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.     

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.     

Differential effects of polyunsaturated fatty acids on sterol synthesis rates in adult and fetal tissues of the hamster: consequence of altered sterol balance

Cholesterol is necessary for the proper growth and development of the fetus. Consequently, disruptions in cholesterol biosynthesis lead to abnormal fetal development. It has been shown that in cells exposed to polyunsaturated fatty acids (PUFA), the expressions of genes and activities of enzymes involved in cholesterol synthesis are reduced. Similarly, we found that adult male hamsters fed PUFA-enriched diets had an approximately 60% reduction in in vivo hepatic sterol synthesis rates. If fetal tissues respond to PUFA in the same manner as do adult livers, then maternal dietary PUFA could lead to a reduction in fetal sterol synthesis rates and possibly abnormal development. To investigate the impact of maternal dietary fatty acids on fetal sterol synthesis rates, female hamsters were fed diets enriched in various fatty acids before and throughout gestation. In vivo sterol synthesis rates were measured in fetuses at mid- and late gestation. At both gestational stages, dietary PUFA had no effect on fetal sterol synthesis rates. This lack of effect was not a consequence of a lack of PUFA enrichment in fetal fatty acids or the lack of PUFA receptor expression in the fetus. We hypothesize that the fetus may experience a dysregulation of sterol synthesis as the result of the fetus being in a negative sterol balance; the PUFA-induced suppression of sterol synthesis in the adult male hamster liver was ablated by creating a net negative sterol balance across the adult hepatocyte.     

Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain

Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.     

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.     

Transport of maternal cholesterol to the fetus is affected by maternal plasma cholesterol concentrations in the Golden Syrian hamster

The fetus has a high requirement for cholesterol and synthesizes cholesterol at elevated rates. Recent studies suggest that fetal cholesterol also can be obtained from exogenous sources. The purpose of the current study was to examine the transport of maternal cholesterol to the fetus and determine the mechanism responsible for any cholesterol-driven changes in transport. Studies were completed in pregnant hamsters with normal and elevated plasma cholesterol concentrations. Cholesterol feeding resulted in a 3.1-fold increase in the amount of LDL-cholesterol taken up by the fetus and a 2.4-fold increase in the amount of HDL-cholesterol taken up. LDL-cholesterol was transported to the fetus primarily by the placenta, and HDL-cholesterol was transported by the yolk sac and placenta. Several proteins associated with sterol transport and efflux, including those induced by activated liver X receptor, were expressed in hamster and human placentas: NPC1, NPC1L1, ABCA2, SCP-x, and ABCG1, but not ABCG8. NPC1L1 was the only protein increased in hypercholesterolemic placentas. Thus, increasing maternal lipoprotein-cholesterol concentrations can enhance transport of maternal cholesterol to the fetus, leading to 1) increased movement of cholesterol down a concentration gradient in the placenta, 2) increased lipoprotein secretion from the yolk sac (shown previously), and possibly 3) increased placental NPC1L1 expression.     

The liver plays a key role in whole body sterol accretion of the neonatal Golden Syrian hamster

Neonates have a significant requirement for cholesterol. From -1 to 25 days of age, the liver accrues 6.9 mg cholesterol and the extra-hepatic tissues accrue 107.7 mg cholesterol in the hamster. It is currently unknown if each of these body compartments synthesizes their own cholesterol or if they have alternative source(s) of sterol. Using (3)H(2)O, in vivo hepatic sterol synthesis rates (per g liver per animal) increased between -1 and 5 days of age, decreased by 10 days of age, and increased again by 15 days of age. HMG-CoA reductase (HMGR) expression levels paralleled in vivo synthesis rates. Extra-hepatic sterol synthesis rates followed the same pattern as sterol synthesis rates in the liver. When sterol synthesis rates were converted to the mass of sterol synthesized per day, the liver synthesized 38.9 and the extra-hepatic tissues synthesized 63.9 mg cholesterol in the 26-day neonatal period. Comparing the amount of cholesterol accrued to that synthesized, one can conclude that the liver is a major source of sterol for the whole body during the neonatal period of the hamster. These results may help elucidate the cause(s) of reduced growth rates in neonates with liver disease or in neonates with compromised sterol synthesis rates.     

Adult sterol metabolism is not affected by a positive sterol balance in the neonatal Golden Syrian hamster

Dietary components impact metabolism early in life. Some of the diet-induced effects are long lasting and can lead to various adult-based diseases. In the current studies, we examined the short-term effects of dietary cholesterol on neonatal hepatic sterol metabolism and the long-term effects that those early-life diets had on sterol metabolism in adulthood. Neonatal hamsters began consuming solid food as a supplement to milk by 5 days of age; diets contained 0 or 2% added cholesterol (wt/wt). By 10 days of age, plasma and liver cholesterol concentrations were 3.2- and 2.5-fold greater, respectively, in the neonates fed cholesterol. Hepatic sterol synthesis rates were suppressed 65% in cholesterol-fed neonates compared with control neonates. By 20 days of age, plasma and liver cholesterol concentrations were still greater and sterol synthesis rates were now suppressed maximally in neonates fed cholesterol compared with control neonates. The expression level of an apolipoprotein B-containing lipoprotein receptor (low-density lipoprotein receptor-related protein) was greater and the mature form of the sterol regulatory element-binding protein-2 was similar in livers of 20-day-old control neonates compared with control neonates at 10 days of age. To test whether the change in sterol balance in the neonatal period had a lasting effect on hepatic sterol metabolism, all animals were weaned on a low-cholesterol diet. At 70 days of age, hepatic sterol synthesis rates, plasma lipoprotein and liver cholesterol concentrations, and bile acid pool sizes and compositions were measured. Sterol balance in the adults was similar between animals fed either diet early in life, as demonstrated by a lack of difference in any parameter measured. Thus, even though dietary cholesterol suppressed hepatic sterol synthesis rates dramatically in the neonatal hamster, the change has little impact on sterol balance later in life.