence suggesting causality between sitosterol and coronary artery disease.

Dietary phytosterols, which comprise plant sterols and stanols, reduce plasma Low-Density Lipoprotein-Cholesterol (LDL-C) levels when given 2 g/day. Since this dose has not been reported to cause health-related side effects in long-term human studies, food products containing these plant compounds are used as potential therapeutic dietary options to reduce LDL-C and cardiovascular disease risk. Several mechanisms have been proposed to explain the cholesterol-lowering action of phytosterols. They may compete with dietary and biliary cholesterol for micellar solubilization in the intestinal lumen, impairing intestinal cholesterol absorption. Recent evidence indicates that phytosterols may also regulate other pathways. Impaired intestinal cholesterol absorption is usually associated with reduced cholesterol transport to the liver, which may reduce the incorporation of cholesterol into Very-Low- Density Lipoprotein (VLDL) particles, thereby lowering the rate of VLDL assembly and secretion. Impaired liver VLDL production may reduce the rate of LDL production. On the other hand, significant evidence supports a role for plant sterols in the Transintestinal Cholesterol Excretion (TICE) pathway, although the exact mechanisms by which they promote the flow of cholesterol from the blood to enterocytes and the intestinal lumen remains unknown. Dietary phytosterols may also alter the conversion of bile acids into secondary bile acids, and may lower the bile acid hydrophobic/hydrophilic ratio, thereby reducing intestinal cholesterol absorption. This article reviews the progress to date in research on the molecular mechanisms underlying the cholesterol-lowering effects of phytosterols.

Transintestinal cholesterol excretion (TICE) is a major route for eliminating cholesterol from the body and a potential therapeutic target for hypercholesterolemia. The underlying mechanism, however, is largely unclear, and its contribution to cholesterol disposal from the body is obscured by the counteracting process of intestinal cholesterol reabsorption. To determine the quantity of TICE independent from its reabsorption, we studied two models of decreased intestinal cholesterol absorption. Cholesterol absorption was inhibited either by ezetimibe or, indirectly, by the genetic inactivation of the intestinal apical sodium-dependent bile acid transporter (ASBT; SLC10A2). Both ezetimibe treatment and Asbt inactivation virtually abrogated fractional cholesterol absorption (from 46% to 4% and 6%, respectively). In both models, fecal neutral sterol excretion and net intestinal cholesterol balance were considerably higher than in control mice (5- and 7-fold, respectively), suggesting that, under physiological conditions, TICE is largely reabsorbed. In addition, the net intestinal cholesterol balance was increased to a similar extent but was not further increased when the models were combined, suggesting that the effect on cholesterol reabsorption was already maximal under either condition alone. On the basis of these findings, we hypothesize that the inhibition of cholesterol (re)absorption combined with stimulating TICE will be most effective in increasing cholesterol disposal.

Hypercholesterolemia is a preventable risk factor for atherosclerosis and cardiovascular disease. However, the mechanisms of diosgenin (DG) that promote cholesterol homeostasis and alleviate hypercholesterolemia remain elusive. To investigate the effects and molecular mechanisms of the promotion of cholesterol metabolism by DG, a rat model of hypercholesterolemia was induced by providing a high-fat diet for 4 weeks. After 4 weeks, the rats were intragastrically administered high-dose DG (0.3 g/kg/d), low-dose DG (0.15 g/kg/d) or simvastatin (4 mg/kg/d) once a day for 8 weeks. The serum and hepatic cholesterol were tested, the mRNA and protein expression levels of Niemann-Pick C1-Like 1 (NPC1L1), liver X receptor-α (LXR-α) and the ATP-binding cassette G5/G8 (ABCG5/G8) transporters were measured. The results indicate that DG could reduce body weight, decrease the serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, liver total cholesterol and free cholesterol levels compared to those in the controls. Simultaneously, liver tissue pathological morphology analyses revealed that DG could attenuate hepatic steatosis compared to that in the high-fat diet group. Further investigation demonstrated that DG significantly decreased the expression of NPC1L1 and LXR-α in the intestine and markedly increased the expression of ABCG5/G8 in the liver and intestine. Compared to the high-fat diet group, the rats in the DG-treated groups ameliorated hypercholesterolemia in a dose- and time-dependent manner. These data suggest that DG may not only inhibit intestinal cholesterol absorption by downregulating NPC1L1 but also enhance cholesterol excretion by increasing the expression of ABCG5/G8. DG could be a new candidate for the prevention of hypercholesterolemia.

BACKGROUND/OBJECTIVES Consumption of cholesterol-rich foods, such as eggs, has a minimal effect on circulating cholesterol levels in healthy humans. To gain insight, we investigated whether phospholipids rich in eggs (EPL) interfere with intestinal cholesterol absorption in vivo. MATERIALS/METHODS To investigate the acute effect of EPL on intestinal cholesterol absorption, male C57BL/6J mice were orally administered with 6, 11, or 19 mg of EPL for three days. We also tested the effect of chronic EPL consumption on cholesterol metabolism in the small intestine and the liver in mice with diet-induced hypercholesterolemia. Male C57BL/6J mice were fed a high fat/high cholesterol (HF/HC; 35% fat, 0.25% cholesterol, w/w) diet for 4 weeks to induce hypercholesterolemia, and subsequently the mice were either fed 0, 0.4 or 0.8% (w/w) of EPL for 6 weeks. RESULTS Intestinal cholesterol absorption was significantly decreased by the highest dose of acute EPL administration compared to control. Chronic EPL supplementation did not significantly alter intestinal cholesterol absorption nor plasma levels of total cholesterol and low-density lipoprotein cholesterol. In the small intestine and the liver, EPL supplementation minimally altered the expression of genes which regulate cellular cholesterol levels. CONCLUSION Although chronic EPL consumption was not able to counteract hypercholesterolemia in HF/HC-fed mice, acute EPL administration decreased intestinal cholesterol absorption. This study provides in vivo evidence that acute administration of PLs in eggs prevent cholesterol absorption in the intestine, suggesting a mechanism for a minimal effect of egg consumption on circulating cholesterol levels.

