Changes in Brain Cholesterol Metabolome After Excitotoxicity

Excitotoxicity due to excess stimulation of glutamate receptors in neurons is accompanied by increased Ca²⁺ influx, stimulation of Ca²⁺-dependent enzymes, ATP depletion, increase in lipid peroxidation products, and loss of glutathione. These changes resemble neurochemical alterations in acute neuronal injury (stroke, spinal cord injury, and traumatic brain injury) and chronic neurodegenerative diseases such as Alzheimer’s disease. Intracerebroventricular injection of the potent glutamate analog kainate in rats results in increased cholesterol concentration in the hippocampus at short to medium time intervals, i.e., 3 days–1 week post-injection, as detected by gas chromatography–mass spectrometry in the lesioned hippocampus. This is accompanied by an early increase in levels of cholesterol biosynthetic precursors and increases in both enzymatically derived oxysterols such as 24-hydroxycholesterol and cholesterol oxidation products (COPs) generated by reactive oxygen species, including cholesterol epoxides and 7-ketocholesterol. In contrast to COPs, no change in concentration of the neurosteroid pregnenolone was found after KA injury. Cholesterol and COPs significantly increase exocytosis in cultured PC12 cells and neurons, and both oxysterols and COPs are able to induce cytotoxic and apoptotic injuries in different cell types, including neurons. Together, the findings suggest that increased cholesterol and COPs after KA excitotoxicity could themselves lead to disturbed neuronal ion homeostasis, increased neurotransmitter release, and propagation of excitotoxicity.     

22R-hydroxycholesterol and 9-cis-retinoic acid induce ATP-binding cassette transporter A1 expression and cholesterol efflux in brain cells and decrease amyloid beta secretion

The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of peripheral cholesterol efflux and plasma high density lipoprotein metabolism. In adult rat brain we found high expression of ABCA1 in neurons in the hypothalamus, thalamus, amygdala, cholinergic basal forebrain, and hippocampus. Large neurons of the cholinergic nucleus basalis together with CA1 and CA3 pyramidal neurons were among the most abundantly immunolabeled neurons. Glia cells were largely negative. Because cholesterol homeostasis may have an essential role in central nervous system function and neurodegeneration, we examined ABCA1 expression and function in different brain cell types using cultures of primary neurons, astrocytes, and microglia isolated from embryonic rat brain. The basal ABCA1 mRNA and protein levels detected in these cell types were increased markedly after exposure to oxysterols and 9-cis-retinoic acid, which are ligands for the nuclear hormone liver X receptors and retinoic X receptors, respectively. Functionally, the increased ABCA1 expression caused by these ligands was followed by elevated apoA-I- and apoE-specific cholesterol efflux in neurons and glia. In non-neuronal and neuronal cells overexpressing a human Swedish variant of amyloid precursor protein, 22R-hydroxycholesterol and 9-cis-retinoic acid induced ABCA1 expression and increased apoA-I-mediated cholesterol efflux consequently decreasing cellular cholesterol content. More importantly, we demonstrated that these ligands alone or in combination with apoA-I caused a substantial reduction in the stability of amyloid precursor protein C-terminal fragments and decreased amyloid beta production. These effects of 22R-hydroxycholesterol may provide a novel strategy to decrease amyloid beta secretion and consequently reduce the amyloid burden in the brain.     

油酸对THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1表达和胆固醇流出的影响

以THP 1巨噬细胞源性泡沫细胞为研究对象 ,观察油酸对THP 1巨噬细胞源性泡沫细胞胆固醇流出和三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响 ,以探讨油酸对动脉粥样硬化发生发展的影响。用液体闪烁计数器检测细胞内胆固醇流出 ,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量 ,运用逆转录多聚酶链反应和Western印迹分别检测ABCA1mRNA与ABCA1蛋白的表达 ,采用流式细胞术检测细胞平均ABCA1荧光强度。实验显示油酸引起THP 1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯呈时间依赖性增加 ,而ABCA1蛋白水平、细胞平均ABCA1荧光强度以及apoA I介导的胆固醇流出呈时间依赖性减少 ,细胞内胆固醇增多 ,但ABCA1mRNA没有明显变化。结果表明 ,油酸减少THP 1巨噬细胞源性泡沫细胞ABCA1蛋白水平 ,降低细胞内胆固醇流出 ,增加细胞内胆固醇聚积。     

