Cholesterol Pool Size Research Articles

Abstract 397: Cotinine, but Not Nicotine, Increased ApoA-I Mediated HDL Synthesis in THP-1 Cells

Aim: Cotinine is known as a stable metabolite of nicotine, the plasma half-life is over 20-30 hrs. Here, we evaluated its characteristic properties on the apoA-I mediated HDL generation. Method: Cotinine (5.6 to 11.2μM), or nicotine (5 to 150μM) were supplemented to medium and co-incubated with apoA-I (10μg/mL) for 18 hrs with THP-1 cells to determine their effect on efflux activity. Cellular ACAT accessible cholesterol pool size was determined by [ 14 C]oleic acid labeling of cells for 60 min immediately after removal of the culture medium. The culture medium and the cellular lipids were extracted by organic solvents and cholesterol and phospholipids content were determined. Nicotine was administered orally as an aqueous solution (10 μM) to C57BL/6 mice for 10 days and the plasma lipoprotein profiles were measured by gel-HPLC (tandem TSK-lipopropak XL ) with online enzymatic lipid assay system (Skylight biotech, Inc.). Results: Supplementation of nicotine into the medium showed no effect on the apoA-I mediated cellular cholesterol release from THP-1 cells. On the other hand, cotinine increased apoA-I mediated cellular cholesterol efflux 2.8 fold. ACAT accessible cholesterol pool size was dramatically decreased to 80% and 58% of control with 5 and 10 μM cotinine treatment, respectively, indicating that cotinine effectively increased apoA-I/ABCA1 mediated cellular cholesterol efflux. Mice with oral nicotine administration showed increased plasma HDL by 11.4% (50.1±4.4 and 55.8±3.9 mg/dL for control and nicotine treatment, respectively). Conclusions: Cotinine increased apoA-I mediated cellular cholesterol efflux in THP-1 cells. The cellular ACAT accessible cholesterol pool size reduction is one of the early reactions of apoA-I/ABCA1 mediated cellular cholesterol efflux. Cotinine seemingly modifies cellular factor/factors to induce a change in cholesterol distribution. Mice administrated oral nicotine may convert the compound to cotinine by CYP2A6 and aldehyde oxidase activities in their liver, and which likely increased HDL generation. Further experiments are necessary to examine the direct effect of cotinine on the ABCA1 transporter for increasing HDL assembly system.

Recent insights into the implications of metabolism in plasmacytoid dendritic cell innate functions: Potential ways to control these functions

There are more and more data concerning the role of cellular metabolism in innate immune cells, such as macrophages or conventional dendritic cells. However, few data are available currently concerning plasmacytoid dendritic cells (PDC), another type of innate immune cells. These cells are the main type I interferon (IFN) producing cells, but they also secrete other pro-inflammatory cytokines (e.g., tumor necrosis factor or interleukin [IL]-6) or immunomodulatory factors (e.g., IL-10 or transforming growth factor-β). Through these functions, PDC participate in antimicrobial responses or maintenance of immune tolerance, and have been implicated in the pathophysiology of several autoimmune diseases, as well as in tumor immune escape mechanisms. Recent data support the idea that the glycolytic pathway (or glycolysis), as well as lipid metabolism (including both cholesterol and fatty acid metabolism) may impact some innate immune functions of PDC or may be involved in these functions after Toll-like receptor (TLR) 7/9 triggering. The kinetics of glycolysis after TLR7/9 triggering may differ between human and murine PDC. In mouse PDC, metabolism changes promoted by TLR7/9 activation may depend on an autocrine/paracrine loop, implicating type I IFN and its receptor IFNAR. This could explain a delayed glycolysis in mouse PDC. Moreover, PDC functions can be modulated by the metabolism of cholesterol and fatty acids. This may occur via the production of lipid ligands that activate nuclear receptors (e.g., liver X receptor [LXR]) in PDC or through limiting intracellular cholesterol pool size (by statin or LXR agonist treatment) in these cells. Finally, lipid-activated nuclear receptors (i.e., LXR or peroxisome proliferator activated receptor) may also directly interact with pro-inflammatory transcription factors, such as NF-κB. Here, we discuss how glycolysis and lipid metabolism may modulate PDC functions and how this may be harnessed in pathological situations where PDC play a detrimental role.

