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Correspondence to:
D. M. Waterworth
Remnant particles of triglyceride-rich lipoproteins (RLP) are known to be a strong predictor of atherogenicity. The serum concentrations of remnant-like particle triglyceride (RLPTG) and remnant-like particle cholesterol (RLPC) have been determined in a representative sample of the Czech MONICA study (n = 285). The relationship was investigated between remnant particle triglyceride/cholesterol concentrations and polymorphisms in the genes APOC3 (-482C
Further characterization of this remnant particle phenotype and its genetic determinants may lead to a better understanding of its metabolism and contribution to atherosclerosis.—Waterworth, D. M., J. A. Hubacek, J. Pitha, J. Kovar, R. Poledne, S. E. Humphries, and P. J. Talmud. Plasma levels of remnant particles are determined in part by variation in the APOC3 gene insulin response element and the APOCI;–APOE cluster. J. Lipid Res. 2000. 41: 1103;–1109.
Supplementary key words:
insulin response element, remnant-like triglyceride, remnant-like cholesterol, hepatic lipase, lipoprotein lipase, apolipoprotein B
Remnant lipoproteins are partially catabolized chylomicrons and very low density lipoproteins (VLDLs) that are depleted of triglycerides (TG) and enriched with cholesterol esters. High remnant lipoprotein levels are associated with the presence, severity, and progression of atherosclerosis (reviewed in ref. 1) and have been shown to be an independent predictor of future coronary events in patients with coronary artery disease (CAD) (2). Smaller VLDL and intermediate density lipoprotein (IDL) particles have been consistently shown to have an increased capacity to enter the arterial wall as compared with larger VLDL and chylomicrons (1). The development of immunochemical assays for immunoseparation of remnant particles (using anti-apolipoprotein A-I [apoA-I] and anti-apoB-100 antibodies) has allowed the specific isolation of these particles and determination of their triglyceride and cholesterol content (3) (4). These antibodies allow the removal of most of the apoB-containing lipoproteins (low density lipoprotein [LDL]/VLDL) and apoA-I-containing lipoproteins (chylomicrons and high density lipoprotein [HDL]), leaving behind chylomicron and VLDL remnants that are enriched in apoE. This method has been found to compare favorably with ultracentrifugation and electrophoresis methods for determining triglyceride-rich lipoprotein remnants and remnant-like particle cholesterol (RLPC) concentrations were found to be highly correlated with total VLDL- and IDL-cholesterol and VLDL-TG but not with LDL-cholesterol or LDL-apoB levels (4) (5) (6).
The aim of this study was to identify whether any of a number of candidate gene variants involved in key pathways of remnant lipoprotein metabolism are involved in the determination of the levels of RLPC or remnant-like particle triglyceride (RLPTG) within a representative sample of healthy men and women. The candidate genes chosen for this study were APOE, APOCI, APOC3, APOB, hepatic lipase (LIPE), and lipoprotein lipase (LPL).
ApoE is the major ligand involved in the binding and uptake of remnant particles. There are three major apoE isoforms (E2, E3, and E4) that have been extensively studied and have been found to differentially affect lipid profiles and CAD risk. In particular, the
The apoC-III content of triglyceride-rich lipoproteins (TRL) is a marker of TRL metabolism and clearance of VLDL and chylomicrons, as a greater proportion of apoC-III on HDL particles than on VLDL/chylomicron particles would indicate a recent clearance of VLDL and chylomicrons (14). ApoC-III inhibits lipoprotein lipase (LPL) in vitro (15) and displaces apoE on the particles (16), which may prolong the clearance of these atherogenic particles and their circulatory residence time (14). APOC3 3238C
Lipoprotein lipase (LPL) is abundant in muscle, adipose tissue, and macrophages, and hydrolyzes triglyceride in chylomicrons and VLDL. A common C-to-T transversion converts Ser447 to a premature stop codon, truncating LPL by two amino acids (X447 allele). Subjects in the ECTIM study who carried the X447 had significantly lower LpCIII:B (particles containing both apoC-III and apoB) and LpE:B, both of which represent remnant particles, and VLDL-cholesterol levels (8). Similarily, in German CAD patients who carried an X447 allele had a beneficial lipid profile of lower triglyceride and lower VLDL-triglyceride than S447 subjects (20). In the EARS study, the X447 allele was also found to be associated with reduced fasting and postprandial triglyceride and risk of paternal MI (21).
