Cholesterol Excretion Research Articles

The TBK1/IKKε inhibitor amlexanox improves dyslipidemia and prevents atherosclerosis.

Cardiovascular diseases, especially atherosclerosis and its complications, are a leading cause of death. Inhibition of the noncanonical IκB kinases TANK-binding kinase 1 and IKKε with amlexanox restores insulin sensitivity and glucose homeostasis in diabetic mice and human patients. Here we report that amlexanox improves diet-induced hypertriglyceridemia and hypercholesterolemia in Western diet–fed (WD-fed) Ldlr–/– mice and protects against atherogenesis. Amlexanox ameliorated dyslipidemia, inflammation, and vascular dysfunction through synergistic actions that involve upregulation of bile acid synthesis to increase cholesterol excretion. Transcriptomic profiling demonstrated an elevated expression of key bile acid synthesis genes. Furthermore, we found that amlexanox attenuated monocytosis, eosinophilia, and vascular dysfunction during WD-induced atherosclerosis. These findings demonstrate the potential of amlexanox as a therapy for hypercholesterolemia and atherosclerosis.

Dietary administration of L-carnitine during the fattening period of early feed restricted lambs modifies liver transcriptomic and plasma metabolomic profiles

Early feed restriction of lambs promotes a permanent mitochondrial dysfunction that impairs feed efficiency along the whole life. Dietary L-carnitine, a compound that plays an essential role in energy metabolism transferring long-chain fatty acids to mitochondria for subsequent β-oxidation, has demonstrated positive effects (e.g., improved markers or muscle/heart function) in early feed restricted lambs. The purpose of the present study is to ascertain the underlying mechanisms under these effects using samples of the same lambs in a liver transcriptomic and metabolomic approach. Twenty-two male Merino lambs were separated from the dams for 9 h daily to allow feed restriction during the suckling period. Once weaned, the lambs were allocated to a control group (CTRL, n = 11) being fed ad libitum a complete pelleted diet during the fattening phase, whereas the second group (CARN, n = 11) received the same diet formulated with 3 g/kg of L-carnitine. The most relevant results of this study revealed that the administration of 3 g of L-carnitine/ kg diet during the fattening period of early feed restricted lambs caused no differential expression of genes involved directly in β-oxidation. However the increased expression of genes related to the excretion of cholesterol as bile acids was in agreement with the higher amounts of glycodeoxycholic acid and other intermediates of cholesterol metabolism in the CARN group, and might have contributed to achieve an improved heart function. Moreover, the increased expression of ribosomes and translation pathways in the liver also allowed explaining the positive effects observed in the CARN group. It can be concluded that the improved markers or muscle/heart function achieved by the administration of 3 g of L-carnitine/kg diet to early feed restricted lambs are caused, mainly, by the increased expression of genes related either to ribosomes or cholesterol excretion.

Role of sortilin 1 (SORT1) on fatty acid-mediated cholesterol metabolism in primary calf hepatocytes.

Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism-related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum β-hydroxybutyrate concentration <0.6 mM) and cows diagnosed with clinical ketosis (serum β-hydroxybutyrate concentration >3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid-mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35-45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA.

Great Northern Beans (Phaseolus vulgaris L.) Lower Cholesterol in Hamsters Fed a High-Saturated-Fat Diet

