Accumulation Of Plant Sterols Research Articles

Association of ABCG5 and ABCG8 Transporters with Sitosterolemia.

Sitosterolemia is a rare genetic lipid disorder, mainly characterized by the accumulation of dietary xenosterols in plasma and tissues. It is caused by inactivating mutations in either ABCG5 or ABCG8 subunits, a subfamily-G ATP-binding cassette (ABCG) transporters. ABCG5/G8 encodes a pair of ABC half transporters that form a heterodimer (G5G8). This heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the hepatobiliary and transintestinal secretion of cholesterol and dietary plant sterols to the surface of hepatocytes and enterocytes, promoting the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. In this way, ABCG5/G8 function in the reverse cholesterol transport pathway and mediate the efflux of cholesterol and xenosterols to high-density lipoprotein and bile salt micelles, respectively. Here, we review the biological characteristics and function of ABCG5/G8, and how the mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis, among other features.

FRI094 Case Report: Treatment Of Sitosterolemia With Ezetimibe - 17 Years Of Follow-up

Abstract Disclosure: J.V. Coutinho: None. A.S. Costa: None. M.M. Issa: None. N.P. Paim: None. E.P. Quedas: None. V.S. Nunes: None. A.L. Jorge: None. E.R. Nakandakare: None. A.F. Carrilho: None. Background: Sitosterolemia is a rare autosomal recessive disease caused by inactivating mutations in the ABCG5 or ABCG8 genes. It is characterized by the accumulation of plant sterols in tissues in the form of xanthomas and vascular atheroma. Ezetimibe has been used to decrease serum sterol levels, but its role in xanthomas and arterial atherosclerosis progression remains undetermined. We present a patient diagnosed with sitosterolemia done at age 23 years and followed for 17 years on treatment with ezetimibe 20 mg daily. Clinical case: A 23-year-old man showed typical xanthomas on the extensor surfaces of his hands and Achilles tendons, in addition to splenomegaly and thrombocytopenia. Serum cholesterol and triglyceride levels were normal and sitosterolemia was suspected by a tendon biopsy. The diagnosis was confirmed by high serum concentrations of β-sitosterol and campesterol, and the patient was advised to follow a low plant-sterol diet and to take ezetimibe 20 mg daily. Seven years later, there was no change in xanthoma size, abdominal ultrasound showed normal spleen, and plasma lipid levels were: total cholesterol (TC) 147 mg/dL, HDL cholesterol (HDL) 44 mg/dL, LDL cholesterol (LDL) 61 mg/dL, triglycerides (Tg) 206 mg/dL, desmosterol 0. 137 µg/mL (n < 1.6 µg/mL), lanosterol 0.368 µg/mL (n < 2.2 µg/mL), campesterol 39.65 µg/mL (n < 5.5 µg/mL), β-sitosterol 87.53 µg/mL (n < 1.7 µg/mL) and platelet count 109,000/mL. Seventeen years after diagnosis, at age 40, there was no significant change in xanthoma size and the patient remained without cardiovascular symptoms (normal echocardiography and exercise stress test), however ultrasound showed bilateral carotid-intimal-medial thickening (0.1 on the right and 0.17 on the left, n < 0.1) and serum lipid concentrations were: TC 203 mg/dL, LDL 130 mg/dL, HDL 46 mg/dL, Tg 145 mg/dL, campesterol 21.34 µg/mL (n < 5.5 µg/mL) and β-sitosterol 65.13 µg/mL (n < 1.7 µg/mL). Then, atorvastatin 20 mg daily was added to ezetimibe. In addition, genomic DNA sequencing revealed a compound heterozygous mutation of the ABCG5 gene (NM_022436:exon1:c.64C>T:p.Q22* and NM_022436:exon9:c.1217G>A:p.R406Q). Conclusion: treatment with ezetimibe prevented the growth of xanthomas, however it was not able to normalize plasma levels of campesterol and β-sitosterol. Furthermore, the small carotid intimal-medial thickening found raised the question about the role of plant sterols in atherosclerosis. Presentation: Friday, June 16, 2023

