Conversion Of Cholesterol Research Articles

High soluble endoglin levels regulate cholesterol homeostasis and bile acids turnover in the liver of transgenic mice

AimsIncreased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. Main methodsNine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. Key findingssEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. SignificanceFor the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.

Iron overload reduces synthesis and elimination of bile acids in rat liver

Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.

Effects of dietary ascorbic acid levels on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss)

An 80‐day feeding trial was conducted to evaluate the effects of dietary ascorbic acid (AA) supplementation at different levels (0, 0.2 and 0.4 g/kg l‐ascorbate‐2‐polyphosphate; 7.6, 77.2 and 146.7 mg/kg AA, respectively) on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss). Dietary AA supplementation regardless of inclusion level increased the serum total cholesterol and low‐density lipoprotein (LDL) cholesterol levels and LDL cholesterol/high‐density lipoprotein cholesterol ratio. No significant differences were observed in the serum triiodothyronine and thyroxine levels, faecal cholesterol content, hepatic activity and mRNA expression of acyl‐coenzyme A:cholesterol acyltransferase among the dietary treatments. Dietary AA inclusion increased the faecal bile acid content, hepatic activity and mRNA expression of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and hepatic cholesterol 7α‐hydroxylase mRNA expression, but decreased the hepatic LDL receptor content. High level of AA supplementation (0.4 g/kg) depressed the serum cortisol levels. These results suggest that dietary supplementation with 0.2‒0.4 g/kg l‐ascorbate‐2‐polyphosphate may increase the serum total cholesterol level in rainbow trout. The cholesterol‐raising effect of AA may be due to the increased hepatic cholesterol production and the depressed cholesterol clearance from serum. In addition, dietary AA inclusion also facilitates the hepatic conversion of cholesterol to bile acids.

Abstract LB-295: Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma

Abstract BACKGROUND Tumor cells display higher cholesterol levels compared to normal cells and emerging evidence indicates a pivotal role of cholesterol content in tumor cell proliferation and growth mediated by a dysregulation of key regulators involved in cholesterol homeostasis has been implicated in various cancers. In this study, we assessed the dependency of glioblastoma (GBM) cells to extracellular cholesterol levels for growth. Through analysis of whole-transcriptome databases, we identified the cholesterol homeostasis-related genes whose expression are dysregulated in gliomas. One of the most dramatically down-regulated genes identified encodes cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol via conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC). METHODS Molecular and clinical data was obtained from publicly genomic databases. Immunohistochemistry was applied to assess the protein level of CYP46A1 in tissue microarrays. Functionally, CYP46A1 overexpression was determined using lentiviral constructs in LN229 and a primary glioblastoma cell line (GBM#P3). Cells were characterized in vitro and implanted in vivo to generate orthotopic xenografts in order to assess their molecular status, cell survival, and self-renewal by western blotting, flow cytometry, and extreme limiting dilution assay (ELDA), respectively. RESULTS Down-regulation of CYP46A1 was found in malignant glioma samples compared to normal brain tissue. Importantly, reduced CYP46A1 expression was associated with aggressive clinicopathological features and poor prognosis of GBM patients. In functional assays, restoration of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. Consistent, treatment of GBM cells with 24OHC inhibited tumour growth and induced apoptosis in a concentration dependent manner. RNA-seq data showed that 24OHC regulated LXR and sterol regulatory-element binding protein 1 (SREBP1) signalling activity. Intriguingly, efavirenz (EFV), a widespread used anti-HIV medication that effectively penetrates the blood–brain barrier, has recently been reported as a potent activator of the CYP46A1 enzyme. The therapeutic potential of EFV is currently being assessed in vivo and the data will be presented. CONCLUSIONS Collectively, our findings demonstrate that CYP46A1 is a critical cellular cholesterol regulator in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target in GBM. Citation Format: Mingzhi Han, Shuai Wang, Xingang Li, Jian Wang, Rolf Bjerkvig. Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-295.

