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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1097.)
© 2005 American Heart Association, Inc.

Editorials

ApoA-V

The Regulation of a Regulator of Plasma Triglycerides

Sven-Olof Olofsson

From the Wallenberg Laboratory, University of Göteborg, Sahlgrenska University Hospital, Göteborg, Sweden.

Correspondence to Sven-Olof Olofsson, Wallenberg Laboratory, University of Göteborg, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. E-mail Sven-Olof.Olofsson{at}wlab.gu.se

Apolipoprotein A-V was identified as an open reading frame, approximately 30 kb downstream of the A-I–C-III–A-IV gene cluster, that coded for a 343-aa-long protein with a molecular mass of 39 kDa.¹ Structural analyses have indicated that apoA-V has a multi-domain organization and is relatively hydrophobic/amphipatic.²

See page 1186

ApoA-V is involved in the regulation of plasma triglyceride levels. Thus, human apoA-V transgenic mice have lower triglyceride levels than controls, whereas the knockouts have increased levels.¹ Because hypertriglyceridemia is an independent risk factor for the development of atherosclerosis and cardiovascular diseases, elucidation of the mechanism behind the regulation of apoA-V is of great importance.

Expression of apoA-V is increased by interaction of the heterodimer PPAR (peroxisome proliferator-activator receptor)/RXR (retinoid X receptor) with a "Direct Repeat"1 (DR1) sequence in the apoA-V promoter.^3,4 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Genoux et al⁵ show that the retinoid-related orphan receptors alpha (ROR) 1 and 4 are also involved in the regulation of the apoA-V gene by interaction with the DR1 binding site. This interaction occurs with the human gene but not in mice, which is logical because the motive is not conserved in mice.

ROR has been crystallized with cholesterol in the binding site.⁶ Also, cholesterol stabilizes ROR, and depletion of cellular cholesterol by statins has been shown to modulate the transcriptional activity of ROR.⁶ For a review, see Boukhtouche et al.⁷ However, it remains to be clarified whether cholesterol is a physiological ligand for ROR.

The connection between apoA-V and ROR opens up interesting possibilities, because ROR has been shown to prevent atherosclerosis (see Boukhtouche et al⁷ for review). Thus, both the knockout mice and the natural knockout, the so-called staggerer mice, have an increased frequency of atherosclerosis (see Boukhtouche et al⁷ for review).

ROR has previously been shown to regulate the expression of apoA-I, which may be one of the reasons why the receptor prevents atherosclerosis. Moreover, it has been shown to inhibit inflammatory responses in smooth muscle cells by transcriptional regulation of the I-B gene, which codes for the inhibitor of NF-B transcription factor activity. Thus, ROR interferes with the signaling pathway of NF-B (see Boukhtouche et al⁷ for review). It should also be kept in mind that ROR appears to have an important role in lipid homeostasis in skeletal muscle.⁸ Such an effect may affect the insulin sensitivity, thereby giving rise to a reduced incidence of atherosclerosis.

Because hypertriglyceridemia is an independent risk factor for atherosclerosis, the observation that ROR regulates apoA-V⁵ may indicate that this protein must also be taken into account when explaining the antiatherogenic effect of ROR.

What is the role of apoA-V in the regulation of plasma triglyceride levels? Two mechanisms have been proposed: (1) that apoA-V influences the removal of triglycerides from plasma; and (2) that apoA-V influences the secretion of VLDL.

Degradation of plasma triglycerides is catalyzed by the enzyme lipoprotein lipase (LPL). This enzyme is mainly expressed in adipose tissue and muscle, and acts bound to the proteoglycans on the endothelial cells of the vasculature in these tissues. The enzyme is activated by apoC-II, whereas apoC-III has been shown to inhibit it (see, for example, van Dijk et al⁹ for review).

Based on results in transgenic mice it was concluded that apoA-V induced both an increase in lipolysis, through the increase in the LPL activity, and an elevated removal of VLDL particles.¹⁰ Using adenovirus-mediated gene transfer of apoA-V, it has been shown that the protein decreased the hypertriglyceridemia induced both by an intravenous and an intragastric fat load.¹¹ Thus, the protein not only influences the turnover of VLDL, but also that of chylomicrons. These studies also showed that apoA-V increased the LPL activity in vitro.¹¹ However, other authors have failed to observe an effect of apoA-V on the LPL activity¹² unless proteoglycans are present. These observations led the authors to speculate that apoA-V guides chylomicrons and VLDL to proteoglycan-bound LPL. Taken together, the results from studies in mice indicate that apoA-V can influence the hydrolysis of plasma triglycerides (for review, see also van Dijk et al⁹). There are somewhat divergent opinions about the exact mechanism, however.

Recently, apoA-V was investigated in human plasma.¹³ The results demonstrated that the apolipoprotein was associated with lipoproteins; in addition to VLDL, also with HDL and chylomicrons, but not with LDL. The distribution of apoA-V is actually very similar to that of apoC-III, and it has been suggested that these two proteins influence the triglyceride degradation in opposite directions: apoA-V stimulating the LPL and apoC-III inhibiting it. Interestingly, there have been recent results indicating that apoC-III expression is increased by ROR.¹⁴ Thus, there may be a very intricate balance between apoA-V and apoC-III.