Distribution of hydroxynonenal-modified proteins in the kainate-lesioned rat hippocampus: evidence that hydroxynonenal formation precedes neuronal cell death

Decomposition of lipid peroxides gives rise to a wide range of aldehydes. 4-Hydroxyalkenals and in particular 4-hydroxynonenal (HNE) are often the most toxic products. Frequently, it is unclear at which stage in the tissue injury process HNE is formed, i.e., is it a late stage or an early stage in which HNE contributes to subsequent cell death? The present study was carried out using an antibody to HNE-modified proteins to elucidate the time course and distribution of HNE in the lesioned hippocampus after kainate injections. HNE was absent from normal neurons, but dense staining to HNE was observed in degenerating neurons after kainate injection. The increase in HNE staining occurred as early as 1 d postinjection, at a time when there was no histological evidence of cell death. HNE immunoreactivity was observed in the degenerating CA1 and CA3 fields at 3 d and 1 week postinjection, but was confined to a cluster of neurons at the edge of the degenerating CA fields, at 2 and 3 weeks postinjection. These observations suggest that HNE formation is an early event after this tissue injury, and may contribute to later cell death.     

Origins of intestinal ABCA1-mediated HDL-cholesterol

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.     

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.     

Posthatching evolution of in vivo mevalonate metabolism in chick liver

The in vivo mevalonate incorporation into total nonsaponifiable lipids by chick liver was minimal after hatching and drastically increased between 1-5 days. The hepatic synthesis of different cholesterol precursors emerged sequentially after hatching. Between 1-5 days increased strongly the conversion of mevalonate into squalene and also the formation of oxygenated lanosterol derivatives from squalene. The conversion of squalene became completely active at day 8. Cholesterol formation from lanosterol derivatives was completely activated between 8-11 days. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipids identified as lanosterol derivatives and cholesterol precursors formed from [5-14C]mevalonate in experiments carried out in vivo. Postnatal evolution of these oxysterols may explain the great increase of 3-hydroxy-3-methylglutaryl-CoA reductase activity found in chick liver between 5-11 days, simultaneous or posterior to the diminution of the oxygenated cholesterol precursors.     

转运蛋白ABCG1/4 和ABCA1对脑胆固醇的调节作用

脑是富含胆固醇的器官,机体大约有25%的胆固醇集中在脑组织中.ATP结合盒超家族转运蛋白对脑组织中胆固醇的膜外转运和动态平衡起着重要的调节作用.研究发现,ATP结合盒超家族转运蛋白亚体ABCG1、ABCG4和ABCA1在成体脑组织中存在不同程度的表达,一种或多种亚体的缺失可以导致神经退行性病变.然而,ATP结合盒超家族转运蛋白亚体对脑发育过程中脑胆固醇动态变化的调节缺乏相关性的报道.在本研究中,从低胆固醇饮食喂养的C57BL/6J小鼠中获取出生后不同发育时期的脑组织,对ABCG1、ABCG4和ABCA1的mRNA与蛋白质表达水平进行测定,并对脑组织和血清中ATP结合盒超家族转运蛋白的表达水平与胆固醇水平的相关性进行研究.同时,使用ABCG1、ABCG4单一基因敲除鼠和ABCG1、ABCG4双基因敲除鼠,研究ATP结合盒超家族转运蛋白对与胆固醇合成的相关基因表达的影响以及对脑组织胆固醇代谢的调节作用.结果发现,ABCG1、ABCG4和ABCA1在机体多个器官中均有表达,但ABCG1和ABCG4在小鼠脑组织中表达量最高.在脑组织发育过程中,ABCG1和ABCG4mRNA水平呈现明显的表达时效性,小鼠于出生后42天达到峰值,而ABCA1 mRNA的表达水平无明显变化.血清和脑组织中中酯化型胆固醇水平呈双高峰分布,也于出生后42天达到最高.基因敲除鼠模型显示,单一敲除ABCG1或者ABCG4基因对脑组织胆固醇水平无明显影响,而ABCG1和ABCG4基因的同时缺失导致脑胆固醇水平显著升高,并明显降低胆固醇合成相关基因的表达水平.本研究表明,在脑发育成熟过程中,ATP结合盒超家族转运蛋白亚体ABCG1和ABCG4,而非ABCA1,以调节脑胆固醇的膜外转运;ABCG1和ABCG4互补调控脑胆固醇的动态平衡.     

Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.     

Expression and localisation of brain-type organic cation transporter (BOCT/24p3R/LCN2R) in the normal rat hippocampus and after kainate-induced excitotoxicity

The iron siderophore binding protein lipocalin 2 (LCN2, also known as 24p3, NGAL and siderocalin) may be involved in iron homeostasis, but to date, little is known about expression of its putative receptor, brain-type organic cation transporter (BOCT, also known as BOCT1, 24p3R, NGALR and LCN2R), in the brain during neurodegeneration. The present study was carried out to elucidate the expression of LCN2 and BOCT in hippocampus after excitotoxicity induced by the glutamate analog, kainate (KA) and a possible role of LCN2 in neuronal injury. As reported previously, a rapid and sustained induction in expression of LCN2 was found in the hippocampus after intracerebroventicular injection of KA. BOCT was expressed in neurons of the saline-injected control hippocampus, and immunolabel for BOCT protein was preserved in pyramidal neurons of CA1 at 1 day post-KA injection, likely due to the delayed onset of neurodegeneration after KA injection. At 3 days and 2 weeks after KA injections, loss of immunolabel was observed due to degenerated neurons, although remaining neurons continued to express BOCT, and induction of BOCT was found in OX-42 positive microglia. This resulted in an overall decrease in BOCT mRNA and protein expression after KA treatment. Increased expression of the pro-apoptotic marker, Bim, was found in both neurons and microglia after KA injection, but TUNEL staining indicating apoptosis was found primarily in Bim-expressing neurons, but not microglia. Interaction between LCN2 and BOCT was found by DuoLink assay in cultured hippocampal neurons. Apo-LCN2 without iron caused no significant differences in neuronal Bim expression or cell survival, whereas holo-LCN2 consisting of LCN2:iron:enterochelin complex increased Bim mRNA expression and decreased neuronal survival. Together, results suggest that LCN2 and BOCT may have a role in neuronal injury.     

对氧磷降低RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出

三磷酸腺苷结合盒转运体A1(ABCA1)是体内胆固醇逆向转运的关键环节.对氧磷是广泛使用的有机磷农药的活性代谢产物.研究发现,对氧磷能增加巨噬细胞中胆固醇的堆积,但具体机制还不清楚.以RAW264.7巨噬细胞源性泡沫细胞为研究对象,观察对氧磷对RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出的影响并探讨其机制.结果显示,对氧磷以时间和剂量依赖的方式增加RAW264.7巨噬细胞源性泡沫细胞中总胆固醇、游离胆固醇和胆固醇酯水平,降低ABCA1表达和胆固醇流出,同时对氧磷降低细胞中环磷酸腺苷(cAMP)的水平及腺苷酸环化酶(AC)的活性和增加磷酸二酯酶(PDE)的活性,而cAMP的类似物双丁酰环腺苷酸(dBcAMP)能够阻断对氧磷降低ABCA1表达和部分阻断对氧磷降低胆固醇流出,对氧磷导致的cAMP水平的降低也可被AC激动剂福斯高林(Forskolin)和PDE抑制剂3-异丁基-1-甲基黄嘌呤(IBMX)所阻断.以上结果表明,对氧磷通过cAMP信号通路下调RAW264.7巨噬细胞源性泡沫细胞ABCA1的表达,降低细胞内胆固醇流出和增加细胞内胆固醇堆积.     

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.     

