Cholesterol Ester Content Research Articles

Bilirubin ameliorates murine atherosclerosis through inhibiting cholesterol synthesis and reshaping the immune system

Atherosclerosis is a chronic inflammatory disease caused mainly by lipid accumulation and excessive inflammatory immune response. Although the lipid-lowering and cardioprotective properties of bilirubin, as well as the negative relationship between bilirubin and atherosclerosis, were well documented, it is not yet clear whether bilirubin can attenuate atherosclerosis in vivo. In this study, we investigated the role of bilirubin in improving atherosclerosis. We found that mildly elevated bilirubin significantly reduced the risk factors of atherosclerosis, such as plasma glucose, total cholesterol, and low-density lipoprotein cholesterol, and the formation of atherosclerotic plaques, liver total cholesterol, and cholesterol ester concentration in apolipoprotein E-deficient (ApoE−/−) mice fed a western-type (high fat) diet. It was further found that bilirubin could promote the degradation of 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), a rate-limiting enzyme for endogenous cholesterol synthesis. Using mass cytometry-based high dimensional single cell analysis, we observed a decrease of natural killer cells and an increase of dendritic cells and myeloid-derived suppressor cells, which all are closely associated with atherosclerosis risk factors and contribute to the improvement of atherosclerosis, in ApoE−/− mice treated with bilirubin. By in-depth analysis, modulation of multiple spleen or peripheral blood T cell clusters exhibiting either positive or negative correlations with total cholesterol or low-density lipoprotein cholesterol was detected after bilirubin treatment. In this study, we demonstrate that bilirubin serves as a negative regulator of atherosclerosis and reduces atherosclerosis by inhibiting cholesterol synthesis and modulating the immune system.

Hyperglycemia-Induced Metabolic Reprogramming Mediates a Proatherogenic Phenotype in Healthy Human Monocytes

Introduction: Poor glycemic control is considered an important contributor to cardiovascular disease in patients with diabetes. Episodic hyperglycemia as a surrogate for glycemic variability promotes monocyte adhesion and increases the prevalence of proinflammatory monocytes within atherosclerotic plaques of patients with diabetes. We previously found that acute hyperglycemia-induced a pro-inflammatory phenotype and promoted the development of foamy monocytes by increasing total cholesterol deposition, cholesterol ester, and free cholesterol content by enhancing oxidized LDL uptake. However, the mechanism by which acute hyperglycemia induces monocyte cholesterol deposition and inflammation remains unknown. Methods: Monocytes isolated from healthy individuals (age range 20–40; n=5) were cultured in low (5mM) or high (16.7mM) glucose conditions with or without a glycolysis inhibitor (2-deoxyglucose, 2DG, 5 mM) or an endoplasmic reticulum stress inhibitor (4-phenylbutyric acid, PBA; 20mM) for 6 hrs. After treatment, cytokine release, oxidized LDL uptake, and metabolic assays using Seahorse Technology were performed. Results: Healthy human monocytes exposed under high glucose conditions showed a pro-atherosclerotic phenotype with higher levels of the pro-inflammatory cytokines, TNFα(median of differences 6.34 pg/ml, p=0.002) and IL1β(12.04 pg/ml, p=0.003), and increased oxidized LDL uptake (5062ug Dil-Ox LDL/mg, p=0.001). Furthermore, hyperglycemia resulted in higher levels of glycolysis (basal glycolysis 12.94 pmol/min, p=0.01; basal proton efflux rate 15.5 pmol/min, p=0.03) and mitochondrial respiration (percentage of respiratory capacity 16pmol/min p=0.04), suggesting a significant alteration in the metabolic programming of these monocytes. Treatment with 2-DG or PBA attenuated the pro-atherosclerotic phenotype induced by hyperglycemia, promoting a reduction of cytokine release, a reduction of oxidized LDL uptake, and near normalization of the glycolic rate and mitochondrial respiration, stabilizing cellular bioenergetics. Conclusions: Altogether, our results suggest that monocyte ER stress in response to acute hyperglycemia promotes a hypermetabolic state characterized by a proinflammatory and proatherogenic monocyte phenotype. Therefore, acute hyperglycemia is a potential mechanism promoting atherosclerosis in patients with type 2 diabetes.

