Elsevier

Life Sciences

Volume 73, Issue 11, 1 August 2003, Pages 1463-1477
Life Sciences

Impact of an enriched-cholesterol diet on enzymatic cholesterol metabolism during rabbit gestation

https://doi.org/10.1016/S0024-3205(03)00436-3Get rights and content

Abstract

An appropriate cholesterol homeostasis is vital for the maintenance and the optimal fetal development. The cholesterol is essential for the synthesis of progesterone and 17β-estradiol, hormones that actively participate to sustain gestation. However, the administration of 0.2% enriched cholesterol diet (ECD) during rabbit gestation significantly increased the cholesterol blood profile (total-cholesterol, LDL, HDL, esterified-cholesterol and free-cholesterol) of dams and offspring, and induced a reduction of the offspring weight of 15% as compared to the control group. Enzymes involved in cholesterol metabolism (ACAT, HMG-CoA-reductase and cholesterol-7α-hydroxylase) are greatly influenced by cholesterol profile. We hypothesized that the administration of an ECD during rabbit gestation modifies the activity of those enzymes. Female rabbits (pregnant or not) were fed with a standard diet or an ECD. At term, livers (dams and offspring) and placentas were collected and ACAT, HMG-CoA-reductase and cholesterol-7α-hydroxylase activities were assayed. Our results demonstrate that gestation induced a reduction of ACAT activity (48.9%) in dam's liver and, an augmentation of HMG-CoA-reductase activity (142.4%) whereas it has no effect on cholesterol-7α-hydroxylase activity. The administration of the ECD has no additive effect on ACAT, but significantly reduced the HMG-CoA-reductase activity and cholesterol-7α-hydroxylase activity as compared with the pregnant control group. In placentas the ECD supplementation has an influence for HMG-CoA-reductase activity, where a 43% increased in observed. Any ACAT activity was detected in placenta and the ECD has no influence on the cholesterol-7α-hydroxylase activity. Whereas their offspring's liver present a reduction of ACAT and HMG-CoA-reductase activity. Gestation associated with ECD reduces significantly the HMG-CoA-reductase activity, decreasing the cholesterol synthesis, but placenta seems to compensate this effect by increasing its HMG-CoA-reductase activity.

Introduction

Cholesterol plays a major role in ovarian and placental progesterone and estrogen synthesis [31]. These syntheses are vital for the maintenance of the homeostatic mechanisms implicated in pregnancy maintain [21], [25], [31]. During rabbit gestation, maternal plasmatic concentration of total cholesterol (TC), LDL and HDL decreased in an important manner compared to the values observed in non-pregnant rabbit [27], [32], [40]. Those reductions of TC and LDL are the consequence of the utilization of cholesterol for the hormonal steroid synthesis (progesterone, estrogen and cortisol) that is highly request for the pursuit of a normal gestation [26]. In pregnant Watanabe heritable hyperlipidemic rabbit or pregnant rabbit fed with an ECD (0.2%) [27], an important hypercholesterolemia was observed, throughout gestation, characterized by an augmentation of TC and LDL. This hypercholesterolemia is also observed in their offspring plasma cholesterol, where higher concentration of cholesterol is observed as compared to control offspring, moreover an important lipid accumulation was observed in their placenta [27].

The balance between free and esterified cholesterol influences affects hepatic sterol level, which then influence activities of ACAT (EC 2.3.1.26), HMG-CoA-reductase (EC 1.1.1.34) and cholesterol-7α-hydroxylase (EC 1.14.13.17) [3], [4], [5], [6], [7]. The liver's ACAT esterifies free cholesterol (FC) that was internalized by low-density lipoprotein receptors (LDLr), by a trans-esterification and accumulates it as an esterified cholesterol product in liver [33], [47]. In rats fed with a supplemented cholesterol diet, liver's ACAT activity is increase by 4-fold [34]. The administration of 4-aminopyrazolo (3,4-d) pyrimidine (4-APP), an inhibitor of lipoprotein secretion, induced a reduction of plasmatic TC and liver's ACAT activity [34]. Moreover, this treatment also significantly reduced ACAT expression in a baboon syncytiotrophoblast and the progesterone concentration [19], a vital hormone involved in the maintenance of primate pregnancy that is produced by the ovary and later by the placenta [14].

The HMG-CoA-reductase, an enzyme presents in liver [18] and placenta [6], is implicated in cholesterol synthesis by generating mevalonic acid from acetate and 3-hydroxy-3-methylglutaryl-CoA [18]. Its activity is regulated by hepatic cholesterol concentration, thus when its cholesterol concentration is increased, the hepatic HMG-CoA-reductase activity is reduced [18]. Rats presenting low plasmatic cholesterol, following a treatment with 4-APP, showed a high HMG-CoA-reductase activity [34].

