Conversion Of Cholesterol Research Articles

Identification and characterization of select oxysterols as ligands for GPR17.

G-protein coupled receptor 17 (GPR17) is an orphan receptor involved in the process of myelination, due to its ability to inhibit the maturation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Despite multiple claims that the biological ligand has been identified, it remains an orphan receptor. Seventy-seven oxysterols were screened in a cell-free [35 S]GTPγS binding assay using membranes from cells expressing GPR17. The positive hits were characterized using adenosine 3',5' cyclic monophosphate (cAMP), inositol monophosphate (IP1) and calcium mobilization assays, with results confirmed in rat primary oligodendrocytes. Rat and pig brain extracts were separated by high-performance liquid chromatography (HPLC) and endogenous activator(s) were identified in receptor activation assays. Gene expression studies of GPR17, and CYP46A1 (cytochrome P450 family 46 subfamily A member 1) enzymes responsible for the conversion of cholesterol into specific oxysterols, were performed using quantitative real-time PCR. Five oxysterols were able to stimulate GPR17 activity, including the brain cholesterol, 24(S)-hydroxycholesterol (24S-HC). A specific brain fraction from rat and pig extracts containing 24S-HC activates GPR17 in vitro. Expression of Gpr17 during mouse brain development correlates with the expression of Cyp46a1 and the levels of 24S-HC itself. Other active oxysterols have low brain concentrations below effective ranges. Oxysterols, including but not limited to 24S-HC, could be physiological activators for GPR17 and thus potentially regulate OPC differentiation and myelination through activation of the receptor.

Unhatched bovine blastocysts express all transcripts of the estrogen biosynthetic pathway, but steroid hormone synthesis could not yet be demonstrated

Early embryos of rodent species and rabbits but also farm animals such as pigs, horses and cattle produce estrogens, which are considered important regulators of the implantation process. In cattle, the exact stage at which embryonic estrogen synthesis commences is yet unknown. However, this information is regarded as important to consider a possible role of embryonic estrogens in preimplantation development. Therefore, in this study, we first used quantitative reverse transcription PCR to examine the mRNA expression of the enzymes required for the conversion of cholesterol into free and sulfonated estrogens (CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1), the cholesterol carrier protein STAR, and the estrogen receptors ESR1 and ESR2 in in vitro produced morulae and unhatched blastocysts (d 6–9). Only in the blastocysts, were the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors clearly present, whereas mRNA specific to ESRs was already detectable in the morulae. We also examined the expression of the corresponding enzymes in blastocysts at the protein level. None of the enzymes were detectable by capillary-based western analysis. Immunofluorescence methods were established for the detection of CYP17A1, CYP19A1, and SULT1E1. CYP17A1 was observed in the inner cell mass and trophectoderm, whereas CYP19A1 and SULT1E1 were present only in trophectoderm. An attempt to detect estrogen sulfotransferase activity was unsuccessful. Despite clear evidence that some elements of the estrogen biosynthetic pathway are also present at the protein level, it remains to be clarified whether the enzyme cascade underlying estrogen production is already functional in unhatched blastocysts.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B.mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

Differential tissue expression of sex steroid-synthesizing enzyme CYP11A1 in male Tibetan sheep (Ovis aries)

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.

The structure and characterization of human cytochrome P450 8B1 supports future drug design for nonalcoholic fatty liver disease and diabetes

Human cytochrome P450 8B1 (CYP8B1) is involved in conversion of cholesterol to bile acids. It hydroxylates the steroid ring at C12 to ultimately produce the bile acid cholic acid. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. Herein, the human CYP8B1 protein was generated and used to identify and characterize interactions with a series of azole inhibitors, which tend to be poorly selective P450 inhibitors. Structurally related miconazole, econazole, and tioconazole bound with submicromolar dissociation constants and were effective inhibitors of the native reaction. CYP8B was cocrystallized with S-tioconazole to yield the first X-ray structure. This inhibitor bound in the active site with its azole nitrogen coordinating the heme iron, consistent with inhibitor binding and inhibition assay data. Additionally, the CYP8B1 active site was compared with similar P450 enzymes to identify features that may facilitate the design of more selective inhibitors. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