Low-level laser irradiation in the comprehensive treatment of acute renal damage

Background. The discussed topic is worth attracting attention because of its high mortality rate in patients with acute renal damage which is caused by urological and surgical diseases. The membrane-destructive component in damaged renal tissues is manifested with decreased glomerular filtration rate, increased endotoxin level and lipid aberrations. The standard infusion and antibacterial therapy does not fully compensate the severity of pathological changes.Purpose. To study the effectiveness of low-level laser irradiation at endotoxicosis and lipid metabolism indicators in acute renal damage in acute experimental peritonitis.Material and methods. Lipid peroxidation, membrane-destructive processes in renal tissues as well as lipid metabolism and endotoxicosis were studied in the experiment with dogs. The effectiveness of infusion and antibiotic therapy and combined therapy with laser light were compared.Basic results: the experiment has demonstrated that acute renal damage was caused by systemic inflammatory diseases of non-urological profile. The applied low-level laser irradiation (LLLI) promoted more rapid correction of pathological processes which were manifested with increased concentration of cholesterol esters, total phospholipids, phosphatidylserine; LLLI decreased levels of cholesterol, phosphatidylcholine, triacylglycerol, phosphatidylinositol, triacylglycerol, phosphatidylinositol, diacylglycerol, lysophospholipids, free fatty acids and endotoxicosis parameters.Conclusion: the obtained results reliably indicate a LLLI positive effect at the organism’s potentials to recover when LLLI is included into the comprehensive therapy (under experiment). This is evidently seen in the restoration of lipid component in elements of renal parenchyma and in the decrease of lipid peroxidation activity.

MicroRNA-328-5p Alleviates Macrophage Lipid Accumulation through the Histone Deacetylase 3/ATP-binding cassette transporter A1 pathway.

MicroRNA-328 (miR-328) was reported to protect against atherosclerosis, but its role in foam cell formation remains unknown. The aim of this study was to investigate the effect of miR-328-5p on macrophage lipid accumulation and the underlying mechanisms. The results showed that miR-328-5p expression was robustly decreased in oxidized low-density lipoprotein (ox-LDL)-treated macrophages. Treatment of human acute monocytic leukemia cel (THP-1) macrophage-derived foam cells with a miR-328-5p mimic markedly increased [3 H]-cholesterol efflux, inhibited lipid droplet accumulation, and decreased intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) contents. Upregulation of miR-328-5p also reduced the expression of histone deacetylase 3 (HDAC3) but increased the levels of ATP-binding cassette transporter A1 (ABCA1) in THP-1 macrophage-derived foam cells. Mechanistically, miR-328-5p inhibited HDAC3 expression by directly targeting its 3'UTR, thereby promoting ABCA1 expression and the subsequent cholesterol efflux. Furthermore, miR-328-5p mimic treatment did not affect the uptake of Dil-ox-LDL or the expression of scavenger receptor-A (SR-A), thrombospondin receptor (CD36) and ABCG1. Taken together, these findings suggest that miR-328-5p alleviates macrophage lipid accumulation through the HDAC3/ABCA1 pathway.

APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Lipoprotein Lipase Regulates Microglial Lipid Droplet Accumulation.

