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Role of ApoCs in Lipoprotein Metabolism

Functional Differences Between ApoC1, ApoC2, and ApoC3
Originally published1 Mar 1999https://doi.org/10.1161/01.ATV.19.3.472Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:472–484

    The human apoCs (ie, apoC1, apoC2, and apoC3) are often portrayed as members of 1 consistent protein family because of their similar distributions among lipoprotein classes, their low molecular weights, and coincident purification. The human apoCs are protein constituents of chylomicrons, VLDL, and HDL. In comparison with the intensely studied apoE, apoB, and apoA1, which play important roles in the development of hyperlipidemia and atherosclerosis, only modest attention has been paid so far to the roles of the apoCs in lipoprotein metabolism. Many of the studies regarding the functional properties of apoCs have been hampered by methodological problems dealing with purification, quantification, and their poorly understood association with hyperlipidemia and other lipoprotein disorders. In the past few years, however, new insights into the metabolic properties of apoCs have been provided, in particular by the technologies of transgenesis and gene targeting in mice.

    The present review addresses the influence of apoCs on the major metabolic pathways in lipoprotein metabolism. Therefore, a number of important in vitro and in vivo studies will be discussed that point to a distinct role for each of the individual apoCs in lipoprotein metabolism and human disease.

    APOC Genes

    The genes coding for human apoC1 and human apoC2 are members of a 48-kb gene cluster on chromosome 19 that also includes the APOE and pseudo-APOC1′ genes.12345 It has been reported that the human APOC1 gene is located either 4.323 or 5.34 kb downstream from the APOE gene in the same transcriptional orientation. The APOC1 gene is ≈4.7 kb and is primarily expressed in the liver, but lower amounts are also found to be expressed in the lung, skin, testes, and spleen (Table 1).4 One copy of the APOC1 gene, the so-called pseudo-APOC1′ gene, is located 7.5 kb downstream from APOC1.14 No mRNA products of the pseudo-APOC1′ gene have been detected in any tissue.4APOC2 spans a region of 3.4 kb and is primarily expressed in the liver and intestine678 (Table 1). An additional gene within the APOE/C1/C2 gene cluster, designated the APOC2-linked gene, was first discovered in mice.9 Recently, a similar gene was found in humans.10 On the basis of its properties and location (555 bp upstream from APOC2), this 3.3-kb gene was designated APOC4. RNase protection analysis indicated relatively low APOC4 mRNA levels in the human liver.10

    The regulation of human APOC1 gene expression, together with that of the APOE gene, is under control of an array of elements found throughout the whole APOE/C1/C2/C4 gene cluster (for a review, see References 11 and 121112 ). The hepatic control region (HCR), an element located ≈17 kb downstream from the APOE gene and ≈9 kb downstream from the APOC1 gene, was found to regulate the expression of both APOC1 and APOE genes in the liver.1314 A second hepatic controlling element within the APOE/C1/C2 cluster was identified 27 kb downstream from the APOE gene.15 Recently, it was shown that both HCRs can individually coordinate the hepatic expression of all 4 genes in the APOE/C1/C2/C4 gene cluster and that the presence of at least 1 of the regions is sufficient for significant liver expression of each of the genes.16

    The human APOC3 gene is located in a gene cluster together with the APOA1 and APOA4 genes17 on the long arm of chromosome 11 and is ≈3.1 kb (Table 1).1819202122 The human APOC3 gene is expressed in the liver and intestine and is controlled by positive and negative regulatory elements that are spread throughout the gene cluster.2324252627 Experiments with transgenic animals have allowed the localization of an element controlling the intestinal expression of APOC3, APOA1, and APOA4 in the proximal 5′ human APOC3 region.2829

    Molecular Defects in Human APOC Genes and Their Association With Lipoprotein Disorders

    Little is known about naturally occurring mutations in the human APOC1 gene. So far, only 1 study has reported a case of apoC1 deficiency in patients with familial chylomicronemia30 (Table 2). Because these patients suffered from apoC2 deficiency as well, the chylomicronemia is most likely caused by the apoC2 defect. Remarkably, however, the apoC1/apoC2-deficient patient exhibited markedly decreased levels of cholesterol ester, especially apparent in HDL, which was much more severe than previously reported in cases of apoC2 deficiency.30 These observations suggest that apoC1 deficiency in HDL may modulate lecithin-cholesterol acyltransferase (LCAT) activity, which is known to catalyze the esterification of free cholesterol in plasma.31

    The importance of apoC2 as an activator of lipoprotein lipase (LPL) has unequivocally been demonstrated in patients with genetic defects in the structure or production of apoC2, all of whom display high circulating levels of triglycerides (TGs) and are phenotypically indistinguishable from patients with LPL deficiency.3233343536 As summarized in Table 2, sequence analysis of the APOC2 gene in families with familial hyperchylomicronemia has revealed a variety of molecular defects in this particular gene. In 7 families (Nijmegen, Paris, Barcelona, Japan, Venezuela, Padova, and Bari), a single base change resulted in the introduction of a premature stop that led to the synthesis of truncated forms of apoC2 that were either not secreted or rapidly cleared from the circulation3738394041 (Table 2). A donor splice-site mutation in the first base of the second intron of the APOC2 gene was found in a Hamburg family and in a neonatal Japanese patient (APOC2Hamburg and APOC2Tokyo, respectively). This mutation caused abnormal splicing of APOC2 mRNA and was associated with low levels of apoC2 in plasma.4243 In addition, a variety of single–amino acid substitutions in the APOC2 gene has been described (Table 2) that either resulted in the inability to initiate apoC2 synthesis44 or in the production of nonfunctional apoC2.454647 For 2 APOC2 variants (APOC2SanFrancisco and the APOC2 Lys19→Thr mutation), a direct relationship between this mutant form of apoC2 and lipoprotein abnormalities could not be established.48495051

