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. 2000 Feb;105(4):521-32.
doi: 10.1172/JCI8623.

The role of the LDL receptor in apolipoprotein B secretion

J Twisk  1 , D L Gillian-Daniel, A Tebon, L Wang, P H Barrett, A D Attie
Affiliations

The role of the LDL receptor in apolipoprotein B secretion

J Twisk et al. J Clin Invest. 2000 Feb.

Abstract

Familial hypercholesterolemia is caused by mutations in the LDL receptor gene (Ldlr). Elevated plasma LDL levels result from slower LDL catabolism and a paradoxical lipoprotein overproduction. We explored the relationship between the presence of the LDL receptor and lipoprotein secretion in hepatocytes from both wild-type and LDL receptor-deficient mice. Ldlr(-/-) hepatocytes secreted apoB100 at a 3.5-fold higher rate than did wild-type hepatocytes. ApoB mRNA abundance, initial apoB synthetic rate, and abundance of the microsomal triglyceride transfer protein 97-kDa subunit did not differ between wild-type and Ldlr(-/-) cells. Pulse-chase analysis and multicompartmental modeling revealed that in wild-type hepatocytes, approximately 55% of newly synthesized apoB100 was degraded. However, in Ldlr(-/-) cells, less than 20% of apoB was degraded. In wild-type hepatocytes, approximately equal amounts of LDL receptor-dependent apoB100 degradation occured via reuptake and presecretory mechanisms. Adenovirus-mediated overexpression of the LDL receptor in Ldlr(-/-) cells resulted in degradation of approximately 90% of newly synthesized apoB100. These studies show that the LDL receptor alters the proportion of apoB that escapes co- or post-translational presecretory degradation and mediates the reuptake of newly secreted apoB-containing lipoprotein particles.

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Figures

Figure 1
Figure 1
Multicompartmental model describing secretion and intracellular degradation of apoB. Compartments 1–8, 10, and 12 represent intracellular apoB, whereas compartment 9 represents apoB secreted into the media. Compartment 1 represents the radioactive amino acid tracer pool. Compartments 2–7 represent incorporation of tracer into progressively longer fractional lengths of nascent apoB during translation; compartment 7 represents the first appearance of a pool of full-length protein. ApoB can be lost from compartment 7 via a rapid presecretory degradation pathway or can pass on to compartment 8 or 10. Loss of apoB from compartment 10 occurs through a slow presecretory degradation pathway. Compartment 8 represents the delay in apoB appearance in the media; compartment 12 represents the experimentally observed extended delay in apoB100 appearance in the media relative to apoB48. Loss of apoB from compartment 8 occurs via a degradation pathway that is inhibited by heparin addition. Compartment 9 represents apoB in the media. Experimentally determined apoB radioactivity is indicated by the hatched compartments 7–10.
Figure 2
Figure 2
Determination of apoB secretion rate in the presence and absence of a functional LDL receptor. Hepatocytes from wild-type and Ldlr–/– mice were radiolabeled continuously with [35S]methionine/cysteine, and samples were collected at the times indicated. Cells were lysed, and both media and cell lysates immunoprecipitated with antiserum raised against either apoB or albumin. Samples were separated on 5% or 8% SDS-polyacrylamide gels, respectively, and apoB and albumin radioactivity quantitated. Results are mean ± SEM of duplicate dishes from 5 independent isolations for apoB and 3 independent isolations for albumin. Open circle, intracellular protein; filled circle, secreted protein.
Figure 3
Figure 3
Determination of steady-state levels of apoB mRNA and apoB initial synthesis rates in wild-type and Ldlr–/– hepatocytes. (a) Steady-state levels of apoB mRNA. ApoB mRNA abundance was measured by slot blot. The results are expressed as a ratio of apoB/GAPDH (mean ± SEM; 2 independent isolations, up to 8 samples per isolation). (b) Initial synthesis rates for apoB100, apoB48, and albumin. Samples were treated as described in Figure 2, except that cells were labeled with [35S]methionine/cysteine for 5, 15, and 30 minutes. Results shown are a mean ± SEM of duplicate dishes from 4 independent hepatocyte isolations for apoB, and 3 independent isolations for albumin. Filled bars, wild-type; open bars, Ldlr–/–.
Figure 4
Figure 4
(a) Pulse-chase analysis of apoB. Cells were metabolically labeled for 7.5 minutes with [35S]methionine/cysteine. After a wash, cells were incubated in chase medium supplemented with unlabeled methionine and cysteine for the indicated times, in the presence or absence of heparin (10 mg/mL). Samples were treated as described in Figure 2. The results presented represent the multicompartmental modeling fit to the data (mean data ± SEM) from pulse-chase experiments. Data points represent the observed mean data, and the lines are the best fit to the data generated by the kinetic model. n = 6 independent hepatocyte isolations, duplicate samples, for wild-type; 5 isolations for wild-type with heparin; 3 isolations for Ldlr–/–; and a single isolation for Ldlr–/– with heparin (repeated twice). Open circle, intracellular protein; filled circle, secreted protein. (b) Pulse-chase analysis of albumin. Samples in Figure 4a were split at time of collection, and albumin was immunoprecipitated as described in Figure 2. The results presented are from 6 independent hepatocyte isolations, duplicate samples, for wild-type; 5 isolations for wild-type with heparin; 4 isolations for Ldlr–/–; and 2 isolations for Ldlr–/– with heparin. Open circle, intracellular protein; filled circle, secreted protein.
Figure 5
Figure 5
Determination of apoB and albumin secretion after adenovirus-mediated overexpression of the human LDL receptor. Wild-type and Ldlr–/– hepatocytes were infected with adenovirus containing either the LDL receptor gene, or the gene encoding β-galactosidase (Adβ-gal), both under control of the CMV promoter. After infection, cells were incubated a further 36–48 hours. Cells were radiolabeled for 7.5 minutes with [35S]methionine/cysteine, washed once, and chased for the indicated times. The data shown at each time point represent 40% and 50% of total intracellular and secreted apoB, respectively; 10% of total intracellular and 2% of total secreted albumin are also shown. Results with Ldlr–/– cells are representative of results obtained with adenoviral infection of wild-type hepatocytes (n = 4 independent isolations).
Figure 6
Figure 6
Determination of intracellular apoB and albumin levels after adenovirus-mediated overexpression of the LDL receptor. Adenovirus infection was as described in the legend to Figure 5. Ldlr–/– hepatocytes were radiolabeled for 7.5 minutes with [35S]methionine/cysteine in the presence of heparin (6 mg/mL), washed once, and chased for the indicated times. Chase times are relative to the addition of radiolabel. Results are the mean of duplicate samples from a single hepatocyte isolation and are representative of results obtained with wild-type cells. Error bars represent the variance between duplicate samples; error bars that are not visible are smaller than the symbol. Open circle, intracellular; filled circle, intracellular + Ad LDLR. There was no detectable apoB secretion by 22.5 minutes.
Figure 7
Figure 7
Model of apoB secretion and degradation. Amino acid incorporation into increasingly longer apoB nascent chains is depicted. As apoB transits the secretory pathway, presecretory degradation occurs via both LDL receptor–dependent and –independent means that are either rapid or slow. The nascent lipoprotein particle ultimately reaches the cell surface, at which point the LDL receptor can mediate its reuptake; this results in internalization and subsequent turnover of apoB. This final degradation pathway is inhibited by addition of heparin.
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