The level of apoA-V in plasma is low (mean 157 µg/L). It is clear from these results that there is insufficient apoA-V for it to be present on all VLDL particles; rather, only one of 1000 VLDL particles could contain an apoA-V molecule. This raises the question of whether apoA-V could really have any influence on the turnover of the VLDL pool. In addition, there is an unexpected weak relationship between plasma levels of apoA-V and triglycerides. However, genetic analysis of polymorphisms in the human apoA-V gene demonstrated a strong relationship between the genetics of apoA-V and plasma triglyceride concentration.¹⁵ Thus, the presence of the rare allele was associated with an increase in plasma triglyceride concentration (by 21 mg/dL). Could this mean that apoA-V has its effect during the assembly of VLDL rather than after its secretion? The expression pattern (ie, only in the liver) would certainly argue in favor of this (see van Dijk et al⁹for review).

The assembly of VLDL is a complex process (Figure; for review, see Olofsson et al^16,17) which starts in the rough endoplasmic reticulum (rough ER) during the biosynthesis of apoB-100. The protein is lipidized by MTP (the microsomal triglyceride protein) during its biosynthesis and simultaneous translocation to the lumen of the rough ER, forming a primordial particle (a pre-VLDL). Pre-VLDL is retained in the cell, partly by its interaction with chaperones (BiP and PDI in particular).¹⁸ The pre-VLDL particle cannot be secreted unless more neutral lipids (triglycerides and cholesterol esters) are added. This addition of lipids occurs in two ways. First, a size-dependent lipidation converts pre-VLDL to VLDL 2 (ie, the triglyceride-poor VLDL).¹⁸ Second, VLDL 2 can receive a bulk load of lipids, forming VLDL 1 (the triglyceride-rich VLDL).¹⁸ The prerequisite for this bulk lipidation is that apoB should have a size of at least apoB-48¹⁸ and that the protein is translocated out of the rough ER. This transfer involves an ARF1 (ADP ribosylation factor 1)/COP I (coatomere I)-dependent sorting process from the ERGIC (ER-Golgi intermediate compartment) to the cis-Golgi.¹⁹ This sorting process explains the ARF 1 dependence of VLDL assembly¹⁷

View larger version (33K): [in this window] [in a new window]   The assembly of VLDL and possible levels in the process where apoA-V could influence the secretion of VLDL. For details, see text. Arrow heads point at possible site where apoA-V could influence the assembly process. Thus it could influence the processing of pre-VLDL to VLDL 2 or VLDL 2 to VLDL 1. ApoA-V could also influence the triglyceride pool used for secretion.

Is apoA-V involved somewhere in this process? The available results are mainly based on the secretion of VLDL, usually in triton experiments (triton inhibits LPL).^10–12 Thus, they cannot pinpoint the exact role of apoA-V in the process. Moreover, these results are contradictory: some argue for an influence of apoA-V on the secretion of VLDL, and some argue against this.

One attempt has been made to investigate the intracellular role of apoA-V. Weinberg et al² transfected COS 1 cells with apoA-V and compared its intracellular distribution and secretion to that of albumin and a truncated form of apoB (apoB6.F6). The results demonstrated that although apoB6.F6 and albumin were secreted, apoA-V remained mainly in the cell. Both albumin and apoB6.F6 were translocated to the Golgi, but apoA-V remained in the ER. This may indicate that the main function of the apoA-V protein resides in the ER, and the authors proposed that the protein is involved in modifying the lipidation of apoB-100. An intracellular role for apoA-V would explain its large effect on plasma triglyceride levels despite its low abundance in plasma.

The lipidation of apoB is dependent on the release of fatty acids from triglycerides stored in cytosolic lipid droplets. These fatty acids are then re-esterified into triglycerides that are incorporated into VLDL.²⁰ Indeed there must be separate pools of triglyceride for storage and for secretion. Relatively little is known about the nature and regulation of the triglyceride pool used for secretion. Such a pool is most probably present in the membrane of the microsomes, and it has even been suggested that the lipids bud as lipid droplets into the lumen of the secretory pathway. It has also been suggested that VLDL is formed by the fusion of pre-VLDL with such droplets.^17,20 There are several ways by which this lipidation process could be influenced by amphipathic/hydrophobic proteins such as apoA-V. It is possible that apoA-V can interact with both the amphipathic monolayer of a membrane and the surface of a lipid droplet to modify the structure and availability of this pool for lipoprotein assembly. It is also possible that apoA-V can interact with pre-VLDL or VLDL 2 and can prevent fusion with the lipid droplets, thus reducing the formation of VLDL 1 (Figure). Much work obviously remains to be done for us to understand whether (and how) apoA-V might influence the assembly of VLDL.

PPAR regulates apoA-V through a DR1 site in the gene.^3,4 PPAR is also involved in regulation of the secretion of apoB-100.²¹ Thus, agonists to PPAR increase the production of apoB-100 by preventing its degradation. At the same time, these agonists reduce triglyceride biosynthesis. Thus, more apoB-100 competes for less triglyceride. This leads to a shift from the assembly of VLDL 1 to assembly of VLDL 2.²¹ The mechanism behind the increased production of apoB-100 was found to be increased expression of MTP caused by the interaction between PPAR/RXR and a DR1 site in the MTP promoter.²² This effect of the PPAR agonists on MTP and triglyceride biosynthesis, together with their effect on apoA-V and its potential role as a modulator of apoB-100 lipidation, may be the long-sought mechanism for the lipid lowering fibrates. Thus, PPAR appears to be an important regulator of VLDL assembly. Could the results presented by Genoux et al⁵ in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology indicate that ROR has the same important influence on the assembly process? If so, could this be a new target for treatment against the production of atherogenic lipoproteins?

	References

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