Microglia become increasingly dysfunctional with aging and contribute to the onset of neurodegenerative disease (NDs) through defective phagocytosis, attenuated cholesterol efflux, and excessive secretion of pro-inflammatory cytokines. Dysfunctional microglia also accumulate lipid droplets (LDs); however, the mechanism underlying increased LD load is unknown. We have previously shown that microglia lacking lipoprotein lipase (LPL KD) are polarized to a pro-inflammatory state and have impaired lipid uptake and reduced fatty acid oxidation (FAO). Here, we also show that LPL KD microglia show excessive accumulation of LD-like structures. Moreover, LPL KD microglia display a pro-inflammatory lipidomic profile, increased cholesterol ester (CE) content, and reduced cholesterol efflux at baseline. We also show reduced expression of genes within the canonical cholesterol efflux pathway. Importantly, PPAR agonists (rosiglitazone and bezafibrate) rescued the LD-associated phenotype in LPL KD microglia. These data suggest that microglial-LPL is associated with lipid uptake, which may drive PPAR signaling and cholesterol efflux to prevent inflammatory lipid distribution and LD accumulation. Moreover, PPAR agonists can reverse LD accumulation, and therefore may be beneficial in aging and in the treatment of NDs.

DNA methylation and lipid metabolism: an EWAS of 226 metabolic measures

BackgroundThe discovery of robust and trans-ethnically replicated DNA methylation markers of metabolic phenotypes, has hinted at a potential role of epigenetic mechanisms in lipid metabolism. However, DNA methylation and the lipid compositions and lipid concentrations of lipoprotein sizes have been scarcely studied. Here, we present an epigenome-wide association study (EWAS) (N = 5414 total) of mostly lipid-related metabolic measures, including a fine profiling of lipoproteins. As lipoproteins are the main players in the different stages of lipid metabolism, examination of epigenetic markers of detailed lipoprotein features might improve the diagnosis, prognosis, and treatment of metabolic disturbances.ResultsWe conducted an EWAS of leukocyte DNA methylation and 226 metabolic measurements determined by nuclear magnetic resonance spectroscopy in the population-based KORA F4 study (N = 1662) and replicated the results in the LOLIPOP, NFBC1966, and YFS cohorts (N = 3752). Follow-up analyses in the discovery cohort included investigations into gene transcripts, metabolic-measure ratios for pathway analysis, and disease endpoints. We identified 161 associations (p value < 4.7 × 10−10), covering 16 CpG sites at 11 loci and 57 metabolic measures. Identified metabolic measures were primarily medium and small lipoproteins, and fatty acids. For apolipoprotein B-containing lipoproteins, the associations mainly involved triglyceride composition and concentrations of cholesterol esters, triglycerides, free cholesterol, and phospholipids. All associations for HDL lipoproteins involved triglyceride measures only. Associated metabolic measure ratios, proxies of enzymatic activity, highlight amino acid, glucose, and lipid pathways as being potentially epigenetically implicated. Five CpG sites in four genes were associated with differential expression of transcripts in blood or adipose tissue. CpG sites in ABCG1 and PHGDH showed associations with metabolic measures, gene transcription, and metabolic measure ratios and were additionally linked to obesity or previous myocardial infarction, extending previously reported observations.ConclusionOur study provides evidence of a link between DNA methylation and the lipid compositions and lipid concentrations of different lipoprotein size subclasses, thus offering in-depth insights into well-known associations of DNA methylation with total serum lipids. The results support detailed profiling of lipid metabolism to improve the molecular understanding of dyslipidemia and related disease mechanisms.

Myristica fragrans promotes ABCA1 expression and cholesterol efflux in THP-1-derived macrophages.

Myristica fragrans is a traditional herbal medicine and has been shown to alleviate the development of atherosclerosis. However, the anti-atherogenic mechanisms of M. fragrans are still to be addressed. In this study, we explored the effect of M. fragrans on lipid metabolism and inflammation and its mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction and western blot analysis results showed that M. fragrans promotes cholesterol efflux from THP-1-derived macrophages and reduces intracellular total cholesterol, cholesterol ester, and free cholesterol contents in a dose- and a time-dependent manner. Further study found that liver X receptor alpha (LXRα) antagonist GGPP significantly blocked the upregulation of ABCA1 expression with M. fragrans treatment. In addition, chromatin immunoprecipitation assay confirmed that GATA binding protein 3 (GATA3) can bind to the LXRα promoter, and inhibition of GATA3 led to the downregulation of LXRα and ATP-binding cassette subfamily A member 1 expression. Furthermore, M. fragrans reduced lipid accumulation, followed by decreasing tumor necrosis factor-α, interleukin (IL)-6, and IL-1β and increasing IL-10 produced by THP-1-derived macrophages. Therefore, M. fragrans is identified as a valuable therapeutic medicine for atherosclerotic cardiovascular disease.

