Endogenous Cholesterol Research Articles

Lactobacillus oris improves non-alcoholic fatty liver in mice and inhibits endogenous cholesterol biosynthesis

We previously confirmed that a strain of Lactobacillus oris isolated from the fecal samples of healthy Hainan centenarian having potent lipid-lowering ability in HepG2 cells; and this study was to investigate the effect of the stain on non-alcoholic fatty liver in mice in vivio. The Lactobacillus oris strain isolated from Hainan centenarian fecal samples were frozen stored in our laboratory. Thirty ob/ob mice (10 in each group) were orally gavaged with Lactobacillus oris (Lactobacillus, 5 × 109 cfu), mixed probiotics (Mixed, 5 × 109 cfu, a mixture with known lipid-lowering ability), or culture medium (Control) respectively. Lactobacillus oris isolated from fecal samples of Hainan centenarians showed significantly in vivo lipid lowering ability compared with the controls, and the ability was comparable with mixed probiotics strains in mice The possible mechanisms of lipid-lowering of probiotics and Lactobacillus oris may be associated with HMGR inhibition to suppress the synthesis of endogenous cholesterol; bile acids reabsorption, and intestinal FXR-FGF15 signaling pathways promoting the cholesterol conversion into bile acids secretion.

SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.

Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells.

The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis. Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice. These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.

619-P: An Isocaloric High Saturated Fat Diet Increases Endogenous Cholesterol Production Which Is Reversed by a Protein-Enriched Diet in Normal-Weight Humans

Lathosterol is a marker of endogenous cholesterol synthesis whereas plant sterols (e. g. campesterol) reflect cholesterol absorption. Obesity and type 2 diabetes mellitus are associated with increased endogenous cholesterol production and decreased cholesterol absorption. Only few studies evaluated cholesterol homeostasis under isocaloric nutritional conditions. This study investigated endogenous cholesterol production in healthy humans after consumption of isocaloric diets enriched with carbohydrates (KH) or saturated fat (HF) or protein (HP). 24 normal weight participants (women and men, age 18 - 70) were investigated for 18 weeks. An isocaloric diet rich in carbohydrates (55% carbohydrates, 30% fat, 15% protein, KH) was applied followed by an isocaloric diet rich in saturated fat (40% carbohydrates, 45% fat, 15% protein, HF) and finally followed by an isocaloric diet enriched with proteins (45% carbohydrates, 30% fat, 25% protein, HP); each for 6 weeks. Body weight (BW), serum total and LDL cholesterol and serum lathosterol and campesterol levels were analyzed after the KH period, after one week (HF1) and 6 weeks (HF6) of the HF and after the HP period. BW did not change during the study. Serum total and LDL cholesterol increased from 4.38±0.2 and 2.74±0.2 mmol/L, respectively at KH to 4.82±0.2 and 2.97±0.2 mmol/L, respectively at HF6 (both P<0.01) and were decreased back to KH levels at HP. Serum lathosterol levels increased 1.26-fold and 1.16-fold, respectively at HF1 and HF6 (P<0.05 compared to LF), however decreased by 10.1% at HP (compared to KH levels, P<0.05). Serum campesterol levels moderately decreased at HF1 and HF6 and were restored to KH levels at HP (P<0.01 compared to HF6). These data show that composition of macronutrients are responsible for diet induced elevation of serum cholesterol levels due to an increased endogenous cholesterol production in normal weight humans regardless of the energy content of the diet. Disclosure M.Kruse: None. S.Matysik: None. D.Hoffmann: None. T.Frahnow: None. A.F.Pfeiffer: Advisory Panel; Abbott Diabetes, Speaker's Bureau; Novo Nordisk, Sanofi-Aventis Deutschland GmbH. Funding German Federal Ministry of Education and Research (0315424)

Plant sterols and cholesterol metabolism are associated with five-year cognitive decline in the elderly population

