Radioactive Cholesterol Research Articles

Assessing Cholesterol Efflux on Primary Human Trophoblast Cells.

Cholesterol transport across the placenta must be tightly regulated to avoid a deficiency or an oversupply of cholesterol which is transferred from the mother to the fetus. In trophoblasts, the transport of cholesterol across the cell membrane is mainly mediated by the ATP-binding transporters, ABCA1 and ABCG1. The localization of the transporters at the apical and basal sides of syncytiotrophoblasts has been described. A frequently used method to quantify the amount of cholesterol that cells are capable of exporting is the cholesterol efflux assay. The principle of this assay is that when exogenous [3H]-labeled cholesterol is provided to cultured cells, the efflux of the radioactive cholesterol toward different acceptors in the culture medium is evaluated. Then, the percentage of cholesterol efflux from the cells to the acceptors is calculated. The present work gives an overview on the principle of this assay and a detailed protocol of this technique performed in primary trophoblasts isolated from human term placentas.

SiRNA screening reveals that SNAP29 contributes to exosome release

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

Hepatic Cdkal1 deletion regulates HDL catabolism and promotes reverse cholesterol transport

Background and aimsAssociations between CDKAL1 variants and cholesterol efflux capacity (CEC) have been reported. This study aimed to investigate the effects of Cdkal1 deficiency on high-density lipoprotein (HDL) metabolism, atherosclerosis, and related pathways. MethodsLipid and glucose metabolic profiles, CEC, and in vivo reverse cholesterol transport (RCT) were compared in liver-specific Alb-Cre:Cdkal1fl/fl and Cdkal1fl/fl mice. Aortic atherosclerosis was compared in Apoe−/−Alb-Cre:Cdkal1fl/fl and Apoe−/− mice fed high-fat diets. HDL subclasses and mediators of HDL metabolism from Alb-Cre:Cdkal1fl/fl mice were examined. ResultsHDL-cholesterol level tended to be higher in the Alb-Cre:Cdkal1fl/fl mice (p = 0.050). Glucose and other lipid profiles were similar in the two groups of mice, irrespective of diet. The mean CEC was 27% higher (p = 0.007) in the Alb-Cre:Cdkal1fl/fl mice, as were the radioactivities of bile acids (mean difference 17%; p = 0.035) and cholesterol (mean difference 42%; p = 0.036) from faeces. The radioactivity tendency was largely similar in mice fed a high-fat diet. Atherosclerotic lesion area tended to be smaller in the Apoe−/−Alb-Cre:Cdkal1fl/fl mice than in the Apoe−/− mice (p = 0.067). Cholesterol concentrations in large HDLs were higher in the Alb-Cre:Cdkal1fl/fl mice (p = 0.024), whereas in small HDLs, they were lower (p = 0.024). Endothelial lipase (mean difference 39%; p = 0.002) and hepatic lipase expression levels (mean difference 34%; p < 0.001) were reduced in the Alb-Cre:Cdkal1fl/fl mice, whereas SR-B1 expression was elevated (mean difference 35%; p = 0.007). ConclusionsThe promotion of CEC and RCT in Alb-Cre:Cdkal1fl/fl mice verified the effect of CDKAL1 seen in human genetic data. These phenotypes were related to regulation of HDL catabolism. This study suggests that CDKAL1 and associated molecules could be targets for improving RCT and vascular pathology.