Recent insights into the implications of metabolism in plasmacytoid dendritic cell innate functions: Potential ways to control these functions.

There are more and more data concerning the role of cellular metabolism in innate immune cells, such as macrophages or conventional dendritic cells. However, few data are available currently concerning plasmacytoid dendritic cells (PDC), another type of innate immune cells. These cells are the main type I interferon (IFN) producing cells, but they also secrete other pro-inflammatory cytokines (e.g., tumor necrosis factor or interleukin [IL]-6) or immunomodulatory factors (e.g., IL-10 or transforming growth factor-β). Through these functions, PDC participate in antimicrobial responses or maintenance of immune tolerance, and have been implicated in the pathophysiology of several autoimmune diseases. Recent data support the idea that the glycolytic pathway (or glycolysis), as well as lipid metabolism (including both cholesterol and fatty acid metabolism) may impact some innate immune functions of PDC or may be involved in these functions after Toll-like receptor (TLR) 7/9 triggering. Some differences may be related to the origin of PDC (human versus mouse PDC or blood-sorted versus FLT3 ligand stimulated-bone marrow-sorted PDC). The kinetics of glycolysis may differ between human and murine PDC. In mouse PDC, metabolism changes promoted by TLR7/9 activation may depend on an autocrine/paracrine loop, implicating type I IFN and its receptor IFNAR, explaining a delayed glycolysis. Moreover, PDC functions can be modulated by the metabolism of cholesterol and fatty acids. This may occur via the production of lipid ligands that activate nuclear receptors (e.g., liver X receptor [LXR]) in PDC or through limiting intracellular cholesterol pool size (by statins or LXR agonists) in these cells. Finally, lipid-activated nuclear receptors ( i.e., LXR or peroxisome proliferator activated receptor) may also directly interact with pro-inflammatory transcription factors, such as NF-κB. Here, we discuss how glycolysis and lipid metabolism may modulate PDC functions and how this may be harnessed in pathological situations where PDC play a detrimental role.

Ezetimibe Increases Endogenous Cholesterol Excretion in Humans.

Ezetimibe improves cardiovascular outcomes when added to optimum statin treatment. It lowers low-density lipoprotein cholesterol and percent intestinal cholesterol absorption, but the exact cardioprotective mechanism is unknown. We tested the hypothesis that the dominant effect of ezetimibe is to increase the reverse transport of cholesterol from rapidly mixing endogenous cholesterol pool into the stool. In a randomized, placebo-controlled, double-blind parallel trial in 24 healthy subjects with low-density lipoprotein cholesterol 100 to 200 mg/dL, we measured cholesterol metabolism before and after a 6-week treatment period with ezetimibe 10 mg/d or placebo. Plasma cholesterol was labeled by intravenous infusion of cholesterol-d7 in a lipid emulsion and dietary cholesterol with cholesterol-d5 and sitostanol-d4 solubilized in oil. Plasma and stool samples collected during a cholesterol- and phytosterol-controlled metabolic kitchen diet were analyzed by mass spectrometry. Ezetimibe reduced intestinal cholesterol absorption efficiency 30±4.3% (SE, P<0.0001) and low-density lipoprotein cholesterol 19.8±1.9% (P=0.0001). Body cholesterol pool size was unchanged, but fecal endogenous cholesterol excretion increased 66.6±12.2% (P<0.0001) and percent cholesterol excretion from body pools into the stool increased 74.7±14.3% (P<0.0001), whereas plasma cholesterol turnover rose 26.2±3.6% (P=0.0096). Fecal bile acids were unchanged. Ezetimibe increased the efficiency of reverse cholesterol transport from rapidly mixing plasma and tissue pools into the stool. Further work is needed to examine the potential relation of reverse cholesterol transport and whole body cholesterol metabolism to coronary events and the treatment of atherosclerosis. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01603758.