Dysregulations in cholesterol metabolism are associated with neurodegenerative and vascular pathologies, and dementia. Diet-derived plant sterols (phytosterols) have cholesterol-lowering, anti-inflammatory, and antioxidant properties and may interfere with neurodegeneration and cognitive decline. Here we performed multivariate analysis in 720 individuals enrolled in a population-based prospective study to determine whether circulating cholesterol precursors and metabolites, triglycerides, and phytosterols, are associated with cognitive impairment and decline in the older population. We report specific dysregulations of endogenous cholesterol synthesis and metabolism, and diet-derived phytosterols, and their changes over time associated with cognitive impairment, and decline in the general population. These findings suggest circulating sterols levels could be considered in risk evaluation and are relevant for the development of strategies to prevent cognitive decline in older people.

Relation between single nucleotide polymorphisms in circadian clock relevant genes and cholesterol metabolism

Single nucleotide polymorphisms (SNPs) in circadian clock relevant genes are associated with several metabolic health variables, but little is known about their associations with human cholesterol metabolism. Therefore, this study examined associations between SNPs in ARNTL, ARNTL2, CLOCK, CRY1, CRY2, PER2, and PER3 with the intestinal cholesterol absorption markers campesterol and sitosterol, the endogenous cholesterol synthesis marker lathosterol, and total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) concentrations in 456 healthy individuals from Western European descent. One SNP in ARNTL2 (rs1037924) showed a significant association with lathosterol. Several SNPs in ARNTL (rs4146388, rs58901760, rs6486121), ARNTL2 (rs73075788), CLOCK (rs13113518, rs35115774, rs6832769), and CRY1 (rs2078074) were significantly associated with intestinal cholesterol absorption. Genetic variants in CRY2, PER2, and PER3 were not significantly associated with intestinal cholesterol absorption or endogenous cholesterol synthesis. None of the SNPs were associated with TC or LDL-C, except for one SNP in PER2 (rs11894491) with serum LDL-C concentrations. The findings suggest that various SNPs in ARNTL, ARNTL2, CLOCK and CRY1 play a role in intestinal cholesterol absorption and endogenous cholesterol synthesis, which was not reflected in TC and LDL-C concentrations. The significant associations between SNPs and intestinal cholesterol absorption and endogenous cholesterol synthesis should be validated in other cohorts.

Molecular docking studies of Triphala with catalytic portion of HMG-CoA reductase enzyme

Introduction: Triphala, consisting of three fruits, Phyllanthus emblica L. (Phyllanthaceae), Terminalia bellirica (Gaertn.) Roxb. (Combretaceae), and T. chebula Retz, is a well-recognized Ayurvedic herbal formulation, used for various therapeutic purposes, including the treatment of dyslipidemia. Inhibitory activity against 3‑hydroxy‑3‑methylglutaryl‑coenzyme A (HMG‑CoA) reductase, a rate-limiting enzyme in the endogenous cholesterol synthesis pathway, is an essential target for the management of hypercholesterolemia. This in silico study aimed to investigate the HMG-CoA reductase inhibitory activity of the phytochemical compounds derived from Triphala formulation by employing molecular docking analysis. Methods: Ten phytochemical constituents of Triphala formulation were selectively used for docking study by using the HMG-CoA reductase template (PDB: 1HWK). Docking analysis was performed using AutoDock 4.2. The candidates were ranked by the binding energy parameters. Results: From the docking studies, the phytochemical compounds with HMG-CoA reductase inhibition could be classified into 4 groups, including phytosterols, polyphenols, tannins, and flavonoids. Beta-sitosterol exhibited the highest binding affinity to HMG-CoA reductase with a binding energy of -7.75 kcal/mol. Conclusion: These 10 phytochemical compounds in Triphala potentially exert their cholesterol-lowering effects via inhibition against HMG-CoA reductase. Nonetheless, further in vitro and in vivo experiments should be conducted subsequently to confirm this finding.