BackgroundDietary interventions for high cholesterol, a primary risk factor for cardiovascular disease, are generally considered before prescribing drugs. ObjectiveThis study investigated the effects of whole Great Northern beans (wGNBs) and their hull (hGNB) incorporated into a high-saturated-fat (HSF) diet on cholesterol markers and hepatic/small intestinal genes involved in cholesterol regulation. MethodsEach of the 4 groups of 11 male golden Syrian hamsters at 9 wk old were fed a normal-fat [NF; 5% (wt:wt) of soybean oil], HSF [5% (wt:wt) of soybean oil + 10% (wt:wt) of coconut oil], HSF+5% (wt:wt) wGNB, or HSF+0.5% (wt:wt) hGNB diet for 4 wk. Cholesterol markers and expression of genes involved in cholesterol metabolism and absorption were analyzed from plasma, liver, intestinal, and fecal samples. Data were analyzed by 1-factor ANOVA and Pearson correlations. ResultsCompared with the HSF group, the HSF+wGNB group had 62% and 85% lower plasma and liver cholesterol and 3.6-fold and 1.4-fold greater fecal excretion of neutral sterol and bile acid, respectively (P ≤ 0.05). The HSF+hGNB group had 54% lower plasma triglycerides (P < 0.001) and 53% lower liver esterified cholesterol (P = 0.0002) than the HSF group. Compared with the HSF group, the expression of small intestinal Niemann-Pick C1 like 1 (Npc1l1), acyl-coenzyme A:cholesterol acyltransferase 2 (Acat2), and ATP binding cassette transporter subfamily G member 5 (Abcg5) were 75%, 70%, and 49% lower, respectively, and expression of hepatic 3-hydroxy-3-methylglutaryl CoA reductase (Hmgr) was 11.5-fold greater in the HSF+wGNB group (P ≤ 0.05). ConclusionsConsumption of wGNBs resulted in lower cholesterol concentration in male hamsters fed an HSF diet by promoting fecal cholesterol excretion, most likely caused by Npc1l1 and Acat2 suppression. The hGNB may partially contribute to the cholesterol-lowering effect of the wGNBs.

Lipid metabolism and retinal diseases

The retina has enormous lipids demands and must meet those needs. Retinal lipid homeostasis appears to be based on the symbiosis between neurons, Müller glial cells (MGC), and retinal pigment epithelium (RPE) cells, which can be impacted in several retinal diseases. The current research challenge is to better understand lipid-related mechanisms involved in retinal diseases, such as age-related macular degeneration (AMD) and glaucoma. In a first axis, in vitro and focus on Müller glial cell, we aimed to characterize whether the 24S-hydroxycholesterol (24S-OHC), an overexpressed end-product of cholesterol elimination pathway in neural tissue and likely produced by suffering retinal ganglion cells in glaucoma, may modulate MGC membrane organization, such as lipid rafts, to trigger cellular signalling pathways related to retinal gliosis. We have found that lipid composition appears to be a key factor of membrane architecture, especially for lipid raft microdomain formation, in MGC. However, 24S-OHC did not appear to trigger retinal gliosis via the modulation of lipid or protein composition within lipid rafts microdomains. This study provided a better understanding of the complex mechanisms involved in the pathophysiology of glaucoma. On a second clinical ax, we focused on the lipid-related mechanisms involved in the dysfunction of aging RPE and the appearance of drusenoid deposits in AMD. Using the Montrachet population-based study, we intend to report the frequency of reticular pseudodrusen (RPD) and its ocular and systemic risk factors, particularly related to lipid metabolisms, such as plasma lipoprotein levels, carotenoids levels, and lipid-lowering drug intake. Our study showed that RPD was less common in subjects taking lipid-lowering drugs. Lipid-lowering drugs, such as statins, may reduce the risk of RPD through their effect on the production and function of lipoproteins. This observation highlights the potential role of retinal lipid trafficking via lipoproteins between photoreceptors and retinal pigment epithelium cells in RPD formation. Those findings have been complemented with preliminary results on the analysis of plasma fatty acid (FA) profile, a surrogate marker of short-term dietary lipid intake, according to the type of predominant drusenoid deposit, soft drusen or RPD, in age-related maculopathy. Further research on lipid metabolism in retinal diseases is warranted to better understand the pathophysiology of retinal diseases and develop new promising diagnostic, prognostic, and therapeutic tools for our patients.