Increased lanosterol turnover: a metabolic burden for daunorubicin-resistant leukemia cells.

The cholesterol metabolism is essential for cancer cell proliferation. We found the expression of genes involved in the cholesterol biosynthesis pathway up-regulated in the daunorubicin-resistant leukemia cell line CEM/R2, which is a daughter cell line to the leukemia cell line CCRF-CEM (CEM). Cellular 2H2O labelling, mass spectrometry, and isotopomer analysis revealed an increase in lanosterol synthesis which was not accompanied by an increase in cholesterol flux or pool size in CEM/R2 cells. Exogenous addition of lanosterol had a negative effect on CEM/R2 and a positive effect on sensitive CEM cell viability. Treatment of CEM and CEM/R2 cells with cholesterol biosynthesis inhibitors acting on the enzymes squalene epoxidase and lanosterol synthase, both also involved in the 24,25-epoxycholesterol shunt pathway, revealed a connection of this pathway to lanosterol turnover. Our data highlight that an increased lanosterol flux poses a metabolic weakness of resistant cells that potentially could be therapeutically exploited.Electronic supplementary materialThe online version of this article (doi:10.1007/s12032-015-0717-5) contains supplementary material, which is available to authorized users.

Preparation of intravenous cholesterol tracer using current good manufacturing practices

Studies of human reverse cholesterol transport require intravenous infusion of cholesterol tracers. Because insoluble lipids may pose risk and because it is desirable to have consistent doses of defined composition available over many months, we investigated the manufacture of cholesterol tracer under current good manufacturing practice (CGMP) conditions appropriate for phase 1 investigation. Cholesterol tracer was prepared by sterile admixture of unlabeled cholesterol or cholesterol-d7 in ethanol with 20% Intralipid(®). The resulting material was filtered through a 1.2 micron particulate filter, stored at 4°C, and tested at time 0, 1.5, 3, 6, and 9 months for sterility, pyrogenicity, autoxidation, and particle size and aggregation. The limiting factor for stability was a rise in thiobarbituric acid-reacting substances of 9.6-fold over 9 months (P < 0.01). The emulsion was stable with the Z-average intensity-weighted mean droplet diameter remaining at 60 nm over 23 months. The zeta potential (a measure of negative surface charge protecting from aggregation) was unchanged at -36.2. Rapid cholesterol pool size was 25.3 ± 1.3 g. Intravenous cholesterol tracer was stable at 4°C for 9 months postproduction. CGMP manufacturing methods can be achieved in the academic setting and need to be considered for critical components of future metabolic studies.

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis.

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.

Clinical evaluation of ezetimibe on bile lithogenicity in humans: Use of transnasal endoscopy for bile sampling.

Ezetimibe inhibits cholesterol absorption by blocking Niemann-Pick C1-like 1 proteins (NPC1L1) expressed in the small intestine. Because NPC1L1 is also expressed in human liver, ezetimibe conceivably alters biliary lipid compositions. Here, we performed a clinical trial investigating the effect of ezetimibe on biliary lipids using transnasal endoscopy for bile collection. Eight patients with dyslipidemia enrolled in this study completed blood and bile sampling before and at 3 months after ezetimibe treatment (10 mg/day), and the samples are analyzed. Endoscopic bile sampling was performed safely and painlessly. Serum sterol-based biomarkers declared decreased cholesterol absorption and increased synthesis. On analysis of biliary lipids, four of the eight patients showed relative decrease of cholesterol and increase of bile acids with improved lithogenicity while the remainder exhibited the symmetrical changes. Our data suggests that biliary lithogenicity is not worsened by ezetimibe. The regulation of biliary cholesterol is presumably multifactorial such as body cholesterol pool size and biliary cholesterol reabsorption by NPC1L1 in the liver.

Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice.

Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal(-/-) and matching lal(+/+) mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal(-/-) mice sequestered cholesterol at an average rate of 3.2 mg·day(-1)·animal(-1). The proportion of the body sterol pool contained in the liver of the lal(-/-) mice was 64 vs. 6.3% in their lal(+/+) controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal(-/-) mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal(-/-) mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal(+/+) controls. The rate of cholesterol synthesis in the lal(-/-) mice exceeded that in the lal(+/+) controls by 3.7 mg·day(-1)·animal(-1). Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal(-/-) mice, but their rate of neutral sterol excretion was 59% higher than in their lal(+/+) controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal(-/-) mice was 355 days.

Assessment of whole body cholesterol pool size in Smith‐Lemli‐ Opitz syndrome children using liquid chromatography tandem mass spectrometry

Smith‐Lemli‐Opitz syndrome (SLOS) patients typically exhibit abnormally low cholesterol, high 7‐dehyrocholesterol (DHC) and 8‐DHC levels in tissues and plasma. Therapeutic intervention for SLOS is aimed at maximizing whole body cholesterol pool size (WBCP) while down‐regulating biosynthesis to decrease the buildup of potentially toxic precursors. The study aim was to develop a sensitive liquid chromatography‐tandem mass spectrometry (LC/MS/MS) method for detection of changes in blood isotope cholesterol enrichment over time, for assessment of WBCP in SLOS patients receiving supplemental dietary cholesterol. Subjects (n=13; mean age: 7±1 yr) were given iv [18O]cholesterol (1.0–1.4 mg/kg bodyweight) or D7‐cholesterol (0.9–1.4 mg/kg). Blood samples were collected at baseline and over a 10 wk period, cholesterol extracted from RBC, derivatized with piconyl ester and analyzed using LC/MS/MS. The [M+H] +&gt;[M‐picolinic acid] + transition was chosen for monitoring, with 18O‐cholesterol at m/z 494&gt;369 and D7‐cholesterol at 499&gt;376. WBCP was 427.3 ±52.0 mg/kg bodyweight. Plasma cholesterol level was not related to WBCP (p&gt;0.05), but inversely related to 7DHC and 8DHC (p&lt;0.05). In summary, LC/MS/MS is a sensitive method for stable isotopic assessment of WBCP in humans. This study is the first assessment of WBCP in children and in SLOS patients. The data will be a useful reference for a longitudinal SLOS study.Grant Funding Source : NIH

Modelling approach to simulate reductions in LDL cholesterol levels after combined intake of statins and phytosterols/-stanols in humans

BackgroundTo examine the effects on LDL cholesterol of the combined use of statins and phytosterols/-stanols, in vivo studies and clinical trials are necessary. However, for a better interpretation of the experimental data as well as to possibly predict cholesterol levels given a certain dosing regimen of statins and phytosterols/-stanols a more theoretically based approach is helpful. This study aims to construct a mathematical model to simulate reductions in low-density lipoprotein (LDL) cholesterol in persons who combine the use of statins with a high intake of phytosterols/-stanols, e.g. by the use of functional foods.Methods and ResultsThe proposed model includes the cholesterol pool size in the liver and serum levels of very low-density lipoprotein (VLDL) cholesterol. Both an additional and a multiplicative effect of phytosterol/-stanol intake on LDL cholesterol reduction were predicted from the model. The additional effect relates to the decrease of dietary cholesterol uptake reduction, the multiplicative effect relates to the decrease in enterohepatic recycling efficiency, causing increased cholesterol elimination through bile. From the model, it was demonstrated that a daily intake of 2 g phytosterols/-stanols reduces LDL cholesterol level by about 8% to 9% on top of the reduction resulting from statin use. The additional decrease in LDL cholesterol caused by phytosterol/-stanol use at the recommended level of 2 g/d appeared to be similar or even greater than the decrease achieved by doubling the statin dose.ConclusionWe proposed a simplified mathematical model to simulate the reduction in LDL cholesterol after separate and combined intake of statins and functional foods acting on intestinal (re)absorption of cholesterol or bile acids in humans. In future work, this model can be extended to include more complex (regulatory) mechanisms.