Cardiovascular diseases caused by atherosclerosis have become one of the leading cause of death. The aim of the present study was to explore how oat fiber regulated lipid metabolism and to elucidate its anti-atherosclerotic potential in ApoE−/− mice. Animals were daily fed a high-fat diet without or with 0.8% oat fiber for 18 weeks. Both serum total cholesterol and low-density cholesterol levels decreased in mice received oat fiber administration than those without Oat fiber inhibited hepatic lipid accumulation by downregulating SREBP-1 expression and accelerated clearance of intestinal cholesterol by upregulating SREBP-2 expression. In addition, oat fiber activated cholesterol sensors LXRα, and then strengthened hepatic and intestinal cholesterol efflux by increasing ATP-binding cassette A1 and G1, and decreased intestinal cholesterol absorption by inhibiting Niemann pick C 1 like 1. The findings implicated that oat fiber had an anti-atherosclerotic potential via mechanisms of activating the SREBPs/LXRα pathway and improving lipid metabolism.

Single nucleotide polymorphisms (SNPs) have been associated with cholesterol metabolism and may partly explain large inter-individual variability in intestinal cholesterol absorption and endogenous cholesterol synthesis rates. This cross-sectional study therefore examined whether SNPs in genes encoding for proteins involved in intestinal cholesterol absorption (ABCG5, ABCG8, and NPC1L1) and endogenous cholesterol synthesis (CYP51A1, DHCR7, DHCR24, HMGCR, HSD17B7, LBR, and MSMO1) were associated with intestinal cholesterol absorption markers (total cholesterol (TC) standardized campesterol and sitosterol levels), an endogenous cholesterol synthesis marker (TC-standardized lathosterol levels), and serum low-density lipoprotein cholesterol (LDL-C) concentrations in a European cohort. ABCG5 (rs4245786) and the tag SNP ABCG8 (rs4245791) were significantly associated with serum campesterol and/or sitosterol levels. In contrast, NPC1L1 (rs217429 and rs217416) were significantly associated with serum lathosterol levels. The tag SNP in HMGCR (rs12916) and a SNP in LBR (rs12141732) were significantly associated with serum LDL-C concentrations. SNPs in the cholesterol absorption genes were not associated with serum LDL-C concentrations. SNPs in CYP51A1, DHCR24, HSD17B7, and MSMO1 were not associated with the serum non-cholesterol sterols and LDL-C concentrations. Given the variable efficiency of cholesterol-lowering interventions, the identification of SNPs associated with cholesterol metabolism could be a step forward towards personalized approaches.

Background：Flaxseed peptide (FPs) showed serum cholesterol-lowering activity in SD rats fed a high-cholesterol diet, but the cholesterol-lowering amino acid sequences and mechanism of FPs were still unclear. Methods: FPs were separated via ultrafiltration, and the amino acid sequences of the selected fractions were determined via high-performance liquid chromatography- Electrospray Ionisation - Orbitrap- Mass spectrometry (HPLC-ESI-Orbitrap MS). These peptides then were synthesized by solid-phase synthesis (SPPS). IPPF with the highest CMSR was determined to exist in flaxseed protein by specific antibodies. The effects of IPPF on intestinal cholesterol absorption and hepatic cholesterol metabolism were investigated in Caco-2 cells and HepG2 cells.Results：1 kDa FP5 fraction had the highest cholesterol micelle solubility inhibition rate (CMSR) 72.39% compared with the other ultrafiltration fractions. Then Eleven peptides were identified from FP5. Ile-Pro-Pro-Phe (IPPF), with the highest CMSR 93.47%, was selected to research the cholesterol-lowering mechanism in Caco-2 and HepG2 cells. IPPF significantly reduces the amount of cholesterol transported in Caco2 cells and the amount of total cholesterol in HepG2 cells. IPPF significantly modulated the protein levels of NCP1L1 and ABCG5/8 in Caco2 cells and significantly reduced the mRNA levels of Srebp-2 and Hmgcr in HepG2 cells. Conclusion: IPPF inhibits cholesterol intestinal absorption through modulating the expression of cholesterol transporters in Caco-2 cells and reduces hepatic cholesterol synthesis through inhibiting the SREBP2-regulated mevalonate (HMGCR) pathway in HepG2 cells. IPPF is a new food-derived inhibitor of intestinal cholesterol absorption and hepatic cholesterol synthesis without side effects and provides a nutritional therapy component for hypercholesterolemia.

The prevalence of metabolic disorders characterized by chronic inflammation has been on a sharp rise for decades. As such, tools that address metabolic and inflammatory dysregulation are of great importance. Plant stanols are well-known for reducing intestinal cholesterol absorption and may also have direct anti-inflammatory effects. In this study, our aim was to investigate to what extent the benefits of dietary plant stanol supplementation depend on dietary cholesterol intake in an experimental mouse model for cholesterol-induced metabolic inflammation. Here, we used Ldlr−/− mice transplanted with Npc1nih-derived bone marrow, featuring feature bone marrow-derived immune cells characterized by chronic inflammation induced by lysosomal lipid accumulation. Npc1nih- and Npc1wt-transplanted mice were placed on either a high fat, high cholesterol (HFC) or on a chow diet low in cholesterol, with or without 2% plant stanols supplementation. At the end of the study, the metabolic and inflammatory status of the mice was analyzed. Plant stanol supplementation to the HFC diet reduced liver cholesterol levels and improved lipid metabolism and liver inflammation, particularly in Npc1nih-tp mice. In contrast, plant stanol supplementation to the chow diet did not significantly improve the aforementioned parameters, though similar reductive trends to those in the HFC diet setting were observed regarding liver cholesterol accumulation and liver inflammatory markers. The effects of dietary plant stanol supplementation on dietary cholesterol-induced inflammation are largely dependent on dietary cholesterol intake. Future research should verify whether other models of metabolic inflammation exhibit similar stanol-related effects on inflammation.