    Several lines of evidence have implicated apoC3 as possibly contributing to the development of hypertriglyceridemia. A positive correlation has been observed between plasma apoC3 levels and elevated levels of plasma TGs525354 and VLDL-TGs.55 However, structural mutations in the human APOC3 gene fail to clearly show an association between the mutation and an altered lipid/lipoprotein metabolism. Five genetic variants of apoC3 were identified by the presence of additional bands after isoelectric focusing of VLDL (Table 2). Two of these variants differed from normal apoC3 by their degree of sialylation; ie, 1 was oversialylated56 while the other was not sialylated at all because of a Thr74→Ala mutation at the glycosylation site.575859 Carriers of these mutants were normolipidemic, indicating that the degree of apoC3 sialylation has little or no impact on lipoprotein metabolism. The 3 remaining apoC3 variants represented amino acid substitutions in both the N-terminal and C-terminal domains of apoC3 (Table 2). The Lys58→Glu mutation was associated with low plasma apoC3 concentrations and atypically large HDL.60 The number of carriers for this mutation, however, was too small to demonstrate a direct relationship between the mutation and altered lipoprotein levels. The Asp45→Asn variant was found in a Turkish patient who underwent coronary bypass surgery but failed to show a clear association between the mutation and an abnormal lipoprotein metabolism.61 The APOC3 Gln38→Lys mutation was observed in a boy of Mexican origin, and family studies in 16 individuals who were heterozygous for this APOC3 mutation revealed mildly elevated levels of plasma TGs in these subjects.62 Several studies have also reported a complete apoC3 deficiency in families with an increased prevalence of premature coronary heart disease.6364 In addition, 1 family with apoC3 deficiency demonstrated an increased fractional catabolic rate of VLDL.65 However, in all cases, apoC3 deficiency was associated with an apoA1 deficiency, making it difficult to estimate the exact contribution of the lack of apoC3 to changes in lipoprotein levels.

    In addition to the genetic mutations described above, several restriction fragment length polymorphisms (RFLPs) in or around the human APOC genes have been identified that are associated with lipoprotein disorders or altered plasma lipid concentrations in humans. One population-based, genetic association study has reported an HpaI RFLP in the APOC1 promoter,66 located at a site 317 bp 5′ from the apoC1 transcription initiation site.67 Recently, it has been shown by cell expression analysis that the promoter carrying the HpaI site in combination with the HCR mediates enhanced gene expression.68 These results suggest that under certain conditions, the HpaI promoter variant causes overexpression of APOC1, which may contribute to the development of hyperlipidemia.

    It has been demonstrated that a minor allele (S2) of an SstI RFLP in the APOC3 gene is associated with hypertriglyceridemia in several distinct populations,6970717273747576777879 but not in all.8081 Furthermore, Shoulders et al82 reported that healthy carriers of the S2 allele had higher plasma apoC3 levels than did noncarriers. These results indicate that the S2 allele may influence plasma TG levels through modulation of APOC3 gene expression. However, the SstI RFLP is located in the noncoding region of exon 4 of the APOC3 gene, suggesting that the S2 allele may modulate plasma TG levels by linkage disequilibrium with other functional sequences in or near the APOC3 gene. Dammerman et al79 and Xu et al83 have identified several polymorphic sites in and around the APOC3 gene that show strong allelic association with each other and with the SstI site. A detailed overview of these polymorphic sites has recently been published.84

    Other RFLPs within the APOA1/C3/A4 gene cluster such as XmnI and PstI have also been reported to be associated with hypertriglyceridemia85 or coronary artery disease.86 In 1 study of selected British families, the XmnI RFLP within the APOA1/C3/A4 gene cluster was shown to be linked with familial combined hyperlipidemia (FCH),87 but this finding has not been confirmed by others.8889 FCH is a common inherited disorder of lipid metabolism that is characterized by an overproduction of apoB-100–containing lipoproteins and elevated levels of VLDL and LDL.909192 Recently, it was reported that the XmnI polymorphism together with MspI and SstI aggravated hypercholesterolemia and hypertriglyceridemia in FCH probands; ie, a higher frequency of these minor alleles was associated with elevated plasma cholesterol, TGs, LDL cholesterol, apoB, and apoC3 levels.93 A more detailed analysis of a combination of haplotypes within the APOA1/C3/A4 gene cluster showed 2 different susceptibility loci for FCH within this cluster, consisting of an S2-bearing haplotype behaving as a dominant trait and an X2M2 haplotype behaving as a permissive trait.94 Furthermore, a C1100→T polymorphism in exon 3 of the APOC3 gene was found to be associated with an increased number of VLDL and IDL particles in the circulation of FCH probands.95 Altogether, these results suggest that the APOA1/C3/A4 gene cluster may contribute to FCH in a rather complex genetic manner, thereby acting as a modifier gene rather than representing the primary cause of FCH.