The roles of different pathways in the release of cholesterol from macrophages

Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins. We determined the effect of enriching cells with free cholesterol (FC) on the release of FC to human serum. Loading Fu5AH cells with FC had no effect on fractional efflux, whereas enriching mouse peritoneal macrophages (MPMs) resulted in a doubling of fractional efflux. Efflux from cholesterol-normal MPM and Fu5AH cells to 15 human sera correlated well with HDL parameters. However, these relationships were reduced or lost with cholesterol-loaded MPMs. Using macrophages from scavenger receptor class B type I (SR-BI)-, ABCA1-, and ABCG1-knockout mice, together with inhibitors of SR-BI- and ABCA1-mediated efflux, we were able to quantitate efflux upon loading macrophages with excess cholesterol and to establish the contributions of the various efflux pathways in cholesterol-normal and -enriched cells. The removal of ABCA1 had essentially no effect on the total efflux when cell cholesterol levels were normal. However, in cholesterol-enriched cells, the removal of ABCA1 reduced efflux by 50%. Approximately 20% of the efflux stimulated by FC-loading MPM is attributable to ABCG1. The SR-BI contribution to efflux was small. Another pathway that is present in all cells is aqueous diffusion. Our studies demonstrate that this mechanism is one of the major contributors to efflux, particularly in cholesterol-normal cells.

ABCA1 Overexpression in the Liver of LDLr-KO Mice Leads to Accumulation of Pro-atherogenic Lipoproteins and Enhanced Atherosclerosis

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of (125)I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7alpha1, CYP27alpha1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.

HDL and the progression of atherosclerosis: new insights

Aims Lowering low-density lipoprotein (LDL) cholesterol or raising high-density lipoprotein (HDL) cholesterol can result in significant cardiovascular benefit, both in terms of reduction of events and also, to a variable extent, of atheromatous lesions. LDL and HDL have opposite roles in body cholesterol regulation and, both reduced deposition (LDL reduction) and increased removal (raised HDL) can improve vascular disease. Very recently, studies using recombinant apolipoprotein AI liposomes (in particular with the mutant apo AIMilano) have shown that direct infusion can effectively remove cellular cholesterol and dramatically reduce established atheromatous plaques in animals and in coronary patients. It has thus become of growing interest, the possibility of raising HDL by pharmacological treatments. This review will attempt to investigate existing and novel methodologies for HDL raising. Methods and results HDL raising can be achieved by using drugs active on the peroxisome proliferator activated receptor α (PPAR α) system, particularly using fibrates or n-3 fatty acids and, possibly more effectively, by using nicotinic acid. The activity of this agent has been traditionally linked to a reduced free fatty acid (FFA) mobilization from adipose tissue. More recently, it has been noted that nicotinic acid can activate the PPAR system, by stimulating all three PPAR isoforms (α, γ, δ). This mode of action seems to more effectively explain the striking HDL-raising properties of the drug. Conclusions Newly discovered mechanisms of nicotinic acid can explain, on the one hand, reduced atherosclerosis progression in secondary prevention patients treated with statins and, on the other hand, improved body cholesterol mobilization, effectively reducing cholesterol pool sizes.

Dietary iron overload and induced lipid peroxidation are associated with impaired plasma lipid transport and hepatic sterol metabolism in rats.

Although hemochromatosis is characterized by dramatic morphological and functional alterations in the liver, little is known about the effects of an excess of iron on lipid metabolism. Therefore, we determined the effect of chronic iron overload on plasma lipid profile and lipoprotein composition, as well as on hepatic cholesterol metabolism and biliary sterol output. Rats administered a diet enriched with 3% iron carbonyl for 12 weeks displayed a 30-fold increase in iron (P <.0001) and a 5-fold rise in malondialdehyde (P <.001) in the liver. When compared with pair-fed controls, iron-overload rats showed a significant increase in triglycerides (P <.005), free cholesterol (P <.006), cholesteryl ester (P <.007), and high-density lipoprotein (HDL)-cholesterol (P <. 003). Triglyceride and cholesteryl ester enrichment, protein depletion, size increase, and apolipoprotein composition alterations characterized the very low density lipoprotein (VLDL) and HDL particles of iron-overload rats. Assessment of the activity of intracellular key enzymes for cholesterol homeostasis in these rats disclosed a reduction in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (66%, P <.005) and cholesterol 7alpha-hydroxylase (58%, P <.0004) with an increment of acyl-CoA: cholesterol acyltransferase (62%, P <.002). The lack of optimal enzymatic activity may be a result of marked membrane lipid peroxidation that brings about fluidity drop (P <.029) in microsomes of iron-overload rats (5.00 +/- 0.013) versus controls (8.20 +/- 0. 03), reflected by polarization. A decline of the pool size of cholesterol and bile acids was noted in iron-overload rats during a 6-hour bile drainage. Our results show that experimental iron overload causes marked perturbations in plasma lipid transport and hepatobiliary sterol metabolism. Given the positive correlation of malondialdehyde with most of the altered parameters, iron-catalyzed lipid peroxidation may well be one of the involved mechanisms.

ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

Independent Regulation of Cholesterol Incorporation into Free Apolipoprotein-mediated Cellular Lipid Efflux in Rat Vascular Smooth Muscle Cells

Cholesterol was poorly available to free apolipoprotein (apo)A-I-mediated cellular lipid efflux from cholesterol-loaded rat vascular smooth muscle cells generating cholesterol-poorer pre-beta-HDL particles than those generated from macrophages by the same reaction (Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). The factors known to induce transformation of the smooth muscle cells into a macrophage-like stage were used in order to modulate this reaction, such as human platelet-derived growth factor, macrophage colony-stimulating factor, and phorbol 12-myristate-13-acetate (PMA). When the cells were stimulated by PMA following the pretreatment with platelet-derived growth factor plus macrophage colony-stimulating factor, cholesterol efflux mediated by free apoA-I increased 3-fold without changing phospholipid efflux, resulting in generation of pre-beta-HDL particles more rich in cholesterol. This treatment had only a little or no effect on apparent cellular cholesterol efflux to HDL or lipid microemulsion, respectively. Overall cellular free cholesterol pool size was unaffected by the treatment, and probing by extracellular cholesterol oxidase did not detect gross change in the cellular surface cholesterol. This specific enrichment of cholesterol in the apoA-I-mediated cellular lipid efflux was reversed by protein kinase C inhibitors. Measurement of intracellular cholesterol esterification suggested that PMA induced translocation of intracellular cholesterol to a specific pool for apoA-I-mediated efflux, and a protein kinase C inhibitor reversed this effect.

Pool Size and Concentration of Plasma Cholesterol Are Increased and Tissue Copper Levels Are Reduced during Early Stages of Copper Deficiency in Rats

The progressive development of hypercholesterolemia, enlargement of plasma cholesterol pool size, and alteration in tissue mineral concentrations were determined during the early stages of copper deficiency. Fifty-four weanling male Sprague-Dawley rats were assigned to three dietary Cu treatments: deficient (0.6 micrograms Cu/g diet), marginal (1.6 micrograms Cu/g diet) and adequate (6.6 micrograms Cu/g diet). Six rats from each treatment were killed after 3, 5 and 7 wk of dietary treatment. After only 3 wk of treatment, significantly lower hematocrits and liver Cu concentrations, as well as enlargements of plasma volume, plasma pool size of cholesterol and triacylglycerols and relative heart weight, were evident in rats fed the Cu-deficient and Cu-marginal diets relative to those fed the Cu-adequate diet. In general, these alterations were more pronounced in rats fed the Cu-deficient diet than in rats fed the Cu-marginal diet. Thereafter, the hematocrits and plasma volumes remained relatively constant, but the liver Cu concentration progressively decreased in rats in all treatments throughout the study. In contrast, the enlargements in plasma pool size of cholesterol and triacylglycerols relative to Cu-adequate rats were greater at the end of the study for the Cu-marginal and Cu-deficient rats. Most importantly, in the Cu-marginal rats, significantly greater plasma cholesterol and triacylglycerol pool sizes were detected earlier than were differences in concentrations. Thus, the present study established plasma cholesterol and triacylglycerol pool sizes as superior indices for the early detection of alterations in lipid metabolism in Cu deficiency.