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Cardiovascular Disease Prevention: The Earlier the Better? A Review of Plant Sterol Metabolism and Implications of Childhood Supplementation

Atherosclerosis is the underlying cause of major cardiovascular events. The development of atherosclerotic plaques begins early in life, indicating that dietary interventions in childhood might be more effective at preventing cardiovascular disease (CVD) than treating established CVD in adulthood. Although plant sterols are considered safe and consistently effective in lowering plasma cholesterol, the health effects of early-life supplementation are unclear. Studies suggest there is an age-dependent effect on plant sterol metabolism: at a younger age, plant sterol absorption might be increased, while esterification and elimination might be decreased. Worryingly, the introduction of low-cholesterol diets in childhood may unintentionally favor a higher intake of plant sterols. Although CVD prevention should start as early as possible, more studies are needed to better elucidate the long-term effects of plant sterol accumulation and its implication on child development.

Structural Role of ABCG5/ABCG8 in Sterol Transport

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol homeostasis in eukaryotic organisms, including yeast, plants and mammals. In humans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion of cholesterol and dietary plant sterols into bile and into the gut lumen. Mutations inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive genetic disorder characterized by plant sterol accumulation, hypercholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8 are half ABC transporters;each subunit consists of an N-terminal nucleotide-binding domain (NBD) and a C-terminal transmembrane domain (TMD). The NBDs dimerize to form two catalytically asymmetric nucleotide-binding sites (NBS), one that is catalytically active (NBS2) and the other inactive (NBS1). To understand the structural basis for G5G8-mediated sterol transport we developed a large-scale purification of human G5G8 by exploiting Pichia patoris yeast. We crystallized the transporter in lipid bilayers, solved its structure in a nucleotide-free state at 3.9 Å resolution, and generated the first atomic model of an ABC sterol transporter. G5G8 presents a new structural configuration for the TMD of ABC transporters, which is present in a large and functionally diverse ABC2 superfamily. We discover that the TMD and the NBS are coupled through networks of interactions that differ between NBS1 and NBS2, reflecting the catalytic asymmetry of the transporter. A series of conserved polar residues in the TMD form polar networks that we proposed play a role in transmitting signals from the ATPase catalysis in the NBS to sterol transport on the TMD. Molecular dynamic simulation and long-range coevolution analysis revealed an inward-upward TMD movement that predicts a significant conformational change between the TMD subunits. Thus, the G5G8 structure provides a molecular framework that allows us to propose a mechanistic model for ABC transporter-mediated sterol transport and to analyze the disruptive effects of mutations causing sitosterolemia. The structure will serve as a structural template for homology modelling to a wide range of transport system that is regulated by ABCG transporters and by ABC2 superfamily.

Ezetimibe Reduces Plant Sterol Accumulation and Favorably Increases Platelet Count in Sitosterolemia

To assess if ezetimibe (EZE), a sterol-absorption inhibitor, improves platelet (PLT) count and size relative to its effect on plasma plant sterol (PS) in patients with sitosterolemia (STSL). Patients with STSL (5 males, 3 females, 16-56 years of age) receiving EZE intervention as part of their routine care participated in this study. EZE was discontinued for 14 weeks (off) and then resumed for another 14 weeks (on). Hematology variables along with plasma and red blood cells (RBC) PS and total cholesterol (TC) levels were measured at the end of each phase. EZE increased PLT count (23% ± 9%) and decreased mean PLT volume (MPV; 10% ± 3%, all P < .05). In patients off EZE, PLT counts inversely correlated (r = -0.96 and r = -0.91, all P < .01) with plasma and RBC PS to TC ratio (PS/TC), and MPV positively correlated (r = 0.91, P = .03 and r = 0.93, P = .02) with plasma and RBC PS/TC. EZE reduced plasma and RBC sitosterol (-35% ± 4% and -28% ± 3%), total PS (-37% ± 4% and -28% ± 3%, all P < .0001) levels, and PS/TC (-27% ± 4% and -28% ± 4%, P < .01). EZE reduces plasma and RBC PS levels, while increasing PLT count and decreasing MPV, and thereby may reduce the risk for bleeding in STSL. Plasma PS levels and ABCG5/ABCG8 genes should be analyzed in patients with unexplained hematologic abnormalities.

