Increase In Free Cholesterol Research Articles

Study of lipid metabolic indicators in rowers at different exercise intensities

Sports training in cyclic sports with the predominant manifestation of endurance, including rowing, is associated with processes of adaptation to the load for 1 to 1.5 hours. The sources of energy during long intensive training are not only muscle glycogen, but also fat and fat-like substances. In this connection, the authors studied the processes of restructuringand adaptation of the organism of rowers to different intensities of physical load by lipid metabolism. The study involved 15 athletes (males) aged 18 to 21 years with the sport qualification «Candidate Master of Sports in Rowing». The main fractions of the lipid spectrum were studied: phospholipids, free cholesterol, non-esterified fatty acids, mono-, di-, triacylglycerols, cholesterol esters, as well as the spectrum of lipoprotein fractions: chylomicrons, pre-β-lipoproteins, remnants, β-lipoproteins, α-lipoproteins, a complex of non-esterified fatty acids with albumin. The studies showed that among the main fractions of the lipid spectrum, the concentration of the non-esterified fatty acid complex was higher than normal by 22.6% after the 7-minute exercise and by 49.6% after the 60-minute exercise, i.e. good functioning of the transport form for free fatty acids, which is important for energy exchange, was recorded. There was a 12.8% increase in phospholipids after the 7-minute exercise and a 17.6% increase after the 60-minute exercise. There was also a 25.3% increase in free cholesterol after the 7-minute exercise and a 36.8% increase after the 60-minute exercise. Unesterified fatty acid content was normal (greater than 0.45 g/l) before the test exercise, but increased by 49.1% (p < 0.05) after the 7-minute exercise. At the 60-minute exercise, this increase flattened and decreased by 17.7% (p<0.05) compared to the 7-minute exercise, but remained 22.6% (p<0.05) above the initial value. It is concluded that non-esterified fatty acids make an important contribution to the supply of lipids for oxidation during endurance exercise.

Systemwide effects of ER-intracellular membrane contact site disturbance in primary endothelial cells

Membrane contact sites (MCS) make up a crucial route of inter-organelle non-vesicular transport within the cell. Multiple proteins are involved in this process, which includes the ER-resident proteins vesicle associated membrane protein associated protein A and -B (VAPA/B) that form MCS between the ER and other membrane compartments. Currently most functional data on VAP depleted phenotypes have shown alterations in lipid homeostasis, induction of ER stress, dysfunction of UPR and autophagy, as well as neurodegeneration. Literature on concurrent silencing of VAPA/B is still sparse; therefore, we investigated how it affects the macromolecule pools of primary endothelial cells. Our transcriptomics results showed significant upregulation in genes related to inflammation, ER and Golgi dysfunction, ER stress, cell adhesion, as well as Coat Protein Complex-I and -II (COP-I, COP-II) vesicle transport. Genes related to cellular division were downregulated, as well as key genes of lipid and sterol biosynthesis. Lipidomics analyses revealed reductions in cholesteryl esters, very long chain highly unsaturated and saturated lipids, whereas increases in free cholesterol and relatively short chain unsaturated lipids were evident. Furthermore, the knockdown resulted in an inhibition of angiogenesis in vitro. We speculate that ER MCS depletion has led to multifaceted outcomes, which include elevated ER free cholesterol content and ER stress, alterations in lipid metabolism, ER-Golgi function and vesicle transport, which have led to a reduction in angiogenesis. The silencing also induced an inflammatory response, consistent with upregulation of markers of early atherogenesis. To conclude, ER MCS mediated by VAPA/B play a crucial role in maintaining cholesterol traffic and sustain normal endothelial functions.

PEMT Mediates Hepatitis C Virus-Induced Steatosis, Explains Genotype-Specific Phenotypes and Supports Virus Replication.

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.