Chenodeoxycholic acid suppresses AML progression through promoting lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway and inhibiting M2 macrophage polarization

PurposeBile acids are steroid synthesized in liver, which are essential for fat emulsification, cholesterol excretion and gut microbial homeostasis. However, the role of bile acids in leukemia progression remains unclear. We aim at exploring the effects and mechanisms of chenodeoxycholic acid (CDCA), a type of bile acids, on acute myeloid leukemia (AML) progression. ResultsHere, we found that CDCA was decreased in feces and plasma of AML patients, positively correlated with the diversity of gut microbiota, and negatively associated with AML prognosis. We further demonstrated that CDCA suppressed AML progression both in vivo and in vitro. Mechanistically, CDCA bound to mitochondria to cause mitochondrial morphology damage containing swelling and reduction of cristae, decreased mitochondrial membrane potential and elevated mitochondrial calcium level, which resulted in the production of excessive reactive oxygen species (ROS). Elevated ROS further activated p38 MAPK signaling pathway, which collaboratively promoted the accumulation of lipid droplets (LDs) through upregulating the expression of the diacylglycerol O-acyltransferase 1 (DGAT1). As the consequence of the abundance of ROS and LDs, lipid peroxidation was enhanced in AML cells. Moreover, we uncovered that CDCA inhibited M2 macrophage polarization and suppressed the proliferation-promoting effects of M2 macrophages on AML cells in co-cultured experiments. ConclusionOur findings demonstrate that CDCA suppresses AML progression through synergistically promoting LDs accumulation and lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway caused by mitochondrial dysfunction in leukemia cells and inhibiting M2 macrophage polarization.

Effect of Adding L-carnitine to High-Fat/Low-Protein Diets of Common Carp (Cyprinus carpio) and the Mechanism of Regulation of Fat and Protein Metabolism.

L-carnitine is a low molecular weight amino acid that plays an essential role in the oxidation of long-chain fatty acids. The regulatory effects and molecular mechanisms of L-carnitine on fat and protein metabolism in common carp (Cyprinus carpio) were investigated in this study. Common carp (n = 270) were randomly divided into three groups and fed either (1) common carp diet, (2) high-fat/low-protein diet, or (3) L-carnitine-high-fat/low-protein diet. Growth performance, plasma biochemistry, muscle composition, and ammonia excretion rate were all examined after 8 weeks. Additionally, each group's hepatopancreas was subjected to transcriptome analysis. The results showed that decreasing the feed protein/fat ratio resulted in a considerable increase in feed conversion ratio and a significant decrease in common carp-specific growth rate to 1.19 ± 0.02 (P < 0.05). Similarly, total plasma cholesterol sharply increased to 10.15 ± 2.07, while plasma urea nitrogen, muscle protein, and ammonia excretion levels dropped (P < 0.05). After adding L-carnitine to the high-fat/low-protein diet, it was found that the specific growth rate and protein content of the dorsal muscle increased significantly (P < 0.05). In contrast, the plasma total cholesterol and ammonia excretion rate decreased considerably at most time points after feeding (P < 0.05). The expression of genes in the hepatopancreas differed substantially between the different groups. Through GO analysis, it was demonstrated that L-carnitine increased the ability of fat decomposition by up-regulating the expression of cpt1 in the hepatopancreas and decreased the expression of fasn and elovl6 to reduce the production and extension of lipids. Simultaneously, mtor was more abundant in the hepatopancreas, implying that L-carnitine can increase protein synthesis. According to the findings, adding L-carnitine to high-fat/low-protein diets can stimulate growth by enhancing lipolysis and protein synthesis.

Roles of ABCA1 in cancer.

Studies have indicated that anticancer drugs targeting cholesterol metabolism have clinical significance. From the perspective of the mechanism of cholesterol excretion from cells, ATP-binding cassette (ABC)A1 has an essential role that cannot be ignored. ABCA1 is located on the cell membrane and able to mediate the efflux of lipids, such as intracellular cholesterol, thereby initiating reverse cholesterol transport to reduce the intracellular cholesterol level. Therefore, inducing the expression of ABCA1 may become a new breakthrough point in cancer therapy.

Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion.

