A complex plasma plant sterol locus on mouse chromosome 14 has at least two genes regulating intestinal sterol absorption

We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol absorption.     

Identification of mosquito sterol carrier protein-2 inhibitors

A mosquito sterol carrier protein-2, AeSCP-2, has been shown to aid in the uptake of cholesterol in mosquito cells. The discovery of chemical inhibitors of AeSCP-2 is reported here. AeSCP-2 inhibitors (SCPIs) belong to several chemotypes of hydrophobic compounds. Those inhibitors competed with cholesterol for AeSCP-2, binding with relatively high binding affinities. In cultured insect cells, SCPIs reduced cholesterol uptake by as much as 30% at 1-5 microM concentrations. SCPIs were potent larvicides to the yellow fever mosquito, Aedes aegypti, and to the tobacco hornworm, Manduca sexta, with 50% lethal doses (LD50s) of 5-21 microM and 0.013-15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species.     

The combination of ezetimibe and ursodiol promotes fecal sterol excretion and reveals a G5G8-independent pathway for cholesterol elimination

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.     

A reappraisal of sterol biosynthesis and metabolism in aphids

Aphids of Schizaphis graminum (Rondani) (biotype C) reared on its host-plant, Sorghum bicolor (L.) Moench, sequestered campesterol, stigmasterol and sitosterol. Aphids reared for 72 hr on holidic diets supplemented with [4-¹⁴C]-sitosterol contained both [¹⁴C]-sitosterol and [¹⁴C]-cholesterol, indicating that these aphids are capable of dealkylation at C-24. When aphids were reared on artificial diets containing [2-¹⁴C]-mevalonic acid, no detectable amounts of radioactively labelled desmethyl sterols, nor metabolic intermediates in sterol synthesis (i.e. squalene, 2,3-oxidosqualene, 4,4-dimethyl and 4-monomethyl sterols) were found to accumulate in their tissues. The relevance of these findings to previous research suggesting the ability of aphids, via their symbiotes, to synthesize sterols is discussed.     

昆虫固醇转运蛋白的结构与功能

在昆虫中, 胆固醇不仅是细胞膜的重要成分之一, 也是昆虫蜕皮激素生物合成的前体。由于昆虫体内缺少两种合成胆固醇所必需的关键性酶, 所以昆虫不能自主地从简单的前体化合物从头合成胆固醇, 而必须通过吸收食物中的甾醇转化为胆固醇来满足生长、发育和繁殖的需要。胆固醇在组织和细胞内的运输主要由固醇转运蛋白 (sterol carrier proteins, SCPs) 执行。因此, 对固醇转运蛋白结构与功能的研究对于阐明昆虫中固醇运输具有重要的意义。本文对固醇转运蛋白的基因和蛋白结构、 细胞内表达和定位、 翻译后修饰、 蛋白三维结构、底物特异性和可能的运输途径等方面的研究进展进行了综述, 并对其作为害虫防治分子靶标的可能性进行了初步的讨论。研究发现, 不同物种的SCP蛋白的基因编码形式和蛋白剪切形式不同; 双翅目昆虫埃及伊蚊Aedes aegypti和黑腹果蝇Drosophila melanogaster除了SCP-x基因可编码SCP-x和SCP-2蛋白外, 还有另外的SCP-2和类SCP-2 (SCP-2L)基因编码SCP-2和类SCP-2蛋白; 而鳞翅目昆虫棉贪夜蛾Spodoptera littoralis、 斜纹夜蛾Spodoptera litura和家蚕Bombyx mori中SCP-x 基因的表达和转录方式与脊椎动物的SCP-x 基因类似, 通过转录和翻译后剪切形成SCP-2蛋白。SCP-x和SCP-2蛋白定位于过氧化物酶体。SCP-2蛋白由5个α-螺旋和5个β-折叠组成, 其中α5-螺旋可影响蛋白与底物的结合。SCP-2蛋白以不同的亲和力与固醇、胆固醇衍生物、脂肪酸、脂酰辅酶A和磷脂等化合物结合。超表达斜纹夜蛾SlSCP-x 和SlSCP-2基因可增加细胞对胆固醇的吸收; 而利用RNAi技术抑制幼虫体内SlSCP-x表达, 可导致血淋巴中的胆固醇含量降低, 并导致幼虫生长缓慢, 蜕皮化蛹延迟。     

Inhibitors of sterol synthesis. Chemical syntheses and activities of new derivatives of 15-oxygenated sterols

