Reduced Cholesterol Accumulation Research Articles

Glycine N-Methyltransferase Deficiency Affects Niemann-Pick Type C2 Protein Stability and Regulates Hepatic Cholesterol Homeostasis

Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt(-/-)) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt(-/-) mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with (131)I-labeled 6β-iodocholesterol demonstrated that Gnmt(-/-) mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt(-/-) mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81-105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171-295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.

Phenolic Acids Are in Vivo Atheroprotective Compounds Appearing in the Serum of Rats after Blueberry Consumption

Blueberries (BB) have recently been shown to have cardioprotective effects and to prevent atherosclerosis in rodent models. However, the bioactive compounds in BB responsible for these effects have not yet been characterized. Seven phenolic acids (7PA) were identified as metabolites in the serum of rats fed diets supplemented with 10% freeze-dried BB. In this study, 7PA were evaluated for their potential atheroprotective effects in murine macrophage cell line RAW 264.7. 7PA were found to inhibit LPS-induced mRNA expression and protein levels of pro-inflammatory cytokine TNF-α and IL-6 by reducing MAPK JNK, p38, and Erk1/2 phosphorylation. After treatment with 7PA for 2 weeks, mRNA expression and protein levels of scavenger receptor CD36 were decreased (P<0.05), whereas type A scavenger receptor (SR-A) remained unchanged. Moreover, foam cell formation induced by oxLDL and oxLDL binding to macrophages was also inhibited by 7PA. In addition, 7PA increased (P<0.05) expression and protein levels of ATP-binding cassette transporter A1 (ABCA1), which facilitates cholesterol efflux and reduces cholesterol accumulation in macrophages. In summary, the present study demonstrates that certain phenolic acids are potential in vivo atheroprotective compounds following BB consumption in the rodent model. Because BB contain many phytochemicals, other as yet unidentified bioactive compounds may also be important in preventing atherosclerosis in this model and, possibly, in humans.

Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts

Niemann-Pick type C (NPC) disease is predominantly caused by mutations in the NPC1 protein that affect intracellular cholesterol trafficking and cause accumulation of unesterified cholesterol and other lipids in lysosomal storage organelles. We report the use of a series of small molecule histone deacetylase (HDAC) inhibitors in tissue culture models of NPC human fibroblasts. Some HDAC inhibitors lead to a dramatic correction in the NPC phenotype in cells with either one or two copies of the NPC1(I1061T) mutation, and for several of the inhibitors, correction is associated with increased expression of NPC1 protein. Increased NPC1(I1061T) protein levels may partially account for the correction of the phenotype, because this mutant can promote cholesterol efflux if it is delivered to late endosomes and lysosomes. The HDAC inhibitor treatment is ineffective in an NPC2 mutant human fibroblast line. Analysis of the isoform selectivity of the compounds used implicates HDAC1 and/or HDAC2 as likely targets for the observed correction, although other HDACs may also play a role. LBH589 (panobinostat) is an orally available HDAC inhibitor that crosses the blood-brain barrier and is currently in phase III clinical trials for several types of cancer. It restores cholesterol homeostasis in cultured NPC1 mutant fibroblasts to almost normal levels within 72 h when used at 40 nM. The findings that HDAC inhibitors can correct cholesterol storage defects in human NPC1 mutant cells provide the potential basis for treatment options for NPC disease.

Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.

Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells

Niemann-Pick type C disease (NPC) is a lysosomal storage disorder causing accumulation of unesterified cholesterol in lysosomal storage organelles. Recent studies have shown that hydroxypropyl-beta-cyclodextrin injections in npc1(-/-) mice are partially effective in treating this disease. Using cultured fibroblasts, we have investigated the cellular mechanisms responsible for reduction of cholesterol accumulation. We show that decreased levels of cholesterol accumulation are maintained for several days after removal of cyclodextrin from the culture medium. This suggests that endocytosed cyclodextrin can reduce the cholesterol storage by acting from inside endocytic organelles rather than by removing cholesterol from the plasma membrane. To test this further, we incubated both NPC1 and NPC2 mutant cells with cholesterol-loaded cyclodextrin for 1 h, followed by chase in serum-containing medium. Although the cholesterol content of the treated cells increased after the 1-h incubation, the cholesterol levels in the storage organelles were later reduced significantly. We covalently coupled cyclodextrin to fluorescent dextran polymers. These cyclodextrin-dextran conjugates were delivered to cholesterol-enriched lysosomal storage organelles and were effective at reducing the cholesterol accumulation. We demonstrate that methyl-beta-cyclodextrin is more potent than hydroxypropyl-beta-cyclodextrin in reducing both cholesterol and bis(monoacylglycerol) phosphate accumulation in NPC mutant fibroblasts. Brief treatment of cells with cyclodextrins causes an increase in cholesterol esterification by acyl CoA:cholesterol acyl transferase, indicating increased cholesterol delivery to the endoplasmic reticulum. These findings suggest that cyclodextrin-mediated enhanced cholesterol transport from the endocytic system can reduce cholesterol accumulation in cells with defects in either NPC1 or NPC2.

Allopregnanolone treatment delays cholesterol accumulation and reduces autophagic/lysosomal dysfunction and inflammation in Npc1−/− mouse brain

Niemann–Pick Type C (NPC) disease is a devastating developmental disorder with progressive and fatal neurodegeneration. Previous work has shown that a single injection of the neurosteroid allopregnanolone at postnatal day 7 significantly prolonged lifespan of Npc1−/− mice. However, the cellular/molecular basis for this beneficial effect remains undefined. Here, we further characterized the effect of allopregnanolone treatment on cholesterol accumulation, a pathological hallmark of NPC, as well as on autophagic/lysosomal dysfunction, myelination and inflammation in Npc1−/− mouse brains. At 1 month postnatal, accumulation of filipin-labeled unesterified cholesterol was clearly evident not only in neurons but also in microglia in untreated mutant mice, but was mostly absent in allopregnanolone-treated animals. Brain levels of the lysosomal enzymes cathepsins B and D were significantly higher in Npc1−/− than in wild-type mice. Levels of LC3-II, an autophagy marker, were also increased in mutant mouse brain as compared to wild-type mouse brain. Both changes were significantly reduced by allopregnanolone treatment. Injection of the neurosteroid also significantly reduced astrocyte proliferation and microglial activation. Furthermore, allopregnanolone treatment significantly enhanced myelination in mutant mice. Taken together, our results clearly show that allopregnanolone treatment not only reduces cholesterol accumulation and improves autophagic/lysosomal function but also enhances myelination and reduces inflammation. These results provide further support for the potential usefulness of allopregnanolone for treating NPC disease.

Inhibitory Effects of Grape Seed Procyanidins on Foam Cell Formation in Vitro

Human and animal studies have demonstrated that procyanidin-rich diets reduce the risk of cardiovascular diseases and atherosclerosis. Some beneficial effects have been attributed to the well-known antioxidant activity of procyanidins. This study investigated another potential corrective role of procyanidins in cholesterol flux and inflammation in macrophage-derived foam cells. RAW 264.7 macrophages were cultured with moderately oxidized LDL (oxLDL), minimally oxidized LDL (moxLDL), or LPS (0.5 microg/mL) and oxLDL (LPS + oxLDL) to induce foam cells. Then, cells were treated with procyanidins derived from grape seed (PE, 45 microg/mL) for the last 12 h of incubation with the different lipoproteins (25 microg/mL). After lipid extraction, it was determined that total and esterified cholesterol and triglyceride accumulations in foam cells were increased by lipoprotein treatment but reduced by PE incubation. To asses the effect of PE on gene expression, the relative mRNA levels of CD36, ABCA1, iNOS, COX-2, and IkappaBalpha were determined by RT-PCR. It was shown that PE reduced the oxLDL scavenger receptor expression (CD36) and enhanced ATP-binding cassette A1 (ABCA1) expression, a key regulator of macrophage cholesterol efflux. PE also down-regulated inflammatory-related genes such as inducible nitric oxide synthase (iNOS) and kappa beta inhibitor-alpha (IkappaBalpha) without modifying COX-2 expression. In conclusion, evidence is provided that procyanidins may attenuate the development of foam cell formation by reducing cholesterol accumulation and modulating the expression of key genes in cholesterol flux and inflammation.