Abnormal lipid metabolism leads to the development of hyperlipidemia, a common cause of multiple chronic disorders, including cardiovascular disease (CVD), obesity, diabetes, and cerebrovascular disease. Low-density lipoprotein cholesterol (LDL-C) currently remains the primary target for treatment of hyperlipidemia. Despite the advancement of treatment and prevention of hyperlipidemia, medications used to manage hyperlipidemia are limited to allopathic drugs, which present certain limitations and adverse effects. Increasing evidence indicates that utilization of phytochemicals and plant-based whole foods is an alternative and promising strategy to prevent hyperlipidemia and CVD. The current review focuses on phytochemicals and their pharmacological mode of actions for the regulation of LDL-C and prevention of CVD. The important molecular mechanisms illustrated in detail in this review include elevation of reverse cholesterol transport, inhibition of intestinal cholesterol absorption, acceleration of cholesterol excretion in the liver, and reduction of cholesterol synthesis. Moreover, the beneficial effects of plant-based whole foods, such as fresh fruits, vegetables, dried nuts, flax seeds, whole grains, peas, beans, vegan diets, and dietary fibers in LDL-C reduction and cardiovascular health are summarized. This review concludes that phytochemicals and plant-based whole foods can reduce LDL-C levels and lower the risk for CVD.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

Hypercholesterolemia is a prevalent metabolic disorder that has been implicated in the development of steroid-targeted cancers. However, the link between hypercholesterolemia and urinary bladder cancer (UBC), a non–steroid-targeted cancer, remains unresolved. Here we show that diet-induced and Ldlr deficiency–induced hypercholesterolemia enhances both UBC stemness and progression. Inhibition of intestinal cholesterol absorption by ezetimibe reversed diet-induced hypercholesterolemia and cancer stemness. As a key component in hypercholesterolemic sera, oxidized low-density lipoprotein (ox-LDL), but not native low-density lipoprotein-cholesterol or metabolite 27-hydroxycholesterol, increased cancer stemness through its receptor CD36. Depletion of CD36, ectopic expression of an ox-LDL binding–disabled mutant form of CD36(K164A), and the neutralization of ox-LDL and CD36 via neutralizing antibodies all reversed ox-LDL–induced cancer stemness. Mechanistically, ox-LDL enhanced the interaction of CD36 and JAK2, inducing phosphorylation of JAK2 and subsequently activating STAT3 signaling, which was not mediated by JAK1 or Src in UBC cells. Finally, ox-LDL levels in serum predicted poor prognosis, and the ox-LDLhigh signature predicted worse survival in patients with UBC. These findings indicate that ox-LDL links hypercholesterolemia with UBC progression by enhancing cancer stemness. Lowering serum ox-LDL or targeting the CD36/JAK2/STAT3 axis might serve as a potential therapeutic strategy for UBCs with hypercholesterolemia. Moreover, elevated ox-LDL may serve as a biomarker for UBC. Significance: This study demonstrates how hypercholesterolemia-induced oxidized LDL promotes urinary bladder cancer stemness via a CD36/STAT3 signaling axis, highlighting these factors as biomarkers and potential therapeutic targets of aggressive disease.

Background: Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations.Methods: To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE.Results: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels.Conclusions: These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia.Impact: Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

Background and aim: Phytosterols (PS) are recommended by European Guidelines for the treatment of hypercholesterolemia. This study aims to evaluate the lipid-lowering activity of a PS supplement (STEROLIP® ESI) in subjects with hypercholesterolemia and the influence of genetic variants involved in cholesterol absorption in the modulation of the individual response to PS. Methods: 60 subjects suffering from hypercholesterolemia were randomized to PS supplement STEROLIP® 1.6g/day or to placebo for 6 weeks, and subsequently they were all switched to the supplementation with PS for another 6 weeks. At baseline and every 3 weeks, anthropometric measures and lipid profile were collected. All patients were also genotyped for three genetic polymorphisms involved in intestinal cholesterol absorption (APOE, NPC1L1 and ABCG8). Results: After 3 weeks, TC and LDL-C were significantly lower in PS group. In the 28 patients treated for 12 weeks with PS, we obtained the highest effect after 9 weeks (-8.3% for TC and -12% for LDL-C). We observed a greater reduction of TC and LDL-C in subjects carrying the E4 allele of APOE gene, in homozygous subjects for the rare allele of NPC1L1 rs2072183 polymorphism and in heterozygous carriers of rare allele rs11887534 in ABCG8 gene than in other genotypes. Conclusion: This study confirms the lipid-lowering effect of PS that is maintained even in the midterm. We highlighted that genetic variants influencing the intestinal cholesterol absorption can contribute to the inter-individual variability of the response of PS supplementation.

Structures of NPC1L1 reveal how NPC1L1 senses the cholesterol level by conformational changes of the sterol-sensing domain., Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo–electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.

Hypercholesterolemia is a preventable risk factor for atherosclerosis and cardiovascular disease. However, the mechanisms wherebycis-palmitoleic acid (cPOA) andtrans-palmitoleic acid (tPOA) promote cholesterol homeostasis and ameliorate hypercholesterolemia remain elusive. To investigate the effects ofcPOA andtPOA on cholesterol metabolism and its mechanisms, we induced hypercholesterolemia in mice using a high-fat diet and then intragastrically administeredcPOA ortPOA once daily for 4 weeks.tPOA administration reduced serum cholesterol, low-density lipoprotein, high-density lipoprotein, and hepatic free cholesterol and total bile acids (TBAs). Conversely,cPOA had no effect on these parameters except for TBAs. Histological examination of the liver, however, revealed thatcPOA ameliorated hepatic steatosis more effectively thantPOA.tPOA significantly reduced the expression of 3-hydroxy-3-methyl glutaryl coenzyme reductase (HMGCR), LXRα, and intestinal Niemann-Pick C1-Like 1 (NPC1L1) and increased cholesterol 7-alpha hydroxylase (CYP7A1) in the liver, whereascPOA reduced the expression of HMGCR and CYP7A1 in the liver and had no effect on intestinal NPC1L1. In summary, our results suggest thatcPOA andtPOA reduce cholesterol synthesis by decreasing HMGCR levels. Furthermore,tPOA, but notcPOA, inhibited intestinal cholesterol absorption by downregulating NPC1L1. Both high-dosetPOA andcPOA may promote the conversion of cholesterol into bile acids by upregulating CYP7A1.tPOA andcPOA prevent hypercholesterolemia via distinct mechanisms.

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel pentapeptide from bovine milk β-lactoglobulin which lowers cholesterol levels. However, the molecular mechanisms underlying the suppression of intestinal cholesterol absorption by IIAEK are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in Caco-2 cells in a human intestinal model. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1) protein. Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered IIAEK common binding amino acid sequence, GFYLFVEGGR. Transfection of IAP siRNA counteracted the decrease in ABCA1 mRNA levels in Caco-2 cells. IIAEK significantly increased IAP mRNA and protein levels, and significantly decreased ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway with a specific activation of IAP and down-regulation of intestinal ABCA1.