1965-P: Glucagon Receptor Agonism Stimulates Body Weight Loss in Antibiotic Treatment in Obese Mice

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss in diet-induced obese (DIO) mice. Glucagon’s role as a primary counterregulatory hormone to insulin action has long received scientific attention, yet its broader therapeutic potential is an evolving research interest. We reported that chronic glucagon receptor (GCGR) activation increased energy expenditure and plasma bile acids (BA) in an farnesoid X receptor (FXR) dependent manner. Glucagon stimulates the conversion of cholesterol to BAs, with an increase in cholic and chenodeoxycholate species, critical regulators of FXR activity. Gut microbiota are known to alter the chemical structure of bile acids to produce secondary and tertiary BA. Thus, in these studies we tested the hypothesis that GCGR agonism regulates the gut microbiome to alter circulating BAs and stimulate FXR. GCGR agonism stimulated a profound change in the gut microbiome of DIO mice. These changes were largely associated with an increase in Actinobacteria and a decrease in Deferribacteria phylums. Assessment of functional microbiome composition via PICRUSt uncovered significant impacts on 130 out of 160 total KEGG pathways, including those involved in BA synthesis. To investigate the role of the gut microbiome on GCGR-stimulated weight loss we treated DIO mice with an antibiotic (AB) cocktail (ampicillin, vancomycin, metronidazole, neomycin, and amphotericin B) or vehicle. Mice were then treated with the GCGR agonist, IUB288, or vehicle for 14d to stimulate weight-loss. Surprisingly, AB-treated mice lost a similar amount of body-weight when treated with IUB288, suggesting that the gut microbiome may be dispensable for the weight loss effect. However, post hoc analysis of the cecal microbiome identified only a partial ablation of microbiota in AB-treated mice, with unexpected increases in Proteobacteria and Tenericutes. Thus, it is possible that GCGR-agonism acts via one of the resistant microbes to regulate weight loss. Disclosure T. Kim: None. J.P. Antipenko: None. S. Nason: None. N. Presedo: None. W.J. Van Der Pol: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. C.D. Morrow: None. K.M. Habegger: Consultant; Self; Glyscend, Inc., Novo Nordisk Inc. Research Support; Self; Glyscend, Inc. Stock/Shareholder; Self; Glyscend, Inc. Funding National Institutes of Health

Milk Polar Lipids Reduce Cholesterolemia by Decreasing Cholesterol Absorption in Humans (P06-041-19)

ObjectivesNutritional 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. MethodsWe 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 cohort whereas 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 8h-postprandial tests after consuming 0g-, 3g- or 5g-MPL enriched cheese labelled with 2H-cholesterol tracer. Plasma, chylomicrons and ileal efflux were analyzed. ResultsMilk fat enriched with 3 to 5 g MPL induced dose-response 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 2H-cholesterol 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). ConclusionsPresent results suggest that milk polar lipids decrease cholesterol absorption in humans through interactions with sphingomyelin and by increasing conversion of cholesterol to coprostanol. Funding SourcesANR (French National Research Agency, VALOBAB project, ANR-11-ALID-007–01), PHRC-I (French Clinical Research Program, 14–007), CNIEL (French Dairy Interbranch Organization).

Deficient pregnenolone synthesis associated with congenital adrenal hyperplasia and organelle dysfunction.

SummarySteroid hormones are essential for the survival of all mammals. In adrenal glands and gonads, cytochrome P450 side chain cleavage enzyme (SCC or CYP11A1), catalyzes conversion of cholesterol to pregnenolone. We studied a patient with ambiguous genitalia by the absence of Müllerian ducts and the presence of an incompletely formed vagina, who had extremely high adrenocorticotropic hormone (ACTH) and reduced pregnenolone levels with enlarged adrenal glands. The testes revealed seminiferous tubules, stroma, rete testis with interstitial fibrosis and reduced number of germ cells. Electron microscopy showed that the patient’s testicular mitochondrial size was small with little SCC expression within the mitochondria. The mitochondria were not close to the mitochondria-associated ER membrane (MAM), and cells were filled with the microfilaments. Our result revealed that absence of pregnenolone is associated with organelle stress, leading to altered protein organization that likely created steric hindrance in testicular cells.Learning points:Testes revealed seminiferous tubules, stroma, rete testis with interstitial fibrosis and reduced number of germ cells;Testicular mitochondrial size was small with little SCC expression within the mitochondria;Absence of pregnenolone is associated with organelle stress.