CYP27A1 inhibits proliferation and migration of clear cell renal cell carcinoma via activation of LXRs/ABCA1

Cholesterol homeostasis plays an important role in the maintenance of normal body functions. CYP27A1 is a key enzyme known to regulate cholesterol homeostasis, which catalyzes the conversion of cholesterol to 27-HC and has been implicated in the occurrence and metastasis of various cancer types. The present study aimed to explore the regulatory role of CYP27A1 in the development of clear cell renal cell carcinoma (ccRCC). In particular, the effect of CYP27A1 on the proliferation and migration of ccRCC cells was investigated. The construction of a stable 786-O cell line overexpressing CYP27A1/pLVX was mediated by lentiviral infection. The proliferative capacity was assessed using MTT and colony formation. Wound healing assay was used to measure cell migration. Production of intracellular cholesterol and 27-HC was detected by enzyme-linked immunosorbent assay. The LXRs/ABCA1 pathway of cholesterol metabolism regulation was studied by RT-qPCR and Western blotting analysis after cells were treated with stimulation agents of 27-HC or T0901317 and inhibition agents of siRNA or GSK2033. The results revealed that overexpression of CYP27A1 could increase the intracellular production of 27-HC and inhibit the proliferation and migration of 786-O cells. And the treatment of 786-O cells with 27-HC induced a similar effect. CYP27A1/27HC mediated activation of the liver X receptors (LXRs) could up-regulate the expression of ATP-binding cassette transporter A1 (ABCA1), further resulting in the reduction of intracellular cholesterol contents. All of these findings indicated a regulatory role of CYP27A1 in the proliferation and migration of ccRCC, via activating LXRs/ABCA1 to regulate cholesterol homeostasis.

Impact of activating androgen receptor (AR) mutations on AR sensitivity to alternative ligands and response to ODM-208, a selective, first-in-class CYP11A1 inhibitor, in patients with advanced metastatic castration-resistant prostate cancer (mCRPC).

5057 Background: Activating AR mutations ensure a continued AR activation by non-androgen steroid ligands, e.g. progesterone and glucocorticoids. CYP11A1 is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone, from which all steroid hormones (glucocorticoids, mineralocorticoids, and sex steroids) are subsequently derived. ODM-208, an oral, selective inhibitor of CYP11A1, is being evaluated for safety and efficacy as a treatment of mCRPC in the ongoing CYPIDES phase I/II trial in men previously treated with both novel hormonal therapies and taxanes (ClinicalTrials.gov identifier: NCT03436485). Preliminary phase 1 results were previously reported (Fizazi K et al., ASCO GU 2022). Here we confirm that the in-vitro sensitivity of common AR mutations to ODM-208 treatment is mirrored in patient response in CYPIDES phase 1. Methods: ODM-208 was administered at daily doses between 6-150 mg (phase 2 dose: 10 mg) with dexamethasone and fludrocortisone, resulting in maximal suppression in all measured steroids at all doses. AR ligand-binding domain (LBD) mutations (L702H, V716M, W742L, W742C, H875Y, F877L, T878A, T878S, M896T, M896V) were assessed using a BEAMing assay (Sysmex Inostics) from plasma circulating cell-free DNA (cfDNA) collected before the first dose of ODM-208. The activation of wild-type (wt) and LBD mutated AR with various ligands was also studied in vitro using a luciferase reporter assay in AR negative PC3 cells. Results: 17 of 44 patients had at least one AR activating mutation, the most frequent being L702H (n = 11), T878A (n = 10), and H875Y (n = 6). Eleven out of the 16 evaluable patients (68%) with an AR LBD mutation achieved ≥50% reduction in serum PSA compared with 2 out of the 24 evaluable patients (8%) without an AR LBD mutation (P <.0001). The in vitro sensitivity of each of these common AR mutations to a variety of non-androgenic steroids will be presented, along with detailed PSA responses, duration of responses, and safety in mCRPC patients bearing the same mutations. Conclusions: AR activating mutations may permit continued hormone dependence in mCRPC, related to actions of non-androgenic steroid hormones. Treatment with ODM-208 blocked all steroid hormone production and resulted in frequent ≥50% PSA reductions in this group of heavily pretreated mCRPC pts with various AR LBD mutations, some being long-lasting. cfDNA AR mutations are a promising predictive biomarker for ODM-208 efficacy. Dexamethasone did not activate mutated ARs, supporting its selection for glucocorticoid replacement therapy in combination with ODM-208. Clinical trial information: NCT03436485.