Increasing the efficiency of hypolipidemic therapy with the combined use of quercetin in patients with non-alcoholic fatty liver disease on the background of the metabolic syndrome

The article analyzes the results of a set of biochemical indicators in the course of treatment with the combined use of rosuvastatin with quercetin in patients with metabolic syndrome (MS) combined with non-alcoholic fatty liver disease. Changes in blood biochemical parameters have been identified and presented with MS, essential for revealing generalbiological mechanisms development and interrelationship between the components of MS and non-alcoholic fatty liver. The effect of an increase in free cholesterol and triglycerides and activation of freeradical oxidation of lipids followed by theaccumulation of oxidative stress products was noted. In the course of long-term hypolipidemic therapy (90 days), 86 patients with non-alcoholic fatty liver disease on the background of metabolic syndrome were previously divided into 2 groups: a comparison group (45 patients), who weretreated with basic therapy – only rosuvastatin, the main group (41 patients) received quercetin together with rosuvastatin –40 mg 3 times a day, clinical and laboratory-instrumental examinations were carried out. On the 90th day of treatment, positive results in the functional state of the liver and lipid spectrum of the blood wereregistered in all patients. A more significant advantage of the therapeutic combination of rosuvastatin with quercetin was proved. The inclusion of quercetin contributed to reducing the intensity of oxidative stress and enhancing antioxidant protection activity, resultingin a decrease in apoptosis of hepatocytes (cytokeratin-18 level was 1.27 times decreased). The studies have shown the feasibility of combined use of quercetin with rosuvastatin for the prevention of the development and progression of metabolic disorders associated with non-alcoholic fatty liver disease.

Exposure to Deepwater Horizon crude oil increases free cholesterol in larval red drum (Sciaenops ocellatus).

The 2010 Deepwater Horizon oil spill impacted over 2100km of shoreline along the northern Gulf of Mexico, which coincided with the spawning season of many coastal species, including red drum (Sciaenops ocellatus). Red drum develop rapidly and are sensitive to crude oil exposure during the embryonic and larval periods. This study investigates the predictions from recent transcriptomic studies that cholesterol biosynthetic processes are impacted by oil exposure in fish early life stages. We found that red drum larvae exposed for 72h to ΣPAH50 3.55-15.45µg L-1 exhibited significantly increased pericardial area, a cardiotoxicity metric, but the expression of several genes targeted in the cholesterol synthesis pathway was not affected. However, whole-mount staining revealed significant increases in free cholesterol throughout the larval body (ΣPAH50 4.71-16.15µg L-1), and total cholesterol followed an increasing trend (ΣPAH50 3.55-15.45µg L-1). Cholesterol plays a critical role in fish embryo development and ion channel function. Therefore, the disruption of cholesterol homeostasis, as observed here, could play a role in the oil toxicity phenotype observed across many fish species.

Delineating a novel metabolic high triglycerides-low waxes syndrome that affects lipid homeostasis in meibomian and sebaceous glands

Meibomian glands that are embedded in tarsal plates of human eyelids, and sebaceous glands found in the skin, including that of eyelids, are two related types of holocrine glands that produce lipid-rich secretions called meibum and sebum. Pervasive ocular disorders, such as Meibomian gland dysfunction and dry eye, have been linked to changes in the lipid composition of meibum. However, in most described cases the changes were either small, or random, or insufficiently characterized on the molecular level. Here, we present results of comprehensive lipidomic analyses of meibum, tears and sebum of a patient whose secretions were highly abnormal (abnormal meibum, tears and sebum, or AMTS, patient). The lipidomes were characterized on the level of individual lipid species using ultra-high performance liquid chromatography and high resolution mass spectrometry. The major differences between the AMTS patient and normal age- and gender-matched subjects included, among others, severely diminished pools of normal meibomian lipids such as wax esters and cholesteryl esters in meibum and tears, a 2x increase in total cholesteryl esters to wax esters ratio, their skewed molecular profiles, a ~3x increase in free cholesterol to cholesteryl esters ratio, and, most importantly, a 20x to 30x increase in the triglicerides fraction over the norm. Sebum of the AMTS patient was also highly abnormal lacking major wax esters. Notably, the routine blood lipid panel test of the AMTS patient showed no abnormalities. The data imply that the AMTS patient had a severe, previously unreported, metabolic disorder that affected meibogenesis in Meibomian glands and sebogenesis in sebaceous glands. This is, to the best of our knowledge, a first observation of the condition that we have termed High Triglycerides/Low Waxes (HTLW) syndrome.