It is controversial whether atherosclerosis is linked to increased intestinal cholesterol absorption or synthesis in humans. The aim of the present study was to relate atherosclerosis to the measurements of plasma markers of cholesterol synthesis (desmosterol, lathosterol) and absorption (campesterol, sitosterol). In healthy male (n=344), non-obese, non-diabetics, belonging to the city of São Paulo branch of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), we measured in plasma these non-cholesterol sterol markers, together with their anthropometric, dietary parameters, traditional atherosclerotic risk factors, and blood chemistry, coronary arterial calcium score (CAC), and ultrasonographically measured common carotid artery intima-media thickness (CCA-IMT). Cases with CAC>zero had the following parameters higher than cases with CAC = zero: age, waist circumference (WC), plasma total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and non-high density lipoprotein-cholesterol (non HDL-C). Plasma desmosterol and campesterol, duly corrected for TC, age, body mass index (BMI), waist circumference (WC), hypertension, smoking, and the homeostasis model assessment-insulin resistance (HOMA-IR) correlated with CAC, but not with CCA-IMT. The latter related to increased age, BMI, waist circumference (WC), and systolic blood pressure (SBP). Plasma HDL-C concentrations did not define CAC or CCA-IMT degrees, although in relation to the lower tertile of HDL-C in plasma the higher tertile of HDL-C had lower HOMA-IR and concentration of a cholesterol synthesis marker (desmosterol). Present work indicated that increased cholesterol synthesis and absorption represent primary causes of CAD, but not of the common carotid artery atherosclerosis.

The article discusses the current prevalence of hypercholesterolemia in everyday clinical practice in patients of different risk categories and approaches to its correction in the most frequently used clinical guidelines. High prevalence of hypercholesterolemia in practical health care with little change over time is shown. The existing problems of the practical implementation of the principles of a healthy lifestyle as the basis for reducing cardiovascular risk and possible solutions are described. Attention is focused on a low number of patients taking statins, and even less – reaching recommended target cholesterol levels. It is emphasized the validity of lower, compared to previous, target cholesterol levels in National recommendations on the correction of dyslipidemia in patients with proven atherosclerosis-related cardiovascular diseases as well as the feasibility in some cases of combined lipidlowering therapy (statins in combination with intestinal cholesterol absorption blockers, PCSK9 inhibitors, high doses of ethyl eicosopentaenoic acid). It is commented the need to prescribe statins in persons older than 75 years, but with a careful approach. Particular attention is paid to the use of statins in the primary prevention of cardiovascular complications. The validity of prescribing fixed moderate doses of statins for this purpose is discussed, which is both scientifically based and more realistic in practical terms.

Contains fulltext : 206793.pdf (Publisher’s version ) (Open Access) Dietary plant sterols and stanols as present in our diet and in functional foods are well-known for their inhibitory effects on intestinal cholesterol absorption, which translates into lower low-density lipoprotein cholesterol concentrations. However, emerging evidence suggests that plant sterols and stanols have numerous additional health effects, which are largely unnoticed in the current scientific literature. Therefore, in this review we pose the intriguing question "What would have occurred if plant sterols and stanols had been discovered and embraced by disciplines such as immunology, hepatology, pulmonology or gastroenterology before being positioned as cholesterol-lowering molecules?" What would then have been the main benefits and fields of application of plant sterols and stanols today? We here discuss potential effects ranging from its presence and function intrauterine and in breast milk towards a potential role in the development of non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), inflammatory bowel diseases (IBD) and allergic asthma. Interestingly, effects clearly depend on the route of entrance as observed in intestinal-failure associated liver disease (IFALD) during parenteral nutrition regimens. It is only until recently that effects beyond lowering of cholesterol concentrations are being explored systematically. Thus, there is a clear need to understand the full health effects of plant sterols and stanols.

Intestinal cholesterol absorption is a key regulator of systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal cholesterol uptake is mediated by Niemann-Pick C1-like 1 (NPC1L1), a transmembrane protein localized in membrane microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3 synthase (GM3S), in NPC1L1-dependent cholesterol uptake have not been examined previously. Here, we examined NPC1L1-dependent cholesterol uptake in a cell model as well as in wild-type and apoE-deficient mice fed normal or high-cholesterol diets. We showed that NPC1L1-dependent cholesterol uptake was impaired in GM3S-deficient cells and that GM3S deficiency promoted resistance to hypercholesterolemia in both wild-type and apoE-deficient mice fed the high-cholesterol but not the normal diet. Our findings suggest that GM3 and related gangliosides are essential for NPC1L1-mediated intestinal cholesterol absorption and are potential targets for hypercholesterolemia therapy.

The actions of insulin on intestinal cholesterol absorption and lipoprotein secretion are not well understood. Herein, we determined the effects of insulin on the levels of cholesterol transporter scavenger receptor, class B, type I (SR-BI), cellular cholesterol uptake, intracellular lipid accumulation, and lipoprotein secretion in a cellular model of human intestinal epithelium. Methods CaCo-2 cells were cultured to postconfluency in Transwell filters and stimulated with glucose (25 mM) in the presence or absence of insulin (100 nM) at their basolateral surface. SR-BI mRNA and protein levels were quantified by quantitative reverse transcription-PCR and immunoblot, respectively. Polarized localization of SR-BI was determined by cell surface proteins biotinylation and streptavidin precipitation. Activities of PI3K, AKT, mTOR, and SR-BI were pharmacologically antagonized. Cholesterol uptake was assessed by NBD-cholesterol incorporation. Apolipoprotein (apo) B concentration was quantified by ELISA. Subcellular localization of neutral lipids (BODIPY) and SR-BI (immunofluorescence) was determined by confocal microscopy. Results In polarized CaCo-2 cells, insulin increased SR-BI at the mRNA and protein levels. SR-BI was exclusively present at apical cell surface, as indicated by biotinylation and confocal microscopy analysis. Glucose did not modify SR-BI abundance or subcellular localization. Effects of insulin on SR-BI levels were abrogated by PI3K, AKT, or mTOR pharmacological antagonism. Cholesterol uptake, neutral lipid abundance, and apo B secretion were increased by insulin in CaCo-2 cells, and these effects were prevented by SR-BI pharmacological antagonism with block lipid transport-1. Conclusions insulin promotes cholesterol uptake, intracellular lipid store, and apo B-containing lipoproteins secretion by SR-BI-dependent mechanisms in a model of human intestinal epithelium.