Many studies also demonstrated that HMG-CoA-reductase activity could be modulated by the plasmatic concentration of 17β-estradiol [9], [11], a hormone known to increase significantly during the last days of rabbit gestation [29]. Moreover, it is well known that during fetal development, cholesterol is synthesized in liver at a relatively higher rate [44] since cholesterol is an integral part of membrane in all tissues [50]. Woollett [50] demonstrated that during the last trimester of the golden Syrian hamster gestation, offspring required more then 0.73 mg of cholesterol/ day for a normal development [50].

Björkhem et al. [5] showed the bile acids formation occurs in human liver homogenate. This synthesis begins by the conversion of cholesterol into 7α-hydroxycholesterol by the hepatic cholesterol-7α-hydroxylase [1], [28], [30], [37]. Subbiah and Hassan [45] showed in rat, that dams bile acids synthesis occurs during late gestation in dams and increases rapidly after birth. In rat and rabbit liver, the production of 7α-hydroxycholesterol acts as a down regulator of cholesterol-7α-hydroxylase, representing the limiting step in the bile acids synthesis [20], [22], [51]. Rabbit fetus is capable of bile acids synthesis as early as the 22nd day of gestation [8], and total bile acid pool is about 169.6 ± 40 mg/g at the 26th day. In newborn rabbit, the total bile acids pool nearly doubles compared to the amount noted in fetal rabbits [46].

In this study, we evaluated the effects of ECD and gestation on ACAT, HMG-CoA-reductase and cholesterol-7α-hydroxylase activities in rabbits. Moreover, we evaluated the lipid profile of plasmatic and tissue (liver and placenta). Therefore, the aim of this study was to characterize the activity of these enzymes in liver of dam and offspring, as well as in placenta, in relation to enriched cholesterol or standard diets.

Section snippets

Drugs and materials

Cholesterol was purchased from ICN (Aurora, United States), acepromazine maleate (Atravet) and ketamin chlorhydrate were purchased from Ayerst (Montréal, Canada). Xylazin was obtained from Bayer (Etobicoke, Canada) and euthanyl was bought from MTC (Cambridge, Canada). The [14C]-oleyl-CoA, [14C]-cholesterol, [14C]-HMG-CoA came from Amersham Pharmacia Biotech (Baie d'Urfée, Canada). BSC biodegradable cocktail scintillant for aqueous samples was purchased from Amersham Biosciences (Baie d'Urfée,

Enzymatic activity

For the ACAT activity, microsomes (100 μg) were added to 100 μl of phosphate buffer (0.1 M K2HPO4 (pH 7.4) and 1 mM EDTA·Na2 containing 0.22% free fatty acids albumin) containing 31 μM of cholesterol (solubilized in 0.1% tween-80). Reaction was triggered by the addition of 26 μM [14C]-oleyl-CoA (specific activity 57 nCi/nmol), incubated for 15 minutes at 37 °C and stopped by the addition of 50 μl of methanol [15].

For the HMG-CoA-reductase activity, 50 μg of microsomes were preincubated for 5

Effects of gestation and ECD on plasmatic cholesterol

Results show that administration of an ECD to non-pregnant or pregnant groups increase all lipid fractions. In non-pregnant rabbits, TC, EC and FC were increased by about 7-fold, whereas in dams these increase were less important. In dams, lipoprotein fractions (LDL and HDL) were also observed as compared with SD dams group (Table 1). Interestingly, the administration of an ECD in non-pregnant group does not modify TG concentration, but when it is associated with gestation, this lipid fraction

Discussion

In this study, rabbits fed with an ECD have extremely high plasmatic level of cholesterol. These high levels are usually found in animal lacking LDL receptor. Homozygous male and female mice lacking LDL receptors (LDLR-/- mice) were viable and fertile, but their plasma TC levels were twofold higher than those of wild-type litter-mates, owing to a seven- to nine-fold increase in intermediate density lipoproteins (IDL) and LDL without a significant change in HDL. Their plasma TG levels were also

Acknowledgements

This work was supported by grants from NSERC of Canada (Julie Lafond) and a studentship for M. Alain Montoudis from FCAR-Santé (Canada, Québec). We thank Louise Brissette PhD and Louise Martin-Falstrault for their technical support for cholesterol analysis. We also wish to thank Dr. Louiza Belkacemi and Marie-Claude Charest for helpful comments on the manuscript.

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