Original Article

Dietary fat clearance is modulated by genetic variation in apolipoprotein A-IV gene locus

Correspondence to: Francisco Perez-Jimenez.

	ABSTRACT

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Previous studies have shown that the A-IV-347Ser polymorphism is associated with the variability in low density lipoprotein (LDL)-cholesterol response to dietary therapy. The present study was designed to evaluate the association of this polymorphism with the individual variability observed in postprandial lipemic response. This polymorphism was characterized in 50 healthy male subjects homozygous for the apolipoprotein (apo)E3 allele. All subjects were subjected to a vitamin A-fat load test. Blood was drawn at time 0 and every hour over a period of 11 hours. Cholesterol and triglycerides (TG) in plasma and lipoprotein fractions of CH, TG, and retinyl palmitate (RP) were determined. Data from the postprandial lipemia revealed that subjects with the A-IV-347Ser allele (n = 14) have a lower postprandial response in total TG (P < 0.025), large triglyceride rich lipoproteins (TRL) TG (P < 0.02), and small-TRL TG levels (P < 0.007), and a higher postprandial response in large-TRL apoA-IV (P < 0.006) and apoB-100 (P < 0.041) levels than subjects homozygous for the A-IV-347Thr subjects (n = 36).

In conclusion, the modifications observed in postprandial lipoprotein metabolism associated with this polymorphism within the apoA-IV gene locus may be involved in the variability in LDL-CH response observed in subjects consuming high saturated fat diets.—Ostos, M. A., J. Lopez-Miranda, J. M. Ordovas, C. Marin, A. Blanco, P. Castro, F. Lopez-Segura, J. Jimenez-Pereperez, and F. Perez-Jimenez. Dietary fat clearance is modulated by genetic variation in apolipoprotein A-IV gene locus. J. Lipid Res. 1998. 39: 2493–2500.

Supplementary key words: postprandial lipemia, apolipoprotein A-IV, (A-IV-347Ser) polymorphism, triglycerides, retinyl palmitate

	INTRODUCTION

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The current recommendations for lipid and lipoprotein measurements call for blood samples to be obtained after at least 12 h fast. However, the fasting state does not represent the most common physiological status of individuals in developed societies, characterized by the consumption of frequent meals. Several reports in the fifties and sixties have shown an association between postprandial lipemia and risk of atherosclerosis (1) (2). In 1979 Zilversmit (3) proposed the important role of triglyceride (TG)-rich lipoproteins in the atherosclerotic process. Since then, the study of postprandial lipoprotein metabolism has been the focus of increased interest (4) (5) (6). The physiological basis of the wide interindividual variability in postprandial lipid response is complex. It has been shown that sex (7), diet (8), physical exercise (9), age (10), body mass index (BMI) (11), smoking (12), alcohol (13), and genetics (14) (15) are in part responsible for this variability.

Apolipoprotein (apo) A-IV is a plasma glycoprotein with a molecular mass of 46,000 Da consisting of 376 amino acid residues. It is synthesized primarily in the enterocytes of the small intestine during fat absorption. Several roles have been assigned to this apolipoprotein. It has been shown to participate in the absorption of dietary fat (16) (17), in TG transport (18), and in reverse cholesterol transport (19). Moreover, apoA-IV appears to modulate the activity of lecithin:cholesterol acyltransferase (LCAT) (20) and cholesteryl ester transfer protein (CETP) (21). ApoA-IV is secreted into mesenteric lymph in the chylomicrons (22) (23), where it is exchanged for the apolipoprotein (apo) C-II from high density lipoprotein (HDL) (24). ApoC-II is a cofactor required for lipoprotein lipase (LPL) activity (25) (26), which is necessary for the hydrolysis of triglycerides contained in chylomicrons. Thus, apoA-IV may regulate the clearance of TG-rich lipoproteins of intestinal origin.

The gene coding for apoA-IV is located in the long arm of chromosome 11 (27) (28). Several polymorphisms at this gene locus have been described and various associations with plasma lipid levels have been reported. One of the most frequent mutations is an A to T substitution that changes the threonine (ACT) residue at position 347 of the mature apoA-IV to serine (TCT), thereby eliminating a restriction site for the enzyme HinfI (29) (30). The frequency of this allele has been studied in several populations and it ranges from 0.16 to 0.21 (31) (32). Recently, we have demonstrated that carriers of the apoA-IV-347Ser allele have larger increases in low density lipoprotein (LDL)-cholesterol and apoB levels after consumption of a fat-rich diet (33). In this study, we have examined whether the effects of this mutation on LDL-cholesterol response to diet may be the result of a modification of postprandial lipid metabolism.