Measures of high-density lipoprotein function in men and women with severe aortic stenosis

BackgroundCalcification of the aortic valve is a common heart valve disorder, in some cases leading to clinically impactful severe aortic stenosis (AS). Sex-specific differences in aortic valve calcification (ACV) exist, with women having a lower burden of calcification than men as measured by computed tomography; however, the pathophysiological mechanism that leads to these differences remains unclear.MethodsUsing cultured human Tamm-Horsfall protein 1 (THP-1) macrophages and human aortic valve interstitial cells, the effects of high-density lipoprotein (HDL) particles isolated from the plasma of men and women with severe AS were studied for cholesterol efflux capacity (CEC).ResultsHDL-CEC was assessed in 46 patients with severe AS, n = 30 men, n = 16 women. ATP-Binding Cassette A1 (ABCA1)-mediated HDL-CEC was measured from human cultured THP-1 macrophages to plasma HDL samples. Women with severe AS had more ABCA1-mediated HDL-CEC, as compared to men (8.50 ± 3.90% cpm vs. 6.80 ± 1.50% cpm, P = 0.04). HDL pre-β1 and α-particles were higher in woman than in men by spectral density, (pre-β1 HDL, 20298.29 ± 1076.15 vs. 15,661.74 ± 789.00, P = 0.002, and α-HDL, 63006.35 ± 756.81 vs. 50,447.00 ± 546.52, P = 0.03). Lecithin-cholesterol acyltransferase conversion of free cholesterol into cholesteryl esters was higher in women than men (16.44 ± 9.11%/h vs. 12.00 ± 8.07%/h, P = 0.03).ConclusionsSex-specific changes in various parameters of HDL-CEC were found in patients with severe AS. Sex-based modifications in HDL functionality by HDL-CEC might account for the reduced burden of calcification in women vs. men with severe AS. Therefore, future studies should target sex-related pathways in AS to help to improve understanding and treatment of AS.Graphical abstractSex specifc differences in AVC and differences associated with HDL function in men and women with severe AS. When compared to men, women had higher preβ-HDL and α-HDL migrating particles, higher cholesterol efflux to HDL, and higher lecithin cholesterol acyl transferase (LCAT) activity, possibly indicating that improved reverse cholesterol transport may be protective against worsened calcification.

Connecting the Dots Between Hypercholesterolemia and Alzheimer's Disease: A Potential Mechanism Based on 27-Hydroxycholesterol.

Alzheimer’s disease (AD), the most common cause of dementia, is a complex and multifactorial disease involving genetic and environmental factors, with hypercholesterolemia considered as one of the risk factors. Numerous epidemiological studies have reported a positive association between AD and serum cholesterol levels, and experimental studies also provide evidence that elevated cholesterol levels accelerate AD pathology. However, the underlying mechanism of hypercholesterolemia accelerating AD pathogenesis is not clear. Here, we review the metabolism of cholesterol in the brain and focus on the role of oxysterols, aiming to reveal the link between hypercholesterolemia and AD. 27-hydroxycholesterol (27-OHC) is the major peripheral oxysterol that flows into the brain, and it affects β-amyloid (Aβ) production and elimination as well as influencing other pathogenic mechanisms of AD. Although the potential link between hypercholesterolemia and AD is well established, cholesterol-lowering drugs show mixed results in improving cognitive function. Nevertheless, drugs that target cholesterol exocytosis and conversion show benefits in improving AD pathology. Herbs and natural compounds with cholesterol-lowering properties also have a potential role in ameliorating cognition. Collectively, hypercholesterolemia is a causative risk factor for AD, and 27-OHC is likely a potential mechanism for hypercholesterolemia to promote AD pathology. Drugs that regulate cholesterol metabolism are probably beneficial for AD, but more research is needed to unravel the mechanisms involved in 27-OHC, which may lead to new therapeutic strategies for AD.