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

SAT-354 Characterization of Cell Death Induced by SOAT1 Inhibition in Adrenocortical Carcinoma Cells

Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). We have found that mitotane inhibits Sterol-O-Acyl transferase 1 (SOAT1) which leads to depletion of cholesterol esters and increase of free cholesterol in the ACC cell line NCI-H295. We demonstrated activation of endoplasmic reticulum response pathway that resulted in decreased cell viability. Aim: To characterize the mechanisms underlying cell death resulting from SOAT1 inhibition with inhibitors mitotane, ATR101, AZD3988 and Sandoz58-035. Methods: SOAT1 inhibition was quantified in vitro, ER-stress marker expression by qPCR, cell viability by cell titer glo assay, lipid composition by GC-MS, and lipid peroxidation with BODIPY 581/591 C11. Cell death in NCIH295R cells induced by mitotane was determined by mitotane treatment alone and in combination with either the pan caspase inhibitor zvad-fmk, necroptose inhibitor necrostatin-1, ferroptosis inhibitor liproxstatin or ferroptosis activators erastin and RSL-3. Results: Mitotane and ATR101 but not AZD3988 and Sandoz58-035 induced lipid peroxidation in NCI-H295R cells in a concentration dependent manner. ATR101, AZD3988 and Sandoz58-035 were inhibitors of SOAT1 with IC50 of 3 nM, 13 nM and 0.9 nM. Correspondingly, expression of ER-stress marker CHOP and splicing of X-box protein 1 mRNA was activated by mitotane, ATR101 and poorly by AZD3988. Mitotane and ATR101 but not AZD3988 and Sandoz58-035 treatment induced the accumulation of free cholesterol in NCI-H295 cells after 6h. Cell viability was impaired with mitotane, ATR101 and AZD3988 but not Sandoz58-035. None of the inhibitors could reverse the effects of mitotane while synergistic effects were observed for RSL3. Erastin was shown to reverse the effects of mitotane treatment. Conclusion: Although SOAT1 inhibition was confirmed for all compounds, downstream effects on ER-stress markers and cell viability exhibit marked differences. Hence it is likely that targets different from SOAT1 are relevant for in vitro cytotoxic activity of mitotane and ATR101. Caspases do not seem to be predominantly involved in mitotane-induced cell death. Although we observed a synergistic effect of mitotane with RSL3, ferroptosis inhibitors were not able to block mitotane-induced cell death. The precise mechanism of cell death and immunogenic cell death pathways require further investigation.

Alveolar macrophages from tuberculosis patients display an altered inflammatory gene expression profile

Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.

Feeding-fasting dependent recruitment of membrane microdomain proteins to lipid droplets purified from the liver.

Lipid droplets (LDs) are cellular stores of neutral fat that facilitate lipid and protein trafficking in response to metabolic cues. Unlike other vesicles, the phospholipid membrane on the LD is a monolayer. Interestingly, this monolayer membrane has free cholesterol, and may therefore contain lipid microdomains that serve as a platform for assembling proteins involved in signal transduction, cell polarity, pathogen entry etc. In support of this, cell culture studies have detected microdomain-associated “raftophilic” proteins on LDs. However, the physiological significance of this observation has been unclear. Here we show that two proteins (Flotillin-1 and SNAP23) that bind to membrane microdomains associate differently with LDs purified from rat liver depending on the feeding/fasting state of the animal. Flotillin-1 increases on LDs in the fed state, possibly because LDs interact with the endoplasmic reticulum (ER), facilitating supply of flotillin-1 from ER to LDs. Interestingly, this increase in flotillin-1 is correlated with an increase in free cholesterol on the LDs in fed state. In opposite behaviour to flotillin-1, SNAP23 increases on LDs in the fasted state and this appears to mediate LD-mitochondria interactions. Such LD-mitochondria interactions may provide fatty acids to mitochondria for promoting beta-oxidation in hepatocytes in response to fasting. Our work brings out physiologically relevant aspects of lipid droplet biology that are different from, and may not be entirely possible to replicate and study in cell culture.