Piperine is the major pungent ingredient in black and white peppers. The present study investigated plasma cholesterol-lowering activity of piperine and associated underlying mechanism in hypercholesterolemia hamsters. Thirty-two hamsters were assigned into four groups receiving one of four diets namely a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), and the two HCD diets containing 0.03% (PL) and 0.06% (PH) piperine, respectively, for 6 weeks. Results showed that PL and PH diets significantly reduced plasma total cholesterol (TC) by 16–17%, and non-high-density lipoprotein cholesterol (non-HDL-C) by 24–25%, respectively. This was accompanied by 84–109% increase in fecal excretion of neutral sterols with down-regulation on gene expression of intestinal Niemann-Pick C1-like 1 protein (NPC1L1), acyl-CoA:cholesterol acyltransferase 2 (ACAT2), and microsomal triacylglycerol transport protein (MTP). It was concluded that plasma TC-lowering activity of piperine was mediated by reducing the intestinal cholesterol absorption via inhibition on gene expression of intestinal NPC1L1, ACAT and MTP.

Dietary non-digestible carbohydrates are perceived to improve health via gut microbiota-dependent generation of products such as short-chain fatty acids (SCFA). In addition, SCFA are also precursors for lipid and cholesterol synthesis potentially resulting in unwanted effects on lipid metabolism. Inulin is a widely used model prebiotic dietary fiber. Inconsistent reports on the effects of inulin on cholesterol homeostasis have emerged in humans and preclinical models. To clarify this issue, the present study aimed to provide an in-depth characterization of the effects of short-chain (sc)- and long-chain (lc)- inulin on cholesterol synthesis, absorption and elimination in mice. Feeding wildtype C57BL/6J mice diets supplemented with 10% (w/w) of either sc- or lc-inulin for two weeks resulted in approximately 2.5-fold higher fecal SCFA levels (P

Cholesterol is essential for numerous biologic functions and processes, but an excess of intracellular cholesterol can be toxic. Intestinal cholesterol absorption is a major determinant of plasma cholesterol level. The liver X receptor (LXR) is a nuclear receptor known for its activity in cholesterol efflux and reverse cholesterol transport. In this study, we uncovered a surprising function of LXR in intestinal cholesterol absorption and toxicity. Genetic or pharmacologic activation of LXRα-sensitized mice to a high-cholesterol diet (HCD) induced intestinal toxicity and tissue damage, including the disruption of enterocyte tight junctions, whereas the same HCD caused little toxicity in the absence of LXR activation. The gut toxicity in HCD-fed LXR-KI mice may have been accounted for by the increased intestinal cholesterol absorption and elevation of enterocyte and systemic levels of free cholesterol. The increased intestinal cholesterol absorption preceded the gut toxicity, suggesting that the increased absorption was not secondary to tissue damage. The heightened sensitivity to HCD in the HCD-fed LXRα-activated mice appeared to be intestine-specific because the liver was not affected despite activation of the same receptor in this tissue. Moreover, heightened sensitivity to HCD cannot be reversed by ezetimibe, a Niemann-Pick C1-like 1 inhibitor that inhibits intestinal cholesterol absorption, suggesting that the increased cholesterol absorption in LXR-activated intestine is mediated by a mechanism that has yet to be defined.

Background Fatty acids have been shown to modulate intestinal cholesterol absorption in cells and animals, a process that is mediated by several transporter proteins. Of these proteins, Niemann-Pick C1-Like 1 (NPC1L1) is a major contributor to this process. The current study investigates the unknown mechanism by which fatty acids modulate cholesterol absorption. Methods We evaluated the effects of six fatty acids palmitic acid (PAM), oleic acid (OLA), linoleic acid (LNA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cholesterol uptake and transport in human enterocytes Caco-2 cells, and on the mRNA expression levels of NPC1L1, others proteins (ABCG5, ABCG8, ABCA1, ACAT2, MTP, Caveolin 1, Annexin-2) involved in cholesterol absorption, and SREBP-1 and SREBP-2 that are responsible for lipid metabolism. Results The polyunsaturated fatty acids (PUFAs), especially for EPA and DHA, dose-dependently inhibited cholesterol uptake and transport in Caco-2 monolayer, while saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) had no inhibitory effects. EPA and DHA inhibited cholesterol absorption in Caco-2 monolayer might be caused by down-regulating NPC1L1 mRNA and protein levels, which were associated with inhibition of SREBP-1/− 2 mRNA expression levels. Conclusion Results from this study indicate that functional food containing high PUFAs may have potential therapeutic benefit to reduce cholesterol absorption. Further studies on this topic may provide approaches to control lipid metabolism and to promote health. Electronic supplementary material The online version of this article (10.1186/s12944-018-0675-y) contains supplementary material, which is available to authorized users.

Background Hyperlipidemia characterized of elevated serum lipid levels is a prevalent disease frequently resulting in cardiovascular disease (CVD). Berberine and evodiamine are herbal products of traditional Chinese herb Coptis chinensis and Evodia rutaecarpa, which are indicated to exert regulation of lipid metabolism. Therefore, the objective of this study was to investigate the lipid-lowering effect of berberine and evodiamine combination in hyperlipidemic rats. Method The rat model of hyperlipidemia was established by providing high-fat-diet (HFD) for 4 weeks. Berberine (BB), evodiamine (EV), and their combination (BB + EV) were orally administered to HFD induced rats for 4 weeks. Body weight, food utilization, histopathology of liver tissues, lipid profiles of serum and liver were measured. Gas chromatography (GC) analysis was applied to examine the level of plasma total cholesterol and ß- Sitosterol (BS) to estimate cholesterol absorption activity. Furthermore, intestinal NPC1L1, ACAT2, and ApoB48 protein expressions were evaluated by immunohistochemical assay. Result According to the results, decreased levels of serum cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), as well as hepatic TC were showed in hyperlipidemic rats treated by combination of berberine and evodiamine. GC analysis indicated that the elevated plasma BS was significantly ameliorated by BB, EV, and BB + EV. In addition, immunohistochemical analysis revealed that BB + EV treatment down-regulated the expressions of intestinal NPC1L1 and ACAT2, and ApoB48 in HFD induced rats. Conclusion Based on the above results, combination of berberine and evodiamine exerted a promising preventive effect on hyperlipidemia, partially through inhibiting intestinal absorption of cholesterol.

