Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001).These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.     

Transfer of maternal cholesterol to embryo and fetus in pregnant mice

Cholesterol is essential for antenatal development. However, the transport of maternal cholesterol to the embryo has not been sufficiently studied, and that to the fetus is still controversial. To this end, a 1 mg dose of [3,4-(13)C(2)]cholesterol was injected daily into pregnant mice and the labeled cholesterol was measured by gas chromatography-mass spectrometry. After venous injections from days 10 to 17 of gestation, [(13)C]cholesterol levels in total ((12)C and (13)C) cholesterol were increased to 5.1% and 2.8% in maternal and fetal plasma, respectively. Labeled cholesterol was identified in the liver, kidneys, and intestines, but not in the brain, of the fetus. After injections from days 1 to 8, [(13)C]cholesterol levels were increased to 12.4% and 8.0% of total cholesterol in maternal plasma and the embryo, respectively. The level of 11.5% in the yolk sac was higher than that in the embryo. Intrauterine transfer of maternal cholesterol to the embryo as well as the fetus was evident in mice, and both the placenta and the yolk sac appear to be sites of intermediate passage in murine pregnancy.     

Maternal high density lipoproteins affect fetal mass and extra-embryonic fetal tissue sterol metabolism in the mouse

Previous studies have shown that antibodies to cubulin, a receptor on the yolk sac that binds high density lipoproteins (HDL) and cobalamin, induce fetal abnormalities. Mice with markedly low concentrations of plasma HDL-cholesterol (HDL-C) give birth to healthy pups, however. To establish whether maternal HDL-C has a role in fetal development, sterol metabolism was studied in the fetus and extra-embryonic fetal tissues in wild-type and apolipoprotein A-I-deficient mice (apoAI-/-). Maternal HDL-C content was markedly greater in apoAI+/+ mice prior to pregnancy and at 13 days into gestation. By 17 days into gestation, HDL-C content was similar between both types of mice. Fetuses from apoAI (-/- x -/-) matings were 16;-25% smaller than control mice at 13 and 17 days of gestation and contained less cholesterol. The differences in size and cholesterol content were not due to a lack of cholesterol synthesis or apoA-I in the fetus. In the yolk sac and placenta, sterol synthesis rates were approximately 50% greater in the 13-day-old apoAI-/- mice as compared to the apoAI+/+ mice. Even though synthesis rates were greater, cholesterol concentrations were 22% lower in the yolk sac and similar in the placenta of apoAI-/- mice as compared to tissues of wild-type mice. These data suggest that a difference in maternal HDL-C concentration or composition can affect the size of the fetus and sterol metabolism of the yolk sac and placenta in the mouse.     

Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR α/β ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-α or -γ ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.     

Impact of maternal circulating cholesterol and gestational diabetes mellitus on lipid metabolism in human term placenta

Maternal hypercholesterolemia (HC) during pregnancy and gestational diabetes mellitus (GDM) are associated with disturbance of fetal development which may also modify key features of placental functions. In this study, we evaluated the impact of maternal hypercholesterolemia on placental cholesterol and lipid metabolism in 59 women classified in two groups according to the median concentration of plasma total cholesterol (6.42 mM). The impact of GDM was also evaluated on the metabolism of placentas obtained from 7 insulin-treated GDM and 7 non-GDM women. We showed that high maternal circulating cholesterol is associated with a significant increase in the LDL-cholesterol, ApoB-100 and triglyceride concentrations in the maternal blood. However the level of cholesterol in the venous cord blood and placenta remains unchanged in response to modification in maternal cholesterol profile. The levels of Fatty acid synthase (FAS) and SREBP-2 expressions in placenta are significantly increased in the HC group while expression of both sterol regulatory element-binding proteins-1 (SREBP-1) and HMG-CoA reductase (HMGR) are not modified. GDM is not associated with modification in the maternal lipid profile but it increases the concentration of inflammatory cytokines (IL-1beta and TNF-alpha) in placenta which correlates with a dramatic induction of FAS expression without affecting the expression of mature SREBPs proteins. In conclusion, our study suggests that in placenta, expressions of key proteins involved in de novo lipid synthesis are affected by changes in maternal metabolism (HC and GDM) that may subsequently affect fetal development.     

Stanol esters decrease plasma cholesterol independently of intestinal ABC sterol transporters and Niemann-Pick C1-like 1 protein gene expression

Possible mechanisms for the cholesterol-lowering effects of plant stanol esters were addressed by feeding hamsters diets containing stanol esters, cholesterol, or cholestyramine/lovastatin. ABCA1, ATP binding cassette G1 (ABCG1), ABCG5, ABCG8, and Niemann-Pick C1-like 1 (NPC1L1) mRNA levels were then estimated in duodenum, jejunum, and ileum. Plasma cholesterol was decreased by 36% and 94% in animals fed stanol esters and cholestyramine/lovastatin, respectively. Cholesterol feeding increased plasma cholesterol by 2.5-fold. Plasma plant sterols were unchanged by stanol ester feeding but became undetectable by feeding cholestyramine/lovastatin. Cholesterol and stanols accumulated in enterocytes of animals fed cholesterol and stanol esters, respectively. ABCG5 and ABCG8 mRNA levels were decreased by stanol esters and cholestyramine/lovastatin. Cholesterol feeding markedly increased ABCA1 and ABCG1 expression and modestly increased ABCG5/ABCG8. NPC1L1 mRNA was not significantly altered by any of the diets. ABCG1, ABCG5, ABCG8, and NPC1L1 mRNAs were highest in cells of the upper villus, whereas ABCA1 mRNA was highest in cells of the lower villus. The results suggest that cholesterol lowering effect of stanol esters is unrelated to changes in mRNA levels of intestinal ABC sterol transporters or NPC1L1. Cholesterol flux regulates ABC expression but not NPC1L1. The different localization of ABCA1 suggests a different function for this protein than for ABCG1, ABCG5, ABCG8, and NPC1L1.     

