Cholesterol Uptake In Caco-2 Cells Research Articles

Laminarin Reduces Cholesterol Uptake and NPC1L1 Protein Expression in High-Fat Diet (HFD)-Fed Mice.

Aberrantly high dietary cholesterol intake and intestinal cholesterol uptake lead to dyslipidemia, one of the risk factors for cardiovascular diseases (CVDs). Based on previous studies, laminarin, a polysaccharide found in brown algae, has hypolipidemic activity, but its underlying mechanism has not been elucidated. In this study, we investigated the effect of laminarin on intestinal cholesterol uptake in vitro, as well as the lipid and morphological parameters in an in vivo model of high-fat diet (HFD)-fed mice, and addressed the question of whether Niemann-Pick C1-like 1 protein (NPC1L1), a key transporter mediating dietary cholesterol uptake, is involved in the mechanistic action of laminarin. In in vitro studies, BODIPY-cholesterol-labeled Caco-2 cells were examined using confocal microscopy and a fluorescence reader. The results demonstrated that laminarin inhibited cholesterol uptake into Caco-2 cells in a concentration-dependent manner (EC50 = 20.69 μM). In HFD-fed C57BL/6J mice, laminarin significantly reduced the serum levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). It also decreased hepatic levels of TC, TG, and total bile acids (TBA) while promoting the excretion of fecal cholesterol. Furthermore, laminarin significantly reduced local villous damage in the jejunum of HFD mice. Mechanistic studies revealed that laminarin significantly downregulated NPC1L1 protein expression in the jejunum of HFD-fed mice. The siRNA-mediated knockdown of NPC1L1 attenuated the laminarin-mediated inhibition of cholesterol uptake in Caco-2 cells. This study suggests that laminarin significantly improves dyslipidemia in HFD-fed mice, likely by reducing cholesterol uptake through a mechanism that involves the downregulation of NPC1L1 expression.

Cholesterol-Lowering Effects of Asperidine B, a Pyrrolidine Derivative from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178: A Potential Cholesterol Absorption Inhibitor.

Isolated secondary metabolites asperidine B (preussin) and asperidine C, produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, were found to exhibit inhibitory effects against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and oxidative stress in an in vitro assay. Whether or not the known pyrrolidine asperidine B and the recently isolated piperidine asperidine C have lipid-lowering effects remains unknown. Thus, this study aimed to investigate the hypocholesterolemic effects of asperidines B and C and identify the mechanisms involved in using in vitro, ex vivo, and in vivo models. The results show that both compounds interfered with cholesterol micelle formation by increasing bile acid binding capacity, similar to the action of the bile acid sequestrant drug cholestyramine. However, only asperidine B, but not asperidine C, was found to inhibit cholesterol uptake in Caco-2 cells by up-regulating LXRα without changing cholesterol transporter NPC1L1 protein expression. Likewise, reduced cholesterol absorption via asperidine-B-mediated activation of LXRα was also observed in isolated rat jejunal loops. Asperidine B consistently decreases plasma cholesterol absorption, similar to the effect of ezetimibe in rats. Therefore, asperidine B, the pyrrolidine derivative, has therapeutic potential to be developed into a type of cholesterol absorption inhibitor for the treatment of hypercholesterolemia.

Phenolic acids from Coffea arabica L. suppress intestinal uptake of glucose and cholesterol.

The This study assessed the hypoglycaemic and hypocholesterolaemic effects of coffee fruit extracts and their active compounds involved in cholesterol and glucose uptake, as well as the inhibitory activity of α-glucosidase and α-amylase, the physiochemical changes of cholesterol micelles, and the uptake of glucose and cholesterol in Caco-2 cells. The results revealed that coffee green dry (CGD) coffee yellow dry (CYD), and coffee red bean dry (CRBD) extracts inhibited α-amylase activity by approximately 49% whereas caffeine and caffeic acid, showed high potency to inhibit α-amylase activity by 94.49% and 93.35%, respectively. In addition, coffee red pulps dry (CRPD) and citric acid inhibited αglucosidase by 29% and 46%, respectively. The extracts (1 mg/mL) and their active compounds (200 µM) inhibited glucose uptake in Caco-2 cells. All coffee fruit extracts inhibited cholesterol uptake in Caco-2 cells by approximately 40%, and they decreased the micellar solubility of cholesterol, except for coffee red fresh (CRF) and coffee red dry (CRD). Moreover, CYD strongly increased the size of cholesterol micelles. These findings demonstrate the potential effect of coffee extracts on anti-diabetic activity and their cholesterol-lowering activity according to the inhibitory activity of α-glucosidase and α-amylase and the uptake of glucose and cholesterol in Caco-2 cells.