High cholesterol intake remodels cholesterol turnover and energy homeostasis in Nile tilapia (Oreochromis niloticus)

The roles of dietary cholesterol in fish physiology are currently contradictory. The issue reflects the limited studies on the metabolic consequences of cholesterol intake in fish. The present study investigated the metabolic responses to high cholesterol intake in Nile tilapia (Oreochromis niloticus), which were fed with four cholesterol-contained diets (0.8, 1.6, 2.4 and 3.2%) and a control diet for eight weeks. All fish-fed cholesterol diets showed increased body weight, but accumulated cholesterol (the peak level was in the 1.6% cholesterol group). Then, we selected 1.6% cholesterol and control diets for further analysis. The high cholesterol diet impaired liver function and reduced mitochondria number in fish. Furthermore, high cholesterol intake triggered protective adaptation via (1) inhibiting endogenous cholesterol synthesis, (2) elevating the expression of genes related to cholesterol esterification and efflux, and (3) promoting chenodeoxycholic acid synthesis and efflux. Accordingly, high cholesterol intake reshaped the fish gut microbiome by increasing the abundance of Lactobacillus spp. and Mycobacterium spp., both of which are involved in cholesterol and/or bile acids catabolism. Moreover, high cholesterol intake inhibited lipid catabolic activities through mitochondrial β-oxidation, and lysosome-mediated lipophagy, and depressed insulin signaling sensitivity. Protein catabolism was elevated as a compulsory response to maintain energy homeostasis. Therefore, although high cholesterol intake promoted growth, it led to metabolic disorders in fish. For the first time, this study provides evidence for the systemic metabolic response to high cholesterol intake in fish. This knowledge contributes to an understanding of the metabolic syndromes caused by high cholesterol intake or deposition in fish.

Statins in Children, an Update.

Since lipid abnormalities tend to progress from childhood to adulthood, it is necessary to early identify and treat children and adolescents with dyslipidemia. This is important in order to reduce the cardiovascular risk, delay the development of fatty streaks, slow the progression of atherosclerosis and reverse atherosclerotic plaques. Together with therapeutic lifestyle changes, statins are the most common lipid-lowering drugs. By inhibiting the endogenous cholesterol synthesis in the liver, statins increase the catabolism of LDL-C, reduce VLDL-C, IDL-C and TG and modestly increase HDL-C. Regardless of their lipid-lowering effect, statins have also pleiotropic effects. Statins have increasingly been prescribed in children and adolescents and mounting evidence suggests their beneficial role. As with adults, in children, several studies have demonstrated that statin therapy is efficient at lowering lipid levels and reducing CIMT progression and cumulative estimated atherosclerotic burden in children. Statins are generally very well-tolerated in both adults and children and adverse events are quite uncommon. When evaluating the need and the timing for statin treatment, the presence of several factors (secondary causes, familial history, additional risk factors) should also be considered. Before initiating statins, it is imperative for clinical practitioners to consult patients and families and, as with any new medication therapy, to monitor patients taking statins. Despite being safe and effective, many children with lipid disorders are not on statin therapy and are not receiving the full potential benefit of adequate lipid-lowering therapies. It is therefore important that clinicians become familiar with statins.

A Transient Inflammatory Response Induced by Lipopolysaccharide Infusion Lowers Markers of Endogenous Cholesterol and Bile Acid Synthesis in Healthy Normocholesterolemic Young Men.