    Further evidence that APOC3 overexpression may underlie hypertriglyceridemia in humans comes from studies with fibrates, a hypotriglyceridemic class of drugs. Fibrates effectively decrease the apoC3 synthesis rate in humans54 as well as APOC3 mRNA levels in isolated human hepatocytes and rat livers via a peroxisome proliferator–activated receptor–dependent pathway.969798

    In summary, the characterization of mutations in the APOC2 gene of patients with hyperchylomicronemia has clearly established an important role for apoC2 as an activator of LPL. In contrast, the mechanisms underlying the hyperlipidemia and hypertriglyceridemia that are suggested as being associated with genetic mutations and polymorphisms of the APOC1 and APOC3 genes remain largely unknown.

    ApoC Proteins

    Nucleotide sequence analysis has indicated that apoC1 is synthesized with a 26-residue signal peptide that is cleaved cotranslationally in the rough endoplasmic reticulum.99 The remaining single-chain polypeptide of 57 amino acid residues has a molecular mass of 6.6 kDa (Table 1).100101 ApoC1 has a high content of lysine (16 mol%) and contains no histidine, tyrosine, cysteine, or carbohydrate.102 It has been demonstrated that residues 7 to 24 and 35 to 53 of apoC1 are important for the binding to lipoproteins.102 The plasma concentration of apoC1 in humans is ≈6 mg/dL.103

    ApoC2 is synthesized with a 22-residue signal peptide that is cleaved cotranslationally in the rough endoplasmic reticulum.104 The remaining single polypeptide chain of 79 amino acid residues has a calculated molecular mass of 8.8 kDa.6104105106 The structure of apoC2 is predicted to contain 3 helical regions between residue 13 to 22, 29 to 40, and 43 to 52, which are thought to be involved in phospholipid binding.107 Studies using synthetic peptides of apoC2 have shown that LPL interacts with the COOH-terminal amino acids 56 to 79 of apoC2.108 In line with these data, deletion of the COOH-terminal tetrapeptide residues 76 through 79 impairs the ability of the protein to activate LPL.109 ApoC2 is present in human plasma at a concentration of ≈4 mg/dL.8

    ApoC3 is synthesized in the liver and in minor quantities by the intestine as a 99–amino acid peptide. After removal of the 20–amino acid signal peptide in the endoplasmic reticulum, a mature apoC3 protein of 79 amino acids comprises a molecular mass of 8.8 kDa (Table 1).110 Thrombin cleavage of apoC3 results in an N-terminal domain, residues 1 to 40, and a C-terminal domain, residues 41 to 79, corresponding to the products of exons 3 and 4, respectively.111 Structural analysis demonstrated that the binding of apoC3 to surface phospholipids of lipoproteins is mediated by an amphipathic helix at residues 50 to 69 residing in the C-terminal domain of apoC3.112 Isoelectric focusing separates apoC3 into 3 isoforms that differ in their degree of O-linked sialylation at the threonine residue in position 74: apoC3–0 (no sialic acid), apoC3-1 (1 mol sialic acid), and apoC3-2 (2 mol sialic acid).113114115 ApoC3 is the most abundant C apolipoprotein in human plasma, at a concentration of ≈12 mg/dL.116

    Little has been reported on how and in which form apoCs are secreted into plasma. Studies by Roghani and Zannis59 have shown that cell clones expressing the APOC3 gene exclusively secrete the desialylated form of apoC3 (apoC3-2), suggesting that apoC3-2 must be desialylated after secretion in plasma to produce the monosialo (apoC3-1) and asialo (apoC3-0) forms. Furthermore, it was shown that the intracellular glycosylation of apoC3 is not an absolute prerequisite for its secretion and ability to associate with plasma lipoproteins.59 Although it has been reported that nascent apoCs are largely secreted in the lipid-poor form by different cell lines in vitro,59116 it is likely due to their high affinity toward lipid surfaces that apoCs rapidly associate with VLDL and HDL in plasma.117118119 A detailed study by Gibson et al120 showed that apoC3 was found in the broad distribution of particles the size of VLDL, on particles slightly larger than LDL, and on particles slightly larger than HDL. It has been reported that in the fasting state, apoCs are mainly associated with HDL, whereas in the fed state, they preferentially redistribute to the surface of chylomicron and VLDL particles.121 Similarly, release of LPL and hepatic lipase in subjects intravenously injected with heparin induced a shift in the distribution of apoC2 and apoC3 from VLDL to particles slightly larger than HDL.122 At least for apoC3, there is also a nonexchangeable pool present on both VLDL and HDL that accounts for 30% to 60% of the total apoC3 mass in each lipoprotein fraction.123124

    The relatively low human APOC4 gene expression in the liver and the total lack of the apoC4 protein in human plasma (Table 1) suggest that apoC4 plays no major role in lipoprotein metabolism. The apoC4 protein sequence was predicted to comprise 127 amino acid residues, which contain a putative 25-residue signal peptide and 2 potential amphipathic α-helical domains.10 In other species such as the rabbit, it has been demonstrated that apoC4 is secreted at a more substantial level.125 The rabbit apoC4 protein is synthesized as a 124–amino acid protein that includes a typical signal peptide of 27 residues and has a molecular weight of ≈14 kDa. The mature rabbit apoC4 protein of 97 amino acids is primarily associated with VLDL and HDL.125