Evidence of Disturbed Gut Microbial Metabolic Activity in Pediatric Crohn’s Disease

AbstractBackgroundDysbiosis, that is, disturbed gut microbial balance, is well documented in Crohn’s disease (CD). We aimed at studying CD-linked dysbiosis in children by analyzing fecal microbe-associated characteristics, previously not reported in children.MethodsThis observational study included 28 children with active CD and healthy controls. We assessed the following three indicators of gut microbiota metabolism in the feces: the presence of tryptic activity, the conversion of cholesterol to coprostanol, and the conversion of bilirubin to urobilinogen.ResultsThe fecal tryptic activity was significantly higher in children with active CD compared to the control group (P &amp;lt; 0.01). The fecal coprostanol of the CD children was close to zero and differed significantly from the controls (P &amp;lt; 0.001). Furthermore, the children with CD had very low fecal urobilinogen, differing significantly from the control group (P &amp;lt; 0.001).ConclusionsThe significant differences in levels of fecal bacterial metabolites in patients with active CD compared to healthy controls reflect major perturbation of gut microbial functions and have not previously been reported in children. This fits well with the prevailing concept of a dysbiotic gut microbiota in CD and may have important clinical implications by bringing the dysbiosis back into balance.

Advances in understanding the regulatory mechanism of cholesterol 7α-hydroxylase

The conversion of cholesterol to bile acids (BAs) contributes to the elimination of total cholesterol from the body. In addition, manipulating BA homeostasis by modulating cholesterol 7α-hydroxylase (CYP7A1) may affect the metabolic processing of cholesterol, exerting therapeutic effects on hypercholesterolemia and cardiovascular diseases. Multiple mechanisms (such as various nuclear receptors and regulatory factors) are involved in CYP7A1 modulation. Recently, microRNAs, protein degradation pathways, and the gut microbiota have been identified to participate in these sophisticated networks. In this review, research progress on the regulatory mechanism of CYP7A1 is summarized.

Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli.

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3nmol pregnenolone/h per 1mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR-Adx-P450scc to P450scc-Adx-AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05-0.62mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

Effectiveness of using ursodeoxycholic acid in reducing incidence of gallstone formation after sleeve gastrectomy

Background: Obese persons are at risk for cholesterol gallstones because of high saturation of cholesterol in their bile. About 75% of gallstones are of cholesterol type. To reduce the risk for gallstone formation, policies of prophylactic cholecystectomy are proposed. However, this is not widely accepted. A routine cholecystectomy during bariatric surgery is recommended by some centers. Ursodeoxycholic acid enhances the conversion of cholesterol to bile acids. It also enhances cholesterol transport as liquid crystals. Furthermore, it has an inhibitory action on the prostaglandins and biliary glycoprotein, which is the possible explanation for its action on decreasing the saturation index. The objective was the effectiveness of using Ursodeoxycholic Acid (UDCA) in reducing incidence of gallstone formation after sleeve gastrectomy.Methods: 50 morbid obese patients divided into 2 groups. Group A: 30 patients who received UDCA post-operative. (5 male and 25 female) with mean age 34.8year. Patients had a body mass index (BMI) with mean BMI 46.9kg/m2. Group B: 20 patients without receiving UDCA post-operative. (2 male and 18 female) with mean age 32.9year. Patients had a mean of BMI 47.3kg/m2.Results: Using UDCA has significant role in prophylaxis against development of gall stone formation. Patients who received UDCA developed gall stones by a percentage 0% (0/30) while patients who hadn’t received UDCA developed gall stones by a percentage 4.7% (3/20).Conclusions: The use of UCDA effectively reduced the incidence of gall stone formation after laparoscopic sleeve gastrectomy (LSG) in patient with morbid obesity.

A sulfated polysaccharide from Gracilaria Lemaneiformis regulates cholesterol and bile acid metabolism in high-fat diet mice.