Effect of dietary fat selection on plasma cholesterol synthesis in older, moderately hypercholesterolemic humans.

To study factors controlling plasma cholesterol levels, the effect of dietary fat type on cholesterol synthesis was examined in 15 hypercholesterolemic subjects (low-density lipoprotein [LDL] cholesterol > 130 mg.dL-1) consuming over a period of 32 days (1) a baseline diet (36% kcal as fat: 15% saturated, 15% monounsaturated, and 6% polyunsaturated fat; 180 mg cholesterol.1000 kcal-1) and diets meeting National Cholesterol Education Program step 2 criteria (30% kcal as fat, < or = 7% saturated fat, 80 to 85 mg cholesterol/Mcal), where two thirds of the fat was either (2) olive, (3) corn, or (4) canola oil. Plasma total, LDL, and high-density lipoprotein (HDL) cholesterol and triglyceride levels were determined at the end of each period. Cholesterol fractional synthesis rate (FSR) was also measured as the deuterium (D) incorporation into plasma total cholesterol relative to body D2O level (1.2 g D2O.kg-1 estimated body water) over 24 hours. Absolute synthesis rates (ASRs) were determined as the product of FSR and rapid turnover cholesterol pool size. Plasma total and LDL cholesterol levels declined significantly (P < .005) on all plant-oil diets compared with the baseline diet; however, triglyceride levels were not different. FSRs were higher (P < .05) for the corn oil (0.0665 +/- 0.0097 pool.d-1) compared with baseline (0.0412 +/- 0.0060 pool.d-1) and olive oil (0.0409 +/- 0.0052 pool.d-1) but not canola oil (0.0492 +/- 0.0072 pool.d-1) diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of smooth muscle cells to assembly of high density lipoproteins with extracellular free apolipoproteins and to reduction of intracellularly accumulated cholesterol.

Removal of intracellularly accumulated cholesterol by lipid-free human apolipoproteins (apo) A-I and A-II was studied for aortic smooth muscle cells (SMC) of rat, monkey and rabbit, human skin fibroblasts (FB), and mouse peritoneal macrophages (MP). The reaction generated high density lipoprotein (HDL)-like lipoproteins as did those and other helical apolipoproteins with MP, causing efflux of cellular cholesterol. From FB and MP, the maximum efflux rates with apoA-I and A-II per 24 h were as much as 30% of the apparent maximum efflux rate of prelabeled cellular cholesterol to human HDL. From rat SMC these rates were 7.2 and 6.8%, respectively, being independent of cellular cholesterol content. Those from monkey and rabbit SMC were also very low. When standardized for the initial cellular unesterified cholesterol pool size, the maximum efflux rates/24 h were 5.4 and 5.0% for apoA-I and A-II from rat SMC and even less from monkey and rabbit SMC in contrast to 42.4 and 39.7% from FB, and 53.0 and 45.5% from MP, respectively. The standardized apparent maximum efflux to HDL was 76% from rat SMC, 45 and 31% from monkey and rabbit SMC, 139% from FB and 166% from MP. Accordingly, the reaction with free apolipoproteins caused significant net reduction of cellular cholesterol, predominantly in cholesteryl ester, in FB and MP, but not in SMC. While the efflux Km with apoA-I and A-II were 7.5 and 4.5 micrograms/ml for MP, those for SMC and FB were both 1 microgram/ml or lower, as low as 1/1500 and 1/500 of their plasma concentrations, respectively. The apparent efflux Km for HDL were, on the other hand, all in the range of 36 to 65 micrograms of protein/ml for SMC, FB, and MP, showing that the mode of cholesterol exchange of these cells with lipoprotein surface is not significantly different from each other. Thus, peripheral cells such as FB may provide a significant source of HDL by interacting with extracellular free apolipoproteins in interstitial fluid, reducing intracellularly accumulated cholesterol. However, SMC seem very resistant to this interaction, suggesting that atheromatous lesions predominantly consisting of SMC are resistant to regression.