Inflammation is associated with changes in plasma lipids, lipoproteins, and cholesterol efflux capacity (CEC). It is unknown if the changes in lipids and lipoproteins during inflammation are related to changes in cholesterol absorption, synthesis, and bile acid synthesis. We, therefore, examined the effects of acute lipopolysaccharide (LPS)-induced transient systemic inflammation on lipids, lipoproteins, CEC, and markers of cholesterol metabolism. We also evaluated whether markers for cholesterol metabolism at baseline predict the intensity of the inflammatory response. Eight healthy young subjects received LPS infusion, and blood was sampled for the following 24 h. In addition to lipids, lipoproteins, and CEC, we also measured markers for cholesterol absorption and synthesis, bile acid synthesis, and inflammation. Compared with baseline, plasma total cholesterol, low-density lipoprotein cholesterol, and CEC decreased, while triglycerides increased in the 24 h following LPS infusion. TC-standardized levels of cholesterol synthesis markers (lathosterol, lanosterol, and desmosterol) and a bile acid synthesis marker (7α-OH-cholesterol) also decreased, with no changes in cholesterol absorption markers (campesterol, sitosterol, and cholestanol). Baseline TC-standardized levels of desmosterol and 7α-OH-cholesterol were positively correlated with concentrations of various inflammatory markers. Changes in TC-standardized desmosterol and 7α-OH-cholesterol were negatively correlated with concentrations of inflammatory markers. LPS infusion reduced endogenous cholesterol synthesis and bile acid synthesis in healthy young men.

Коронавирусная инфекция и дислипидемия — есть ли связь?

Dyslipidemia is one of the most common comorbidities in patients infected with the SARS-CoV-2 coronavirus. In this review based on the up- to-date publications the authors assess the potential impact of dyslipidemia on the clinical course and prognosis in COVID-19 infected patients, as well as the viral infection effects on the lipid profile in patients with dyslipidemia. The COVID-19 driven inflammatory response leads to an increase in high-density lipoprotein (HDL) levels, modulation of apolipoproteins, increase in serum amyloid protein A concentration, and a decrease in apolipoprotein A-I, M and E levels, thus affecting the antioxidant, anti-inflammatory and immunomodulatory properties of HDL. The use of statins in patients with COVID-19 may mitigate the risk of lipid disorders. By inhibiting endogenous cholesterol synthesis, statins reduce its amount in lipid rafts and therefore may limit the entry of the SARS-CoV-2 virus into host cells. Moreover, statins, by reducing the overexpression of proinflammatory cytokines, can counteract COVID-19-associated cytokine storm. KEYWORDS: COVID-19, dyslipidemia, obesity, treatment, lipoproteins, statins, fibrates. FOR CITATION: Akhmedov V.A. Coronavirus disease and dyslipidemia — is there an association? Russian Medical Inquiry. 2023;7(1):36– 40 (in Russ.). DOI: 10.32364/2587-6821-2023-7-1-36-40.

Bile acid-binding capacity of peptide extracts obtained from chicken blood hydrolysates using HPLC

Bile acids are involved in the modulation of various metabolic processes facilitating the biliary excretion of endogenous and exogenous cholesterol. The objective of this study was to determine the glycocholic acid binding capacity (BC) of chicken blood hydrolysates using an optimized RP-HPLC methodology. Samples were hydrolysed using a combination of five different enzymes. Alcalase and Protamex hydrolysates presented the highest BC, with mean values of 20.09% and 20.61%, respectively. Subsequently, both hydrolysates were ultrafiltered to obtain fractions >10 kDa, between 10 and 3 kDa, and <3 kDa, and the highest BC values were obtained for peptide fractions >10 kDa. Finally, the protein fragments (MW > 10 kDa) potentially responsible for BC were identified by LC-MS/MS. The results confirmed the relation of BC with the molecular weight of the peptides generated, suggesting that certain protein fragments generated from chicken blood could contribute to a positive impact on health by interfering with cholesterol metabolism.

Activation of Transposable Elements in Human Skeletal Muscle Fibers upon Statin Treatment.