This study aimed to evaluate the regulation of lipid metabolism and mechanism of a sulfated polysaccharide from Gracilaria Lemaneiformis (GLP). The results showed that GLP significantly decreased the hepatic total cholesterol (TC) and triglyceride (TG) levels in the serum of high-fat diet mice and prevented body weight gain and alleviated the accumulation of fat. Furthermore, GLP may accelerate the conversion of cholesterol to bile acids by promoting the expression of LxRα and CYP7A1 genes and regulating the relative abundance of Bacteroides, Ruminococcus_1 and Lactobacillus. Pearson's correlation analysis showed that the relative abundance of Alistipes, Prevotellaceae_UCG-001, and Corprococcus_1 was negatively associated with the hydrophobic BAs CDCA and DCA levels, while the relative abundance of Lachnospiraceae_NK4A136_group and Roseburia was positively correlated with the hydrophilic BAs UDCA and TUDCA levels. This study suggests that GLP can be used as a functional food for regulating cholesterol metabolism.

CYP46A1 expression modulates mitochondrial function and integrity in Niemann-Pick type C disease

CYP46A1 expression modulates mitochondrial function and integrity in Niemann-Pick type C disease

RNAi of Sterol Methyl Transferase1 Reveals its Direct Role in Diverting Intermediates Towards Withanolide/Phytosterol Biosynthesis in Withania somnifera.

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S-adenosyl l-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes.

Rosuvastatin improves the FGF19 analogue NGM282-associated lipid changes in patients with non-alcoholic steatohepatitis

NGM282, an engineered analogue of the gut hormone FGF19, improves hepatic steatosis and fibrosis biomarkers in patients with non-alcoholic steatohepatitis (NASH). However, NGM282 increases serum cholesterol levels by inhibiting CYP7A1, which encodes the rate-limiting enzyme in the conversion of cholesterol to bile acids. Herein, we investigate whether administration of a statin can manage the cholesterol increase seen in patients with NASH receiving treatment with NGM282. In this phase II, open-label, multicenter study, patients with biopsy-confirmed NASH were treated with subcutaneous NGM282 once daily for 12 weeks. After 2 weeks, rosuvastatin was added in stepwise, biweekly incremental doses to a maximum of 40 mg daily. Both drugs were continued until the end of treatment at week 12. We evaluated plasma lipids, lipoprotein particles and liver fat content. In 66 patients who received NGM282 0.3 mg (n = 23), NGM282 1 mg (n = 21), or NGM282 3 mg (n = 22), circulating cholesterol increased from baseline at week 2. Initiation of rosuvastatin resulted in rapid decline in plasma levels of total cholesterol and low-density lipoprotein cholesterol. At week 12, reductions from baseline in total cholesterol levels of up to 18% (p <0.001), low-density lipoprotein cholesterol of up to 28% (p <0.001), triglycerides of up to 34% (p <0.001) and an increase in high-density lipoprotein cholesterol of up to 16% (p <0.001), with similar changes in lipoprotein particles, were observed in these patients. Robust decreases from baseline in 7alpha-hydroxy-4-cholesten-3-one (p <0.001) and liver fat content (p <0.001) were also observed. Rosuvastatin was safe and well-tolerated when co-administered with NGM282 in patients with NASH. In this multicenter study, NGM282-associated elevation of cholesterol was effectively managed with rosuvastatin. Co-administration of rosuvastatin with NGM282 may be a reasonable strategy to optimize the cardiovascular risk profile in patients with NASH. Non-alcoholic steatohepatitis (NASH) represents a large and growing public health concern with no approved therapy. NGM282, an engineered analogue of the gut hormone FGF19, reduces liver fat, liver injury and inflammation in patients with NASH. However, NGM282 increases cholesterol levels. Here we show that co-administration of a statin can manage the cholesterol increase seen in patients with NASH receiving treatment with NGM282, producing a favorable overall lipid profile.