Elevated cholesterol uptake by T cells is a biomarker for the efficacy of immune checkpoint inhibitors in mouse models for triple negative breast cancer

Abstract Predicting the outcome of cancer immunotherapy can help avoid the cost and side effects of ineffective treatment and help develop effective treatment strategies. T cells take up cholesterol during activation, which is necessary for their proliferation. However, there is no literature linking the uptake of cholesterol as a biomarker for T cell activation and less so as a biomarker for immune checkpoint inhibitor therapy (ICI). Our research group has developed a novel radioactive cholesterol analog called 18F-FNP-59, which can be used to measure the uptake of cholesterol in live cells and within living organisms. We hypothesize that elevated cholesterol influx in T cells of the tumor microenvironment is linked to T cell activation and predicts the success of cancer immunotherapy in triple negative breast cancer (TNBC) models. We found that the elevated uptake of exogenous labeled cholesterol analogs functions as a biomarker for T cell activation. When comparing ICI-responsive E0771 tumors to non-responsive AT3 tumors, we found that the uptake of a fluorescent cholesterol analog was significantly higher in the T cells of the ICI-responsive tumors both in vitro and in vivo. Using the 18F-FNP-59 compound, we found that the uptake of cholesterol by T cells was further enhanced in ICI-responding tumors that received anti-PD-1 treatment. These results suggest that the uptake of exogenous cholesterol analogs by tumor-infiltrating T cells may be able to predict T cell activation and the success of ICI therapy. In future studies, we will expand the tumor models and types of cancers that we observe. Additionally we will examine the relationship between T cell cholesterol trafficking and invasion of T cells into the tumor using imaging techniques.

Characterization of cholesterol efflux capacity in diabetic and non-diabetic patients with coronary artery disease: comparison between acute coronary syndrome and stable coronary artery disease

Abstract Introduction Type 2 diabetic patients more likely exhibit a lower high-density lipoprotein (HDL) level. Given a greater glycation and oxidative stress in diabetic subjects, these atherogenic characteristics could cause dysfunctional HDL including a reduced cholesterol efflux capacity (CEC), which may account for an increased risk of diabetic macrovascular disease including acute coronary syndrome (ACS). However, it remains to be fully elucidated characteristics of HDL-mediated CEC in type 2 diabetic patients, in association with clinical presentation of coronary artery disease (CAD). Purpose To characterize CEC in CAD subjects with type 2 diabetes mellitus. Methods The current study prospectively analyzed 87 statin-naive patients with CAD. CEC was measured by using the collected apolipoprotein B-depleted serum. Liquid scintillation counting (Perkin-Elmer Analytical Sciences, MA, US) was used to quantify the efflux of radioactive cholesterol from J774 cells. Clinical characteristics and CEC were compared in diabetic and non-diabetic subjects. Results The averaged HbA1c in diabetic patients was 6.7±1.2, and 66.7% of them achieved HbA1c &amp;lt;7.0%. Diabetic subjects more likely exhibited a history of hypertension and dyslipidemia, and multi-vessel disease (Table). Moreover, a lower CEC level was observed in diabetic patients, accompanied by a lower HDL-C and apolipoprotein A-I levels with a higher level of triglyceride (Table). HDL-C (r=0.62, p-value&amp;lt;0.01) and Apolipoprotein A-I (r=0.70, p-value &amp;lt;0.01) were associated with CEC, whereas there was no significant difference in CEC between subjects with HbA1c &amp;lt;7.0% vs. ≥7.0% (0.74±0.07 vs. 0.78±0.08, p=0.22). On multivariate analysis, type 2 diabetes mellitus was an independent contributor to CEC &amp;lt;0.79 (median) (HR=2.75, 95% CI: 1.11–6.82, p=0.03). Interestingly in particular, CEC was substantially lower in diabetic patients with ACS compared to those with stable CAD (Figure). By contrast, clinical presentation of CAD did not affect CEC in non-diabetic subjects (Figure). Conclusions A lower CEC level was observed in subjects with type 2 diabetes mellitus. In particular, this HDL functionality was profoundly diminished in those presenting ACS. Our findings suggest functionality of HDL as a potential therapeutic target in diabetic patients experiencing ACS. Funding Acknowledgement Type of funding sources: None. Table 1Figure 1

Lipid transfer to high‐density lipoproteins in coronary artery disease patients with and without previous cerebrovascular ischemic events