    Interaction of ApoCs With Receptors and Enzymes Involved in Lipoprotein Metabolism

    Studies in the early 1980s have demonstrated that enrichment of chylomicrons and VLDL with a mixture of apoCs significantly inhibits their uptake by the isolated, perfused rat liver.126127128129130131132133 In line with these studies, it was shown that the apoE-mediated uptake of TG-rich emulsions by HepG2 cells and rat hepatocytes in culture was effectively inhibited by apoC3 and apoC1.131133 Ligand blotting assays showed that apoC1 and apoC2 inhibit the apoE-mediated binding of β-VLDL to the low density lipoprotein receptor (LDLR)–related protein (LRP), apoC1’s being a more effective inhibitor than apoC2.134135 As shown in Table 3, apoC3 had no effect on the binding affinity of β-VLDL to LRP.135 It is suggested that the inhibitory action of apoC1 on lipoprotein binding to LRP was due to displacement of apoE from the lipoprotein particle. In line with these results, it was shown that synthetic peptides corresponding to the lipid-binding domain of apoC1 were also able to displace significant amounts of apoE from β-VLDL and inhibit the binding of β-VLDL to LRP.136 Sehayek and Eisenberg137 reported that apoC1 and apoC2 impaired the apoE-mediated binding of VLDL to the LDLR in cultured fibroblasts (Table 3). In line with the LRP ligand blotting assays, the strongest inhibition of lipoprotein binding to the LDLR was observed with apoC1. In this study, it was concluded that the inhibition of lipoprotein binding to the LDLR occurred through masking or altering the conformation of apoE by apoC1 rather than through displacement of apoE, as suggested by Weisgraber et al.135

    Previous studies have shown that apoC3 completely abolishes the apoB-mediated binding of lipoproteins to the LDLR (see Table 3). It is suggested that this inhibitory action of apoC3 on lipoprotein binding was due to a masking of the receptor domain of apoB by apoC3.138139 An inhibitory effect was also observed for apoC2, whereas apoC1 did not inhibit apoB-mediated binding of lipoproteins to the LDLR.139 Recent studies have shown that apoCs can also interfere with the binding of lipoproteins to other lipoprotein receptors, including the VLDL receptor140 and lipolysis-stimulated receptor.141 The binding of lipoproteins to the VLDL receptor was completely inhibited by apoC1,140 whereas apoC3 specifically inhibited the binding of chylomicrons and VLDL to the lipolysis-stimulated receptor.141

    Numerous in vitro studies have investigated the influence of apoCs on the LPL-mediated lipolysis of TG-rich lipoproteins. As shown in Table 3, apoC2 is an essential activator of LPL. However, at high protein concentrations, apoC2 was demonstrated to inhibit LPL activity rather than stimulate it.142 The mechanism by which apoC2 activates LPL is not fully understood.143144 It has been suggested that apoC2 activates LPL after binding of LPL to phospholipids on the surface of TG-rich lipoproteins. On the other hand, apoC2 may also bind directly to LPL. Recent studies by Olivecrona and Beisiegel145 showed that the lipid binding domain of apoC2 is essential for activation of LPL.

    Studies in the early 1970s have indicated that both apoC1 and apoC3 inhibit LPL activity142146147148149 (Table 3). In a study with hypertriglyceridemic patients, it was shown that apoC3 was 1 of the most specific inhibitors of LPL.150 Further in vitro kinetic analysis with bovine LPL and purified apoC3 demonstrated that apoC3 displays noncompetitive inhibitory properties against both apoC2 and triolein, indicating that apoC3 exerts its inhibitory effect directly on LPL.150 In line with these results, McConathy et al151 used synthetic polypeptide fragments of apoC3 and observed that the N-terminal domain of apoC3 is primarily responsible for inhibition of LPL activity. Studies by Ginsberg et al65 showed that sera from subjects deficient for both apoC3 and apoA1 were able to normally activate human milk LPL at increasing volumes of sera, whereas normal sera effectively inhibits LPL activity at increasing concentrations. Furthermore, addition of purified apoC3 to the apoC3/A1-deficient sera progressively reduced maximal levels of LPL activity, suggesting that apoC3 inhibits the LPL-mediated lipolysis of TG-rich lipoproteins.

    In addition to LPL, it has been demonstrated that apoCs can act on several other enzymes involved in lipoprotein processing (see Table 3). In vitro, high concentrations of apoC3 have been shown to inhibit hepatic lipase (HL).152 In line with this study, apoC3 inhibited the lipolysis of TG emulsions by heparin-immobilized HL in the presence of apoE.153 An inhibitory effect on the HL-mediated lipolysis of TG emulsions was also observed for apoC2, although to a lesser extent than with apoC3.153 In the latter study, however, the inhibitory action of apoC3 and apoC2 may have been due to interference of the apoCs with the apoE-mediated binding of the substrate to the lipase-loaded heparin-Sepharose column rather than a direct inhibitory action of the apoCs on HL itself.