Vasculoprotective properties of plasma lipoproteins from brown bears (Ursus arctos)

Plasma cholesterol and triglyceride (TG) levels are twice as high in hibernating brown bears (Ursus arctos) than healthy humans. Yet, bears display no signs of early stage atherosclerosis development when adult. To explore this apparent paradox, we analyzed plasma lipoproteins from the same 10 bears in winter (hibernation) and summer using size exclusion chromatography, ultracentrifugation, and electrophoresis. LDL binding to arterial proteoglycans (PGs) and plasma cholesterol efflux capacity (CEC) were also evaluated. The data collected and analyzed from bears were also compared with those from healthy humans. In bears, the cholesterol ester, unesterified cholesterol, TG, and phospholipid contents of VLDL and LDL were higher in winter than in summer. The percentage lipid composition of LDL differed between bears and humans but did not change seasonally in bears. Bear LDL was larger, richer in TGs, showed prebeta electrophoretic mobility, and had 5–10 times lower binding to arterial PGs than human LDL. Finally, plasma CEC was higher in bears than in humans, especially the HDL fraction when mediated by ABCA1. These results suggest that in brown bears the absence of early atherogenesis is likely associated with a lower affinity of LDL for arterial PGs and an elevated CEC of bear plasma.

Sterol-O-acyltransferase-1 has a role in kidney disease associated with diabetes and Alport syndrome

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. Invivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

Tribotechnical Characteristics of Mated Metals in Lubricants Containing Cholesterol Compounds

It was found that during the friction interaction of 45 grade steel on 45 grade steel, the behavior of the friction coefficient and temperature in the dynamic contact area directly depends ent the concentration of cholesterol esters in the lubricant medium and the contact time of the mating surfaces. It is noted that the lowest values of the studied values are realized with an increase in the concentration of cholesterol esters in the lubricant medium, namely, at their 100% concentration in the lubricant composition.

Loss of hepatic Mboat7 leads to liver fibrosis

ObjectiveThe rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis...

SUN-664 Effects of Acute Hyperglycemia on Monocytes in Type 2 Diabetes

Background: Type 2 diabetes is linked to accelerated atherosclerosis, however the mechanisms behind this association are unclear. Inadequate glycemic control is an important contributor for cardiovascular disease (CVD). Episodic hyperglycemia as a surrogate for glycemic variability (GV) promotes monocyte adhesion and increases proinflammatory monocytes within plaques of patients with diabetes. Monocytes are precursors to macrophages which play a vital role in atherosclerosis and are capable of delivering cholesterol content to plaques. Therefore, elucidating the mechanism by which acute hyperglycemia promotes proatherogenic monocytes in diabetes can lead to novel therapies slowing atherosclerosis progression in these patients. Methods: To assess the effects of acute hyperglycemia on monocyte cholesterol metabolism, we isolated monocytes from 28 patients with Type 2 diabetes cultured for 2 and 6 hours in normoglycemic (90mg/dL) or hyperglycemic media (300mg/dL). Cholesterol content was measured by lipid extraction and fluorometric cholesterol oxidase assay. Cholesterol uptake was assessed by incubating monocytes with Dil-oxidized LDL (Dil-OxLDL). Cholesterol synthesis was performed by incubating monocytes with 2-14C-Acetate in both glycemic conditions for 6 hours followed by lipid saponification, and separated via TLC on silica gel plates. Cholesterol efflux was assessed by labeling cells with 3H-Cholesterol-OxLDL in both glycemic conditions for 6 hours followed by APO-A1 or HDL stimulation for 4 hours. Results: Acute hyperglycemia for 2 and 6 hours induced foamy monocytes with increased total cholesterol content (22 and 32%, respectively), cholesterol ester (40% and 43%, respectively), and free cholesterol content (14% and 25%, respectively) due to enhanced DiI-OxLDL uptake. There was no significant increase in monocyte cholesterol synthesis or cholesterol efflux after stimulation with HDL or APO A-1. Conclusions: Increased monocyte cholesterol content during acute hyperglycemia is a potential mechanism promoting atherosclerosis in patients with Type 2 diabetes.