Some Structural and Chemical Changes in Endocardial Endothelium of Rats in Emotional and Pain Stress Complicated by Hypercholesterolemia

The objective of the research was to study the content of some neutral lipids of endocardial endothelium in rats in relation to structural changes occurring in it, in the co-existence of emotional and pain stress, as well as alimentary hypercholesterolemia. Materials and methods. The electric-impulse model was used for stress modeling. Alimentary hypercholesterolemia was modeled feeding animals an atherogenic diet. The concentration of triacylglycerols, free and esterified cholesterol were examined using the method of thin-layer chromatography performed on silica gel. The concentration of free fatty acids was determined using the radiochemical method. The state of endocardial endothelium was studied with the help of light microscopy; the impression smears obtained from macro preparations of ventricle were analyzed.Results. In co-existence of stress and hypercholesterolemia, significant increase in free cholesterol as well as free fatty acid concentration was noticed. This essentially exceeded the analogical indices under the action of stress only. Structural changes in the endocardium followed by desquamation of separate endotheliocytes were the result of stress reaction. In the action of both pathogenic factors, this process was intensified; layer-by-layer exfoliation of endotheliocytes was observed. Conclusions. In acute emotional and pain stress, changes in lipid spectrum of membrane structures of endocardial endotheliocytes the main manifestation of which is the accumulation of free cholesterol in cells and increase in the levels of free fatty acids take place. The increase in the number of desquamated endothelial cells is the result of stress action as well. Alimentary hypercholesterolemia significantly increases such pathological changes.

Mitotane Revisited: A New Target for an Old Drug

Adrenocortical carcinoma (ACC) is among the deadliest endocrine malignancies. Radical surgical resection of a localized tumor is the only curative option for ACC, but in many patients cancer is diagnosed already at an advanced stage, needing pharmacological treatment. The mainstay of the medical therapy for metastatic ACC is mitotane (o,p -DDD), a derivative of the insecticide dichlorodiphenyl-trichloroethane, which is also used as an adjuvant treatment in patients resected for localized ACC but at a high risk of relapse. Mitotane is an old drug. More than 60 years ago, Nelson and Woodard showed that technical-grade dichlorodiphenildichloroethane caused selective atrophy of the adrenal cortex in the dog (1). Subsequent studies demonstrated that the active substance was o,p -DDD, an impurity in the technical-grade product, which was then introduced in the treatment of ACC (2). Today mitotane is the only approved drug for ACC treatment, even if controversies remain concerning its long-term efficacy (reviewed in reference 3). Mitotane has both an adrenolytic action on ACC cells and inhibits steroid hormone synthesis, with beneficial effects in patients with Cushing’s syndrome. However, mitotane therapy often has important side effects that limit its use in the clinic. Furthermore, for many patients it is difficult to attain and maintain the therapeutic levels of plasma mitotane (between 14 and 20 mg/L) (4). A better knowledge of the mechanism of action of mitotane is then required to develop new drugs that are more efficient and better tolerated by ACC patients. Recent studies showed that mitotane treatment negatively affects mitochondrial respiratory chain activity (5) and induces mitochondrial morphofunctional changes in ACC cells (6), but the mechanisms of those effects remained elusive. A breakthrough in our understanding of how mitotane works as a selective toxic agent for adrenal cells is represented by the study of Sbiera et al (7) published in this issue of Endocrinology. Here the authors show compelling evidence that mitotane treatment rapidly induces endoplasmic reticulum (ER) stress in ACC cells but not in cancer cell lines from tissues other than the adrenal. ER stress generates protein misfolding in the ER, which is sensed by several signaling cascades and triggers a response targeted to limit the effects of ER stress. Collectively this phenomenon is termed the unfolded protein response (UPR) (8). Mitotane-induced ER stress is correlated with the accumulation of toxic lipids inside ACC cells. In particular, increase in free cholesterol and decrease of cholesteryl esters pinpointed the inhibition of sterol-Oacyl-transferase (SOAT) (also known as acyl-coenzyme A cholesterol acyltransferase) activity as a potential mechanism of action of mitotane. In the adrenal, this enzyme has the role to produce stores of esterified cholesterol protecting cells from the damaging effects of free cholesterol. Cholesterol esters can be rapidly made available as substrates for steroidogenesis after ACTH stimulation by the action of hormone-sensitive lipase (9). Consistently with its important role in steroidogenesis, SOAT1 has been shown to be a target for steroidogenic factor-1, an essential transcriptional regulator of steroidogenic genes (10). Based on those results, Sbiera et al (7) show that mitotane indeed inhibits SOAT1 activity in vitro and that its effect correlates with