    ApoCs also appeared to affect LCAT activity (Table 3). Whereas apoA1 is known to be the most powerful LCAT activator, apoC1 was shown to activate LCAT to ≈78% of that of apoA1.154155156 Both apoC2 and apoC3 were reported to inhibit LCAT activity, probably by displacing the activating apolipoproteins from the lipoprotein surface.157 Furthermore, LCAT is also able to esterify lysophosphatidylcholine to phosphatidylcholine.158 This lysolecithin acyltransferase activity was found to be activated by apoC1 as well. In this respect, apoC1 was 70% as effective as apoA1.159

    It has been reported that in a family of baboons with high plasma HDL cholesterol levels, the transfer of cholesteryl ester from HDL to lower-density lipoproteins is inhibited by a 4-kDa protein.160 This 4-kDa protein appeared to correspond to the N-terminal domain of apoC1. Further in vitro studies demonstrated that a synthetic peptide comprising the 38–amino acid N-terminal domain of apoC1 was indeed able to inhibit cholesteryl ester transfer protein (CETP) activity.160 In addition, the 4-kDa protein was associated with apoA1 on HDL and, to a lesser extent, with apoE on VLDL, thereby resulting in modification of these apolipoproteins. From these data, it was hypothesized that an association of the apoC1 fragment with apoA1 on the surface of HDL and with apoE on VLDL may hamper the accessibility of CETP to these substrate lipoproteins.

    Little has been published about the effects of apoC2 and apoC3 on CETP activity. Preliminary studies as discussed by Sparks and Pritchard161 demonstrate that by using recombinant HDL particles, apoC3 stimulates CETP activity (Table 3).

    In summary, in vitro studies have demonstrated that apoCs have an inhibitory or stimulatory effect on a variety of receptors and enzymes involved in lipoprotein metabolism (Table 3). These data suggest a complex role for apoCs in human disease. However, it is important to know which of these in vitro effects extends to the in vivo situation, because several in vitro effects of apoCs on receptors and enzymes may appear nonspecific or secondary, ie, due to the displacement of other activating or inhibiting components of the lipoprotein particle.

    Transgenic Mouse Models Overexpressing or Lacking ApoC1

    Studies relating to the in vivo metabolism of apoCs have been hampered in humans owing to the highly complex nature of lipoprotein metabolism that can be influenced by multiple genetic and environmental factors. To study the in vivo functions of the individual apoCs in lipoprotein metabolism against a defined genetic background and under strictly controlled environmental conditions, several laboratories have created mouse models lacking or overexpressing the respective APOC genes through the technologies of transgenesis and gene targeting. As shown in Table 4, APOC1-transgenic mice were generated by using different DNA constructs that all contained the 154-bp HCR that directs expression of the human APOC1 gene to the liver. Human APOC1–transgenic mice exhibited elevated levels of cholesterol and TGs owing to an accumulation of VLDL-size particles in the circulation.162163164165

    To investigate the mechanisms underlying the hyperlipidemia in human APOC1–transgenic mice, in vivo turnover studies were performed using labeled VLDL. The clearance of both VLDL TG and VLDL apoB was severely hampered in hyperlipidemic human APOC1–transgenic mice,163164165 suggesting that apoC1 interferes with either the lipolysis or hepatic uptake of VLDL. The findings that (1) VLDL from APOC1-transgenic mice bound as efficiently to heparin-Sepharose as did VLDL from wild-type mice,164 (2) the in vitro lipolysis by LPL of VLDL TG fractions isolated from APOC1-transgenic mice was not impaired, and (3) the in vivo extrahepatic lipolysis of VLDL TG in APOC1-transgenic mice was not different from that in wild-type mice165 indicate that apoC1 does not interfere with lipolysis of VLDL TGs in vivo. Furthermore, it was demonstrated that the production rate of VLDL TGs in APOC1-transgenic mice is not different from that in control mice.164165 In conclusion, the elevated lipid levels in the plasma of APOC1-transgenic mice are primarily due to an impaired uptake of VLDL by the liver rather than to an enhanced production or disturbed lipolysis of VLDL.163164165

    Overexpression of apoC1 in LDLR-knockout mice leads to extremely elevated levels of plasma cholesterol and TGs compared with cholesterol and TG levels in LDLR-knockout mice.165 These results suggest that apoC1 inhibits the alternative lipoprotein clearance pathway. The fact that overexpression of the receptor-associated protein (RAP) greatly enhances serum cholesterol and TG levels in LDLR−/− mice whereas it does not alter serum lipid levels in APOC1/LDLR−/− mice indicates that RAP and APOC1 overexpression act on the same pathway in inhibiting the clearance of VLDL remnants by the liver. Because RAP overexpression is known to block LRP, it can be concluded that apoC1 inhibits the uptake of lipoproteins via LRP in vivo, thereby sustaining the in vitro findings that apoC1 is the most efficient apoC for inhibiting the binding of VLDL to the LRP.135136

    The in vitro observation that apoC1 is a potent activator of LCAT suggests that the increases in VLDL/IDL and LDL cholesterol observed in human APOC1–transgenic mice164165 may also partly result from an increase in the cholesterol esterification rate. Increased LCAT activity, as found in transgenic mice overexpressing human LCAT, has been reported to elevate HDL cholesterol esters levels.166167168 However, the findings that the free to total cholesterol ratios were unchanged in APOC1-transgenic mice164 and that HDL cholesterol esters were not significantly elevated in APOC1-transgenic mice compared with wild-type mice165 argue against an LCAT-mediated elevation in cholesterol levels in APOC1-transgenic mice.