BackgroundPatients with coronary artery disease (CAD) and previous ischemic cerebrovascular events (ICVE, ischemic stroke, or transitory ischemic attack) constitute a high‐risk subgroup for cardiovascular outcomes. High‐density lipoprotein cholesterol (HDL‐C) levels are correlated with cardiovascular events. Lipid transfer to HDL affects structure size and HDL subclass profile. Impairment of this transfer could influence ischemic risk seen in patients with CAD + ICVE. The objective was to evaluate the HDL ability to receive the lipids in patients with CAD with or without ICVE.MethodsPatients with CAD + ICVE (n = 60) and patients with CAD only (n = 60) were matched by age, sex, acute coronary syndromes (ACS) event type, and time elapsed between the ACS event and inclusion in the study. Lipid transfer to HDL was evaluated by incubating donor lipid nanoparticles labeled with radioactive unesterified cholesterol (UC) and esterified cholesterol (EC), phospholipid (PL), and triglyceride (TG) with whole plasma. After the chemical precipitation of non‐HDL fractions and nanoparticles, the supernatant was counted for HDL radioactivity.ResultsCAD + ICVE group presented with impaired lipid transfer to HDL for PL (CAD + ICVE: 21.14 ± 2.7% vs CAD: 21.67 ± 3.1%, P = .03), TG (CAD + ICVE: 4.88 ± 0.97% vs CAD: 5.63 ± 0.92%, P = .002), and UC (CAD + ICVE: 5.55 ± 1.19% vs CAD: 6.16 ± 1.14%, P = .009). Lipid transfer to HDL was similar in both groups for EC. Adjusted models showed similar results.ConclusionPatients with CAD and ICVE have reduced lipid transfer to HDL compared to those with CAD only. Dysfunctional HDL may account for the higher incidence of ischemic outcomes observed in this population.

Inhibitory effects of Tiliacora triandra (Colebr.) Diels on cholesterol absorption.

Background Natural supplements and herbal medicines have been attracted to use for managing elevated cholesterol levels. Tiliacora triandra (Colebr.) Diels (TT) or Yanang (in Thai) is commonly used as an ingredient in various types of Thai foods. In this study, we investigated the effect of methanolic TT leaf extract on cholesterol absorption by measuring the uptake and the efflux of cholesterol and cholesterol micellar solubility. In addition, we tested the effect of TT leaf extract on pancreatic lipase activity. Methods The uptake and efflux of cholesterol was determined by quantification of radioactivity in differentiated Caco-2 cells after treatment with radioactive cholesterol. Cholesterol mixed micelles were prepared for cholesterol uptake, efflux and solubility studies. The pancreatic lipase activity was determined using 4-methylumbelliferyl oleate as a substrate. Results Our finding showed that TT extract decreased the uptake of cholesterol by approximately 48% but did not affect the efflux of cholesterol. TT inhibited pancreatic lipase activity with the IC50 at 273.5 μg/mL and also decreased cholesterol micellar solubility. Conclusions These findings suggest that TT leaf extract seems to be a potential candidate as cholesterol-lowering agents.

Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-radiodetection of produced 14C-steroid hormone metabolites

This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid metabolites of the steroidogenic pathway allows for an improved understanding of the various enzymatic mechanisms involved without necessarily being dependent on quantification. Generated radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer (FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be added just before the cells are incubated for 72h in well plates. Based on the obtained HPLC-FSA chromatograms, and confirmation of the observations by studies in the literature, a qualitative time profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

Bisphenol A promotes cholesterol absorption in Caco-2 cells by up-regulation of NPC1L1 expression