Dosvid likuvannia diabetychnoho makuliarnoho nabriaku v patsiientiv iz tsukrovym diabetom 2-ho typu na tli korektsii lipidnoho obminu

Problem. According to current observations, the increase in free cholesterol, low-density lipoprotein and triglyceride levels associated with the risk of diabetic macular edema (DME). Purpose. To study the effectiveness of pharmacological micronized fenofibrate, 145 mg in the complex treatment of patients with type II diabetes with diabetic macular edema. Material and methods. Two groups of patients with diabetes type II and DME were investigated. In the first group, patients received the fenofibrate, 145 mg during 8 months and onetime intraocular injection of aflіbersept; in the second one – only intraocular injection of aflіbersept. Assessment of the retina produced in all terms by visual acuity and retinal thickness of macular area with optical coherence tomography Stratus-3000 with control of the lipid parameters in blood serum. Results. In 8 months in group I, who obtained the drug fenofibrate, 145 mg, marked and more positive trend in ophthalmic and biochemical parameters and improve lipid profile were revealed. Conclusion. The paper presents the results of the comparative analysis to determine the effectiveness of micronized fenofibrate (Traykor®145 mg) in complex microsurgical treatment of DME with the control of the dynamics of changes in lipid parameters of blood serum was done.

Abstract 419: StarD5: Role in a Novel Pathway of Intracellular Cholesterol Transport

Introduction: How cholesterol is moved within the cell via a non-vesicular means and is mobilized against a gradient to the plasma membrane has never been clearly understood. StarD5, a member of the StarD4 subfamily of START (steroidogenic acuteregulatory lipid transfer) domain proteins, is able to bind cholesterol and can be induced by ER stress. Its function, however, has remained a mystery. The objective of this study was to attempt to define the role of StarD5 in intracellular cholesterol metabolism by following its response to ER stress. Methods: StarD5 knockdown cells were obtained by using siRNA, while infection with an adenovirus encoding StarD5 was used to selectively overexpress the protein. Immunoblots were performed to determine knockdown and overexpression of StarD5. Filipin staining and immunocytochemistry were used to determine the localization of free cholesterol within the cells. Apoptosis during ER stress was determined by staining cells with Alexa Fluor-488 Annexin V. Results: Overexpression of StarD5 in THP-1 macrophages led to a rapid redistribution of StarD5 and cholesterol from the Golgi/ER to the plasma membrane. StarD5 knockdown led to a marked increase in free cholesterol within the Golgi and ER in 3T3-L1 cells. Furthermore, thapsigargin-induced apoptosis (which leads to an abnormal accumulation of cholesterol in the ER) was markedly increased with StarD5 knockdown. Conclusion: StarD5 has the ability to respond to different stresses with a directed movement, mobilizing cholesterol between the ER/Golgi to the plasma membrane in order to establish a needed cholesterol membrane concentration gradient amongst intracellular organelles. These findings demonstrate a previously unexplored pathway of intracellular cholesterol transport whose baseline function is induced by ER-stress; likely as a means to stabilize the cell during periods of stress by mobilizing excess cholesterol from the ER and stabilizing the plasma membrane through cholesterol induced changes in permeability and fluidity.