Metabolic engineering in food crops to enhance ascorbic acid production: crop biofortification perspectives for human health.

Ascorbic acid (AsA) also known as vitamin C is considered as an essential micronutrient in the diet of humans. The human body is unable to synthesize AsA, thus solely dependent on exogenous sources to accomplish the nutritional requirement. AsA plays a crucial role in different physiological aspects of human health like bone formation, iron absorption, maintenance and development of connective tissues, conversion of cholesterol to bile acid and production of serotonin. It carries antioxidant properties and is involved in curing various clinical disorders such as scurvy, viral infection, neurodegenerative diseases, cardiovascular diseases, anemia, and diabetes. It also plays a significant role in COVID-19 prevention and recovery by improving the oxygen index and enhancing the production of natural killer cells and T-lymphocytes. In plants, AsA plays important role in floral induction, seed germination, senescence, ROS regulation and photosynthesis. AsA is an essential counterpart of the antioxidant system and helps to defend the plants against abiotic and biotic stresses. Surprisingly, the deficiencies of AsA are spreading in both developed and developing countries. The amount of AsA in the major food crops such as wheat, rice, maize, and other raw natural plant foods is inadequate to fulfill its dietary requirements. Hence, the biofortification of AsA in staple crops would be feasible and cost-effective means of delivering AsA to populations that may have limited access to diverse diets and other interventions. In this review, we endeavor to provide information on the role of AsA in plants and human health, and also perused various biotechnological and agronomical approaches for elevating AsA content in food crops.

SOAT1: A Suitable Target for Therapy in High-Grade Astrocytic Glioma?

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.

An Aggressive form of Cerebrotendinous Xanthomatosis

This 34-year-old man presented to our clinic with progressive gait difficulties and swelling of his tendons. On examination, he had bilateral ataxia, upper motor neuron signs, and tumors in his tendons (Figure 1 A-C). Brain MRI showed diffuse Encephalopathy (Figure 1, D-F). Cerebrotendinous Xanthomatosis was diagnosed after genetic testing confirmed the presence of pathogenic variants on the CYP27A1 gene. Cerebrotendinous Xanthomatosis is a rare, potentially treatable autosomal recessive disorder characterized by impairment of the hepatic conversion of cholesterol to chenodeoxycholic acid, leading to massive deposition of cholestanol in organs. It presents with xanthomas, juvenile cataracts, and progressive neurological dysfunction (1).

27-Hydroxycholesterol Binds GPER and Induces Progression of Estrogen Receptor-Negative Breast Cancer

Simple SummaryBreast cancer is the most common cancer in women, and there is a known link between high cholesterol levels and breast cancer. However, how cholesterol impacts breast cancer is poorly understood, particularly in the case of an aggressive form of cancer known as estrogen receptor negative breast cancer. Using cells in culture and models of breast tumors in mice, we have determined that an abundant metabolite of cholesterol known as 27-hydroxycholesterol stimulates estrogen receptor negative breast cancer growth. We have also determined how 27-hydroxycholesterol stimulates the growth, identifying a new mechanism of action of 27-hydroxycholesterol. These new findings may explain the link between high cholesterol and estrogen receptor negative breast cancer, and they may lead to the development of new therapies for a type of breast cancer that presently lacks specific treatments.Cholesterol affects the proliferation of breast cancer (BC) and in particular of estrogen receptor-negative (ER−) BC. Cholesterol is converted to 27-hydroxycholesterol (27HC), which promotes the growth of ER+ BC. Potentially, 27HC can be involved in cholesterol-dependent ER− BC proliferation. Stable MDA-MB-231 silenced clones for CYP7B1 (27HC metabolizing enzyme) show an increased basal proliferation rate, which is not observed in the presence of lipoprotein-deprived serum. Furthermore, the treatment of SKBR3, MDA-MB-231 and MDA-MB-468 with 27HC increased cell proliferation that was prevented by G15, a selective G Protein-Coupled Estrogen Receptor (GPER) inhibitor, suggested this receptor to be a potential 27HC target. Binding experiments demonstrate that 27HC is a new ligand for GPER. We show that ERK1/2 and NFκB are part of the 27HC/GPER pathway. The stable silencing of GPER prevents NFκB activation and reduces basal and 27HC-dependent tumor growth. Additionally, conditioned medium from ER− BC cells treated with 27HC promotes tube formation, which does not occur with CM from GPER silenced cells. Collectively, these data demonstrate that cholesterol conversion into 27HC promotes ER− BC growth and progression, and the expression of GPER is required for its effects.