Background: Atorvastatin reduces low-density lipoprotein cholesterol (LDL-C) levels and the risk of cardiovascular events, but whether the addition of ezetimibe (EZE), a non-statin drug that reduces intestinal cholesterol absorption, can reduce the rate of cardiovascular events further, and if there any sex differences, is not known. Aim: To evaluate the effects of atorvastatin and EZE combination in acute coronary syndrome (ACS) patients on the incidence of composite endpoint in short-term follow-up and to assess differences according their gender. Methods: We conducted a 16-week, single-centre, prospective, randomised, open-label clinical trial involving 323 patients who had been hospitalised for an ACS within the preceding 14 days. They received atorvastatin 20 mg for 28 days, and after that 292 patients who had LDL-C levels ≥ 1.81 mmol/L were randomised to EZE 10 mg/day co-administered with atorvastatin therapy (EZE + statin) or double their current atorvastatin dose. The primary endpoint was a composite of cardiovascular death, nonfatal myocardial infarction, unstable angina requiring rehospitalisation, coronary revascularisation (≥ 30 days after randomisation), or nonfatal stroke. Results: The Kaplan-Meier event-free survival rate at 16 weeks was 88.1% in the EZE + statin group patients and 77.0% in the atorvastatin monotherapy group (absolute risk reduction: 11.1 percentage points; hazard ratio: 2.099; 95% confidence interval: 1.165–3.781; p = 0.014). The log rank test indicated that there was not a statistically significant difference between male and female survival rates in both treatment groups (p = 0.897). Conclusions: The results of our study demonstrated that when added to statin therapy, EZE resulted in improved cardiovascular outcomes, and the response to atorvastatin and EZE combination was similar for both men and women.

Chronic kidney disease (CKD) patients have a high burden of cardiovascular disease. In the general population, lipid metabolism disorders, which cause the initiation and progression of atherosclerotic vascular changes, are major targets for preventive and therapeutic strategies in cardiovascular medicine. However, data from large cohort studies and from clinical trials suggest that the treatment guidelines on cardiovascular disease prevention and therapy cannot uncritically be transferred from individuals with intact renal function to CKD patients. Thus, unlike in the general population, neither plasma levels of HDL-cholesterol, nor the key parameter of HDL-cholesterol function-that is, cholesterol efflux capacity-predicts future cardiovascular events. Therefore, HDL-cholesterol should presently not be considered as therapeutic target in CKD patients. In contrast, lowering of LDL-cholesterol has been shown to reduce cardiovascular events at least among nondialysis CKD patients. The cardiovascular benefit of targeting LDL-cholesterol among dialysis CKD patients is less evident. We strongly believe that at least some subgroups of dialysis patients may profit from such treatment, particularly those with highest baseline LDL-cholesterol. Finally, as CKD patients have been characterized to have rather high intestinal cholesterol absorption, and relatively low hepatic cholesterol synthesis, substituting combined statin/ezetimibe treatment for statin monotherapy may be of particular benefit for nephrologic patients.

Purpose of review To discuss recent advances in research focused on intestinal lipid handling. Recent findings An important strategy in reducing atherosclerosis and risk of cardiovascular events is to increase the rate of reverse cholesterol transport, including its final step; cholesterol excretion from the body. The rate of removal is determined by a complex interplay between the factors involved in regulation of intestinal cholesterol absorption. One of these factors is a process known as transintestinal cholesterol excretion. This pathway comprises transport of cholesterol directly from the blood, through the enterocyte, into the intestinal lumen. In humans, this pathway accounts for 35% of cholesterol excretion in the feces. Mechanistic studies in mice revealed that, activation of the bile acid receptor farnesoid X receptor increases cholesterol removal via the transintestinal cholesterol excretion pathway as well as decreases plasma cholesterol and triglyceride providing an interesting target for treatment of dyslipidemia in humans. The physical chemical properties of bile acids are under control of farnesoid X receptor and determine intestinal cholesterol and triglyceride solubilization as well as absorption, providing a direct link between these two important factors in the pathogenesis of cardiovascular disease. Besides bile acids, intestinal phospholipids are important for luminal lipid solubilization. Interestingly, phospholipid remodeling through LPCAT3 was shown to be pivotal for uptake of fatty acids by enterocytes, which may provide a mechanistic handle for therapeutic intervention. Summary The importance of the intestine in control of cholesterol and triglyceride homeostasis is increasingly recognized. Recently, novel factors involved in regulation of cholesterol excretion and intestinal triglyceride and fatty acid uptake have been reported and are discussed in this short review.

Objective— Ezetimibe improves cardiovascular outcomes when added to optimum statin treatment. It lowers low-density lipoprotein cholesterol and percent intestinal cholesterol absorption, but the exact cardioprotective mechanism is unknown. We tested the hypothesis that the dominant effect of ezetimibe is to increase the reverse transport of cholesterol from rapidly mixing endogenous cholesterol pool into the stool. Approach and Results— In a randomized, placebo-controlled, double-blind parallel trial in 24 healthy subjects with low-density lipoprotein cholesterol 100 to 200 mg/dL, we measured cholesterol metabolism before and after a 6-week treatment period with ezetimibe 10 mg/d or placebo. Plasma cholesterol was labeled by intravenous infusion of cholesterol-d 7 in a lipid emulsion and dietary cholesterol with cholesterol-d 5 and sitostanol-d 4 solubilized in oil. Plasma and stool samples collected during a cholesterol- and phytosterol-controlled metabolic kitchen diet were analyzed by mass spectrometry. Ezetimibe reduced intestinal cholesterol absorption efficiency 30±4.3% (SE, P P =0.0001). Body cholesterol pool size was unchanged, but fecal endogenous cholesterol excretion increased 66.6±12.2% ( P P P =0.0096). Fecal bile acids were unchanged. Conclusions— Ezetimibe increased the efficiency of reverse cholesterol transport from rapidly mixing plasma and tissue pools into the stool. Further work is needed to examine the potential relation of reverse cholesterol transport and whole body cholesterol metabolism to coronary events and the treatment of atherosclerosis. Clinical Trials Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT01603758.