High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

Rats with high aerobic capacity display enhanced transcriptional adaptability and upregulation of bile acid metabolism in response to an acute high-fat diet.

Rats selectively bred for the high intrinsic aerobic capacity runner (HCR) or low aerobic capacity runner (LCR) show pronounced differences in susceptibility for high‐fat/high sucrose (HFHS) diet‐induced hepatic steatosis and insulin resistance, replicating the protective effect of high aerobic capacity in humans. We have previously shown multiple systemic differences in energy and substrate metabolism that impacts steatosis between HCR and LCR rats. This study aimed to investigate hepatic‐specific mechanisms of action via changes in gene transcription. Livers of HCR rats had a greater number of genes that significantly changed in response to 3‐day HFHS compared with LCR rats (171 vs. 75 genes: >1.5‐fold, p < 0.05). HCR and LCR rats displayed numerous baseline differences in gene expression while on a low‐fat control diet (CON). A 3‐day HFHS diet resulted in greater expression of genes involved in the conversion of excess acetyl‐CoA to cholesterol and bile acid (BA) synthesis compared with the CON diet in HCR, but not LCR rats. These results were associated with higher fecal BA loss and lower serum BA concentrations in HCR rats. Exercise studies in rats and mice also revealed higher hepatic expression of cholesterol and BA synthesis genes. Overall, these results suggest that high aerobic capacity and exercise are associated with upregulated BA synthesis paired with greater fecal excretion of cholesterol and BA, an effect that may play a role in protection against hepatic steatosis in rodents.

Central nervous system cholesterol metabolism in health and disease.

Cholesterol is a ubiquitous and essential component of cellular membranes, as it regulates membrane structure and fluidity. Furthermore, cholesterol serves as a precursor for steroid hormones, oxysterol, and bile acids, that are essential for maintaining many of the body's metabolic processes. The biosynthesis and excretion of cholesterol is tightly regulated in order to maintain homeostasis. Although virtually all cells have the capacity to make cholesterol, the liver and brain are the two main organs producing cholesterol in mammals. Once produced, cholesterol is transported in the form of lipoprotein particles to other cell types and tissues. Upon formation of the blood-brain barrier (BBB) during embryonic development, lipoproteins cannot move between the central nervous system (CNS) and the rest of the body. As such, cholesterol biosynthesis and metabolism in the CNS operate autonomously without input from the circulation system in normal physiological conditions. Nevertheless, similar regulatory mechanisms for maintaining cholesterol homeostasis are utilized in both the CNS and peripheral systems. Here, we discuss the functions and metabolism of cholesterol in the CNS. We further focus on how different CNS cell types contribute to cholesterol metabolism, and how ApoE, the major CNS apolipoprotein, is involved in normal and pathophysiological functions. Understanding these basic mechanisms will aid our ability to elucidate how CNS cholesterol dysmetabolism contributes to neurogenerative diseases.

Increased cholesterol absorption is associated with In-stent-restenosis after stent implantation for stable coronary artery disease

Background and aimsBlood cholesterol levels are regulated by competing mechanisms of cholesterol synthesis, absorption and excretion. Plant sterols are natural constituents of plants, are not synthesized in humans, and serve as markers for cholesterol absorption. Ezetimibe lowers the intestinal absorption of cholesterol and plant sterols. We analyzed the associations of differences in cholesterol metabolism, in particular increased cholesterol absorption, and the occurrence of in-stent restenosis (ISR) in patients with stable coronary artery disease. MethodsElective stent implantation of de novo stenosis was conducted in 59 patients (74.6 % males, 67.2 ± 9.6 years). Cholesterol and non-cholesterol sterols were quantified in serum samples by gas chromatography or mass spectrometry. ISR was assessed by optical coherence tomography (OCT) and quantitative angiography (QCA) after six months. ResultsMarkers for cholesterol absorption (e.g. campesterol-to-cholesterol) were positively associated with ISR measured by QCA (%diameter stenosis, late lumen loss) and OCT (proliferation volume, %area stenosis), whereas markers for cholesterol synthesis (e.g. lathosterol-to-cholesterol) were negatively associated with ISR (%area stenosis: r = -0.271, p = 0.043). There was no association between ISR and total cholesterol, LDL, HDL, triglycerides. Markers for cholesterol absorption (e.g. campesterol-to-cholesterol) were significantly lower in ezetimibe-treated patients compared to patients on a statin only (1.29 ± 0.69 vs. 2.22 ± 1.23; p = 0.007). Combined lipid-lowering with ezetimibe plus statin reduced ISR compared to statin only (13.7 ± 10.4 vs. 22.5 ± 12.1 %diameter stenosis, p = 0.015). ConclusionsDifferences in cholesterol metabolism, more specifically increased cholesterol absorption, are associated with ISR.