Hepatic lipase (LIPE) is a lipolytic enzyme that is mainly found anchored by heparan sulfate proteoglycans (HSPG) to the vascular endothelium in the liver on hepatocytes and is involved in the hydrolysis of phospholipids and triglycerides of chylomicron remnants. LIPE may influence remnant removal by the hydrolysis of chylomicron phospholipids, unmasking apoE and thereby enhancing binding to the LRP receptor (22). LIPE may also act as a ligand for chylomicron-remnant binding to the liver (23). A variant (-480C
ApoB is the major structural apoprotein of lipoproteins synthesized in the liver (apoB-100) and in the intestine (apoB-48), and apoB-100 acts as a ligand for the LDL receptor. SP-24 is a common variant of the apoB 27-residue signal peptide (SP-27), caused by the deletion of three hydrophobic amino acids. This variant has been shown to confer a secretion-defective phenotype in yeast (26). While other APOB variants are markers for apoB catabolism, variation in the apoB signal peptide is thought to affect the production and secretion of apoB-containing lipoprotein. Subjects who carried one or more SP-24 alleles have significantly reduced circulating levels of both postprandial large chylomicron remnants and large very low density lipoproteins after an oral fat tolerance test (27).
These candidate gene variants were determined in this representative sample of the Czech Slav population to examine their specific association, if any, with both remnant particle triglyceride and cholesterol levels.
Population sample
Biochemical and anthropomorphic measures
Polymorphism detection
Statistical analysis
General characteristics
Associations with RLPTG levels
The only other genes found to modulate RLPTG levels significantly were APOE (P = 0.009) and APOCI -317-321ins (P = 0.01) and all three univariate associations (APOC3, APOCI, and APOE) are shown in Fig 1 and Table 2. As with APOC3 -482C
These three significantly associated gene variants were subsequently entered as factors in pairwise combinations into an ANOVA to determine if any nonindependent genetic effects were present (sex and BMI were included as covariates). In a model containing both APOC3 -482C
Associations with RLPC levels
Are these associations specific to remnant particles?
Reports emphasizing the importance of remnant particles as a risk factor for atherosclerosis (1) (2) led us to investigate possible genetic influences on this important phenotype. The ability to measure remnant particles in samples from epidemiological studies has made it feasible to assess the genetic factors that determine their serum concentration. Of the seven candidate gene variants chosen to study, only APOC3 -482C
The raising effect of the APOC3 -482C
It was not surprising that no significant effect was observed on RPLC by APOC3 -482C
Analysis of the associations between RLPTG/RLPC and APOE-CI is complicated by the high degree of linkage disequilibrium that exists between these two genes. The extent of linkage disequilibrium was similar to that previously observed in European-Americans (10) with the APOCI -317-321ins allele being in linkage disequilibrium with both the
Elevated RLPTG was observed in both the
The presence of the APOCI -317-321ins allele has been shown in vitro to increase expression of APOCI by 50%. Elevated APOCI levels have been shown to inhibit both apoE-mediated uptake of triglyceride-rich emulsions in perfused rat liver and remnant clearance in apoC-I transgenic mice (13). Thus, a direct inhibitory mechanism would most likely explain the high levels of RLPTG and RLPC observed in APOCI -317-321ins/ins subjects. This effect appeared to be recessive, with no obvious effect in heterozygous carriers.