Sterol 27-hydroxylase (CYP27A1) is a key enzyme in bile acids (BAs) biosynthesis and a regulator of cholesterol metabolism. Cyp27a1/Apolipoprotein E double knockout (DKO) mice fed with western diet (WD) are protected from atherosclerosis via up-regulation of hepatic Cyp7a1 and Cyp3a11. Since feeding BAs ameliorates metabolic changes in Cyp27a1 KO mice, we tested BAs feeding on the development of atherosclerosis in DKO mice. DKO mice were fed for 8 weeks with WD containing 0.1% cholic acid (CA) (WD-CA) or chenodeoxycholic acid (CDCA) (WD-CDCA). Atherosclerotic lesions, plasma lipoprotein composition and functionality, hepatic lipid content, BAs amount and composition, expression of genes involved in lipid metabolism and BA signaling in liver and intestine as well as intestinal cholesterol absorption were assessed. Hepatic Cyp7a1 and Cyp3a11 expression were reduced by 60% after feeding with both WD-CA and WD-CDCA. After feeding with WD-CA we observed a 40-fold increase in the abundance of atherosclerotic lesions in the aortic valve, doubling of the levels of plasma total and low density lipoprotein cholesterol and halving of the level of high density lipoprotein cholesterol. Furthermore, in these mice plasma cholesterol efflux capacity decreased by 30%, hepatic BA content increased 10-fold, intestinal cholesterol absorption increased 6-fold. No such changes were observed in mice fed with WD-CDCA. Despite similar reduction on Cyp7a1 and Cyp3a11 hepatic expression, CA and CDCA have a drastically different impact on development of atherosclerosis, plasma and hepatic lipids, BAs composition and intestinal absorption. Reduced cholesterol absorption contributes largely to athero-protection in DKO mice.

Cardiovascular diseases (CVDs) are the main cause of mortality worldwide. Risk factors of CVD can be classified into modifiable (smoking, hypertension, diabetes, hypercholesterolemia) through lifestyle changes or taking drug therapy and not modifiable (age, ethnicity, sex and family history). Elevated total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels have a lead role in the development of coronary heart disease (CHD), while high levels of high-density lipoprotein-cholesterol (HDL-C) seem to have a protective role.The current treatment for dyslipidemia consists of lifestyle modification or drug therapy even if not pharmacological treatment should be always considered in addition to lipid-lowering medications.The use of lipid-lowering nutraceuticals alone or in association with drug therapy may be considered when the atherogenic cholesterol goal was not achieved.These substances can be classified according to their mechanisms of action into natural inhibitors of intestinal cholesterol absorption, inhibitors of hepatic cholesterol synthesis and enhancers of the excretion of LDL-C. Nevertheless, many of them are characterized by mixed or unclear mechanisms of action.The use of these nutraceuticals is suggested in individuals with borderline lipid profile levels or with drug intolerance, but cannot replace standard lipid-lowering treatment in patients at high, or very high CVD risk.Nutraceuticals can also have vascular effects, including improvement in endothelial dysfunction and arterial stiffness, as well as antioxidative properties. Moreover, epidemiological and clinical studies reported that in patients intolerant of statins, many nutraceuticals with demonstrated hypolipidemic effect are well tolerated.

Low-density lipoprotein lowering with statins has convincingly and consistently proven to reduce cardiovascular events in both primary and secondary prevention. However, despite high-dose statin therapy, residual cardiovascular risk remains and many patients also do not tolerate statins. Ezetimibe was initially projected as a frontline alternative to statin. It is an intestinal cholesterol absorption inhibitor with modest LDL lowering effects. But, major studies failed to demonstrate any beneficial effect of CV outcomes, and the drug was relegated to oblivion. IMPROVE-IT, a contemporary, large, and well-designed trial, unequivocally demonstrated reduction in CV outcomes with ezetimibe when added to statin therapy. The benefits are seen in both sexes, elderly, CKD, diabetes mellitus, and in patients with prior CABG. It also reduces biomarkers and induces plaque regression like statins. The drug has now established itself as an add-on therapy to statin when monotherapy fails to achieve LDL goals and when it is not tolerated. The combination therapy has excellent safety and efficacy record. It has now been endorsed by major guidelines too in management of dyslipidemia. Yes, ezetimibe can indeed improve cardiovascular outcomes!

Objectives: Nutritional strategies can play a major role in the management of cholesterolemia, notably in postmenopausal women at risk of CVD. Interest has recently grown on the potential health benefits of milk polar lipids (MPL).We showed that isolipidic enrichment of the diet with MPL improved several lipid CV risk factors but underlying mechanisms remained unclear. We hypothesized that MPL reduce intestinal cholesterol absorption in humans. Methods:We performed a double-blind randomized controlled trial in 58 postmenopausal women with fasting HDL-cholesterol < 1.6 mM.They were subjected to a 4-week dietary intervention with daily consumption of a cream-cheese containing 12 g of milk fat including either 0 g (control, n = 19), 3 g (n = 19) or 5 g (n = 20) of MPL. Before and after each intervention, blood lipids were measured in the whole cohortwhereas fecal lipids and coprostanol were analyzed in a subgroup (n = 7–9 per group). A proof-of-concept mechanistic crossover study was also carried out in 4 ileostomized subjects who performed 8hpostprandial tests after consuming 0g-, 3g- or 5g-MPL enriched cheese labelled with 2H-cholesterol tracer. Plasma, chylomicrons and ileal efflux were analyzed. Results: Milk fat enriched with 3 to 5 g MPL induced doseresponse reductions in serum total cholesterol (up to −6.8% in 5 g group, p < 0.05), LDL-cholesterol (−8.7%, p < 0.05) and HDL/total-cholesterol ratio (p < 0.001), compared to the control that had no effect. Fecal excretion of coprostanol increased after MPL supplementation (p < 0.05, 3g- and 5g-MPL vs control), and the fecal coprostanol/cholesterol ratio was inversely correlated with serum total- and LDL-cholesterol after intervention (r = −0.5, p < 0.05). In ileostomized subjects, postprandial accumulation of 2Hcholesterol in plasma and chylomicrons was reduced after 3 to 5 g MPL consumption (p < 0.05, vs control). Both cholesterol and milk sphingomyelin increased in ileal efflux after MPL enriched cheeses (p < 0.05). Conclusions: Present results suggest that milk polar lipids decrease cholesterol absorption in humans through interactions with sphingomyelin and by increasing conversion of cholesterol to coprostanol. Funding Sources: ANR (French National Research Agency, VALOBAB project, ANR-11-ALID-007–01), PHRC-I (French Clinical Research Program, 14–007), CNIEL (French Dairy Interbranch Organization).