Extra virgin coconut oil (Cocos nucifera L.) exerts anti-obesity effect by modulating adiposity and improves hepatic lipid metabolism, leptin and insulin resistance in diet-induced obese rats

Few studies have addressed the effect of extra virgin coconut oil (E-VCO) in obesity. Therefore, this study investigated the anti-adiposity effect of E-VCO focusing on biochemical parameters and liver health of obese rats. E-VCO contains caprilyc, capric and lauric fatty acids and rutin, 3,4-dihydroxybenzoic acid, salicylic acid as major phenolic compounds. A total of 32 male Wistar rats were randomized into four groups: healthy; healthy treated with E-VCO; obese; obese treated with E-VCO. The rats consumed a control or cafeteria diet for 16 weeks. The treated groups received 3,000 mg/kg of E-VCO via gavage. E-VCO reduced body mass and adiposity index and improved hormonal parameters. E-VCO also decreased the accumulation of liver fat, hepatic cholesterol and triglyceride levels, increased faecal cholesterol excretion and decreased faecal triglycerides excretion. E-VCO had an anti-adiposity effect and reversed changes in liver and adipose tissues induced by obesity with improvements in metabolic parameters.

Blood lipids lowering effect of medicinal plants

Hyperlipidemia refers to elevated levels of lipids and cholesterol in the blood. It plays an important role in the development of atherosclerosis, the main cause of death in the world. Medicinal plants can lower blood lipids by many mechanisms included inhibition of the expression of fatty acid synthase, decreasing free fatty acid release, inhibition of HMG-CoA reductase, increasing the fecal excretion of fat and cholesterol, inhibition of the activity of pancreatic lipase and inhibition of cholesterol absorption. The current review will highlight the hypolipidemic effects of medicinal plants as promising effective and safe therapies.

The Association between Serum Total Bile Acid Level and Long-Term Prognosis in Patients with Coronary Chronic Total Occlusion Undergoing Percutaneous Coronary Intervention.

Background and Aims Bile acids, the key products for elimination of cholesterol, play an important role in coronary artery disease (CAD). However, few studies focused on the roles of more accessible serum total bile acids (TBA) in the prediction of adverse cardiovascular events for coronary chronic artery occlusion (CTO). The aim of this study was to explore the potential relationship between serum TBA and long-term prognosis in patients with CTO undergoing percutaneous coronary intervention (PCI). Methods Baseline TBA was determined in 613 patients with CTO after PCI in the present study. All patients were divided into 3 groups according to the median (3.5 μmol/l) and the normal upper limit of the TBA (10 μmol/l). The primary endpoint was all-cause mortality, and the secondary endpoint was major adverse cardiovascular events (MACE). Results Average age in this study was 65.44 ± 9.94 years old. The median of TBA was 3.5 (2.1-6.1) μmol/l. Over a median follow-up of 33.5 months, compared to those with below 3.5 μmol/l TBA, 3.5 ~ 10 μmol/l TBA was associated with significantly reduced risk for the MACE (hazard ratio (HR): 0.59, 95% confidence interval (CI): 0.40 to 0.88; p = 0.009) even after adjustment for baseline variables. However, TBA did not predict all-cause mortality and cardiovascular death. Spline analyses showed an L-shaped relationship of the serum TBA with the incidence of MACE. Conclusions Moderate fasting serum TBA level has a predictive value for MACE even after adjusting for lifestyle and clinical risk factors in CTO patients undergoing PCI.