Cyanidin-3-rutinoside acts as a natural inhibitor of intestinal lipid digestion and absorption

BackgroundCyanidin-3-rutinoside (C3R), a naturally occurring anthocyanin, possesses anti-oxidant, anti-hyperglycemic, anti-glycation and cardioprotective properties. However, its mechanisms responsible for anti-hyperlipidemic activity have not been fully identified. The aim of the study was to investigate the lipid-lowering mechanisms of C3R through inhibition of lipid digestion and absorption in vitro.MethodsThe inhibitory activity of C3R against pancreatic lipase and cholesterol esterase was evaluated using enzymatic fluorometric and enzymatic colorimetric assays, respectively. An enzyme kinetic study using Michaelis-Menten and the derived Lineweaver-Burk plot was performed to understand the possible types of inhibition. The formation of cholesterol micelles was determined using the cholesterol assay kit. The bile acid binding was measured using the colorimetric assay. The NBD cholesterol uptake in Caco-2 cells was determined using fluorometric assay. The mRNA expression of cholesterol transporter (Niemann-Pick C1-like 1) was determined by RT-PCR.ResultsThe results showed that C3R was a mixed-type competitive inhibitor of pancreatic lipase with the IC50 value of 59.4 ± 1.41 μM. Furthermore, C3R (0.125–1 mM) inhibited pancreatic cholesterol esterase about 5–18%. In addition, C3R inhibited the formation of cholesterol micelles and bound to primary and secondary bile acid. In Caco-2 cells, C3R (12.5–100 μM) exhibited a significant reduction in cholesterol uptake in both free cholesterol (17–41%) and mixed micelles (20–30%). Finally, C3R (100 μM) was able to suppress mRNA expression of NPC1L1 in Caco-2 cells after 24 h incubation.ConclusionsThe present findings suggest that C3R acts as a lipid-lowering agent through inhibition of lipid digestion and absorption.

Spirogyra neglecta inhibits the absorption and synthesis of cholesterol in vitro

BackgroundSpirogyra neglecta (SN) has many nutritional benefits and it is commonly used to ameliorate different human conditions including inflammation, gastric ulcer, hyperglycemia, and hyperlipidemia. However, the mechanism of the hypocholesterolemic effect of SN still remains unclear. Therefore, the present study was aimed to evaluate the effect of SN extract particularly on cholesterol absorption and synthesis mechanisms. MethodsFor cholesterol absorption, the uptake of cholesterol was measured by using tritium radiolabeling of cholesterol in Caco-2 cells. Bile acid binding, micelles size, and cholesterol solubility were analyzed in in vitro assays, while cholesterol synthesis was evaluated by using a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase assay kit. ResultsSN extract was found to decrease cholesterol uptake in Caco-2 cells and decreased the solubility of cholesterol in micelles. The SN extract bound to taurocholate, taurodeoxycholate, and glycodeoxycholate bile acids, and increased micelles size. SN has also demonstrated an inhibitory effect on HMG-CoA reductase (HMGR) enzymatic activity. For further experimentation, the treatment combination of SN and ezetimibe (0.04mg/mL) showed a greater significant reduction in cholesterol uptake than the extract alone. ConclusionThese observations suggested that inhibitory cholesterol absorption effects of SN could be mediated through the modulation of size and solubility of cholesterol micelles, resulting in interference of cholesterol uptake. In addition, SN inhibited the rate limiting step of cholesterol synthesis. This study provides supporting evidence for the potential usage of SN as a cholesterol lowering agent.

Hypolipidemic activity of common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum Gaertn.) buckwheat.