    In addition to hyperlipidemia, it has recently been reported that APOC1-transgenic animals exhibit several abnormalities, consisting of elevated plasma free fatty acid levels, epidermal hyperplasia and hyperkeratosis, atrophic sebaceous glands, lack of sebum, and (subcutaneous) adipose tissue.169 These results suggest an additional role for apoC1 in epidermal lipid synthesis as well as adipose tissue formation.

    Because transgenic mice overexpressing APOC1 develop hyperlipidemia, a hypolipidemic phenotype was expected in ApoC1-knockout mice. It was, however, surprising to observe that ApoC1-knockout mice had normal serum lipid levels on a chow diet (Table 4).170 Only when fed a high-fat and high-cholesterol diet did apoC1-deficient mice develop hypercholesterolemia. In vitro binding experiments revealed that apoC1-deficient VLDL was a poor competitor for LDL binding to the LDLR, suggesting that total apoC1 deficiency leads to an impaired receptor-mediated clearance of remnant lipoproteins.170 Later, these results were confirmed in a more detailed characterization of these ApoC1-knockout mice, demonstrating that an impaired in vivo hepatic uptake of VLDL is the primary metabolic defect in apoC1-deficient mice.171

    In summary, whereas overexpression of human APOC1 in transgenic mice predominantly inhibits the uptake of VLDL particles by the liver, the absence of endogenous mouse ApoC1 in mice appears to have the same effect, though to a lesser extent. It has been suggested that apoC1 may impair VLDL clearance either directly, by a specific interaction between apoC1 and the hepatic receptor, or indirectly, as caused by an apoC1-induced displacement of apoE from the lipoprotein particle.164165 On the other hand, it is suggested that the impaired interaction of apoC1-deficient VLDL with hepatic receptors is due to an enrichment of the VLDL particle with apoA1 and apoA4.170171

    Transgenic Mice Overexpressing Human ApoC2

    Transgenic mice overexpressing human APOC2 were generated by using a vector containing the human APOC2 gene joined to a cytochrome P450 CYPIA1 promoter172 (Table 4). This promoter is normally silent in intrauterine life but can lead to transgene expression after administration of β-naphthoflavone. Strikingly, transgenic mice overexpressing human apoC2 were hypertriglyceridemic, due to an accumulation of TG-rich VLDL particles in the circulation. This hypertriglyceridemia was shown to be caused by impaired clearance of VLDL TGs.172 This finding suggests that high levels of apoC2 interfere with either the peripheral lipolysis of VLDL or the uptake of the VLDL particle by the liver. The observation that APOC2-transgenic mice accumulate large, TG-rich VLDLs and have only minimally elevated levels of plasma cholesterol is most consistent with a defective LPL-mediated hydrolysis of VLDL TGs in these mice rather than an impaired hepatic VLDL uptake. The observation that VLDL isolated from APOC2-transgenic mice showed decreased binding affinity to heparin-Sepharose suggests that these lipoprotein fractions may be less accessible to cell surface–bound LPL172 and therefore sustains the hypothesis that excess apoC2 on the VLDL particle inhibits LPL activity in vivo. These results are in striking contrast to the human studies discussed earlier, in which it was shown that apoC2 is the physiological activator of LPL. Altogether, these data suggest that apoC2 may play a complex role in plasma TG metabolism; ie, apoC2 activates LPL, most likely at low protein concentrations, whereas at high protein levels, apoC2 directly inhibits VLDL lipolysis.

    Transgenic Mouse Models Overexpressing or Lacking ApoC3

    Two laboratories have reported the generation of human APOC3–transgenic mice by using DNA fragments of different sizes, both of which resulted in high levels of human APOC3 mRNA in the liver and intestine173174 (Table 4). Human APOC3–transgenic mice exhibited very elevated levels of VLDL TGs. Recently, it was reported that mouse ApoC3–transgenic mice are also hypertriglyceridemic.175 Human and mouse APOC3–transgenic mice had impaired clearance of VLDL TGs, concomitant with a decreased VLDL apoE to apoC ratio.174175176 Because crossbreeding of human APOC3–transgenic mice with human APOE–overexpressing transgenic mice normalizes plasma TG levels,174175 it was concluded that the delayed clearance of VLDL TGs in APOC3-transgenic mice was due to the low amount of apoE relative to apoC3 on the VLDL particle. More recent studies, however, have shown that the hypertriglyceridemia in APOC3-transgenic mice is most probably caused by an excess of apoC3 rather than by the apoC3-induced displacement of apoE. ApoE-knockout mice normally accumulate large amounts of VLDL that is enriched in cholesterol ester but relatively poor in TG.177 Crossbreeding of ApoE-knockout mice with transgenic mice overexpressing human apoC3 resulted in a massive accumulation of TG-rich VLDL-size particles,178 indicating that it is the amount of apoC3 that causes hypertriglyceridemia.