High cholesterol levels have been linked to a high risk of cardiovascular diseases, and preventative pharmacological care to lower cholesterol levels is critically important. Statins, which are hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are drugs used to reduce the endogenous cholesterol synthesis, thus minimizing its pathophysiological effects. Despite the proven benefits, statins therapy is known to cause a number of skeletal muscle disorders, including myalgia, myopathy and myositis. The mechanisms underlying such statin-induced side effects are unknown. Recently, a group of genes and molecular pathways has been described to participate in statin-induced myopathy, caused by either simvastatin or rosuvastatin, although the mechanism by which changes in gene regulation occur was not studied. Transposable Elements (TEs), repetitive elements that move within the genome, are known to play regulatory roles in gene expression; however, their role in statin-induced muscle damage has not been studied. We analyzed the expression of TEs in human skeletal fiber cells treated with either simvastatin or rosuvastatin, as well as their respective controls, and identified TEs that change their expression in response to the treatment. We found that simvastatin resulted in >1000 differentially expressed (DE) TEs, whereas rosuvastatin resulted in only 27 DE TEs. Using network analysis tools, we predicted the impact of the DE TEs on the expression of genes and found that amongst the genes potentially modulated by TEs, there are some previously associated to statin-linked myopathy pathways (e.g., AKT3). Overall, our results indicate that TEs may be a key player in the statin-induced muscle side effects.

Limb-girdle muscular dystrophy type 2B causes HDL-C abnormalities in patients and statin-resistant muscle wasting in dysferlin-deficient mice

Limb-girdle muscular dystrophy (MD) type 2B (LGMD2B) and Duchenne MD (DMD) are caused by mutations to the Dysferlin and Dystrophin genes, respectively. We have recently demonstrated in typically mild dysferlin- and dystrophin-deficient mouse models that increased plasma cholesterol levels severely exacerbate muscle wasting, and that DMD patients display primary dyslipidemia characterized by elevated plasma cholesterol and triglycerides. Herein, we investigate lipoprotein abnormalities in LGMD2B and if statin therapy protects dysferlin-deficient mice (Dysf) from muscle damage. Herein, lipoproteins and liver enzymes from LGMD2B patients and dysferlin-null (Dysf) mice were analyzed. Simvastatin, which exhibits anti-muscle wasting effects in mouse models of DMD and corrects aberrant expression of key markers of lipid metabolism and endogenous cholesterol synthesis, was tested in Dysf mice. Muscle damage and fibrosis were assessed by immunohistochemistry and cholesterol signalling pathways via Western blot. LGMD2B patients show reduced serum high-density lipoprotein cholesterol (HDL-C) levels compared to healthy controls and exhibit a greater prevalence of abnormal total cholesterol (CHOL)/HDL-C ratios despite an absence of liver dysfunction. While Dysf mice presented with reduced CHOL and associated HDL-C and LDL-C-associated fractions, simvastatin treatment did not prevent muscle wasting in quadriceps and triceps muscle groups or correct aberrant low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) protein expression. LGMD2B patients present with reduced serum concentrations of HDL-C, a major metabolic comorbidity, and as a result, statin therapy is unlikely to prevent muscle wasting in this population. We propose that like DMD, LGMD2B should be considered as a new type of genetic dyslipidemia.

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

Real-World Clinical Experience of Rosuvastatin as a Lipid-Lowering Therapy for Primary and Secondary Prevention of Cardiovascular Events (REAL ROSE).

Background Rosuvastatin effectively reduces endogenous cholesterol synthesis and low-density lipoprotein (LDL) cholesterol and increases high-density lipoprotein (HDL) cholesterol. This study aimed to evaluate the clinical characteristics of patients and treatment patterns of rosuvastatin as a lipid-lowering therapy for primary and secondary prevention of cardiovascular events in Indian settings. Methods This real-world, retrospective multi-centric observational study included patients aged >18 years who received treatment with a rosuvastatin/rosuvastatin-based combination. Demographic and data about concomitant diseases and medications were recorded. Results Out of 1,816 patients, the majority were men (66.2%); the mean age was 54.1 years. The patients prescribed rosuvastatin for primary and secondary prevention of cardiovascular events were 71.9% and 28.1%, respectively. Rosuvastatin 10 mg (56.8%) was the most commonly prescribed dose. For primary prevention, 10 mg (65.0%) was the most preferred dose, and for secondary prevention, 20 mg (54.3%) was the most preferred dose. Rosuvastatin treatment significantly (pre- vs. post-treatment) reduced the levels of total cholesterol (227.2 vs. 178.4 mg/dL), triglycerides (212.6 vs. 154.4 mg/dL), and LDL cholesterol (167.0 vs. 125.6 mg/dL), and increased HDL cholesterol levels (40.7 vs. 44.3 mg/dL) (p<0.0001). A total of 1,196 patients received combination therapy with rosuvastatin (aspirin, 34.0%, and fenofibrate, 21.9%). Adverse events were reported in 0.4% of the study population (leg pain, nausea, muscle cramps/pain, bleeding, and myalgia). Conclusion This study demonstrated the clinical effectiveness and safety of moderate- to high-intensity rosuvastatin (5-40 mg) for primary and secondary prevention of cardiovascular events in the Indian population. A primary prevention strategy with statins can reduce cardiovascular events and associated morbidity and mortality.