We have attempted to ascertain the specificity of these associations by comparing the magnitude of the effects of these associated gene variants between the remnant particles and their respective total lipid levels. As these associations were generally reflected in the total lipid level, we attempted to test for any specific effect on remnant particles by adjusting the remnant particle levels for the respective total cholesterol or TG level (as well as BMI and sex) by regression. This analysis indicated that the effects associated with the APOC3 and APOE-CI variants on RLPTG were not independent of their effects on triglyceride levels, suggesting a simple linear relationship between the two variables. However, there was some evidence of specific effects on RLPC by APOE and APOCI that were not reflected in effects on total cholesterol, suggesting a nonlinear relationship between the two variables. This suggests that the RLP may be specifically enriched for cholesterol in subjects who carry either
This study has allowed us to perform a preliminary screen of putative candidate genes involved in the remnant particle phenotype and we have been able to indicate which genetic variants warrant further examination. Clearly APOC3, APOCI, and APOE are the strongest candidates for effects on clearance of these particles, but much larger studies will be required to delineate whether these effects are specific to the APOE or APOCI region because of the high degree of linkage disequilibrium that exists between these variants. Xu and co-workers (10) attempted to differentiate between these gene effects by examining another ethnic group (African-American) in which linkage disequilibrium between APOE and APOCI is weaker; however, their results are difficult to interpret as the different genetic background may also contribute to and interact with the various lipid phenotypes studied. However, because of the important predictive power of remnant particles with respect to atherosclerosis (1) (2), the elucidation of the mechanisms by which genetic variation modulate this phenotype will be of clinical importance.
D.M.W., P.J.T., and S.E.H. are funded by the British Heart Foundation (RG/95007, PG/95190, and PG/96184). This work was partially supported by the Grant Agency of the Czech Republic (Grant No. 306/96/K220). J.A.H. was supported by Bristol Myers Squibb. The apoA-I and apoB antibodies were a gift from Dr. K. Nakajima (Kumamoto, Japan).
Manuscript received September 28, 1999; and in revised form February 4, 2000
Abbreviations:
apo, apolipoprotein; BMI, body mass index; CAD, coronary artery disease; IDL, intermediate density lipoprotein; IRE, insulin response element; LDL, low density lipoprotein; LRP, LDL-related protein; MADGE, microtiter array diagonal gel electrophoresis; MI, myocardial infarction; NEFA, nonesterified fatty acids; oligo, oligonucleotide; RLP, remnant-like particle; RLPC, remnant-like particle cholesterol; RLPTG, remnant-like particle triglyceride; TG, triglyceride; TRL, triglyceride-rich lipoprotein; VLDL, very low density lipoprotein
Copyright © 2000 by Lipid Research, Inc.
Original Article
Plasma levels of remnant particles are determined in part by variation in the APOC3 gene insulin response element and the APOCI;–APOE cluster
D. M. Waterwortha,
J. A. Hubaceka,b,
J. Pithab,
J. Kovarb,
R. Poledneb,
S. E. Humphriesa, and
P. J. Talmuda
a Centre for Cardiovascular Genetics, RFUCLMS, London, WC1E 6JJ UK
b Institute of Clinical and Experimental Medicine, 14000 Prague, Czech Republic
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
T/3238CG), APOE (
2/3/4), APOCI (-317-321ins), APOB (signal peptide), hepatic lipase (LIPE, -480CT), and lipoprotein lipase (LPL, S447X). Univariate analysis showed significant effects on RLPTG associated only with the APOE genotype (P = 0.009), the APOC3 -482CT genotype (P = 0.018), and the APOCI -317-321ins (P = 0.014) genotype and significant effects on RLPC with APOE (P = 0.01) and APOCI -317-321ins (P = 0.021). The raising effect of the APOE genotype for both remnant cholesterol and triglyceride was confined to the 2/4 (n = 6) and 4/4 (n = 3) groups, and thus when the 2/4 group was omitted in order to analyze by allele (2+/3+/4+), significance was lost (P = 0.6). There was strong linkage disequilibrium between the APOE and APOCI alleles (
2, P < 0.001) and a multivariate ANOVA of RLPTG with all three significantly associated variants as factors demonstrated that while the APOC3 -482CT effect was independent of the others (P = 0.003), the APOCI -317-321ins and APOE effects were not. This was also true for the APOCI -317-321ins and APOE effects on RLPC. To assess whether APOE-CI effects on RLPC were independent of their effects on total cholesterol and triglyceride levels, multiple linear regression was used. Using multiple linear regression, it appeared that the APOE-CI effects on RLPC were independent of their effects on plasma cholesterol, but the effects of APOC3 and APOE-CI on RLPTG could not be separated from their effects on plasma Tg levels.
INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4 allele has been associated with context-dependent (gender, diet, and ethnic origin) higher levels of triglyceride and low density lipoprotein-cholesterol (LDL-C) and risk for atherosclerosis (7) (8) (9). The 2 allele has been shown to be associated with high levels of TG but low levels of LDL-cholesterol, and was found to be associated with low risk of myocardial infarction (MI) (8) (9). APOE is closely linked to APOCI on chromosome 19 and an HpaI site (-317 bp to APOCI) has shown an ethnically distinct pattern of linkage disequilibrium with the APOE gene variants (10). ApoC-I is a constituent of triglyceride-rich lipoproteins and has been shown to displace apoE from triglyceride-rich emulsions and interfere with their hepatic clearance. ApoC-I also interferes with the binding of ß-VLDL to the LDL-related protein (LRP) receptor (11) and of VLDL/IDL to LDL receptors (12). Transgenic mice overexpressing apoC-I developed hyperlipidemia but apoC-I knockout mice exhibited normal lipid levels when fed a chow diet and developed hypercholesterolemia only on a high-fat and high-cholesterol diet (reviewed in ref. 13). The HpaI site is produced by a 4-bp CGTT insertion and has been shown to increase gene transcription in vitro, and was found to be associated with a disordered lipoprotein metabolism in a population-based study (10). G (SstI site) has been investigated in a large number of studies and possession of the rare 3238G allele has generally been found to be associated with high triglyceride levels and CAD risk (reviewed in ref. 17). The -482CT site is situated in an insulin responsive element (IRE) and the presence of the -482T allele has been shown to reduce responsiveness to insulin in vitro, resulting in inappropriate expression of apoC-III (18). In a study of native Canadians, the -482T allele was associated with significantly increased triglycerides in both men and women (19). T) in the LIPE promoter has been found to influence LIPE activity in CAD patients (24), fasting lipid concentrations and pre- and postprandial LpCIII:B levels in the EARSII group of young healthy men, such that carriers of the -480T had higher levels of apoC-III:B particles (25).
MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
The 129 men and 153 women, aged 25;–64 years, were randomly selected in 1988 from the 1% representative Czech population sample (Benesov region) according to the protocol of the MONICA study (28) and reinvestigated in 1996. The response rate of this selected group was 72% and all subjects were white. The origins of the Czech Slav population are not clear but frequency differences of common genetic variants between the Czechs and the neighboring Germanic population suggest that the the two groups may be genetically distinct (R. Poledne and J. A. Hubacek, unpublished observations, 1999). The MONICA study was a World Health Organization (WHO) project for monitoring risk factors for cardiovascular diseases in 26 countries. Written informed consent was obtained from the study participants and the study design was approved by a local ethics committee.
Information was obtained in the clinic on entry into the study on age, body mass index (BMI), smoking, and menopausal status of the women. Seven men and four women were receiving lipid-lowering medication, 19 men and 29 women were receiving diuretics, and 29 men and 42 women were receiving ß-blockers. Four men and 15 women were receiving a lipid-lowering diet and no women were receiving hormone replacement therapy. Lipids were obtained after an overnight fast. Remnant particles were immunoseparated from the remaining lipoproteins and their serum concentration determined in the supernatant as remnant-like particle triglyceride (RLPTG) and remnant-like particle cholesterol (RLPC) according to the method of Nakajima et al. (3) in the WHO regional reference laboratory (Institute of Clinical and Experimental Medicine, Prague, Czech Republic).