BackgroundBisphenol A (BPA), an commonly exposed environmental chemicals in humans, has been shown to have a hypercholesterolemic effect with molecular mechanism not clear. Since intestinal cholesterol absorption plays a major role in maintaining total body cholesterol homeostasis, the present study is to investigate whether BPA affects cholesterol absorption in the intestinal Caco-2 cells. Methods: The Caco-2 cells were pretreated with BPA at different concentrations for 24 h and then incubated with radioactive micellar cholesterol for 2 h. The absorption of radioactive cholesterol was quantified by liquid scintillation. The expression of Niemann-Pick C1-like 1 (NPC1L1) and sterol regulatory element binding protein-2 (SREBP-2) was analyzed by Western blot and qPCR.ResultsWe found that confluent Caco-2 cells expressed NPC1L1, and the absorption of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. We then pretreated the cells with 0.1–10 nM BPA for 24 h and found that BPA at 1 and 10 nM doses promoted cholesterol absorption. In addition, we found that the BPA-induced promotion of cholesterol absorption was associated with significant increase in the levels of NPC1L1 protein and NPC1L1 mRNA. Moreover, the stimulatory effects of BPA on cholesterol absorption and NPC1L1 expression could be prevented by blockade of the SREBP-2 pathway.ConclusionsThis study provides the first evidence that BPA promotes cholesterol absorption in the intestinal cells and the stimulatory effect of BPA is mediated, at least in part, by SREBP-2-NPC1L1 signaling pathway.

Retrograde cholesterol transport in the human Caco-2/TC7 cell line: a model to study trans-intestinal cholesterol excretion in atherogenic and diabetic dyslipidemia.

The dyslipidemia associated with type 2 diabetes is a major risk factor for the development of atherosclerosis. Trans-intestinal cholesterol excretion (TICE) has recently been shown to contribute, together with the classical hepatobiliary route, to fecal cholesterol excretion and cholesterol homeostasis. The aim of this study was to develop an in vitro cell model to investigate enterocyte-related processes of TICE. Differentiated Caco-2/TC7 cells were grown on transwells and incubated basolaterally (blood side) with human plasma and apically (luminal side) with lipid micelles. Radioactive and fluorescent cholesterol tracers were used to investigate cholesterol uptake at the basolateral membrane, intracellular distribution and apical excretion. Our results show that cholesterol is taken up at the basolateral membrane, accumulates intracellularly as lipid droplets and undergoes a cholesterol acceptor-facilitated and progressive excretion through the apical membrane of enterocytes. The overall process is abolished at 4°C, suggesting a biologically active phenomenon. Moreover, this trans-enterocytic retrograde cholesterol transport displays some TICE features like modulation by PCSK9 and an ABCB1 inhibitor. Finally, we highlight the involvement of microtubules in the transport of plasma cholesterol from basolateral to apical pole of enterocytes. The human Caco-2/TC7 cell line appears a good in vitro model to investigate the enterocytic molecular mechanisms of TICE, which may help to identify intestinal molecular targets to enhance reverse cholesterol transport and fight against dyslipidemia.

Abstract 17824: Gemcabene and Atorvastatin Alone and Combined Markedly Reduce LDL-C in LDL Receptor-deficient Mice, a Model of Homozygous Familial Hypercholesterolemia

Background: Gemcabene (Gem) is a late-stage Phase 2 clinical candidate being evaluated for the reduction of low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemia (HoFH) patients. Combination therapy includes statins as standard of care to treat this genetic disorder. LDL-deficient (LDLr) mice have been widely used with lipid-lowering agents to demonstrate LDL-C lowering with good translation to clinical efficacy. We have previously shown that atorvastatin (Atorva) reduces LDL-C and apoB in the LDLr deficient mice and in casein-fed (endogenous hypercholesterolemic) rabbits. Experiment and Results: In the current study, we assessed Gem and Atorva effects alone and in combination on reduction of LDL-C in chow-fed LDLr deficient mice, an animal model of HoFH. Baseline LDL-C and total cholesterol were 246±20 mg/dL and 329±23 mg/dL in these mice Female LDLr deficient mice (n=10) were treated with Atorva alone (60mg/kg/day), Gem alone (60/mg/kg/day) or the agents combined (Atorva + Gem)(each at 60 mg/kg/day) for 14 days. Following treatments with Atorva, Gem, and Atorva+Gem, fasting LDL-C reduced by 22%, 55% and 72%, respectively, and total cholesterol by 21%, 47%, and 58%, respectively; showing additional reduction of 50% LDL-C by Gem on top of Atorva alone. Since LDLr are absent in these mice, similar to HoFH patients, the LDL-C lowering by Gem could be due to reduced VLDL production rates. Indeed, our studies in rat hepatocytes and in apoB100-only mice (apobec-1 knockout) showed reduced radioactivity in cholesterol and triglyceride fractions in the hepatocytes and in the plasma and liver of mice administered 14C acetate, suggesting cholesterol and fatty acid synthesis inhibition as a mechanism of LDL-C lowering. Gem also enhances the clearance of VLDL in normal rats. We propose both mechanisms may contribute to LDL-C reduction in LDLr deficient mice. A more detailed kinetic study of Gem in LDLr deficient models is underway to verify these proposed mechanisms. Conclusions: Gemcabene alone and combined with atorvastatin significantly (55-72%) reduced LDL-C in this predictive in-vivo HoFH disease model. These findings suggest that treatment with Gem on top of standard of care therapy by statins may benefit HoFH patients.