Effects of a Disrupted Blood-Brain Barrier on Cholesterol Homeostasis in the Brain

The presence of the blood-brain barrier (BBB) is critical for cholesterol metabolism in the brain, preventing uptake of lipoprotein-bound cholesterol from the circulation. The metabolic consequences of a leaking BBB for cholesterol metabolism have not been studied previously. Here we used a pericyte-deficient mouse model, Pdgfb(ret/ret), shown to have increased permeability of the BBB to a range of low-molecular mass and high-molecular mass tracers. There was a significant accumulation of plant sterols in the brains of the Pdgfb(ret/ret) mice. By dietary treatment with 0.3% deuterium-labeled cholesterol, we could demonstrate a significant flux of cholesterol from the circulation into the brains of the mutant mice roughly corresponding to about half of the measured turnover of cholesterol in the brain. We expected the cholesterol flux into the brain to cause a down-regulation of cholesterol synthesis. Instead, cholesterol synthesis was increased by about 60%. The levels of 24(S)-hydroxycholesterol (24S-OHC) were significantly reduced in the brains of the pericyte-deficient mice but increased in the circulation. After treatment with 1% cholesterol in diet, the difference in cholesterol synthesis between mutants and controls disappeared. The findings are consistent with increased leakage of 24S-OHC from the brain into the circulation in the pericyte-deficient mice. This oxysterol is an efficient suppressor of cholesterol synthesis, and the results are consistent with a regulatory role of 24S-OHC in the brain. To our knowledge, this is the first demonstration that a defective BBB may lead to increased flux of a lipophilic compound out from the brain. The relevance of the findings for the human situation is discussed.

Sitosterolemia: diagnosis, investigation, and management.

Sitosterolemia is a rare autosomal recessively inherited disease caused by mutations affecting ABCG5 or ABCG8, which are located on human chromosome band 2p21. Around 100 cases have been reported in the literature. Sitosterolemic patients typically exhibit a 30-fold to 100-fold increase in plasma concentrations of plant sterols. The clinical manifestations include xanthomas, premature atherosclerosis, hemolytic anemia, and macrothrombocytopenia. It is noteworthy that abnormal hematological parameters may be the only clinical feature of sitosterolemic patients, suggesting that sitosterolemia may be more frequent than previously thought. Severe accumulation of plant sterols in mouse models of sitosterolemia induced complex cardiac lesions, anemia, and macrothrombocytopenia, disrupted adrenal and liver cholesterol homeostasis, and caused infertility and hypertriglyceridemia. It remains unclear whether all disease traits are present in sitosterolemic patients. The drug ezetimibe appears to be effective in reducing plasma plant sterol levels, promotes xanthoma regression, and improves the cardiovascular and hematological signs in sitosterolemic patients.

Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels.

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.

Sitosterolaemia: role of different pathology disciplines in its diagnosis and management

Introduction Sitosterolaemia is a rare autosomal recessive disorder caused by mutations of ABCG5 and ABCG8, which leads to progressive accumulation of plant sterols within the body. We describe a patient with sitosterolaemia who presented with stoma-tocytosis and macrothrombocytopenia. Case report A pre-operative full blood count was checked on a 30-year-old Turkish woman (from parents as first degree cousin) with known history of idiopathic thrombocytopenia. Review of this blood film together with those from the past 2 years revealed a persistent picture of stomatocytosis and macrothrombocytopenia. Literature suggests these features can be from sitosterolaemia. There was history of migraine, oral contraceptive pill related acute hepatocellular hepatitis and hyperlipidaemia (total cholesterol 6.1 mmol/L, LDL-C 4.0 mmol/L, triglyceride 0.9 mmol/L - not on OC pills). There was no history of statin therapy and no family history of sitosterolaemia or premature cardiovascular diseases. Examination only revealed bilateral xanthelasma. Results Total plasma plant sterol was markedly raised (1.3 mmol/ L; NR Conclusions Sitosterolaemia can present exclusively with stoma-tocytosis and macrothrombocytopenia.1 Whether they represent a subgroup with a different prognosis is unknown.

ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/−) and ABCG5/G8-deficient (G5/G8−/−) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/− and G5/G8−/− mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8−/− mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/− (1.3-fold) and G5/G8−/− mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/− (1.8-fold) and G5/G8−/− mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/− and G5/G8−/− than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-3H] oleate, was 5.8-fold higher in G5/G8−/− than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8−/− than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.

Mass‐Spec Identification of Human Genetic Disease

Oxysterols are metabolites of cholesterol that play important roles in the brain, immune system, and liver. Mutations in four of seven genes that produce oxysterols cause learning defects, immunoglobulin abnormalities, and liver failure. To identify mutations in the remaining three genes, a mass spectrometry (MS)‐based method was developed to quantify sterols and oxysterols in small volumes (~200 μl) of human serum. Every procedural step was optimized, including organic solvents used to extract total lipids from serum, partial purification of sterols and oxysterols by aminopropyl silica chromatography, derivatization prior to gas chromatography‐MS, and resolution by ultra‐HPLC prior to quantification via isotope‐dilution electrospray or atmospheric pressure ionization MS. To test the method, levels of 60 sterols and oxysterols were determined in sera from 200 volunteers from the Cooper Aerobics Clinic in North Dallas. The power of the approach to detect mutations was illustrated by identification of an undiagnosed case of sitosterolemia (abnormal accumulation of plant sterols). Sterol and oxysterol levels are now being measured in ~3,500 individuals from the Dallas Heart Study, a cohort whose genomic DNAs and clinical histories are maintained on campus. Oxysterol biosynthetic and catabolic genes will be sequenced in individuals with oxysterol levels in the lowest and highest 5th percentiles and the consequences of mutations identified determined from clinical histories.

Cerebral Accumulation of Dietary Derivable Plant Sterols does not Interfere with Memory and Anxiety Related Behavior in Abcg5−/− Mice

Plant sterols such as sitosterol and campesterol are frequently applied as functional food in the prevention of atherosclerosis. Recently, it became clear that plasma derived plant sterols accumulate in murine brains. We questioned whether plant sterols in the brain are associated with alterations in brain cholesterol homeostasis and subsequently with brain functions. ATP binding cassette (Abc)g5−/− mice, a phytosterolemia model, were compared to Abcg5+/+ mice for serum and brain plant sterol accumulation and behavioral and cognitive performance. Serum and brain plant sterol concentrations were respectively 35–70-fold and 5–12-fold increased in Abcg5−/− mice (P < 0.001). Plant sterol accumulation resulted in decreased levels of desmosterol (P < 0.01) and 24(S)-hydroxycholesterol (P < 0.01) in the hippocampus, the brain region important for learning and memory functions, and increased lanosterol levels (P < 0.01) in the cortex. However, Abcg5−/− and Abcg5+/+ displayed no differences in memory functions or in anxiety and mood related behavior. The swimming speed of the Abcg5−/− mice was slightly higher compared to Abcg5+/+ mice (P < 0.001). In conclusion, plant sterols in the brains of Abcg5−/− mice did have consequences for brain cholesterol metabolism, but did not lead to an overt phenotype of memory or anxiety related behavior. Thus, our data provide no contra-indication for nutritional intake of plant sterol enriched nutrition.