Severe HDL deficiency due to novel defects in the ABCA1 transporter

The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. Patients were referred to the clinic because of almost complete HDL deficiency. The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system.

Conjugated Linoleic Acid Isomers Reduce Cholesterol Accumulation in Acetylated LDL‐Induced Mouse RAW264.7 Macrophage‐Derived Foam Cells

Synthetic activators of peroxisome proliferator-activated receptors (PPAR)-alpha and -gamma are capable of reducing macrophage foam cell cholesterol accumulation through the activation of genes involved in cholesterol homeostasis. Since conjugated linoleic acids (CLA) were also demonstrated to activate PPARalpha and PPARgamma in vivo and in vitro, we tested the hypothesis that CLA are also capable of reducing macrophage foam cell cholesterol accumulation. Thus, mouse RAW264.7 macrophage-derived foam cells were treated with CLA isomers, c9t11-CLA and t10c12-CLA, and linoleic acid (LA), as reference fatty acid, and analyzed for the concentrations of free and esterified cholesterol, cholesterol efflux and expression of genes involved in cholesterol homeostasis (CD36, ABCA1, LXRalpha, NPC-1, and NPC-2). Treatment with c9t11-CLA and t10c12-CLA, but not LA, lowered cholesterol accumulation, stimulated acceptor-dependent cholesterol efflux, and increased relative mRNA concentrations of CD36, ABCA1, LXRalpha, NPC-1, and NPC-2 (P < 0.05). In conclusion, the present study showed that CLA isomers reduce cholesterol accumulation in RAW264.7 macrophage-derived foam cells presumably by enhancing lipid acceptor-dependent cholesterol efflux.

A‐002, a secretory phospholipase A2 inhibitor, reduces cholesterol accumulation in aorta and aortic cytokines in guinea pigs

Secretory phospholipase A2 enzymes (sPLA2) are involved with the generation of proinflammatory molecules such as leukotrienes and prostaglandins and are associated with atherosclerotic plaques. In this study we tested the effects of an sPLA2 inhibitor (A‐002) on aortic inflammatory cytokines and aortic lipid accumulation in a guinea pig atherosclerosis model. For this purpose, 24 male Hartley guinea pigs (12 per group) were fed a diet containing 0.25% dietary cholesterol to induce atherosclerosis, for 3 months. Animals were randomized into placebo (PL) or treated (A‐002) group, and gavaged daily either with vehicle (10% acacia, PL) or A‐002 (150 mg/kg/d, A‐002). At the end of 12 weeks, guinea pigs were sacrificed and blood was collected to measure plasma lipids and lipoprotein subfractions. The aorta was harvested for histological evaluation, measurement of inflammatory cytokines and lipid accumulation. Aortic cytokines including Interleukin (IL)‐2, IL‐10, IL‐12 and GMSCF measured by use of antibodies and Luminex technique were significantly lower (p &lt; 0.05) in the A‐002 group. In addition, the A‐002 group had 26.5% lower cholesterol accumulation in the aorta (p &lt; 0.05), but not in plasma, in association with a reduction in atherosclerotic lesion content in the aortic sinus. From these studies we conclude that A‐002 was effective in reducing atherosclerosis, as indicated by reductions in aortic cholesterol content, inflammatory cytokines and lesion content. [Study supported by Anthera Pharmaceuticals, Inc., San Mateo, CA].