Role of ABCG1 and ABCA1 in regulation of neuronal cholesterol efflux to apolipoprotein E discs and suppression of amyloid-beta peptide generation

Maintenance of an adequate supply of cholesterol is important for neuronal function, whereas excess cholesterol promotes amyloid precursor protein (APP) cleavage generating toxic amyloid-beta (Abeta) peptides. To gain insights into the pathways that regulate neuronal cholesterol level, we investigated the potential for reconstituted apolipoprotein E (apoE) discs, resembling nascent lipoprotein complexes in the central nervous system, to stimulate neuronal [3H]cholesterol efflux. ApoE discs potently accelerated cholesterol efflux from primary human neurons and cell lines. The process was saturable (17.5 microg of apoE/ml) and was not influenced by APOE genotype. High performance liquid chromatography analysis of cholesterol and cholesterol metabolites effluxed from neurons indicated that <25% of the released cholesterol was modified to polar products (e.g. 24-hydroxycholesterol) that diffuse from neuronal membranes. Thus, most cholesterol (approximately 75%) appeared to be effluxed from neurons in a native state via a transporter pathway. ATP-binding cassette transporters ABCA1, ABCA2, and ABCG1 were detected in neurons and neuroblastoma cell lines and expression of these cDNAs revealed that ABCA1 and ABCG1 stimulated cholesterol efflux to apoE discs. In addition, ABCA1 and ABCG1 expression in Chinese hamster ovary cells that stably express human APP significantly reduced Abeta generation, whereas ABCA2 did not modulate either cholesterol efflux or Abeta generation. These data indicate that ABCA1 and ABCG1 play a significant role in the regulation of neuronal cholesterol efflux to apoE discs and in suppression of APP processing to generate Abeta peptides.     

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Effect of apolipoprotein A-I on ATP binding cassette transporter A1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells

The accumulation of lipoprotein cholesterol in theartery wall is thought to be an important factor in thedevelopment of atherosclerosis. After retentionand modi-fication in arteries, atherogenic lipoproteins are taken upby macrophages, bringing about macrophage-derived foamcells. High-density lipoprotein (HDL) plays a role in trans-porting cholesterol from peripheral tissues to the liver.The elevated level of HDL is associated with a decreasein atherosclerosis and the apolipoproteins to remo…     

Wogonin preventive impact on hippocampal neurodegeneration,inflammation and cognitive defects in temporal lobe epilepsy

Previous studies demonstrated that the pathophysiological changes after temporal lobe epilepsy (TLE) such as oxidative stress, inflammatory reaction contribute to cognitive defect and neuronal damage. The present study was conducted to evaluate the anticonvulsant effect of wogonin ameliorates kainate-induced TLE, and to investigate the mechanism underlying these effects. Rats were divided into control, wogonin, kainate, and wogonin-pretreated kainate groups. The rat model of TLE was induced by unilateral intrahippocampal injection of 0.4 ug/ul of kainate. The results showed that the cognitive function in TLE rats was significantly impaired, and wogonin treatment improved cognitive function in the Morris water maze (MWM). H & E staining and TUNEL staining showed obvious damage in the hippocampus of TLE rats, and wogonin alleviated the damage. To evaluate the oxidative stress, the expression of MDA and GSH in plasma were detected. Nrf-2 and HO-1 mRNA expression in the hippocampus were detected. The levels of MDA in plasma increased in TLE rats, and the levels of GSH in plasma and Nrf-2, HO-1 in the brain decreased. Treatment with wogonin alleviated these changes. We also detected the mRNA expression of inflammatory mediators like IL-1β, TNF-α, and NF kB in the brain. The inflammatory reaction was significantly activated in the brain of TLE rats, and wogonin alleviated neuroinflammation. We detected the mRNA expression of Bcl-2, Bax, caspase-3, in the hippocampus. The levels of Bcl-2 decreased in TLE rats, Bax and caspase-3 increased, while wogonin alleviated these changes. The present study indicated that wogonin exerted a noticeable neuroprotective effect in kainate-induced TLE rats.     

Hypoxanthine induces cholesterol accumulation and incites atherosclerosis in apolipoprotein E‐deficient mice and cells

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)‐deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP‐binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H₂O₂ also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N‐acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE‐siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE‐deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.