Capsaicin regulates dyslipidemia by altering the composition of bile acids in germ-free mice.

The improvement of lipid metabolism by capsaicin (CAP) has been extensively studied, mostly with respect to the vanilloid type 1 (TRPV1) ion channel and intestinal flora. In this study, a model was established in germ-free mice by using resiniferatoxin (RTX) to ablate TRPV1 ion channels. Bile acid composition, blood parameters, and colonic transcriptome analyses revealed that CAP could improve dyslipidemia caused by high-fat diet even in the absence of TRPV1 ion channels and intestinal flora. CAP fed to germ mice decreased the concentrations of low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), fasting blood glucose and fasting insulin, increased the concentration of high-density lipoprotein (HDL-C), and decreased the levels of plasma endotoxin and pro-inflammatory factor interleukin 6 (IL-6). Furthermore, CAP could affect both classical and alternative pathways of cholesterol conversion by changing the composition of bile acids, reducing the concentrations of glycocholic acid (GCA), ursodeoxycholic acid (UDCA) and glycochenodeoxycholic acid (GCDCA). First, changing the composition of bile acids inhibited the expression of colon Fgf15. CAP promoted the expression of Cyp7a1 (Cytochrome p450, family 7, subfamily a, and polypeptide 1) in the liver, and thus reduced TC and TG levels. In addition, it could change the composition of bile acids and increase the expression of Cyp7b1 (Cytochrome p450, family 7, subfamily b, and polypeptide 1) in the colon, increase Cyp7b1 protein in the liver and thus inhibit fat accumulation. In conclusion, CAP could alter the composition of bile acids and promote the conversion of cholesterol to bile acids, thereby improving lipid metabolism abnormalities caused by a high-fat diet.

HDL Structure.

HDL has various protein components, including enzymes, complement components, apolipoproteins, protease inhibitors, etc. In addition to proteins, lipids are also a significant component of HDL. These components and their structure determine the function of HDL. HDL is heavily involved in the acute response phase, complement regulation phase, hemostasis phase, immune response phase, and protease inhibition phase. Among the apolipoproteins, the predominant component is Apo A-I, which confers various atherogenic activities to HDL. Apo A-II, Apo-C, Apo-D, Apo-F, Apo-H, Apo-J, and Apo-O, which can bind free fatty acids, regulate the activity of many proteins involved in HDL metabolism, inhibit lipid transfer, and control the endogenous coagulation cascade. A major functional component is the enzyme LCAT, which helps catalyze the conversion of cholesterol to plasma-based lipoproteins and then to cholesteryl esters. Another enzyme associated with HDL is human paraoxonase, calcium-, PON1-, PON2-, and PON3-dependent lactone enzyme with catalytic activity, including reversible binding to substrates. PAF-AH is a phospholipase with lipoprotein properties, and HDL and LDL particles are commonly bound to plasma PAF-AH for circulation. As for lipid components, PC is an essential phospholipid subclass and may be a biomarker for constitutive inflammation. Sphingolipids, such as sphingomyelin and ceramide, also play an indispensable role in HDL function. In different physiological and pathological stages and plasma environments, HDL can exhibit different structural features, such as discoid HDL and spherical rHDL.