ACAT-selective and Nonselective DGAT1 Inhibition

Acyl-coenzyme A: cholesterol O-Acyltransferase (ACAT) and Acyl-coenzyme A: diacylglycerol O-acyltransferase (DGAT) enzymes play important roles in synthesizing neutral lipids, and inhibitors of these enzymes have been investigated as potential treatments for diabetes and other metabolic diseases. Administration of a Acyl-coenzyme A: diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor with very limited cellular selectivity over ACAT resulted in significant adrenocortical degenerative changes in dogs. These changes included macrosteatotic vacuolation associated with adrenocyte cell death in the zonae glomerulosa and fasciculata and minimal to substantial mixed inflammatory cell infiltration and were similar to those described previously for some ACAT inhibitors in dogs. In the mouse, similar but only transient adrenocortical degenerative changes were seen as well as a distinctive nondegenerative reduction in cortical fine vacuolation. In the marmoset, only the distinctive nondegenerative reduction in cortical fine vacuolation was observed, suggesting that the dog, followed by the mouse, is the most sensitive species for cortical degeneration. Biochemical analysis of adrenal cholesterol and cholesteryl ester indicated that the distinctive reduction in cortical fine vacuolation correlated with a significant reduction in cholesteryl ester in the mouse and marmoset, whereas no significant reduction in cholestryl ester, but an increase in free cholesterol was observed in dogs. Administration of a DGAT1 inhibitor with markedly improved selectivity over ACAT to the marmoset and the mouse resulted in no adrenal pathology at exposures sufficient to cause substantial DGAT1 but not ACAT inhibition, thereby implicating ACAT rather than DGAT1 inhibition as the probable cause of the observed adrenal changes. Recognizing that the distinctive nondegenerative reduction in cortical fine vacuolation in the mouse could be used as a histopathological biomarker for an in vivo model of the more severe changes observed in dogs, the mouse has subsequently been used as a model to select DGAT1 inhibitors free of adrenocortical toxicity.

SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to high-density lipoprotein 3

BackgroundThe single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated.ResultsWhen apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI.ConclusionsWe found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.

Lipids-induced Apoptosis Is Aggravated by Acyl-coenzyme A: Cholesterol Acyltransferase Inhibitor

To investigate the role of acyl-coenzyme A: cholesterol acyltransferase inhibitor (ACATI) in apoptosis induced by lipids and whether lipids-induced apoptosis is accompanied by increase of free cholesterol in endoplasmic reticulum (ER), in order to further understand the mechanism of lipids-induced apoptosis in advanced atherosclerosis. Human vascular smooth muscle cells (VSMCs) and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages were used. Tritiated thymidine incorporation was applied to detect cell proliferation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release. 4',6-diamidino-2-phenylindole (DAPI) staining, caspase-3, -7 assay, and Annexin-V/propidium iodide (PI) staining were used to detect apoptosis. High performance liquid chromatography was used in intracellular free cholesterol and cholesterol ester assay. ER free cholesterol was quantified. Different lipids had different effects on proliferation and cytotoxicity of VSMCs. 25-hydroxycholesterol (25OHC) had biphasic effects on the proliferation of VSMCs. At low concentration, it stimulated cell proliferation, but turned to proliferation inhibition as concentration reached 15 mug/mL. 25OHC and acetylated low density lipoprotein (AcLDL) could respectively induce apoptosis in human VSMCs and PMA differentiated THP-1 macrophages, which was aggravated by ACATI, accompanied by increase of intracellular free cholesterol content. There was also an increase of cholesterol content in ER with AcLDL-induced apoptosis in THP-1 macrophages. Lipids could induce apoptosis, accompanied by increase of intracellular free cholesterol content, which could be augmented by ACATI, suggesting that insults resulting in ER free cholesterol rise might be the initiator of apoptosis.