Fetal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in offspring cockerels with modification of promoter DNA methylation

In documents, maternal betaine modulates hypothalamic cholesterol metabolism in chicken posthatchings, but it remains unclear whether this effect can be passed on by generations. In present study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings (F1) were raised under the same condition until sexual maturation. Both the control group and the betaine group used artificial insemination to collect sperm from their cockerels. Fertilized eggs were incubated, and the hatchlings of the following generation (F2) were raised up to 64 D of age. F2 cockerels in betaine group showed significantly (P < 0.05) lower body weight, which was associated with significantly decreased (P < 0.05) hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic expression of cholesterol biosynthetic genes, SREBP2 and HMGCR, were significantly downregulated (P < 0.05), together with cholesterol conversion-related and excretion-related genes, CYP46A1 and ABCA1. These changes coincided with a significant downregulation in mRNA expression of regulatory neuropeptides including brain-derived neurotrophic factor, neuropeptide Y, and corticotropin-releasing hormone. Moreover, genes involved in methyl transfer cycle were also modified. Betaine homocysteine methyltransferase (P < 0.05) was downregulated, yet DNA methyltransferase1 tended to be upregulated (P = 0.06). S-adenosyl methionine/S-adenosylhomocysteine ratio was higher in the hypothalamus of betaine-treated F2 cockerels, which was associated with significantly modified CpG methylation on the promoter of those affected genes. These results suggested that betaine might regulate central cholesterol metabolism and hypothalamic expression of genes related to brain function by altering promoter DNA methylation in F2 cockerels.

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

Ethanolic extract of propolis from the Malaysian stingless bee Geniotrigona thoracica inhibits formation of THP-1 derived macrophage foam cells

Propolis is resinous materials with various therapeutic potentials used by bees to protect hive. Propolis extracted from honey bees may inhibit atherosclerosis, but no study has been performed using propolis extracted from Indo-Malayan stingless bees. The study aims to investigate the effect of the ethanolic extract of propolis (EEP) from Geniotrigona thoracica (G. thoracica) on the formation of macrophage-derived foam cells. Propolis from G. thoracica was extracted using 80% ethanol. The optimal concentrations of EEP to treat THP-1 derived macrophages were determined. THP-1 cells were differentiated into macrophages using 100 ng/mL of Phorbol 12-myristate-13-acetate for 4 days. THP-1 derived macrophages were then treated with 80 µg/mL of oxidized low-density lipoprotein (oxLDL) and 20 µg/mL of EEP at 6, 24, and 48 hours. The lipid droplets were determined by Oil Red O staining, and the amount of cholesterol ester in the cells was quantified. Concentrations of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) secreted by treated cells were measured. Twenty µg/mL of EEP was selected for cell treatment. Microscopic observation showed that EEP reduced lipid droplet aggregation in the cytosol of THP-1 derived macrophages-treated oxLDL. Similarly, cholesterol ester contents in oxLDL-treated THP-1 derived macrophages were also significantly reduced with the addition of EEP. EEP treatment also significantly reduced TNF-α and IL-1β secretion in the supernatant of oxLDL-treated THP-1 derived macrophages. In conclusion, EEP inhibited the formation of THP-1 derived macrophage foam cells by suppressing TNF-α and IL-1β secretion, which normally is upregulated in atherosclerotic plaques.

Adrenal cortex hypoxia modulates aldosterone production in heart failure

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated β3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced β3-adrenergic receptor expression. Hypoxia-mediated β3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.