Impact of colonic fermentation on sterols after the intake of a plant sterol-enriched beverage: A randomized, double-blind crossover trial

Cholesterol microbial transformation has been widely studied using invitro fermentation assays, but less information is available on the biotransformation of plant sterols (PS). Theexcretion percentage of animal sterols (AS) (67-73%) is considerably greater than that of PS (27-33%) in feces from healthy humans following a Western diet. However, a lower content of AS in feces from subjects following a vegetarian, vegan or low-fat animal diet has been seen when compared to omnivorous subjects. Although only one human study has reported fecal sterol excretion after the consumption of PS-enriched food (8.6gPS/day), it was found that the target group showed an increase in the excretion of cholesterol and a 57% decrease in its metabolites compared to the control group. Evaluation of the impact of a PS-enriched milk based fruit beverage intake on fecal sterol excretion and the microbial conversion of sterols in postmenopausal women with mild hypercholesterolemia. Forty postmenopausal women participated in a randomized, double-blind, crossover study with two beverages, with a PS-enriched (2gPS/day) or without. The women were divided in two groups: 20 women consumed the PS-enriched beverage and the other 20 women consumed a placebo (without PS) beverage for 6 weeks. After a four-week washout period, the type of beverage was exchanged and consumed for another 6 weeks. Feces were collected at the start (0 and 10 weeks) and end of each intervention period (6 and 16 weeks), and fecal sterols were determined by capillary gas chromatography with mass spectrometry. The intake of the PS-enriched beverage modified the fecal sterol excretion profile. A significant increase mainly in PS and their metabolites versus the placebo intervention period was observed. Although the same effect was not observed in the case of AS, a tendency towards increased cholesterol and decreased coprostanol (the main metabolite of cholesterol) was recorded after PS-enriched beverage intake versus placebo. Furthermore, the PS-enriched beverage also modified the microbial conversion of sterols. In this context, an important decrease in the conversion percentage of cholesterol in 16 women (between 11% and 50%) and of sitosterol in 24 women (between 15% and 61%) was observed. The results obtained suggest that the microbiota could preferably use PS as a substrate, when present in a greater proportion compared with cholesterol. Besides, a lower sitosterol and cholesterol conversion trend would mean that intake of the PS-enriched beverage could modulate the metabolic activity of the gut microbiota. Therefore, further studies on the impact of PS-enriched foods upon gut microbiota modulation are needed. Clinical Trial Registry Number: NCT02065024 listed on the NIH website: ClinicalTrials.gov. Clinical Trial Registry Name: Food Matrix and Genetic Variability as Determinants of Bioavailability and Biological Effects of Beta-cryptoxanthin and Phytosterols (foodmagenpol). The full trial protocol is available upon request to the corresponding author.

Aberrant Splicing Is the Pathogenicity Mechanism of the p.Glu314Lys Variant in CYP11A1 Gene.

Context: The cholesterol side chain cleavage enzyme (CYP11A1) catalyzes the conversion of cholesterol to pregnenolone, the first rate-limiting step of steroidogenesis. CYP11A1 mutations are associated with primary adrenal insufficiency (PAI) as well as disorders of sex development (DSD) in 46,XY patients.Objective: To define the pathogenicity mechanism for the p.Glu314Lys variant, previously reported, and found in four additional patients with CYP11A1 deficiency.Subjects and Methods: DNA of four patients presenting with delayed PAI and/or 46,XY DSD were studied by Sanger or Massively Parallel sequencing. Three CYP11A1 mutations were characterized in vitro and in silico, and one by mRNA analysis on testicular tissue.Results: All patients were compound heterozygous for the previously described p.Glu314Lys variant. In silico studies predicted this mutation as benign with no effect on splicing but mRNA analysis found that it led to incomplete exon 5 skipping. This mechanism was confirmed by minigene experiment. The protein carrying this mutation without exon skipping should conserve almost normal activity, according to in vitro studies. Two other mutations found in trans, the p.Arg120Gln and p.Arg465Trp, had similar activity compared to negative control, consistent with the in silico studies.Conclusions: We provide biological proof that the p. Glu314Lys variant is pathogenic due to its impact on splicing and seems responsible for the moderate phenotype of the four patients reported herein. The present study highlights the importance of considering the potential effect of a missense variant on splicing when it is not predicted to be disease causing.