Macrophages Create a Lysosomal Synapse to Digest Aggregated Lipoproteins

Atherosclerosis is the underlying cause of the majority of heart attacks and strokes. Lipoprotein accumulation and degradation by monocyte-derived macrophages is a central event in the pathogenesis of atherosclerosis. Data show that the vast majority of low density lipoprotein (LDL) in atherosclerotic plaques is aggregated and avidly bound to the subendothelial matrix.We employ various microscopy and biochemical techniques in both cell culture and animal models to investigate the mechanism of uptake of aggregated LDL (agLDL) by macrophages. We show that macrophages create an extracellular, acidic compartment where the cells contact the agLDL, and lysosomal contents are delivered to these compartments, thereby forming an extracellular hydrolytic compartment - a lysosomal synapse. Active acidification of these specialized compartments and aggregate catabolism were observed by fluorescence ratiometric time-lapse imaging of an in vitro cell culture model. Biochemical assays, employing radiolabeled agLDL demonstrated an increase in free cholesterol in aggregates contained in the lysosomal synapse. This cholesterol can be delivered to the cell, initiating the process of macrophage cholesterol loading and ultimately causing progression of the atherosclerotic plaque.Although in vitro systems provide insight into macrophage uptake of lipoproteins, ultimately we must strive to understand the function and behavior of macrophages in intact animals. Relatively little is known of the fate of monocytes once they migrate into atherosclerotic lesions. To this end, we employ intravital multiphoton microscopy in mouse models of atherosclerosis to allow observation of macrophages within the artery wall.Our studies elucidate the mechanism of a novel pathway for catabolism of agLDL by macrophages. Better understanding of the mechanisms by which macrophages interact with lipoproteins in the subendothelium may lead to new approaches to inhibit lipid accumulation in macrophages and thus, may be of therapeutic value in preventing atherosclerosis.

Altered Cholesterol Ester Cycle in Skin Fibroblasts from Patients with Alzheimer's Disease

Intracellular cholesterol metabolism was reported to modulate amyloid-beta (Abeta) generation in Alzheimer's disease (AD). Results presented herein demonstrated that, like brain cells, cultured skin fibroblasts from AD patients contained more cholesterol esters than fibroblasts from healthy subjects. Particularly, Oil Red-O, Nile Red, and filipin staining highlighted higher levels of neutral lipids which responded to inhibitors of acyl-coenzyme A:cholesterol acyl-transferase (ACAT-1), associated with an increase in free cholesterol. ACAT-1 mRNA levels increased significantly in AD fibroblasts, whereas those of sterol regulatory element binding protein-2, neutral cholesterol ester hydrolase, and ATP-binding cassette transporter member 1 were markedly down-regulated. Instead, mRNA levels of low-density lipoprotein receptor, hydroxy-methyl-glutaryl-coenzyme A reductase, caveolin-1, and amyloid-beta protein precursor (AbetaPP) were virtually unchanged. Notably, mRNA levels of both beta-site AbetaPP-cleaving enzyme 1 (BACE1) and neprilysin were significantly down-regulated. An increase in Abeta(40) and Abeta(42) immunostaining and a decrease in BACE1 active form were also found in AD versus control fibroblasts. Altogether, these findings support the hypothesis that the derangement of cholesterol homeostasis is a systemic alteration involving central but also peripheral cells of AD patients, and point to cholesterol ester levels in AD fibroblasts as an additional metabolic hallmark useful in the laboratory and clinical practice.

Macrophages Create an Acidic Extracellular Hydrolytic Compartment to Digest Aggregated Lipoproteins

A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake. We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing of portions of the compartments, allowing formation of an "extracellular" proton gradient. An increase in free cholesterol is observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein. A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.