    From in vitro binding studies, it was suggested that excess apoC3 inhibits the binding of VLDL to the LDLR.174175 However, the prolonged residence time of the predominantly enlarged, TG-rich VLDL particles in APOC3-transgenic mice implies that apoC3 impairs the hydrolysis of VLDL TGs. In line with this observation, VLDL isolated from APOC3-transgenic mice displayed decreased binding affinity to heparin-Sepharose.164175 In addition, the observations that apoC3-deficient mice are protected from postprandial hypertriglyceridemia and exhibit reduced serum lipid levels compared with control mice also points to an inhibitory action of apoC3 on VLDL lipolysis.179

    Transgenic Mice Overexpressing Human ApoC4

    The recently identified human APOC4 gene was overexpressed in transgenic mice180 (Table 4). Under normal conditions, the APOC4 gene is poorly expressed in human liver, most likely as a consequence of a TATA-less promoter.10 Therefore, to enhance liver expression of the human APOC4 gene in mice, a vector was constructed containing human APOC4 cDNA and the HCR element under control of the human APOE gene promoter. Human APOC4–transgenic mice were hypertriglyceridemic compared with their nontransgenic littermates, owing to an accumulation of TG-rich VLDL particles. Because there was little change in serum cholesterol levels in these transgenic mice, apoC4 may interfere with the clearance of VLDL TGs via an inhibitory effect on lipolysis in a way similar to that discussed for apoC2 and apoC3.180 The fact that apoC4 is totally absent in human plasma indicates no major modulating role for apoC4 in VLDL TG metabolism in humans.

    Conclusions

    Clinical evidence, as well as in vitro data and in vivo work on transgenic mouse models, have demonstrated that each of the individual human apoCs effectively modulates lipoprotein metabolism. As schematically depicted in panel A of the Figure, apoC1 inhibits the uptake of TG-rich lipoproteins via hepatic receptors, particularly the LRP. As a consequence, the presence of apoC1 on the lipoprotein particle may prolong their residence time in the circulation and subsequently facilitate their conversion to LDL.

    ApoC2 is an important activator of LPL and is required for efficient lipolysis of TG-rich lipoproteins in the circulation. The total absence of apoC2 or defects in its structure severely hamper LPL-mediated lipolysis of TG-rich lipoproteins, resulting in strongly elevated levels of plasma TGs. In contrast, excess apoC2 on the lipoprotein particle has been demonstrated to inhibit LPL-mediated hydrolysis of TGs (panel B of the Figure).

    At least from in vivo studies with APOC3-transgenic mice, it appears that apoC3 inhibits the lipolysis of TG-rich lipoproteins by hampering the interaction of these lipoproteins with the heparan sulfate proteoglycan–LPL complex (panel C of the Figure). Subsequently, the poorly lipolyzed apoC3-containing lipoprotein particles may accumulate in plasma because of their lower binding affinity to hepatic receptors as a consequence of their lipid composition, large size, or the presence of apoC3 on the particle. These results suggest that the amount of apoC3 on the lipoprotein particle is a strong modulator of plasma TG metabolism and may contribute to hypertriglyceridemia in the human population.

    Several in vitro studies have shown that apoCs can also modulate enzymes that are involved in the transport of cholesterol from extrahepatic tissues to the liver (the Figure). Although these specific functions remain to be established in vivo, it has been demonstrated that apoC1 can effectively activate LCAT. In contrast, both apoC2 and apoC3 have been reported to inhibit LCAT activity, most likely as a consequence of displacing the activating components of the HDL particle. CETP, which mediates the transfer of cholesterol ester from HDL to apoB-containing lipoprotein particles, was shown to be inhibited by apoC1, whereas apoC3 was reported to activate this process.

    In conclusion, human apoCs have been demonstrated to have distinct effects on the major metabolic pathways in lipoprotein metabolism, implying that changes in human APOC gene expression may play an important role in the etiology of human hyperlipidemias.

    Figure 1.

    Figure 1. Schematic representation of the effects of apoC1 (A), apoC2 (B), and apoC3 (C) on the major metabolic pathways in lipoprotein metabolism. The stimulatory (↑) and inhibitory action (↓) of the individual apoCs on lipoprotein lipolysis, clearance, and hepatic uptake is depicted.

    Table 1. Properties of Human APOC Genes and Proteins

    PropertiesAPOC1APOC2APOC3APOC4
    Chromosomal localization19q13.219q13.211q23-qter19q13.2
    Size of gene, kb4.73.43.13.3
    Tissue expressionLiver, lung, skin, testis, spleenLiver, intestineLiver, intestineLow amounts in liver
    Length of mature protein, No. of amino acids577979102
    Molecular mass, kDa6.68.88.8N
    Plasma concentration, mg/dL6412ND
    ReferencesLauer et al4 Curry et al103Wei et al7 Das et al8Protter et al18 Nestel and Fidge116Allan et al10

    Chromosomal localization, tissue-specific expression, and other biological properties of the human APOC genes and proteins are depicted. N indicates not reported; ND, not detectable.