Polymerase chain reaction (PCR) conditions and restriction enzyme digestions were as follows (oligonucleotides [oligos] were synthesized by GIBCO-BRL [Gaithersburg, MD]): APOC3 -482CT; forward 5'-GGT GCT GGG AGG GGC GGT GAG AGC TCA GCC-3', reverse 5'-CCC TCC ACC AGC CCC AAG CCC GGA ACA CAG-3'. The PCR was carried out in 30 µL containing 200 ng of genomic DNA, 100 ng of each primer, 0.2 mM dNTPs (U.S. Biochemical, Cleveland, OH), 2.75 mM MgCl2, NH4 polymerase buffer (from Bioline, UK), and 0.25 units of Taq polymerase (GIBCO-BRL). After an initial denaturation for 5 min, denaturing was at 95°C for 1 min, with a combined annealing and extension step of 72°C for 1 min for 35 cycles. The PCR product was digested with 2 units of MvaI (MBI Fermentas, Amherst, NY) overnight at 37°C. The rare allele was detected by the presence of a cutting site. Polymorphisms in the APOC3 3238CG (29), APOE (30), APOCI -317-321ins (31), LIPE -480CT (24), LPL (S447X) (21), and APOB (signal peptide) (32) genes were determined as described previously, except that for the APOC3, LIPE, and LPL PCRs, the fragments were separated by 5;–10% polyacrylamide microtiter array diagonal gel electrophoresis (MADGE) (33).
Data for each genotype was tested for deviation from Hardy;–Weinberg equilibrium, using a 2 test in EXCEL. Linkage disequilibrium (
) was calculated with EXCEL and the method of Chakravarti et al. (34). Statistical analysis was performed with the SPSS (Chicago, IL) package. RLPTG and RLPC were both log-transformed for analysis. Differences between geometric means were tested by analysis of variance (ANOVA) (unique method) and included sex and BMI as covariates. The assumption of equal variance in the differerent genotype groups was tested and was not found to be violated in any of the comparisons. Age, smoking, and menopausal status were not found to be significant predictors of RLPTG or RLPC and were therefore not subject to adjustment. Linear regression analysis was used in an attempt to differentiate between the effect on total lipid and remnant particle lipid. Log RLPTG was regressed for log triglyceride, BMI, and sex, and the resulting residuals were tested for association with genotype by ANOVA. Likewise, log RLPC was regressed for cholesterol, BMI, and sex. No appreciable difference was observed in any of the associations when drugs (ß-blockers, diuretics, and fibrates) or a lipid-lowering diet was entered into the ANOVA as covariates. A value of P < 0.05 was selected for a significance threshold.
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The general characteristics of the sample are shown in Table 1. Men and women were similar with respect to age and BMI, but RLPTG was 114% higher (P = 0.0001) and RLPC 29.3% higher (P = 0.09) in the men than the women. For all variants the distribution of genotypes was as expected for Hardy;–Weinberg equilibrium. The rare allele frequencies and 95% confidence interval (95%CI) in the Czech sample were not significantly different in men and women and were as follows: APOCI -317-321ins, 0.175 (0.14;–0.21); APOB SP-24, 0.319 (0.28;–0.36); APOC3 -482T, 0.339 (0.30;–0.38); APOC3 3238G, 0.123 (0.10;–0.15); LIPE -480T, 0.209 (0.18;–0.24); LPL X447, 0.094 (0.07;–0.12); 2, 0.081 (0.06;–0.10); 3, 0.826 (0.79;–0.86); and 4, 0.094 (0.07;–0.12).
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Table 1.
General characteristics of the study population
For all the analyses there was no heterogeneity of effect between men and women (P = 0.57) and data were analyzed for the group as a whole to increase the power of the study. For the APOC3 gene, univariate analysis (with sex and BMI as covariates) showed ( Fig 1 and Table 2) that APOC3 (-482CT) was significantly associated with RLPTG (P = 0.02), with the raising effect (largely) confined to the -482TT group (TT 25.6% higher than CC group). There was no significant relationship observed with the APOC3 3238CG (SstI) site (P = 0.58), even though the two variants are in strong allelic association ( = 0.49, P < 0.001).
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Figure 1.
Geometric means and SEM of RLPTG by genotype, with univariate ANOVA P values.
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Table 2.