Convergence of Nanotechnology and Cardiovascular Medicine

Advances in the emergence of biological probes, materials, and analytical tools limited to the nanoscale size range, collectively referred to as 'nanotechnology', are increasingly being applied to the understanding and treatment of the major pathophysiological problems in cardiovascular medicine. Analytical techniques based on high-resolution microscopy and molecular-level fluorescence excitation processes capable of detecting nanoscale interactions have been used to elucidate cardiovascular pathology. Nanotechnology has also significantly impacted diagnostic intervention in cardiology, with the use of nanoparticles as contrast agents, for targeted biomedical imaging of vulnerable plaques, for detection of specific pathologic targets signaling the onset of atherosclerosis, and for tracking inflammatory events. Real-time nanoscale biosensors can be used to measure cardiovascular biomarkers, and nanopore sequencing has the potential to speed up the analysis of gene expression in cardiovascular disease. Potential therapeutic applications include the use of nanomaterials in cardiovascular devices, for delivery of drugs and bioactive molecules, or in novel technologies for reducing cholesterol accumulation and for dissolving clots.

Cholesterol Accumulation Is Increased in Macrophages of Phospholipid Transfer Protein-Deficient Mice

Phospholipid transfer protein (PLTP) is a multifunctional, extracellular lipid transport protein that plays a major role in lipoprotein metabolism and atherosclerosis. Recent in vivo studies suggested that unlike systemic PLTP, macrophage-derived PLTP would be antiatherogenic. The present study aimed at characterizing the atheroprotective properties of macrophage-derived PLTP. Peritoneal macrophages were isolated from PLTP-deficient and wild-type mice and their biochemical characteristics were compared. It is shown that macrophages isolated from PLTP-deficient mice have increased basal cholesterol content and accumulate more cholesterol in the presence of LDL compared with wild-type cells. Cholesterol parameters in macrophages of PLTP-deficient mice were normalized by dietary alpha-tocopherol supplementation. The antiatherogenic properties of macrophage-derived PLTP are related at least in part to its ability to reduce cholesterol accumulation in macrophages through changes in the alpha-tocopherol content and oxidative status of the cells.

Old Drug, New Tricks

See related article, pages 156–165 Ironically, even though there are currently only a few drugs to specifically treat low high-density lipoprotein (HDL), numerous drugs have been observed to inadvertently alter HDL levels.1 This has been particularly noted for some antihypertensive drugs. Because they are used long-term, any positive or negative effect of antihypertensive drugs on lipids and lipoproteins can significantly impact on their effectiveness in preventing cardiovascular disease. Recently, advances in our understanding of HDL metabolism have helped us to unravel the mechanism of action of these and other drugs that unexpectedly alter HDL levels. This work has largely been led by the laboratory of Shinji Yokoyama and colleagues, who have previously identified the mechanism of action of three drugs on HDL metabolism. Probucol, which has been used to treat patients at risk for cardiovascular disease, is an antioxidant and lowers LDL, but it also lowers HDL. Probucol is now known to decrease HDL by inhibiting the activity of the ABCA1 transporter,2 a key protein involved in reverse cholesterol transport pathway and in HDL biogenesis.3 Verapamil, a calcium channel blocker, which can be used as antihypertensive and antiarrhythmic agent, and fenofibrate, a lipid-lowering drug, both raise HDL by increasing the gene expression of the ABCA1 transporter.4,5 In this issue of Circulation Research ,6 Iwamoto et al describe the mechanism of action of a fourth agent that unexpectedly alters HDL, namely the antihypertensive agent doxazosin. The results of this study raise the possibility of a new strategy for developing drugs to increase HDL. Doxazosin (Cardura) is a quinazoline based compound that is sold by Pfizer and is a long lasting selective inhibitor of the α-1a subtype of adrenergic receptors. It blocks the binding of norepinephrine to adrenergic receptors in the autonomic nervous system, which leads …