Untargeted and Targeted Metabolomics Reveal the Underlying Mechanism of Aspirin Eugenol Ester Ameliorating Rat Hyperlipidemia via Inhibiting FXR to Induce CYP7A1.

Hyperlipidemia is an important lipid disorder and a risk factor for health. Aspirin eugenol ester (AEE) is a novel synthetic compound which is made up of two chemical structural units from aspirin and eugenol. Therapeutic effect of AEE on hyperlipidemia has been confirmed in animal model. But the action mechanism of AEE on hyperlipidemia is still poorly understood. In this study, we investigated the effects of AEE on liver and feces metabolic profile through UPLC-Q-TOF/MS-based untargeted metabolomics in hyperlipidemia hamster induced with high fat diet (HFD), and the effects of AEE on the expression of genes and proteins related to cholesterol and bile acid (BA) in HFD-induced hyperlipidemia SD rat. The concentrations of 26 bile acids (BAs) in the liver from hyperlipidemia SD rat were also quantified with the application of BA targeted metabolomics. The results of untargeted metabolomics showed that the underlying mechanism of AEE on hyperlipidemia was mainly associated with amino acid metabolism, glutathione metabolism, energy metabolism, BA metabolism, and glycerophospholipid metabolism. AEE induced the expression of the BA-synthetic enzymes cholesterol 7α-hydroxylase (CYP7A1) by the inhibition of BA nuclear receptor farnesoid X receptor (FXR) in liver, which resulted in accelerating the conversion of cholesterol into bile acids and excrete in feces. The results of BA targeted metabolomics showed that AEE elevated the glycine-conjugated BA level and decreased the tauro-conjugated BA level. In conclusion, this study found that AEE decreased FXR and increased CYP7A1 in the liver, which might be the possible molecular mechanisms and targets of AEE for anti-hyperlipidemia therapies.

Abstract 11554: Measures of High-Density Lipoprotein Function and Composition in Men and Women with Severe Aortic Valve Stenosis

Introduction and Hypothesis: Aortic valve calcification (AVC), is the intrinsic mechanism of valvular obstruction in severe aortic stenosis (AS), sharing similarities with atherosclerosis. There are differences in AVC by sex, with women having a lower burden of calcification than men, but the mechanism for this is unclear. Improved HDL functionality has been associated with protection against coronary artery calcification, but its relationship to AVC is less studied. Methods: We evaluated measures of high-density lipoprotein (HDL) functionality in 46 patients with severe AS, n= 30 men, n=16 women. ATP-Binding Cassette A1 (ABCA1) mediated cholesterol efflux capacity (CEC) was measured from human cultured Tamm-Horsfall protein 1 (THP-1) macrophages to apoB-depleted patient plasma samples. Lipid profile, lecithin-cholesterol acyltransferase activity (LCAT), fast protein liquid chromatography (FPLC), HDL size, and small pre-β1 and α-HDL particles, were measured in apolipoprotein B-depleted patient plasma. Proteomic analysis was used to explore sex-based differences in the HDL proteome. Results: Women with severe AS had higher ABCA1-mediated CEC, as compared to men (n=46, p=0.04). We found differences in FPLC profiles between sexes in severe AS along with a reduction in preβ and α-particles in men vs women by spectral counts, (preβ-HDL, 15661.74±789.00 vs 20298.29±1076.15, p=0.002), and (α-HDL, 50447.00± 546.52 vs 63006.35±756.81, p=0.03). Conversion of free cholesterol into cholesteryl esters by LCAT was decreased in men vs. women (12.00±8.07 %/h vs 16.44±9.11 %/h, p=0.03). We identified 108 HDL-associated proteins in a representative man and woman, of which, 15 were found only in the female sample Conclusions: Sex-specific differences in measures of HDL functionality were found in patients with severe AS. Future studies should consider whether sex-based differences in HDL functionality might account for the decreased burden of calcification in women vs. men with severe AS.

A Novel Sialoglycopeptide from Gadus morhua Eggs Prevents Liver Fibrosis Induced by CCl4 via Downregulating FXR/FGF15 and TLR4/TGF-β/Smad Pathways.