Non-cholesterol sterols are validated biomarkers for intestinal cholesterol absorption and endogenous cholesterol synthesis. However, their use in metabolic disturbances has not been systematically explored. Therefore, we conducted a systematic review to provide an overview of non-cholesterol sterols as markers for cholesterol metabolism in different metabolic disorders. Potentially relevant studies were retrieved by a systematic search of three databases in July 2018 and ninety-four human studies were included. Cholesterol-standardized levels of campesterol, sitosterol and cholestanol were collected to reflect cholesterol absorption and those of lathosterol and desmosterol to reflect cholesterol synthesis. Their use as biomarkers was examined in the following metabolic disorders: overweight/obesity (n = 16), diabetes mellitus (n = 15), metabolic syndrome (n = 5), hyperlipidemia (n = 11), cardiovascular disease (n = 17), and diseases related to intestine (n = 16), liver (n = 22) or kidney (n = 2). In general, markers for cholesterol absorption and synthesis displayed reciprocal patterns, showing that cholesterol metabolism is tightly regulated by the interplay of intestinal absorption and endogenous synthesis. Distinctive patterns for cholesterol absorption or cholesterol synthesis could be identified, suggesting that metabolic disorders can be classified as ‘cholesterol absorbers or cholesterol synthesizers’. Future studies should be performed to confirm or refute these findings and to examine whether this information can be used for targeted (dietary) interventions.

Hypercholesterolemia is highly associated with cardiovascular diseases (CVDs) such as atherosclerosis. Our previous study showed that extracts of crabapple fruit may decrease serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) in high-fat diet-induced obese mice. To investigate which bioactive component is responsive for cholesterol-lowering effect in crabapples and whether the component has effects on atherosclerosis, three different chemical fractions were isolated from ethanol extract of crabapples, and obese C57BL/6 mice fed with a high-fat diet and arteriosclerotic low-density lipoprotein receptor deficiency (LDLR−/−) mice fed with a western-diet (WD) were involved in our pharmacological studies. The results showed that ethyl acetate extract of crabapple (EAC) significantly decreased serum TC in both DIO and LDLR−/− mice. EAC also improved obesity, insulin resistance, lipid accumulation in the liver tissue in the DIO mice and prevented atherosclerotic plaque formation in LDLR−/− mice. Mechanistic study showed that EAC increased cholesterol excretion in the feces of DIO and LDLR−/− mice, and elevated CYP7A1 mRNA in HepG2 cell and inhibited cholesterol accumulation in the cell membrane. Our data suggest that EAC may lower serum TC and block atherosclerotic plaque via the inhibition of intestinal cholesterol absorption and TC synthesis in the liver, and EAC may be used as a dietary supplement on hypercholesterolemia and atherosclerosis.

Hypercholesterolemia promotes atherosclerosis and precise regulation of cholesterol homeostasis is essential. Besides risk factor for cardiovascular disease, abnormalities in cholesterol metabolism have been associated with type 2 diabetes. Cholesterol homeostasis in the body is maintained by de novo synthesis. Furthermore, intestinal cholesterol absorption has recently been considered as an important control point in cholesterol homeostasis. Important insights have been gained into the mechanisms of transport of cholesterol from the intestinal lumen into the enterocytes. Several transporter proteins that appear to be key players in the control of the cholesterol absorption from the intestinal lumen have been identified. Here, we review intestinal cholesterol absorption and the mechanisms underlying alterations in cholesterol absorption under physiological conditions and in diseases such as diabetes mellitus.

The aim of pharmacological lipid modification is to reduce low-density lipoprotein cholesterol (LDL-C) as a means of either secondary or primary prevention of cardiovascular disease. Statins are the first-line therapy for pharmacological lipid modification. Ezetimibe is a drug which reduces LDL-C by selectively inhibiting intestinal cholesterol absorption. This provides an alternative pharmacological approach to that of statin therapy to reduce LDL-C. Ezetimibe has been shown to significantly reduce levels of LDL-C and recently, as demonstrated in the IMPROVE-IT trial, to reduce the rate of cardiovascular events in high-risk patients. Ezetimibe therefore has an important role in pharmacological lipid modification. In this paper, we examine the body of research behind ezetimibe and assess its current clinical applications in different patient subgroups.

Cholesterol homeostasis is maintained through a balance of de novo synthesis, intestinal absorption, and excretion from the gut. The small intestine contributes to cholesterol homeostasis by absorbing and excreting it, the latter of which is referred to as trans-intestinal cholesterol efflux (TICE). Because the excretion efficiency of endogenous cholesterol is inversely associated with the development of atherosclerosis, TICE provides an attractive therapeutic target. Thus, elucidation of the mechanism is warranted. We have shown that intestinal cholesterol absorption and TICE are inversely correlated in intestinal perfusion experiments in mice. In this review, we summarized 28 paired data sets for absorption efficiency and fecal neutral sterol excretion, a surrogate marker of TICE, obtained from 13 available publications in a figure, demonstrating the inverse correlation were nearly consistent with the assumption. We then offer a bidirectional flux model that accommodates absorption and TICE occurring in the same segment. In this model, the brush border membrane (BBM) of intestinal epithelial cells stands as the dividing ridge for cholesterol fluxes, making the opposite fluxes competitive and being coordinated by shared BBM-localized transporters, ATP-binding cassette G5/G8 and Niemann-Pick C1-like 1. Furthermore, the idea is applied to address how excess plant sterol/stanol (PS) intake reduces circulating cholesterol level, because the mechanism is still unclear. We propose that unabsorbable PS repeatedly shuttles between the BBM and lumen and promotes concomitant cholesterol efflux. Additionally, PSs, which are chemically analogous to cholesterol, may disturb the trafficking machineries that transport cholesterol to the cell interior.

Hypercholesterolemia accelerates atherosclerosis, and extensive research has been undertaken to ameliorate this abnormality. Plant sterols have been shown to inhibit cholesterol absorption and lower plasma cholesterol level since the 1950s. This ingredient has recently been reappraised as a food additive that can be taken daily in a preclinical period to prevent hypercholesterolemia, considering that cardiovascular-related diseases are the top cause of death globally even with clinical interventions. Intestinal cholesterol handling is still elusive, making it difficult to clarify the mechanism for plant sterol-mediated inhibition. Notably, although the small intestine absorbs cholesterol, it is also the organ that excretes it abundantly, via trans-intestinal cholesterol efflux (TICE). In this review, we show a model where the brush border membrane (BBM) of intestinal epithelial cells stands as the dividing ridge for cholesterol fluxes, making cholesterol absorption and TICE inversely correlated. With this model, we tried to explain the plant sterol-mediated inhibitory mechanism. As well as cholesterol, plant sterols diffuse into the BBM but are effluxed back to the lumen rapidly. We propose that repeated plant sterol shuttling between the BBM and lumen promotes cholesterol efflux, and plant sterol in the BBM may disturb the trafficking machineries that transport cholesterol to the cell interior.