Effects of new combinative antioxidant FeAOX-6 and α-tocotrienol on macrophage atherogenesis-related functions

Pivotal role in atherogenesis is played by macrophages, which are early site for lipid accumulation and mediate the inflammatory and immune response in the intima. Epidemiological evidence indicates that natural antioxidants reduce the risk of heart disease, but, so far, supplementation studies have failed to confirm any protective effects of these compounds against cardiovascular disease. This study evaluated the effects of the natural antioxidant α-tocotrienol and of the newly designed compound, FeAOX-6, which combines antioxidant structural features of both tocopherols and carotenoids into a single molecule, on macrophage functions involved in foam cell formation. FeAOX-6 or α-tocotrienol induce a strong dose-dependent reduction of cholesterol and reduce cholesterol accumulation in human macrophages. The extent of the reduction found with α-tocotrienol was greater than that induced by FeAOX-6 and did not correlate with their respective antioxidant capacities. Treatment of HMDM with α-tocotrienol or FeAOX-6 enhanced also tumor necrosis factor-α secretion. These results are consistent with a reduction in scavenger receptor activity, but we found that antioxidant treatment did not affect cholesterol uptake from modified LDL. The effects on release on pro-inflammatory prostanoid precursors, PGE 2 and cytokine suggest a variety of metabolic responses that are both dependent on antioxidant compounds and macrophages activation status.

ATP-Binding Cassette Cholesterol Transporters and Cardiovascular Disease

A hallmark of atherosclerotic cardiovascular disease (CVD) is the accumulation of cholesterol in arterial macrophages. Factors that modulate circulating and tissue cholesterol levels have major impacts on initiation, progression, and regression of CVD. Four members of the ATP-binding cassette (ABC) transporter family play important roles in this modulation. ABCA1 and ABCG1 export excess cellular cholesterol into the HDL pathway and reduce cholesterol accumulation in macrophages. ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. All 4 transporters are induced by the same sterol-sensing nuclear receptor system. ABCA1 expression and activity are also highly regulated posttranscriptionally by diverse processes. ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. ABCG5 or ABCG8 mutations can cause sitosterolemia, in which patients accumulate cholesterol and plant sterols in the circulation and develop premature CVD. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excess cholesterol in macrophages, and manipulating mouse macrophage ABCA1 expression affects atherogenesis. Overexpressing ABCG5 and ABCG8 in mice attenuates diet-induced atherosclerosis in association with reduced circulating and liver cholesterol. Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and inhibit transcription of all 4 transporters. Thus, impaired ABC cholesterol transporters might contribute to the enhanced atherogenesis associated with common inflammatory and metabolic disorders. Their beneficial effects on cholesterol homeostasis have made these transporters important new therapeutic targets for preventing and reversing CVD.

Reduced cholesterol accumulation by leptin deficient (ob/ob) mouse macrophages

Although presenting many aspects of the metabolic syndrome, leptin deficient (ob/ob) mice do not spontaneously develop atherosclerosis. To examine the role of leptin in foam cell formation we analyzed ob/ob leukocyte inflammation markers and macrophage cholesterol accumulation. Resident and thioglycollate (TG) elicited peritoneal cells of ob/ob and wildtype mice were studied. Activation markers, scavenger receptors (SR) and cholesterol accumulation were analyzed using flow cytometry and Taqman analysis. Cytokines, haptoglobin, adiponectin and amyloid A levels were analyzed with ELISA. Macrophages of ob/ob mice had reduced expression of MHC class II, CD11b, CD40, SR-A and CD36 and reduced cholesterol accumulation in vitro. Plasma haptoglobin was increased and T-cell IFNgamma was reduced in ob/ob mice. Peritoneal TG instillation induced an unexpectedly weak inflammatory response in ob/ob mice. The ob/ob mice had a reduced inflammatory response and reduced macrophage cholesterol accumulation in vitro. The data suggest decreased foam cell formation and atherosclerosis development in ob/ob mice.