	MATERIAL AND METHODS

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Human subjects
Fifty healthy male subjects, 36 homozygous for the most common allele, AIV-347Thr allele (T/T) and 14 carriers of the A-IV-347Ser allele (S/+) (2 homozygous and 12 heterozygous), were studied. They ranged in age from 18 to 49 years. None of them had liver, renal, or thyroid disease or diabetes. All subjects were selected to have the apoE 3/3 genotype to avoid allele effects of this gene locus on postprandial lipemia (14). Of the 36 subjects with the T/T genotype, 8 presented the 360His mutation of apoA-IV, whereas only one of the S/+ subjects presented this mutation. None of the subjects were taking medication or vitamins known to affect plasma lipids. Their fasting plasma lipid, lipoprotein, and apolipoprotein levels, and age and body mass index (BMI) by apoA-IV alleles are shown in Table 1. All studies were carried out in the Research Unit of the Reina Sofia University Hospital. The experimental protocol was approved by the Human Investigation Review Committee at the Reina Sofia University Hospital.

Vitamin A fat-loading test
After a 12-h fast, subjects were given a fatty meal enriched with 60,000 units of vitamin A per m² of body surface area. The fatty meal consisted of 2 cups of whole milk, eggs, bread, bacon, cream, walnuts, and butter and it was consumed within 20 min. This meal provided 1 g of fat and 7 mg of cholesterol per kg body weight, and it contained 60% of calories as fat, 15% protein, and 25% carbohydrates. After the meal, subjects were not allowed to consume any calorie-containing food for 11 h. Blood samples were drawn before the meal, every hour until the 6th hour, and every 2nd hour and 30 min until the 11th hour.

Lipoprotein separations
Blood was collected in tubes containing EDTA to give a final concentration of 0.1% EDTA. Plasma was separated from red cells by centrifugation at 1,500 g for 15 min at 4°C. The chylomicron fraction of triglyceride-rich lipoproteins (large TRL) was isolated from 4 ml of plasma overlayered with 0.15 M NaCl, 1 mM EDTA (pH 7.4, d 1.006 g/mL) by a single ultracentrifugal spin (28,000 g, 30 min, 4°C) in a 50 type rotor (Beckman Instruments, Fullerton, CA). Chylomicrons, contained in the top layer, were removed by aspiration after cutting the tubes and the infranatant was centrifuged at a density of 1.019 g/mL for 24 h at 115,000 g in the same rotor. The non-chylomicron fraction of TRL (also referred to as small-TRL) was removed from the top of the tube. The small-TRL fraction contains both VLDL and IDL. All operations were done in subdued light. Large and small TRL fractions were stored at -70°C until assayed for retinyl palmitate (RP).

Lipid analysis
Cholesterol and triglycerides in plasma and lipoprotein fractions were assayed by enzymatic procedures (34) (35). ApoA-I and apoB were determined by turbidimetry (36). HDL-cholesterol was quantitated by analyzing the supernatant obtained after precipitation of a plasma aliquot with dextran sulfate-Mg²+, as described by Warnick, Benderson, and Albers (37). LDL-cholesterol was calculated as the difference between the cholesterol at the bottom of the tube after ultracentrifugation at 1.019 g/mL and the HDL-cholesterol.

Retinyl palmitate assay
The retinyl palmitate (RP) content of large and small TRL fractions was determined using a method previously described (38). Briefly, different volumes of the fractions (100 µl for chylomicrons and 100–500 µl for remnants) were placed in 13 x 100 mm glass tubes. The total volume in each tube was adjusted, as necessary, to 500 µl using normal saline. Retinyl acetate (40 ng in 200 µl of mobile phase buffer) was added to each tube as internal standard. Five hundred µl of methanol was added, followed by 500 µl of the mobile phase buffer for a total volume of 1.7 ml. The mobile phase buffer was freshly prepared each day by combining 90 ml hexane, 15 ml n-butyl chloride, 5 ml acetonitrile, and 0.01 ml acetic acid (82:13:5 by volume with 0.01 ml of acetic acid). The tubes were thoroughly mixed after each step. The final mixture was centrifuged at 350 g for 15 min (room temperature) and the upper layer was carefully removed by aspiration and placed into individual autosampler vials. The autoinjector was programmed to deliver 100 µl per injection and a new sample every 10 min in a custom pre-packed silica column SupelcoSil LC-SI (5 µm, 25 cm x 4.6 mm ID) provided by Supelco Inc. The flow was maintained at a constant rate of 2 ml/min, and the peaks were detected at 330 nm. Peaks of RP and retinyl acetate were identified by comparing the retention time with a purified standard (Sigma, St. Louis, MO), and the RP concentration in each sample was expressed in terms of the ratio of the area under the RP peak to the area under the RA peak (39). Here too, all operations were performed in subdued light.