HDL activates expression of genes stimulating cholesterol efflux in human monocyte-derived macrophages

High density lipoproteins (HDL) are key components of reverse cholesterol transport pathway. HDL removes excessive cholesterol from peripheral cells, including macrophages, providing protection from cholesterol accumulation and conversion into foam cells, which is a key event in pathogenesis of atherosclerosis. The mechanism of cellular cholesterol efflux stimulation by HDL involves interaction with the ABCA1 lipid transporter and ensuing transfer of cholesterol to HDL particles. In this study, we looked for additional proteins contributing to HDL-dependent cholesterol efflux. Using RNAseq, we analyzed mRNAs induced by HDL in human monocyte-derived macrophages and identified three genes, fatty acid desaturase 1 (FADS1), insulin induced gene 1 (INSIG1), and the low-density lipoprotein receptor (LDLR), expression of which was significantly upregulated by HDL. We individually knocked down these genes in THP-1 cells using gene silencing by siRNA, and measured cellular cholesterol efflux to HDL. Knock down of FADS1 did not significantly change cholesterol efflux (p = 0.70), but knockdown of INSIG1 and LDLR resulted in highly significant reduction of the efflux to HDL (67% and 75% of control, respectively, p < 0.001). Importantly, the suppression of cholesterol efflux was independent of known effects of these genes on cellular cholesterol content, as cells were loaded with cholesterol using acetylated LDL. These results indicate that HDL particles stimulate expression of genes that enhance cellular cholesterol transfer to HDL.

Modulation of cholesterol-related sterols during Eimeria bovis macromeront formation and impact of selected oxysterols on parasite development

Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1.2%) supplementation. To analyze the impact of extensive E. bovis proliferation on host cellular sterol metabolism we here compared the sterol profiles of E. bovis-infected primary endothelial host cells grown under optimized (1.2% FCS) and non-optimized (10% FCS) cell culture conditions. Therefore, several sterols indicating endogenous de novo cholesterol synthesis, cholesterol conversion and sterol uptake (phytosterols) were analyzed via GC–MS-based approaches. Overall, significantly enhanced levels of phytosterols were detected in both FCS conditions indicating infection-triggered sterol uptake from extracellular sources as a major pathway of sterol acquisition. Interestingly, a simultaneous induction of endogenous cholesterol synthesis based on increased levels of distinct cholesterol precursors was only observed in case of optimized parasite proliferation indicating a parasite proliferation-dependent effect. Considering side-chain oxysterols, 25 hydroxycholesterol levels were selectively found increased in E. bovis-infected host cells, while 24 hydroxycholesterol and 27 hydroxycholesterol contents were not significantly altered by infection. Exogenous treatments with 25 hydroxycholesterol, 27 hydroxycholesterol, and 7 ketocholesterol revealed significant adverse effects on E. bovis intracellular development. Thus, the number and size of developing macromeronts and merozoite I production was significantly reduced indicating that these oxysterols bear direct or indirect antiparasitic properties. Overall, the current data indicate parasite-driven changes in the host cellular sterol profile reflecting the huge demand of E. bovis for cholesterol during macromeront formation and its versatility in the acquisition of cholesterol sources.

Liver X receptors in lipid signalling and membrane homeostasis.

Liver X receptors α and β (LXRα and LXRβ) are nuclear receptors with pivotal roles in the transcriptional control of lipid metabolism. Transcriptional activity of LXRs is induced in response to elevated cellular levels of cholesterol. LXRs bind to and regulate the expression of genes that encode proteins involved in cholesterol absorption, transport, efflux, excretion and conversion to bile acids. The coordinated, tissue-specific actions of the LXR pathway maintain systemic cholesterol homeostasis and regulate immune and inflammatory responses. LXRs also regulate fatty acid metabolism by controlling the lipogenic transcription factor sterol regulatory element-binding protein 1c and regulate genes that encode proteins involved in fatty acid elongation and desaturation. LXRs exert important effects on the metabolism of phospholipids, which, along with cholesterol, are major constituents of cellular membranes. LXR activation preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids by inducing transcription of the remodelling enzyme lysophosphatidylcholine acyltransferase 3. The ability of the LXR pathway to couple cellular sterol levels with the saturation of fatty acids in membrane phospholipids has implications for several physiological processes, including lipoprotein production, dietary lipid absorption and intestinal stem cell proliferation. Understanding how LXRs regulate membrane composition and function might provide new therapeutic insight into diseases associated with dysregulated lipid metabolism, including atherosclerosis, diabetes mellitus and cancer.