Lycopene reduces cholesterol absorption through the downregulation of Niemann-Pick C1-like 1 in Caco-2 cells.

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Tomato lycopene has been found to have a hypocholesterolemic effect, and the effect was considered to be related to inhibition of cholesterol synthesis. However, since plasma cholesterol levels are also influenced by the absorption of cholesterol in the gut, the present study is to investigate whether lycopene affects cholesterol absorption in the intestinal Caco-2 cells. The Caco-2 cells were pretreated with lycopene at different concentrations for 24 h and then incubated with radioactive micellar cholesterol for 2 h. The absorption of radioactive cholesterol was quantified by liquid scintillation. The expression of Niemann-Pick C1-like 1 (NPC1L1) and liver X receptor α (LXRα) was analyzed by Western blot and qPCR. We found that lycopene dose dependently inhibited cholesterol absorption and the expression of NPC1L1 protein and NPC1L1 mRNA. The inhibitory effects of lycopene on cholesterol absorption and NPC1L1 expression could be prevented by blockade of the LXRα pathway. This study provides the first evidence that lycopene inhibits cholesterol absorption in the intestinal cells and this inhibitory effect of lycopene is mediated, at least in part, by LXRα-NPC1L1 signaling pathway.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Cholesterol Regulates Prokaryotic Kir Channel by Direct Binding to Channel Protein

Cholesterol is known to play a major role in regulation of variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. In this study, we address the key question of whether cholesterol-induced regulation of ion channel function can be attributed to direct binding of the lipid to the channel protein and our observations provide the first direct evidence that cholesterol binds to an ion channel. Specifically, we show that cholesterol binds to a prokaryotic Kir channel, KirBac1.1 in a saturable way and that there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Furthermore, inhibition of cholesterol-KirBac1.1 binding by trifluoperazine, known to inhibit cholesterol binding to other proteins results in full recovery of the channel activity. Taken together, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a KD of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol–KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol–KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Cholesterol Binding Regulates Prokaryotic Kir Channels

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by specific binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol specifically binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a Km of 390 μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17[]β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel function. We conclude, therefore, that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

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.

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.

Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol

The proliferation and differentiation of normal prostate epithelial cells depends upon the action of androgens produced by the testis. Prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). In the present study, we have determined the potential of prostate cancer cells to synthesize androgens from cholesterol which may be involved in intracrine regulation of AR in advanced stages of the disease. Established androgen-independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B1 (SRB1), steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum-free cultures, the androgen-independent C81 cells secreted approximately 5-fold higher testosterone than C33 cells as determined in the conditioned media by immunoassays. These cells could also directly convert radioactive cholesterol into testosterone which was identified by thin layer chromatography. These results for the first time show that prostate cancer cells in advanced stages of the disease could synthesize androgens from cholesterol and hence are not dependent upon testicular and/or adrenal androgens.