484-P: Acute Hyperglycemia In Vivo Promotes a Proatherogenic Monocyte Phenotype

Diabetes is a chronic inflammatory disease that accelerate cardiovascular disease (CVD). However, the contribution of hyperglycemia to inflammation and CVD risk is unclear. Animal studies have shown that monocytes carry cholesterol to the atherosclerotic plaque. Thus, it is key to determine if hyperglycemia can exert a pro-atherogenic effect in human monocytes in vivo. We performed octreotide euglycemic-hyperglycemic clamps in 21 healthy subjects, 20-45 yo. Monocytes isolated during euglycemia (90 mg/dl) and after 2h of hyperglycemia (300 mg/dl) were evaluated for cholesterol metabolism and monocyte function (adhesion to fibronectin, transwell migration assays). Also, we assessed the effects of hyperglycemia on monocyte cholesterol metabolism and function from 10 healthy controls cultured in euglycemia or high glucose media with or without 2-deoxyglucose (2DG) at 2 and 6 hours. Acute hyperglycemia in vivo increased 37% total monocyte cholesterol content (TC) (p&amp;lt;0.02), 40% cholesterol ester (CE)(p&amp;lt;0.02) and upregulated oxidized low-density lipoprotein (oxLDL) cholesterol uptake by by 24%(p&amp;lt;0.01). Moreover, acute hyperglycemia increased monocyte adhesion and transwell migration (p&amp;lt;0.01) suggesting cholesterol full monocytes carry cholesterol into the vessel wall. In vitro studies confirmed that hyperglycemia increased monocyte adhesion and migration by 20% and monocyte TC by 20% and CE by 40% at 2 and 6 hours (p&amp;lt;0.03) by increasing oxLDL or LDL cholesterol uptake. Hyperglycemia didn’t affect monocyte cholesterol biosynthesis or cholesterol efflux induced by HDL or APOA1. Addition of 2DG cultured monocytes in both glucose conditions prevented hyperglycemia induced monocyte adhesion migration and monocyte CE and TC content at 2 and 6 hours. Thus, acute hyperglycemia in vivo without hyperinsulinemia induce monocyte glycolysis increasing monocyte cholesterol uptake, total cholesterol content it activates monocytes adhesion and migration, all key components to accelerate atherosclerosis. Disclosure R.M. Zhang: None. J. Oh: None. X. Lin: None. P. Stone: None. K.J. Wharton: None. A. Arbelaez: None. C. Bernal-Mizrachi: None. Funding National Heart, Lung, and Blood Institute (R01HL094818-06); National Institute of Neurological Disorders and Stroke (P01NS080675)

Hepatic VLDL secretion: DGAT1 determines particle size but not particle number, which can be supported entirely by DGAT2

We investigated whether, in view of its activity being expressed on both aspects of the endoplasmic reticulum (ER; dual membrane topology), diacylglycerol acyltransferase 1 (DGAT1) plays a distinctive role in determining the triglyceride (TAG) content of VLDL particles secreted by the liver. Mice in which the DGAT1 gene was specifically ablated in hepatocytes (DGAT1-LKO mice) had the same number of VLDL particles (apoB concentration) in the plasma 1 h after Triton 1339 treatment, but these particles were approximately half the size of VLDL particles secreted by control mice and had a proportionately decreased content of TAG, with normal cholesterol and cholesteryl ester contents. Analyses of purified microsomal fractions prepared from 16 h fasted control and DAGT1-LKO mice showed that the TAG/protein ratio in the ER was significantly lower in the latter. Electron micrographs of these livers showed that those from DGAT1-LKO mice did not show the increased lipid content of the smooth ER shown by control livers. The effects of DGAT1- and DGAT2-specific inhibitors on apoB secretion by HepG2 cells showed that DGAT1 is not indispensable for apoB secretion and demonstrated redundancy in the ability of the two enzymes to support apoB secretion. Therefore, our findings show that DGAT1 is essential for the complete lipidation and maturation of VLDL particles within the lumen of the ER, consistent with its dual topology within the ER membrane. In the mouse, DGAT2 can support apoB secretion (particle number) even when TAG availability for full VLDL lipidation is restricted in the absence of DGAT1.