Conjugated linoleic acid isomers reduce blood cholesterol levels but not aortic cholesterol accumulation in hypercholesterolemic hamsters

The aim of the present study was to characterize plasma lipids and lipoprotein cholesterol and glucose concentrations in hamsters fed either cis-9,trans-11 CLA (9c,11 tCLA); trans-10,cis-12 CLA (10t,12c CLA); or linoleic acid (LA) on the accumulation of aortic cholesterol in hypercholesterolemic hamsters. One hundred male F1B strain Syrian Golden Hamsters (Mesocricetus auratus) (BioBreeders Inc., Watertown, MA) approximately 9 wk of age were housed in individual stainless steel hanging cages at room temperature with a 12-h light/dark cycle. Hamsters were given food and water ad libitum. Following a 1-wk period of acclimation, the hamsters were fed a chow-based (nonpurified) hypercholesterolemic diet (HCD) containing 10% coconut oil (92% saturated fat) and 0.1% cholesterol for 2 wk. After an overnight fast, the hamsters were bled and plasma cholesterol concentrations were measured. The hamsters were then divided into 4 groups of 25 based on similar mean plasma VLDL and LDL cholesterol (nonHDL-C) concentrations. Group 1 remained on the HCD (control). Group 2 was fed the HCD plus 0.5% 9c,11t CLA isomer. Group 3 was fed the HCD plus 0.5% 10t,12c CLA isomer. Group 4 was fed the HCD plus 0.5% LA. Compared with the control, both CLA isomers and LA had significantly lower plasma total cholesterol and HDL cholesterol concentrations (P < 0.001) after 12 but not 8 wk of treatment and were not significantly different from each other. Also, both CLA isomers had significantly lower plasma nonHDL-C concentrations (P < 0.01) compared with the control after 12 but not 8 wk of treatment and were not significantly different from each other or the LA-fed hamsters. Plasma TG concentrations were significantly higher (P < 0.004) with the 10t, 12c CLA isomer compared with the other treatments at 8 but not at 12 wk of treatment. Plasma TG concentrations were also significantly lower (P < 0.03) with the 9c,11t CLA isomer compared with the control at 12 wk of treatment. Also, the 10t,12c CLA isomer and LA had significantly higher plasma glucose concentrations compared with the control and 9c,11t CLA isomer (P < 0.008) at 12 wk of treatment, whereas at 8 wk, only the LA treatment had significantly higher plasma glucose concentrations (P < 0.001) compared with the 9c,11t CLA isomer. Although liver weights were significantly higher in 10t,12c CLA isomer-fed hamsters, liver total cholesterol, free cholesterol, cholesterol ester, and TG concentrations were significantly lower in these hamsters compared with hamsters fed the control, 9c,11t CLA isomer, and LA diets (P< 0.05). The 9c,11t CLA isomer and LA diets tended to reduce cholesterol accumulation in the aortic arch, whereas the 10t,12c CLA isomer diet tended to raise cholesterol accumulation compared with the control diet; however, neither was significant. In summary, no differences were observed between the CLA isomers for changes in plasma lipids or lipoprotein cholesterol concentrations. However, the 9c,11t CLA isomer did appear to lower plasma TG and glucose concentrations compared with the 10t,12c CLA isomer. Such differences may increase the risk of insulin resistance and type 2 diabetes in humans when the 10t,12c CLA isomer is fed separately.