Platelet Activation In Sitosterolemia Mice

AbstractAbstract 2022 Introduction:Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can be corrected by treatment with the sterol-absorption inhibitor, ezetimibe. Because the mechanism by which accumulated plant sterols affects platelet size, production, and function is incompletely understood, we further analyzed these traits in Abcg5- and Abcg8-deficient mice in an animal model of sitosterolemia. Methods:Blood was collected every 1–2 weeks and CBC monitored in Abcg5- and Abcg8-deficient mice that had been fed either a high or low plant sterol diet. Expression of platelet receptors was analyzed by both flow cytometry and Western blotting. GPIbα null mice were used as controls since they have similarly enlarged platelets. Platelet microparticles were analyzed by labeling with a mAb specific for GPIIb and Annexin V. Plasma samples were analyzed for von Willebrand factor (VWF) antigen and multimer patterns. Results:Following onset of a high sterol diet, the platelet count decreased over a two week period to less than 30% of that of normal controls in both Abcg5-/- and Abcg8-/- mice. At the same time, the mean platelet volume gradually increased over a 4 week period from a normal value of approximately 7 fl to approximately 10 fl. In addition, an increased number of platelet-derived microparticles were detected in Abcg5-/- and Abcg8-/- mice kept on high sterol diet over a 9 week period, suggesting that low-grade platelet activation was occurring in these mice. Unexpectedly, the surface expression of the GPIIb integrin subunit was decreased in both Abcg5-/- and Abcg8-/- mouse platelets compared to that of similarly large platelets derived from GPIbα-null (Bernard-Soulier) platelets. Intracellular staining revealed that the GPIIb in Abcg8-/- platelets was being internalized and retained within the cell. Both surface-bound and total platelet fibrinogen was increased in Abcg5-/- and Abcg8-/- mice fed a high sterol diet. Finally, plasma VWF antigen levels decreased by approximately 50% within two weeks following switching Abcg5-/- mice from a low-fat to a high-fat diet. Multimer analysis showed loss of high molecular weight multimers in some of the plasma samples. Conclusions:This study demonstrates a heightened state of platelet activation in a murine model of sitosterolemia, leading to VWF and fibrinogen binding, with concomitant internalization of GPIIb. This might explain, at least in part, the cause of macrothorombocytopenia and absence of ristocetin-induced platelet aggregation in sitosterolemia patients. Platelet activation may also lead to calpain activation, resulting filamin A and talin degradation and microparticle production. Future studies aims at identifying the mechanism by which plant sterols or their metabolites elicit these effects on platelets may provide important clues to the ability of dietary lipids to affect platelet activation and the development of atherosclerosis. Disclosures:Newman:New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.

P285 FIRST DEMONSTRATION OF THE LOCALIZATION OF HEPATIC apoB100- AND INTESTINAL apoB48-CONTAINING LIPOPROTEIN PARTICLES IN HUMAN ATHEROSCLEROTIC PLAQUES USING DUAL-STAINING IMMUNOFLUORESCENCE

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/−) and ABCG5/G8-deficient (G5/G8−/−) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/− and G5/G8−/− mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8−/− mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/− (1.3-fold) and G5/G8−/− mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/− (1.8-fold) and G5/G8−/− mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/− and G5/G8−/− than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-3H] oleate, was 5.8-fold higher in G5/G8−/− than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8−/− than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.

Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1: a review

1. Whole-body sterol (cholesterol and xenosterol) balance is delicately regulated by the gastrointestinal tract and liver, which control sterol absorption and excretion, respectively, in addition to the contribution to the cholesterol pool by whole-body cholesterol synthesis. In the past ten years enormous strides have been made not only in establishing that specific transporters mediate the entry and exit of sterols and how these may regulate selective sterol access to the body pools, but also in how these pathways operate to integrate these physiological pathways.2. The entry of sterols from the gastrointestinal and biliary canalicular lumen into the body is mediated by NPC1L1, which was discovered by a novel method, via a genomics–bioinformatics approach.3. Identification of the genetic basis responsible for causing sitosterolaemia, characterized by plant sterol accumulation, led to the identification of two half-transporters (ABCG5 and ABCG8) that normally efflux plant sterols (and cholesterol) into the intestinal and biliary lumen for faecal excretion.4. The objective of this review is to provide up-to-date knowledge on genomics, proteomics and function of these two transporter systems.

Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r=0.74, P<0.001 for lathosterol and r=0.88, P<0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r= -0.47, P=0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.