Liver fibrosis plays a critical role in liver disease progression. A sialoglycopeptide from the Gadus morhua eggs (Gm-SGPP) was identified having a 7000 Da molecular weight with a core pentasaccharide structure and osteogenesis activity. However, whether Gm-SGPP is beneficial to liver fibrosis remains unknown. In this study, mice with liver fibrosis were intraperitoneally injected with 2.5% CCl4 (10 mL/kg) and orally administered with Gm-SGPP (500 mg/kg) for 30 days. Results showed that Gm-SGPP alleviated oxidative liver damage and lipid metabolism disorder and reduced hepatocyte necrosis and lipid droplet accumulation. Notably, we found that Gm-SGPP increased the number and changed the composition of bile acids via increasing cholesterol 7a-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) expression, which caused inhibition of ileum farnesoid X receptor (FXR) expression and accelerated the cholesterol conversion. Cholesterol accumulation is a risk factor for liver fibrosis. Masson staining showed that Gm-SGPP significantly reduced the degree of collagen deposition. Western blotting further suggested that Gm-SGPP downregulated the key gene of the toll-like receptor 4 (TLR4)-mediated transforming growth factor-β (TGF-β)/Smad pathway. To our best knowledge, this is the first report that Gm-SGPP prevented liver fibrosis via attenuating cholesterol accumulation. Our present results provide new ideas for the Gadus morhua egg's high-value utilization.

Helicobacter pylori cholesterol-α-glucosyltransferase manipulates cholesterol for bacterial adherence to gastric epithelial cells

ABSTRACT Helicobacter pylori infection is associated with several gastrointestinal diseases, including gastritis, peptic ulcers, and gastric cancer. Infection of cells with H. pylori is dependent on lipid rafts, which are cholesterol-rich microdomains located in the cell membrane. H. pylori cholesterol-α-glucosyltransferase (CGT) catalyzes the conversion of membrane cholesterol to cholesteryl glucosides, which can be incorporated into the bacterial cell wall, facilitating evasion from immune defense and colonization in the host. However, the detailed mechanisms underlying this process remain to be explored. In this study, we discovered for the first time that H. pylori CGT could promote adherence to gastric epithelial cells in a cholesterol-dependent manner. Externalization of cell membrane phosphatidylserine (PS) is crucial for enhancement of binding of H. pylori to cells by CGT and for cytotoxin-associated gene A (CagA)-induced pathogenesis. Furthermore, exogenous cholesterol interferes with the actions of H. pylori CGT to catalyze cellular cholesterol, which impedes bacterial binding to cells and attenuates subsequent inflammation, indicating that the initial attachment of H. pylori to cells is closely dependent on host cholesterol. These results provide evidence that CGT contributes to H. pylori infectivity and it may serve as a key target for the treatment of H. pylori-associated diseases.

Cholesterol-to-Coprostanol Conversion by the Gut Microbiota: What We Know, Suspect, and Ignore.

Every day, up to 1 g of cholesterol, composed of the unabsorbed dietary cholesterol, the biliary cholesterol secretion, and cholesterol of cells sloughed from the intestinal epithelium, enters the colon. All cholesterol arriving in the large intestine can be metabolized by the colonic bacteria. Cholesterol is mainly converted into coprostanol, a non-absorbable sterol that is excreted in the feces. Interestingly, cholesterol-to-coprostanol conversion in human populations is variable, with a majority of high converters and a minority of low or inefficient converters. Two major pathways have been proposed, one involving the direct stereospecific reduction of the Δ5 double bond direct while the indirect pathway involves the intermediate formation of 4-cholelesten-3-one and coprostanone. Despite the fact that intestinal cholesterol conversion was discovered more than a century ago, only a few cholesterol-to-coprostanol-converting bacterial strains have been isolated and characterized. Moreover, the responsible genes were mainly unknown until recently. Interestingly, cholesterol-to-coprostanol conversion is highly regulated by the diet. Finally, this gut bacterial metabolism has been linked to health and disease, and recent evidence suggests it could contribute to lower blood cholesterol and cardiovascular risks.