Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

https://doi.org/10.1016/S1388-1981(98)00004-3Get rights and content

Abstract

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.

Introduction

LDL is a cholesterol-rich lipoprotein produced in vivo via very-low-density lipoprotein (VLDL) delipidation cascade [1], [2]. Apolipoprotein (apo) B-100, the protein moiety of low-density lipoprotein (LDL), plays an important role in the intracellular assembly of the precursor VLDL [3], [4]. In contrast with the other apolipoproteins, which exchange intensively among lipoprotein particles, apo B-100 remains part of the lipoprotein structure during the catabolic process, until the particles are removed from the bloodstream [2]. In this last step, apo B-100 is the ligand for the cellular recognition and uptake of LDL by the specific LDL receptor (LDLR) on the cell plasma membrane [3], [5].

In rats, apo B-100 is synthesized predominantly by the liver, whereas apo B-48, a shorter isoform, is mainly produced by the intestine [3]. The latter actually results from an editing mechanism of the apo B-100 mRNA [6], [7]. Apo B-100 is a large molecule with polar and apolar domains allowing both the association of the protein with lipids in LDL particle, as well as the interaction of the lipoprotein with LDLR [8], [9]. Previous investigations have used protein-free microemulsions resembling LDL, designated LDE, to study LDL metabolism [10], [11]. It has been shown that LDE can be removed from circulation through interaction of the apo E, adsorbed by LDE particles, with LDLR [11], [12].

In this study, we have evaluated the influence of apo B-100 on LDL metabolism, by modeling the LDL lipid structure with LDE. LDE labeled with radioactive lipid analogues and associated with apo B-100 (LDE-apoB) was injected into the bloodstream of control rats and rats pre-treated with 17α-ethinylestradiol. Administration of this hormone is known to increase LDLR activity [13]. Following injection, plasma kinetics and organ uptake of the LDE-apoB were determined and compared with the metabolic parameters of the LDE and native LDL.

Section snippets

Animals and treatments

Male Wistar rats, weighing 250–350 g, were fed on a standard commercial chow, with free access to food and water. One group of rats was injected subcutaneously a daily dose of 17α-ethinylestradiol (5 mg/kg body wt.) for 5 days before the experiment [13]. Control rats received 0.5 ml of propyleneglycol in equivalent periods.

Preparation of LDE

LDE was prepared from lipid mixture composed of 33% cholesteryl oleate (Nu Check Prep, Elysian, USA), 66% egg phosphatidylcholine (Lipid Products, Surrey, UK) and 1% glyceryl

Results

The chemical analysis of the microemulsions showed that lipid composition of LDE-apoB was similar to that of LDE (Table 1). Both LDE and LDE-apoB microemulsions and native LDL presented compositional data comparable to that previously reported [10], [11], [12], [17], [29], [30]. Cholesteryl ester (CE) label of both LDE and LDE-apoB presented a similar distribution profile on density gradient ultracentrifugation. About 80% of particles floated between 1.020 g/ml and 1.065 g/ml (Fig. 1). Protein

Discussion

The lipid structure of LDL can be artificially modeled by very well defined procedures [10], [11], [31], [32]. These protein-free microemulsion models mimic the lipid physical behavior of native LDL. In previous studies, we had demonstrated that the metabolism of LDE had some similarities with the metabolism of endogenous LDL in rats [11] and in human subjects [12]. We have hypothesized that in the plasma compartment, LDE is capable of assimilating apolipoproteins from the plasma lipoproteins,

Acknowledgements

The authors are grateful to Ricardo Omoto, Edson L. Silva and Edilberto P. Oliveira for help with the experiments, and Prof. Sonia Q. Doi (USUHS, USA) for revising the manuscript. This study was supported by grants from FAPESP, São Paulo, Brazil. M.H.H. and R.C.M. are recipients of award research scholarships from CNPq, Brasilia, Brazil.

References (51)

  • G.R. Bartlett

    Phosphorus assay in column chromatography

    J. Biol. Chem.

    (1959)
  • M.A. Markwell et al.

    A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples

    Anal. Biochem.

    (1978)
  • D.M. Neville

    Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system

    J. Biol. Chem.

    (1971)
  • M.T. Walsh et al.

    Reassembly of low-density lipoproteins

    Methods Enzymol.

    (1986)
  • D.P. Via et al.

    Cholesteryl ester-rich microemulsions: stable protein-free analogs of low density lipoproteins

    J. Lipid Res.

    (1982)
  • R.E. Reisinger et al.

    Phospholipid/cholesteryl ester microemulsions containing unesterified cholesterol: model systems for low density lipoproteins

    J. Lipid Res.

    (1990)
  • T.G. Redgrave et al.

    Metabolism of protein-free lipid emulsion models of chylomicrons in rats

    Biochim. Biophys. Acta

    (1985)
  • T.G. Redgrave et al.

    Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

    Biochim. Biophys. Acta

    (1987)
  • B. Lundberg et al.

    Preparation of biologically active analogs of serum low-density lipoprotein

    J. Lipid Res.

    (1984)
  • R.E. Pitas et al.

    Cell surface receptor binding of phospholipid-protein complexes containing different ratios of receptor-active and -inactive E apoprotein

    J. Biol. Chem.

    (1980)
  • P.C.N. Rensen et al.

    Particle size determines the specificity of apolipoprotein E-containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo

    J. Lipid Res.

    (1997)
  • E. Sehayek et al.

    Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway

    J. Biol. Chem.

    (1991)
  • M. Bertolotti et al.

    Effect of hypocholesterolemic doses of 17α-ethinyl estradiol on cholesterol balance in liver and extrahepatic tissues

    J. Lipid Res.

    (1996)
  • K. Hayashi et al.

    Metabolic changes in lipids of rat plasma and hepatocytes induced by 17α-ethinyl estradiol treatment

    Biochim. Biophys. Acta

    (1986)
  • P.L. Colvin

    Estrogen increases low-density lipoprotein receptor-independent catabolism of apolipoprotein B in hyperlipidemic rabbits

    Metabolism

    (1996)
  • Cited by (0)

    View full text