Co-Amorphous Formation of Simvastatin-Ezetimibe: Enhanced Physical Stability, Bioavailability and Cholesterol-Lowering Effects in LDLr-/-Mice.

Hypercholesterolemia is one of the independent risk factors for the development of cardiovascular diseases such as atherosclerosis. The treatment of hypercholesterolemia is of great significance to reduce clinical cardiovascular events and patient mortality. Simvastatin (SIM) and ezetimibe (EZE) are commonly used clinically as cholesterol-lowering drugs; however, their treatment efficacy is severely affected by their poor water solubility and low bioavailability. In this study, SIM and EZE were made into a co-amorphous system to improve their dissolution, oral bioavailability, storage stability, and cholesterol-lowering effects. The SIM-EZE co-amorphous solids (CO) were prepared successfully using the melt-quenched technique, and the physicochemical properties of CO were characterized accordingly, which exhibited improved physical stability and faster dissolution release profiles than their physical mixture (PM). In the pharmacokinetic study, the SIM-EZE CO or PM was given once by oral gavage, and mouse blood samples were collected retro-orbitally at multiple time points to determine the plasma drug concentrations. In the pharmacodynamic study, low-density lipoprotein receptor-deficient (LDLr−/−) mice were fed with a high-fat diet (HFD) for two weeks to establish a mouse model of hypercholesterolemia. Using PM as a control, we investigated the regulation of CO on plasma lipid levels in mice. Furthermore, the mice feces were collected to determine the cholesterol contents. Besides, the effect of EZE on the NPC1L1 mRNA expression level in the mouse intestines was also investigated. The pharmacokinetics results showed that the SIM-EZE CO has improved bioavailability compared to the PM. The pharmacodynamic studies showed that SIM-EZE CO significantly increased the cholesterol-lowering effects of the drugs compared to their PM. The total cholesterol excretion in the mouse feces and inhibitory effect on NCP1L1 gene expression in the mouse intestines after being given the SIM-EZE CO were more dramatic than the PM. Our study shows that the SIM-EZE CO prepared by the melt-quenched method can significantly improve the stability, bioavailability, and cholesterol-lowering efficacy with excellent development potential as a new drug formulation.

Decrease in Aflatoxin M1 Concentration in Milk during Cholesterol Removal by Application of β-Cyclodextrin.

Approximately one-third of humankind is chronically exposed to the carcinogenic aflatoxin M1 contained in milk. As β-cyclodextrin is frequently used in the food industry, its effect on aflatoxin M1 concentration was investigated during cholesterol removal from milk due to the similarity among the physicochemical properties of aflatoxin M1 and cholesterol. Moreover, the elimination of cholesterol using β-cyclodextrin has been successfully applied in many studies without any substantial effect on the quality of the treated milk. Therefore, milk samples were spiked with aflatoxin M1 within the range from 0.20 to 2.00 µg/kg, and cholesterol removal was carried out by 2.0% (w/w) β-cyclodextrin addition, as this concentration is enough for the sufficient removal of cholesterol. It was found that the mean cholesterol concentration decreased by 92.3%, while the aflatoxin M1 concentration decreased to 0.53 ± 0.04 µg/kg, i.e., by 39.1% after treatment (n = 2). This mitigation procedure itself is easy and inexpensive and thus is fully applicable with a high potential for complete decontamination of aflatoxin M1 milk. This method will therefore considerably improve the food safety issues associated with aflatoxin M1 presence in milk and dairy products.