Mean (SE) lipids and RLPTG and RLPC by genotype T, no significant genotype;–gender interactions were observed (APOE, P = 0.95; APOCI, P = 0.86). Further scrutiny of the data showed that the significant effects of APOE and APOCI on RLPTG were derived primarily from a large raising effect in rare genotypes, with the 2/4 group (n = 6) having 30.8% higher and the 4/4 group (n = 3) having 77.3% higher RLPTG levels than the 3/3 group, and the APOCI -317-321ins/ins group (n = 7) having 42.5% higher levels than the -317-321del/del group. Therefore, when APOE was analyzed by alleles (2, 3, 4) instead of genotype, using the standard procedure of omitting the 2/4 group and grouping into 2+, 3+, and 4+, significance was lost (P = 0.34). The possible nonindependence of the APOE and APOCI findings may result from the strong linkage disequilibrium between the two variants (Pearson 2 test, P < 0.0001), reflected in the fact that four of the seven APOCI -317-321ins/ins subjects also carry either 2/4 or 4/4. No significant gene;–gene interactions were present between any of the variants investigated on RLPTG. There was no significant association between RLPTG and variation examined at the APOB, LIPE, or LPL loci. T and APOCI -317-321ins, both genetic effects remained significant (P = 0.013 and P = 0.012, respectively), as did the combination of APOC3 -482CT and APOE (P = 0.003 and P = 0.006, respectively). However, when APOE and APOCI -317-321ins were entered pairwise in both orientations, significance was lost in the variant that was entered second, regardless of the order (APOE, P = 0.009 and APOCI, P = 0.18, respectively/APOCI, P = 0.01 and APOE, P = 0.29, respectively), thus confirming that the effects of APOCI -317-321ins and APOE were not independent of each other.
Univariate analysis for cholesterol content of RLP of each genetic variant showed significant results for APOCI -317-321ins, where the ins/ins group had 43% higher RLPC than the del/del group, and for the APOE gene, where the 2/4 group (n = 6) and the 4/4 group (n = 3) had, respectively, 38 and 75.7% higher RLPC than the 3/3 group (P = 0.02 and P = 0.01, respectively). There was no significant association with APOC3 -482CT and RLPC (P = 0.28), although a similar trend as seen with RLPTG was noted ( Fig 2 and Table 2). None of the other variants investigated showed a significant association with RLPC. As with RLPTG, no significant gene;–gender or gene;–gene interactions were observed. In a multivariate ANOVA with RLPC, effects of the APOCI and APOE variants were not found to be independent of each other, significance being retained once again only in the variant entered first (APOE, P = 0.01 and APOCI, P = 0.27, respectively/APOCI, P = 0.004 and APOE, P = 0.34, respectively). The similar findings for the genetic associations with RLPC and RLPTG were substantiated by the strong correlation observed between the two measures (r = 0.84, P < 0.001).
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Figure 2.
Geometric means and SEM of RLPC by genotype, with univariate ANOVA P values.
As cholesterol and RLPC, and triglyceride and RLPTG, are strongly correlated (r = 0.53, P < 0.001; r = 0.79, P < 0.001, respectively), the question arose whether the observed effects of these variants on remnant particles were a reflection of the total cholesterol and triglyceride levels, or whether they were specific to the remnant particle TRL fraction. For each genotype, means and significance values for all four measures are shown in Table 2. Similar relationships were observed in almost all cases between the remnant fraction and total triglyceride/cholesterol levels, except for APOCI -317-321ins, where a significant relationship with RLPC (P = 0.02) was not seen on total cholesterol levels (P = 0.47) although a similar trend was evident. In an attempt to ascertain whether the genetic effects on RLPTG are independent of their effect on triglyceride, RLPTG was adjusted for triglyceride level by multiple linear regression (including BMI and sex) and ANOVA was performed on the resulting residuals. No significant effects were observed for any of the three associated gene variants (APOE, APOCI -317-321ins, and APOC3 -482CT) on the adjusted RLPTG (data not shown). A similar analysis was performed for RLPC and total cholesterol and significant effects were still observed for APOE (P = 0.017) and APOCI (P = 0.043) (data not shown). Adjusting RLPC for TG levels resulted in the loss of these significant associations for both APOE (P = 0.4) and APOCI (P = 0.4).
DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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T, APOE (2, 3, 4), and APOCI -317-321ins influenced serum RLPTG concentrations in this sample. However, as expected from their strong genetic relationship, the effect of the APOE and APOCI variants were not independent of each other. The APOE and APOCI variants also significantly influenced serum RLPC concentration in a nonindependent manner. None of the other variants showed any significant effect on remnant particle concentration in this study, although there would be limited power to detect effects of smaller magnitude. T variant on RLPTG was confined to homozygous carriers of the -482T allele and there was no heterogeneity of effect between men and women. There was no effect observed on this phenotype by the APOC3 variant 3238CG (SstI) (17), which is in strong linkage disequilibrium with APOC3 -482CT ( = 0.49, P < 0.001), but it is possible that the lower frequency of the 3238G allele (0.123) as compared with the -482T allele (0.339) could reduce the possibility of detecting significant effects. The -482CT variant is present in an insulin-responsive element and the -482T allele has been shown in vitro to be associated with the loss of insulin-mediated downregulation of APOC3 gene expression, as compared with the -482C allele, where a 40;–50% downregulation by insulin occurs (18). It should be noted that a second variant, -455TC, which was not evaluated in this study, is also present in the IRE, and would also be likely to show an association with RLPTG as it is in strong linkage disequilibrium with the -482CT variant. Both IRE variants were shown to be critical in conferring the response to insulin in vitro (18). Hypothetically, in a postprandial situation, where insulin downregulates apoC-III to release its inhibitory effect on LPL, carriers of the -482T allele could maintain innappropriately high levels of apoC-III and, as a consequence, a reduced triglyceride hydrolysis by LPL and a delayed clearance of triglyceride-rich lipoproteins and RLPs. ApoC-III transgenic mouse studies have suggested that the hypertriglyceridemic effect of apoC-III may be partly mediated by the displacement of apoE from TRL, leading to reduced remnant particle uptake (16). However, hypertriglyceridemia was not corrected in APOC3 tg/APOE null mice, suggesting that the predominant mechanism of apoC-III-induced hypertriglyceridemia may be due to decreased lipolysis at the cell surface (35). Thus, there are a number of possible mechanisms that could explain the effect of APOC3 -482CT on RLPTG, but until the exact functions of apoC-III have been elucidated, whether via LPL inhibition and/or apoE displacements, or possibly another mechanism, it is not possible to speculate further. T, as cholesterol levels are not generally affected by APOC3 variants, although a similar trend, as with RLPTG, was observed. The two measures of RLPTG and RLPC were highly correlated, but the data suggest that this variant affects the triglyceride content of the remnant particles more than the cholesterol content. 2 and 4 alleles; specifically four of the seven APOCI -317-321ins/ins subjects also carried either an 2 or an 4 allele. Variants in both APOE and APOCI can provide plausible mechanisms for modulating remnant particle concentrations and this data set is insufficiently powered to differentiate between them, but it could also be the case that both variants contribute to the remnant particle phenotype. It is also possible that other, as yet unknown variants in the APOE-CI-CII gene cluster (36), which are in allelic association with those we have investigated, could be responsible for these observed effects. 2/4 and 4/4 groups, reflecting previous findings of raised triglyceride in carriers of 2 and 4 alleles (8) (9). However, mean levels of RLPTG were similar in the group of subjects who carried at least one 3 allele compared with the 3/3 group, suggesting a dominant protective effect of the 3 allele. The highest levels were observed in the 4/4 group, suggesting that high remnant particle concentrations may be contributing to the increased risk of CAD commonly observed in this group (37). However the small sizes of the 2/4 and 4/4 groups in this study limit the interpretation of this result. A similar pattern was observed with RLPC, an effect that is probably due to the strong correlation between the two measures of RLPC and RLPTG. 4/4, 2/4, or APOCI -317-321ins/ins. These associations with RLPC were not, however, independent of TG level, as evidenced by the loss of significant association with APOCI and APOE when RLPC was adjusted for TG.
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