Abstract 15254: Acta2 Missense Variant Increases Atherosclerotic Plaque Burdern by Heat Shock Factor 1-Driven Augmented Phenotypic Modulation of Smooth Muscle Cells

ACTA2 encodes the contractile protein smooth muscle α-actin expressed in smooth muscle cells (SMCs). A heterozygous pathogenic variant in ACTA2 , p.R149C, predisposes to premature coronary artery disease (CAD) in the absence of risk factors. Recent studies have determined that SMCs contribute to atherosclerotic plaque formation after undergoing phenotypic modulation, characterized by de-differentiation and increased expression of macrophage, fibroblast, stem, and osteogenic markers. This SMC modulation is driven in part by exogenous cholesterol entering the endoplasmic reticulum (ER) and activating ER stress, specifically PERK signaling. To determine how a mutation in SM α-actin predisposes to atherosclerosis, Acta2 R149C/+ mice were crossed into Apoe -/- mice and fed a high fat diet for 3 months. Despite no differences in serum lipid levels, the Acta2 R149C/+ Apoe -/- mice have a 3-fold increased plaque burden compared to Apoe -/- mice. SMCs explanted from Acta2 R149C/+ mice have phenotypic modulation at baseline while WT SMCs do not. We determined that misfolding of the R149C SMα-actin activates heat shock factor 1 (HSF1), which increases endogenous cholesterol biosynthesis through increasing HMG-CoA reductase expression. The increase in endogenous cholesterol levels induces ER stress, activates PERK, and augments both modulation of SMCs and atherosclerotic plaque formation. ScRNA sequencing confirms increased HSF1 and PERK signaling and modulation in SMCs in the mutant mice. Importantly, pravastatin treatment dramatically lowers plaque burden to similar low levels of plaque burden in both the Acta2 R149C/+ Apoe -/- and Apoe -/- mice (Fig. 1a) . These studies identify a novel mechanism by which mutant SM α-actin increases endogenous cholesterol biosynthesis and drives atherosclerotic plaque formation, a mechanism that also informs as to why statins are effective in individuals at risk for CAD who do not have elevated plasma cholesterol levels (Fig. 1b) .

ODP037 Effects of DHCR24 Inhibitor on Excessive Adrenal Steroidogenesis in db/db mouse