Whey protein hydrolysate alleviated atherosclerosis and hepatic steatosis by regulating lipid metabolism in apoE-/- mice fed a Western diet

Whey protein hydrolysate (WPH) has been proved to possess various biological activities associated with the amelioration of cardiovascular disease (CVD). The objective of this study was to investigate the anti-atherosclerotic and hepatoprotective effects of WPH on apolipoprotein E knockout (apoE-/-) mice fed with a Western diet for 15 weeks. Results revealed that WPH markedly inhibited the development of atherosclerotic lesions in the aorta and steatosis injury in the liver. The serum lipid and inflammation levels were both reduced after WPH supplemented in apoE-/- mice. In addition, WPH inhibited the lipid accumulation in the liver, thereby decreasing the hepatic inflammation level and oxidative stress injury. Mechanism investigation revealed that WPH down-regulated the expression of cholesterol biosynthesis genes while up-regulated the expression of cholesterol uptake and excretion genes in the liver. Meanwhile, the de novo lipogenesis was inhibited while the fatty acids β-oxidation was activated in the liver by WPH supplementation. Notably, the n-3 polyunsaturated fatty acid (PUFA)/n-6 PUFA ratio in serum and liver of the WPH-H group were 2.69-fold (p < 0.01) and 3.64-fold (p < 0.01) higher than that of the Model group. Collectively, our results proved WPH possesses potent anti-atherosclerotic and hepatoprotective activities and has the potential to be used as a novel functional ingredient for the management of CVD.

Abstract 439: Abcg5 Abcg8 Are Essential To Maintain Sterol Balance When Challenged With Dietary Cholesterol In Mice

Background: ABCG5/ABCG8 (G5/G8) is the primary biliary cholesterol transporter and accounts for &gt;70% of biliary cholesterol secretion. Mice and humans maintain fecal cholesterol excretion under a variety of conditions that disrupt biliary cholesterol secretion, indicating the presence of a non-biliary route for cholesterol elimination. Cholesterol can be actively secreted from the intestine, a pathway termed transintestinal cholesterol excretion (TICE). The present study investigates rates of intestinal cholesterol secretion in mice lacking G5/G8 to determine if adaptive responses within the gut facilitate cholesterol excretion in the absence of the primary biliary cholesterol transporter. Methods: Wild-type (WT) and whole body Abcg5/Abcg8 deficient mice (G5/G8 KO) were maintained on a plant sterol free (PSF) diet to prevent the development of experimental sitosterolemia and potential confounding effects of biologically active phytosterols. Mice were anesthetized, the gall bladder cannulated, and the intestine perfused with model bile to simultaneously determine biliary and intestinal cholesterol secretion rates. Sterol balance studies were conducted in mice sequentially maintained on PSF diet, a PSF diet supplemented with 0.2% cholesterol, and standard rodent chow. Mice were allowed to acclimate to each diet for 14 days, then placed on wire-bottom cages for fecal collection. At termination of the experiment, basal bile, plasma, and tissues were collected. Fecal neutral sterols (FNS) were analyzed by GC-MS. Results: Despite a substantial reduction in biliary cholesterol secretion in G5/G8 KO mice, differences in intestinal cholesterol secretion rates were only observed in males, with KO mice excreting less cholesterol into the intestinal lumen. FNS did not differ between genotypes when maintained on the PSF diet. On chow diet, FNS increased 2x in both genotypes, but did not differ across genotypes. When PSF diet was supplemented with 0.2% cholesterol, FNS increased by 3x in WT mice, but were unchanged in G5/G8 KO mice, relative to PSF diet levels. Conclusions: Abcg5/Abcg8 is essential to maintain sterol balance when cholesterol is present in the diet. TICE is unable to compensate for Abcg5/Abcg8 deficiency in cholesterol fed mice.

Abstract 452: Classification And Effect Of Correctors On ABCG8 Sitosterolemia Associated Cytosolic Mutants

Background: Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia. Methods: We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface. Results: Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent. Conclusions: Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators &amp; correctors that have shown to be effective in disease-causing mutations in other ABC transporters.