Phospholipid Transfer Protein Deficiency Impairs Apolipoprotein-B Secretion from Hepatocytes by Stimulating a Proteolytic Pathway through a Relative Deficiency of Vitamin E and an Increase in Intracellular Oxidants

Abstract

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ERto-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wildtype cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.The plasma phospholipid transfer protein (PLTP) 1 is a key participant in the transport of hydrophobic molecules within the circulation. Partially purified PLTP was originally shown in vitro to mediate the transfer and exchange of phospholipids between plasma lipoproteins (1-3). Purified PLTP in vitro also transfers ␣-tocopherol (vitamin E), a naturally occurring hydrophobic anti-oxidant (4). To examine PLTP in vivo, we engineered genetically deficient mice and found that the loss of PLTP lowered plasma high density lipoprotein levels and altered plasma ␣-tocopherol transport, consistent with the previous work in vitro (5). Unexpectedly, however, we also found that PLTP deficiency caused a large impairment in the hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins (6). The net effect of these changes was a decreased susceptibility to atherosclerosis (6). Likewise, it was reported recently that animals overexpressing PLTP exhibit hepatic very low density lipoprotein (VLDL) overproduction (7) and increased athero-susceptibility (8). Associations of plasma PLTP activity with elevated apoB levels (9) and increased cardiovascular risk (10) have been found in humans as well. These findings have led to an interest in PLTP as a potential therapeutic target. Nevertheless, the...

Presecretory oxidation, aggregation, and autophagic destruction of apoprotein-B: A pathway for late-stage quality control

“…The presence of detectable amounts of intracellular apoB-aggregates, even without PUFA supplementation (Figs. 1, 2, and 5), extends our earlier demonstrations of ROS-dependent control of apoB secretion in the basal state, indicating broad physiologic importance (9,10). Thus, the PERPP pathway appears to be not only a major regulator of hepatic apoB secretion but also a means for remarkably late-stage quality control of an endogenous protein after it has left the ER.…”

“…4C) but not in cellular homogenates, we hypothesized a role for intracellular trafficking in LA or IA formation. To test this idea, we blocked apoB exit from the ER, using a combination of brefeldin A and nocodazole (BFA/Noc) (21), which we showed to inhibit PERPP (10). Importantly, BFA/Noc only moderately decreased the intracellular content of lipid peroxides in DHA-treated cells (DHA, 81.4 Ϯ 4.3; DHA plus BFA/Noc, 53.3 Ϯ 4.4 pmol of MDA equivalents per milligram of cell protein; P Ͻ 0.01), but nearly completely blocked the formation of LAs and IAs (Fig.…”

“…Three processes are known (1,7,8): (i) endoplasmic reticulum association degradation (ERAD) mediated by the proteasome and stimulated by severe scarcity of lipids for transfer to apoB during its translocation across the ER (1); (ii) post-ER presecretory proteolysis (PERPP) (8), a nonproteasomal pathway provoked by diverse metabolic factors in vitro and in vivo, including intracellular lipid peroxidation (9 -14); and (iii) receptormediated degradation, also known as reuptake, which occurs via interactions of nascent apoB-particles with LDL receptors (8,10,(15)(16)(17)(18) and heparan sulfate proteoglycans (17,19).…”

“…Both conventional ⍀-6 PUFAs and marine (fish oil) ⍀-3 PUFAs, such as docosahexaenoic acid (DHA), have this effect. Antioxidants inhibit apoB degradation in PUFA-treated cells, and thereby enhance the secretion of atherogenic apoB-lipoproteins in vitro and in vivo (9,10). In the current study, we sought to identify the novel molecular processes by which PUFA-derived lipid peroxides trigger PERPP of newly synthesized apoB.…”

Atheroprotective Potential of Macrophage-Derived Phospholipid Transfer Protein in Low-Density Lipoprotein Receptor-Deficient Mice Is Overcome by Apolipoprotein AI Overexpression

“…Because PLTP plays a major role in the transfer of ␣-tocopherol in vivo, 21,23,24 a second hypothesis is that macrophage-derived PLTP may beneficially alter the distribution of ␣-tocopherol, the main isoform of vitamin E, between lipoproteins and cells of the vascular wall. To test this, we examined whether PLTP deficiency altered vitamin E content in macrophages.…”