Placentation in watersnakes I: Placental histology and development in North American Nerodia (Colubridae: Natricinae)

As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin‐section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia : Colubridae). The pre‐ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid‐ to late‐development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal–fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal‐fetal exchange.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only marginally in fetal livers, fetal bodies, placentas, and yolk sacs when cholesterol concentrations were increased. To begin to elucidate the mechanism responsible for the blunted response of sterol synthesis rates in fetal tissues to exogenous cholesterol, the ratio of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) to Insig-1 was measured in these same tissues since the ratio of SCAP to the Insigs can impact SREBP processing. The fetal tissues had anywhere from a 2- to 6-fold greater ratio of SCAP to Insig-1 than did the adult liver, suggesting constitutive processing of the SREBPs. As expected, the level of mature, nuclear SREBP-2 was not different in the fetal tissues with different levels of cholesterol whereas it was different in adult livers. These findings indicate that the suppression of sterol synthesis to exogenous sterol is blunted in developing tissues and the lack of response appears to be mediated at least partly through relative levels of Insigs and SCAP.     

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.     

Effect of amphotericin B and gestational age on sodium transport across the rat visceral yolk sac placenta in vitro

The transport properties of the rat visceral yolk sac placenta from Days 14.5 to 18.5 of gestation were studied in vitro. All tissues had a positive potential difference, fetal side relative to maternal side, and showed net Na transport towards the fetus. Basal short-circuit current and net Na flux increased rapidly with gestational age over the period studied. Amphotericin B applied to the maternal surface of the yolk sac stimulated current and net Na flux, indicating that the apical membrane Na permeability limited transport and revealing a reserve capacity for transport. Contrary to their basal values, current and Na flux following treatment with amphotericin were independent of gestational age.     

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.     

Yolk sac cholesteryl ester secretion rates can be manipulated in the Golden Syrian hamster: effect of yolk sac cholesterol concentrations

The yolk sac is one of two extra-embryonic fetal tissues that separates the fetal and maternal circulations. The yolk sac can secrete lipoprotein particles to the vitelline vessels, which supply yolk sac-derived nutrients to the embryo. The amount and composition of lipoproteins secreted from the rat yolk sac can be manipulated by fatty acid content and gestational age. The goals of the current studies were to determine, first, if tissue cholesterol concentration could mediate cholesterol secretion rate from the yolk sac and, second, if some of the secreted cholesterol could be derived from the maternal circulation. Golden Syrian hamsters were fed 2% added cholesterol to increase the yolk sac cholesterol concentration. Yolk sac explants secreted similar amounts of triglyceride and apolipoproteins B and E into the media regardless of yolk sac cholesterol concentration. In contrast, yolk sacs with greater cholesterol concentrations secreted 2.3-fold more cholesterol into the media as compared to control yolk sacs; the increase was found mostly as cholesteryl ester. At least part of the secreted cholesterol was maternally derived. These data demonstrate that yolk sac cholesterol concentration influences cholesterol secretion rates, and that at least some of the cholesterol secreted originates from the maternal circulation.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.     

ABCG1-mediated generation of extracellular cholesterol microdomains

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.     

Serine palmitoyltransferase (SPT) deficient mice absorb less cholesterol

Serine palmitoyltransferase (SPT) is the key enzyme for the biosynthesis of sphingolipids. It has been reported that oral administration of myriocin (an SPT inhibitor) decreases plasma sphingomyelin (SM) and cholesterol levels, and reduces atherosclerosis in apoE knockout (KO) mice. We studied cholesterol absorption in myriocin-treated WT or apoE KO animals and found that, after myriocin treatment, the mice absorbed significantly less cholesterol than controls, with no observable pathological changes in the small intestine. More importantly, we found that heterozygous Sptlc1 (a subunit of SPT) KO mice also absorbed significantly less cholesterol than controls. To understand the mechanism, we measured protein levels of Niemann-Pick C1-like 1 (NPC1L1), ABCG5, and ABCA1, three key factors involved in intestinal cholesterol absorption. We found that NPC1L1 and ABCA1 were decreased, whereas ABCG5 was increased in the SPT deficient small intestine. SM levels on the apical membrane were also measured and they were significantly decreased in SPT deficient mice, compared with controls. In conclusion, SPT deficiency might reduce intestinal cholesterol absorption by altering NPC1L1 and ABCG5 protein levels in the apical membranes of enterocytes through lowering apical membrane SM levels. This may be also true for ABCA1 which locates on basal membrane of enterocytes. Manipulation of SPT activity could thus provide a novel alternative treatment for dyslipidemia.