    Table 2. Molecular Defects in the Human APOC Genes

    GeneFamilyMolecular DefectLipoprotein Disorder/AbnormalityProtein in PlasmaReferences
    APOC1NApoC1/C2 deficiencyFamilial chylomicronemia0Dumon and Clerc30
    APOC2NijmegenIntroduction stop codon (Val18)Familial chylomicronemia0Fojo et al37
    Paris2, BarcelonaIntroduction stop codon (Arg19)Familial chylomicronemia0Parrot et al38
    Japan, VenezuelaIntroduction stop codon (Gln2)Familial chylomicronemia0Xiong et al39
    Padova, BariIntroduction stop codon (Tyr37)Familial chylomicronemia0Fojo et al40 Crecchio et al41
    Hamburg, TokyoIntron 2 donor splice defectFamilial chylomicronemia↓Fojo et al42 Okubo et al43
    Paris1Met22→ValFamilial chylomicronemia0Fojo et al44
    TorontoAsp69→ThrFamilial chylomicronemia→Connelly et al45
    St MichaelGln70→ProFamilial chylomicronemia→Connelly et al46
    WakayamaTrp26→ArgFamilial chylomicronemia0Inadera et al47
    San FranciscoGlu38→LysHyperlipidemia→Pullinger et al48
    NLys19→ThrHyperlipidemia→Huff et al49 Hegele et al50 Zysow et al51
    APOC3NLys58→GluHyperalphalipoproteinemia↓von Eckardstein et al60
    TurkeyAsp45→AsnN→Lüttmann et al61
    MexicoGln38→LysMild hypertriglyceridemia→Pullinger et al62

    Mutations affecting the synthesis, secretion, or structure of the respective apoC proteins. The molecular defect, the related lipoprotein disorder or lipoprotein abnormality, and the presence of the apoC protein in plasma are indicated. 0 indicates absence of apoC protein; ↓, low amounts of apoC protein; and →, presence of apoC protein.

    Table 3. Effect of ApoCs on Receptors and Enzymes Involved in Lipoprotein Metabolism

    Receptors and EnzymesApoC1ApoC2ApoC3References
    LRP↓↓↓0Kowal et al134
    Weisgraber et al135
    LDLR
    ApoE mediated↓↓↓↓Sehayek and Eisenberg137
    ApoB mediated0↓↓↓Clavey et al139
    VLDLR↓↓NNJong et al140
    Lipolysis-stimulated receptorN0↓↓Mann et al141
    LPL↓↑↑ or ↓↓↓Havel et al142
    LaRosa et al143
    Ekman and Nilsson-Ehle146
    Brown and Baginsky147
    Wang et al150
    HLN↓↓↓Landis et al153
    LCAT↑↑↓↓Soutar et al154
    Steyrer and Kostner156
    Nishida et al157
    Liu and Subbaiah159
    CETP↓N↑Kushwaha et al160
    Sparks and Pritchard161

    Individual effects of apoC on lipoprotein receptors such as the LDLR, LRP, VLDLR, and Lipolysis-stimulated receptor and enzymes such as LPL, HL, LCAT, and CETP. ↓↓ indicates strong inhibition; ↓, moderate inhibition; 0, no effect; ↑, moderate activation; and ↑↑, strong activation.

    Table 4. APOC-Transgenic Mouse Models

    GeneDNA ConstructTissue ExpressionPhenotypeReferences
    Overexpression
    Human APOC120.8 kb; APOC1/APOC1′/HCRLiverTC↑ TG↑Simonet et al162
    27 kb; APOE2 3L/APOC1/APOC′/HCRLiverTC↑↑ TG↑↑Jong et al163
    10.4 kb; APOC1/HCRLiverTC↑ TG↑Shachter et al164
    18 kb; APOC1/APOC′/HCRLiver/skinTC↑ TG↑Jong et al165169
    Human APOC28.4 kb; CYPIA1 promoter/APOC2Brain, liver, intestineTC↑→ TG↑↑Shachter et al172
    Human APOC36.7 kb; APOC3Liver, intestineTG↑↑Ito et al173
    10.5 kb; APOC3Liver, intestineTC↑→ TG↑↑de Silva et al174
    Mouse ApoC34.7 kb; ApoC3Liver, intestineTC↑ TG↑↑Aalto-Setälä et al175
    Human APOC4APOE promoter/APOC4/HCRLiver, kidney, spleen, brain, lungTC→ TG↑Alan and Taylor180
    Knockout
    Mouse ApoC112 kb; hygro B4 /HSV-tk…TC→ TG↑→van Ree et al170
    Jong et al171
    Mouse ApoC312 kb; Neo4 /HSV-tk…TC↓ TG↓Maeda et al179

    APOC-transgenic and knockout mice were generated by using the constructs as indicated above. TC indicates total cholesterol; TG, triglycerides; ↓, decrease; →, no change; ↑, increase; ↑↑, strong increase; APOC1′, APOC1 pseudogene; APOE

    13L, APOE

    23Leiden gene; CYPIA1, cytochrome P450 IA 1; hygro B

    3, hygromycin B resistance gene; Neo

    4, neomycin resistance gene; and HSV-tk, herpes simplex virus thymidine kinase gene.

    This work was supported by the Netherlands Heart Foundation and the Netherlands Foundation of Scientific Research (projects 97-067 and 903-39-117) (to L.M.H.). We are grateful to Hans van der Boom for excellent technical help.

    Footnotes

    Correspondence to Dr M.C. Jong, TNO–Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden or PO Box 2215, 2301 CE Leiden, The Netherlands. E-mail

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