Pomegranate juice inhibits oxidized LDL uptake and cholesterol biosynthesis in macrophages

Macrophage cholesterol accumulation and foam cell formation are the hallmarks of early atherogenesis. Pomegranate juice (PJ) was shown to inhibit macrophage foam cell formation and development of atherosclerotic lesions. The aim of this study was to elucidate possible mechanisms by which PJ reduces cholesterol accumulation in macrophages. J774.A1 macrophages were preincubated with PJ followed by analysis of cholesterol influx [evaluated as LDL or as oxidized LDL (Ox-LDL) cellular degradation], cholesterol efflux and cholesterol biosynthesis. Preincubation of macrophages with PJ resulted in a significant reduction ( P<.01) in Ox-LDL degradation by 40%. On the contrary, PJ had no effect on macrophage degradation of native LDL or on macrophage cholesterol efflux. Macrophage cholesterol biosynthesis was inhibited by 50% ( P<.01) after cell incubation with PJ. This inhibition, however, was not mediated at the 3-hydroxy-3 methylglutaryl coenzyme A reductase level along the biosynthetic pathway. We conclude that PJ-mediated suppression of Ox-LDL degradation and of cholesterol biosynthesis in macrophages can lead to reduced cellular cholesterol accumulation and foam cell formation.

Melatonin reduces cholesterol accumulation and prooxidant state induced by high cholesterol diet in the plasma, the liver and probably in the aorta of C57BL/6J mice

We examined the hypolipidemic and antioxidative effects of melatonin in plasma, liver and aorta of C57BL/6J mice fed on a high cholesterol (HC) diet. Mice were fed normal mice chow containing 1.5% cholesterol and 0.5% cholic acid for 4 months with or without melatonin (10 mg/L in drinking water) treatment. HC diet was observed to increase cholesterol, triglyceride and diene conjugate (DC) levels in plasma and liver. There was a tendency towards an increase in cholesterol level in the aorta following HC diet. In addition, aortic DC levels were higher than those of control group. No fatty streaks or plaques developed in the aorta of mice following HC diet, but in some sections, derangement of the endothelial layer was detected. Melatonin treatment was found to reduce plasma, liver cholesterol and DC levels as well as liver triglyceride levels in hypercholesterolemic mice. Aortic cholesterol and DC levels were also reduced in hypercholesterolemic mice when given melatonin, although not statistically significant. There were no differences in aortic histopathological findings of mice fed on a HC diet with and without melatonin treatment. In conclusion, our results indicate that melatonin reduces HC diet-induced cholesterol accumulation and prooxidant state in the plasma, liver and probably in the aorta.

Soy Protein Enhances the Cholesterol-Lowering Effect of Plant Sterol Esters in Cholesterol-Fed Hamsters

This study aimed to investigate whether the combination of plant sterol esters (PSE) with soy protein or soy isoflavones may have extra cholesterol-lowering effects. Male hamsters (n=20/group) were fed diets containing (g/100 g diet) (A) 20 casein (control), (B) 0.24 PSE, (C) 20 intact soy protein (replacing casein), (D) 0.02 soy isoflavones, (E) 0.24 PSE plus 20 soy protein (replacing casein), or (F) 0.24 PSE plus 0.02 soy isoflavones, for 5 wk. All diets contained 0.08 g cholesterol/100 g diet. Compared with the control diet, the PSE and soy protein diets significantly lowered the plasma total cholesterol concentration by 13% (P<0.05) and 9% (P<0.05), respectively, whereas the isoflavone diet (D) had no effect. The combination of PSE and soy protein (diet E) decreased plasma total cholesterol by 26% (P<0.05). The decrease in plasma cholesterol concentration was mainly in the non-HDL fraction. In addition, the combination of PSE and soy protein significantly decreased plasma triacylglycerol concentration (37%, P<0.05) and reduced cholesterol accumulation in the liver. The abundance of hepatic LDL-receptors was not influenced by any of the test diets. PSE selectively increased fecal excretion of neutral sterols by 190% (P<0.05), whereas soy protein increased fecal excretion of neutral sterols and bile acids by 66% (P<0.05) and 130% (P<0.05), respectively. The combination of PSE and soy protein increased the fecal excretion of neutral sterols and bile acids compared with PSE and soy protein alone. In conclusion, the combination of PSE and soy protein more dramatically lowers plasma lipids than the individual ingredients.