Abstract Background and purpose Patients with type 2 diabetes have an elevated basal level of cortisol, which has been suggested to be involved in the exacerbation of hyperglycemia and the progression of organ complications. Furthermore, it has been reported that adrenal steroidogenesis increases in db/db mice, an animal model of type 2 diabetes. However, the mechanism of the increased adrenal steroidogenesis in type 2 diabetes is unclear. In this study, we aimed to elucidate the mechanism of increased adrenal steroidogenesis in db/db mice and examine whether it is a therapeutic target. Experiment 1: Methods Gene expression in adrenal glands of 10-week-old male db/db mice and db/+ mice was examined by DNA microarray and real-time PCR. Results In the adrenal glands of db/db mice, expression of Acc and Scd1, which are involved in fatty acid synthesis, was decreased by 3.7-fold and 2.6-fold, respectively, compared with db/+ mice. However, expression of Cpt1α, which is involved in acetyl-CoA production by lipolysis, was increased by 1.8-fold and expression of Dhcr24, the final enzyme of the de novo cholesterol synthesis system, was increased by 2.5-fold. Thus, fatty acid degradation and endogenous cholesterol synthesis had increased in the adrenal glands of db/db mice. Experiment 2: Methods 8-week-old male db/db mice were divided into two groups, PBS administration (P) and 10mg/kg DHCR24 inhibitor (U18666A) administration (U) groups. PBS or U18666A was injected intraperitoneally into mice once a week. 4 weeks later, the mice were euthanized, blood corticosterone was measured by LC-MS/MS, and gene expression in adrenal glands was measured by real-time PCR. Results The blood corticosterone level was 196.6±12.7ng/mL in the U group and 245.8±15.4ng/mL in the P group, which was significantly lower in the U group (unpaired t-test, p&amp;lt;0. 05). mRNA expression of Acc and Scd1 was increased by 3.1-fold and 1.3-fold, respectively, in the U group compared with the P group. Conclusion These results suggest that increased endogenous cholesterol synthesis may be a cause of steroid overproduction in the adrenal glands of db/db mice and a DHCR24 inhibitor suppresses the increase in steroid hormone synthesis. This study indicates that changes in adrenal fatty acid metabolism and endogenous cholesterol synthesis might play a major role in understanding the mechanism of increased adrenal steroidogenesis and finding a new therapeutic target to prevent organ complications of type 2 diabetes. Presentation: No date and time listed

Structural mechanisms of TRPV2 modulation by endogenous and exogenous ligands.

The transient receptor potential vanilloid 2 (TRPV2) ion channel is a polymodal receptor widely involved in many physiological and pathological processes. Despite many TRPV2 modulators being identified, whether and how TRPV2 is regulated by endogenous lipids remains elusive. Here, we report an endogenous cholesterol molecule inside the vanilloid binding pocket (VBP) of TRPV2, with a 'head down, tail up' configuration, resolved at 3.2 Å using cryo-EM. Cholesterol binding antagonizes ligand activation of TRPV2, which is removed from VBP by methyl-β-cyclodextrin (MβCD) as resolved at 2.9 Å. We also observed that estradiol (E2) potentiated TRPV2 activation by 2-aminoethoxydiphenyl borate (2-APB), a classic tool compound for TRP channels. Our cryo-EM structures (resolved at 2.8-3.3 Å) further suggest how E2 disturbed cholesterol binding and how 2-APB bound within the VBP with E2 or without both E2 and endogenous cholesterol, respectively. Therefore, our study has established the structural basis for ligand recognition of the inhibitory endogenous cholesterol and excitatory exogenous 2-APB in TRPV2.

Effect of Coffee on the Bioavailability of Sterols.

Absorption at the intestinal epithelium is a major determinant of cholesterol levels in the organism, influencing the entry of dietary cholesterol and the excretion of endogenous cholesterol. Several strategies are currently being followed to reduce cholesterol absorption, using both pharmacological agents or food ingredients with hypocholesterolemic properties. Coffee has recently been shown to affect cholesterol bioaccessibility, although it has not been shown if this translates into a decrease on cholesterol bioavailability. In this work, coffee obtained with different commercial roasting (light and dark) and grinding (finer and coarser) was evaluated regarding their effect on cholesterol absorption through Caco-2 monolayers, mimicking the intestinal epithelium. The fluorescent dehydroergosterol was used as a sterol model, which was shown to permeate Caco-2 monolayers with a low-to-moderate permeability coefficient depending on its concentration. In the presence of coffee extracts, a 50% decrease of the sterol permeability coefficient was observed, showing their potential to affect sterol bioavailability. This was attributed to an increased sterol precipitation and its deposition on the apical epithelial surface. A higher hypocholesterolemic effect was observed for the dark roasting and finer grinding, showing that the modulation of these technological processing parameters may produce coffees with optimized hypocholesterolemic activity.