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J Clin Invest. 1976 Aug;58(2):368-79.

Synthesis of very low density lipoproteins in the cockerel. Effects of estrogen.

Abstract

The effect of estrogen on the synthesis of plasma very low density lipoproteins (VLDL) in the cockerel was studied both in vivo and in vitro. Synthesis was studied by immunoprecipitation techniques with antisera prepared against VLDL and a major VLDL protein. VLDL were isolated from the plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels by ultracentrifugal flotation at d 1.006 g/ml. After delipidation, the lipid-free proteins (apoproteins) were fractionated on Sephadex G-150 and DEAE-cellulose. Both the hen and the estrogen-treated cockerel VLDL were shown to contain an identical apoprotein with a mol wt of approximately 12,000; the apoprotein is designated fraction B. Reduction and S-carboxy-methylation of fraction B resulted in a reduction of the molecular weight by approximately one-half, indicating a dimer-monomer relationship. Antiserum prepared to the hen VLDL dimer protein gave precipitin lines of complete identity to both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density lipoproteins, and plasma; no precipitin line was formed with either hen or cockerel high density lipoproteins. After a single subcutaneous injection of diethylstilbestrol into the cockerel, plasma VLDL protein, cholesterol, and triglyceride increased, reaching a maximum 24--48 h after hormone administration. Liver slices from similarly treated animals were incubated in vitro in culture medium in the presence of [3H]lysine for 2 h. Immunoprecipitable radioactivity in VLDL increased within 2 h of diethylstilbestrol treatment and reached a maximum at 24 h; VLDL radioactivity returned to base-line levels by 72 h. At the peak of induction, newly synthesized VLDL represented 11% of the total soluble protein synthesized. When actinomycin-D (5 mg/kg) was administered simultaneously with estrogen, the induction of VLDL synthesis was totally inhibited. To determine whether the effect of estrogen on VLDL synthesis was mediated at the level of transcription, partially-purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA activity for fraction B was quantitated in a wheat germ translation system. Fraction B mRNA was found to increase from a low base-line value to a maximum 16-24 h after estrogen treatment, returning towards baseline values at 30 h. At the peak of induction, fraction B constituted 12% of the total protein synthesized. The kinetics of induction of fraction B mRNA activity in the cell-free translation system is very similar to that observed in liver slice experiments. This finding suggests that estrogen stimulates VLDL synthesis, at least partially, by enhancing the accumulation of the mRNA for one of their major apoproteins.

PIP:

In vivo and in vitro studies were undertaken to investigate the mechanism of the induction of the synthesis of very low density lipoproteins (VLDL) by estrogens in the cockerel. VLCL were isolated from plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels. VLCL from both these groups contained an identical apoprotein (Fraction B) with a molecular weight of about 12,000. Reduction and S-carboxy-methylation of this fraction reduced its molecular weight by approximately 50%, thus indicating a dimer-monomer relationship. When antiserum was prepared against the hen VLDL dimer protein, completely identical precipitin lines were found for both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density proteins, and plasma. However, no precipitin line was formed with hen and cockerel high density lipoptoteins. A single sc injection of diethylstilbestrol (DES) into the cockerel increased levels of plasma VLDL protein, cholesterol, and triglyceride, with maximum values occurring 24-48 hours after injection. Immunoprecipitation of liver slices from similarly treated animals showed an increase of radioactivity of VLDL within 2 hours of injection. Values reached a maximum at 24 hours and returned to baseline levles by 72 hours. Newly synthesized VLDL comprised 11% of the total soluble protein synthesized during the period of peak values. Actinomycin-D (5 mg/kg), when administered simultaneously with the estrogen, completely inhibited the induction of VLDL synthesis. In another experiment, partially purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA acitivity for Fraction B was measured in a wheat germ translation system. Values for Fraction B mRNA reached a maximum 16-24 hours after estrogen-treatment and returned to baseline levels by 30 hours. Fraction B represented 12% of the total protein synthesized at the peak of induction. The results suggest that estrogen stimulates the synthesis of VLDL by enhancing the accumulationg of the mRNA of 1 of their major components.

PMID:
182719
PMCID:
PMC333192
DOI:
10.1172/JCI108481
[Indexed for MEDLINE]
Free PMC Article

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