Buckwheat grain has well-balanced nutritional value, whereas its digestibility is relatively low. This review summarizes recent advances in studies on the hypolipidemic activity of buckwheat. The most remarkable function is a powerful hypocholesterolemic activity of buckwheat protein in rats, which is far stronger than that of soy protein. The cholesterol-lowering effect is mediated by mechanisms involving higher excretion of fecal sterols and lower digestibility of buckwheat protein. The insoluble fraction of buckwheat protein associates with cholesterol and reduces micelle cholesterol uptake in caco-2 cells. Furthermore, consumption of buckwheat protein suppresses cholesterol-induced gallstones and body fat in rodents. Buckwheat sprouts also have hypolipidemic activity in rats or type 2 diabetic mice. Tartary buckwheat bran extract reduced the serum level of total cholesterol and triglyceride in hyperlipidemic rats. The consumption of buckwheat seed reduced low-density lipoprotein cholesterol in the pastureland Mongolian population. Taken together, buckwheat may be beneficial for prevention of hyperlipidemia.

Black rice and anthocyanins induce inhibition of cholesterol absorption in vitro

Black rice (Oryza sativa L.) is often associated with blood lipid control. This study systematically assessed the inhibition of cholesterol absorption in vitro by black rice and explored cholesterol-lowering compounds present in this rice. Our results indicated that black rice extracts (BRE), which were aqueous, ethanol extracts and a fraction of macroporous resin caused the reduction of cholesterol absorption by inhibiting pancreatic lipase, decreasing the micellar cholesterol solubility and suppressing cholesterol uptake in Caco-2 cells. The inhibitory activity was positively associated with anthocyanin (cyanidin-3-glucoside (Cy-3-G) and peonidin-3-glucoside (Pn-3-G)) contents of the extracts. Therefore, the cholesterol absorption inhibiting properties of anthocyanins were further explored. The IC50 values of Cy-3-G and Pn-3-G against pancreatic lipase were 42.53 ± 4.45 and 18.13 ± 4.22 μg mL(-1), respectively. Kinetic analysis suggested that the enzymatic inhibitory mode of Cy-3-G and Pn-3-G belonged to the competitive type. In mixed micelles, Cy-3-G and Pn-3-G dose dependently reduced the solubility of cholesterol. Meanwhile, a potential mechanism of cholesterol reduction by anthocyanins was investigated. Results showed that anthocyanins led to precipitation of cholesterol from micellar solution, which may induce the reduction of cholesterol. In Caco-2 cells, Cy-3-G and Pn-3-G (40 μg mL(-1)) exhibited a significant reduction in cholesterol uptake, and the degree of this reduction was almost the same as that observed in the group treated with Ezetimibe at the same concentration. These findings provide important evidence that anthocyanins may partly contribute to the inhibitory effects of black rice on cholesterol absorption, and thus may be applied for the prevention and treatment of hypercholesterolaemia.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

BackgroundCurcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.MethodsCaco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification.ResultsWe found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively.ConclusionCurcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.

Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells

Background Sphingomyelin (SM) is present in dietary products and cell plasma membranes. We previously showed that dietary SM inhibited cholesterol absorption in rats. In the intestinal tract, SM is mainly hydrolyzed by alkaline sphingomyelinase (alk-SMase) to ceramide.Aims We investigated the influence of SM and its hydrolytic products ceramide and sphingosine on cholesterol uptake in intestinal Caco-2 cells.Methods Micelles containing bile salt, monoolein, and (14)C-cholesterol were prepared with or without SM, ceramide,or sphingosine. The micelles were incubated with Caco-2 cells, and uptake of radioactive cholesterol was quantified.Results We found that confluent monolayer Caco-2 cells expressed NPC1L1, and the uptake of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. Incorporation of SM in the cholesterol micelles inhibited cholesterol uptake dose-dependently; 38% inhibition occurred at an equal mole ratio of SM and cholesterol.The inhibition was further enhanced to 45% by pretreating the cholesterol/SM micelles with recombinant alk-SMase, which hydrolyzed SM in the micelles by 85%, indicating ceramide has stronger inhibitory effects on cholesterol uptake. To confirm this, we further replaced SM in the micelles with ceramide and sphingosine, and found that at equal mole ratio to cholesterol, ceramide exhibited stronger inhibitory effect (50% vs 38%) on cholesterol uptake than SM, whereas sphingosine only had a weak effect at high concentrations.Conclusion Both SM and ceramide inhibit cholesterol uptake, the effect of ceramide being stronger than that of SM. Alk-SMase enhances SM-induced inhibition of cholesterol uptake by generating ceramide in the intestinal lumen.

Insoluble Fraction of Buckwheat (Fagopyrum esculentum Moench) Protein Possessing Cholesterol-Binding Properties That Reduce Micelle Cholesterol Solubility and Uptake by Caco-2 Cells

Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.