The chemical syntheses of 5α-cholestane-3β,14α,15β-triol, 5α-cholestane-14α-ol-3,15-dione, 5α-cholestane-3β,14α-diol-15-one, 14α,15α-epoxy-5α-cholestan-3β-ol, and 5α-cholest-8(14)-en-3β-ol-15-one oxime are described. All of these compounds were found to be potent inhibitors of sterol synthesis in cultured mouse L cells. However, the former three compounds had little or no effect on the levels of 3-hydroxy-3-methylgutaryl (HMG)-CoA reductase in the same cells. In contrast, in the case of the latter two compounds, the concentrations required to cause a 50% inhibition of the synthesis of digitonin-precipitable sterols were comparable to those required to cause a 50% reduction in the levels of HMG-CoA reductase in the same cells. 5α-Cholest-8(14)-en-3β-ol-15-one oxime had no effect on serum cholesterol levels when administered to male rats at a level of 0.15% in a cholesterol-free diet.     

Brain sterol dysregulation in sporadic AD and MCI: relationship to heme oxygenase-1

The objective of this study was to ascertain the impact of aging and Alzheimer's disease (AD) on brain cholesterol (CH), CH precursors, and oxysterol homeostasis. Altered CH metabolism and up-regulation of heme oxygenase-1 (HO-1) are characteristic of AD-affected neural tissues. We recently determined that HO-1 over-expression suppresses total CH levels by augmenting liver X receptor-mediated CH efflux and enhances oxysterol formation in cultured astroglia. Lipids and proteins were extracted from postmortem human frontal cortex derived from subjects with sporadic AD, mild cognitive impairment (MCI), and no cognitive impairment ( n  = 17 per group) enrolled in the Religious Orders Study, an ongoing clinical-pathologic study of aging and AD. ELISA was used to quantify human HO-1 protein expression from brain tissue and gas chromatography–mass spectrometry to quantify total CH, CH precursors, and relevant oxysterols. The relationships of sterol/oxysterol levels to HO-1 protein expression and clinical/demographic variables were determined by multivariable regression and non-parametric statistical analyses. Decreased CH, increased oxysterol and increased CH precursors concentrations in the cortex correlated significantly with HO-1 levels in MCI and AD, but not no cognitive impairment. Specific oxysterols correlated with disease state, increasing neuropathological burden, neuropsychological impairment, and age. A model featuring compensated and de-compensated states of altered sterol homeostasis in MCI and AD is presented based on the current data set and our earlier in vitro work.     

Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.     

Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.     

Mechanistic and metabolic studies of sterol 24,25-double bond reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K_m, 3 μM), lanosterol (K_m, 18 μM), and cycloartenol (K_m, 33 μM), to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-²H]lanosterol was transformed by larvae to [24-²H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and ¹H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epimino-lanosterol (IL), was assayed and found to be inhibitory with an I₅₀ of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I₅₀ 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-³H]IL when fed to the larvae. [2-³H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley-Liss, Inc.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells

Transport of the fluorescent cholesterol analog dehydroergosterol (DHE) from the plasma membrane was studied in J774 macrophages (Mphis) with normal and elevated cholesterol content. Cells were labeled with DHE bound to methyl-beta-cyclodextrin. In J774, Mphis with normal cholesterol, intracellular DHE became enriched in recycling endosomes, but was not highly concentrated in the trans-Golgi network or late endosomes and lysosomes. After raising cellular cholesterol by incubation with acetylated low-density lipoprotein (AcLDL), DHE was transported to lipid droplets, and less sterol was found in recycling endosomes. Transport of DHE to droplets was very rapid (t1/2 = 1.5 min after photobleaching) and did not require metabolic energy. In cholesterol-loaded J774 Mphis, the initial fraction of DHE in the plasma membrane was reduced, and rapid DHE efflux from the plasma membrane to intracellular organelles was observed. This rapid sterol transport was not related to plasma membrane vesiculation, as DHE did not become enriched in endocytic vesicles formed after sphingomyelinase C treatment of cells. When cells were incubated with DHE ester incorporated into AcLDL, fluorescence of the sterol was first found in punctate endosomes. After a chase, this DHE colocalized with transferrin in a distribution similar to cells labeled with DHE delivered by methyl-beta-cyclodextrin. Our results indicate that elevation of sterol levels in Mphis enhances transport of sterol from the plasma membrane by a non-vesicular pathway.     

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli/

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067–1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.     

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.     

Molecular basis for sterol transport by StART‐like lipid transfer domains

Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non‐vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a‐c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo‐bound and sterol‐bound forms, at 2.0 Å resolution. It folds into a truncated version of the steroidogenic acute regulatory protein‐related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3‐hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.