Determination of apoB-48 and apoB-100
ApoB-48 and apoB-100 were determined by SDS-polyacrylamide gel electrophoresis as described by Karpe and Hamsten (40). In summary, samples containing isolated lipoprotein fractions were delipidated in a methanol–diethyl ether solvent system and the protein pellet was dissolved in 100–500 µL of 0.15 mol/L sodium phosphate, 12.5% glycerol, 2% sodium dodecyl sulfate (SDS), 5% mercaptoethanol, and 0.001% bromophenol blue, pH 6.8, at room temperature for 30 min followed by denaturation at 80°C for 10 min. Electrophoretic separation was performed using a 3–20% gradient polyacrylamide gel with a vertical Hoefer Mighty Small II electrophoresis apparatus connected to an EPS 400/500 (Pharmacia) power supply. The upper and lower electrophoresis buffers contained 25 mmol/L Tris, 192 mmol/L glycine, and 0.2% SDS adjusted to pH 8.5. ApoB-100 derived from LDL was used as a reference protein and for standard curve dilutions. A dilution curve ranging from 0.10 to 2 µg of apoB-100 was applied to four of the gel lanes. Electrophoresis was run at 60 V for the first 20 min and then at 100 V for 2 h. Gels were fixed in 12% trichloroacetic acid for at least 30 min and stained in 0.2% Coomassie G-250/40% methanol/10% acetic acid for at least 4 h. Destaining was done in 12% methanol/7% acetic acid with four changes of destaining solution for 24 h. Gels were scanned with a videodensitometer scanner (TDI, Madrid, Spain) connected to a personal computer for integration of the signals. Background intensity was calculated after scanning an empty lane. The coefficient of variation for the SDS-PAGE was 7.3% for apoB-48 and 5.1% for apoB-100.

DNA amplification and genotyping
DNA was extracted from 10 ml of EDTA-containing blood. Amplification of a region of the apo A-IV gene was done by polymerase chain reaction (PCR) with 250 ng of genomic DNA and 0.2 µmol of each oligonucleotide primer (P1:5'-GCCCTGGTG CAGCAGATGGAACAGCTCAGG-3' and P2:5'-CATCTGCACCT GCTCCTGCTGCTGCTCCAG-3') in 50 µl (41). DNA was denatured at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 65°C for 1 min, and extension at 70°C for 2 min. PCR product (10 µL) was digested with 5 units of restriction enzyme HinfI (Promega, Madison, WI) in a total volume of 35 µl. Digested DNA was separated by electrophoresis on an 8% non-denaturing polyacrylamide gel at 150 V for 2 h. Bands were visualized after silver staining.

Amplification of a region of 266-bp of the apoE gene was done by PCR with 250 ng of genomic DNA and 0.2 µmol of each oligonucleotide primer (E1, 5'-GAACAACTGACCCCGGTGGCG GAG-3', and E2, 5'-TCGCGGGCCCCGGCCTGGTACACTGCCA-3') and 10% dimethyl sulfoxide in 50 µl. DNA was denatured at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 63°C for 1.5 min, and extension at 72°C for 2 min. PCR products (20 µL) were digested with 10 units of restriction enzyme CfoI (BRL, MD) in a total volume of 35 µl. Digested DNA was separated by electrophoresis on an 8% non-denaturing polyacrylamide gel at 150 V for 2 h. Bands were visualized after silver staining.

ApoA-IV measurement
ApoA-IV was measured in total plasma and in both large and small TRL, in postprandial samples obtained at hour 0, 1, 3, 5, 8:30, and 11, using an ELISA assay. Briefly, polystyrene microtiter plates (Nunc Immunoplate I) were coated with affinity-purified polyclonal apoA-IV antibody (10 µg/ml) in PBS 0.1 M (pH 7.4), 100 µl/well. The plates were covered with acetate plate sealers (ICN) and incubated overnight at room temperature. The next day the solution containing the unbound antibody was removed and the remaining binding sites in the plate were blocked using 0.5% bovine serum albumin (RIA grade BSA, Sigma) and 0.1% NaN₃ in PBS (1 h incubation). Plates were then washed 3 times with PBS containing 0.5% Tween-20 (PBST).

Control and plasma samples were diluted (1:5000) in PBS-BSA. Large and small TRL samples were diluted 1/500 and 1/100, respectively. Two-fold serial dilutions were performed for the plasma standard (standard curve 333.3 ng/ml to 10.4 ng/ml). Controls were prepared in the laboratory by pooling plasma from different individuals. Multiple aliquots were stored at -70°C. Controls were calibrated against a primary standard determined by amino acid analysis.

Aliquots (100 µl) of standards, controls, and plasma samples were added to designated wells in the microtiter plate. Aliquots were diluted and thoroughly mixed immediately before addition. Controls and samples were run in duplicate wells in each plate. After 2 h incubation at 37°C, the contents of the plate were discarded and the plate was washed 3 times with PBST.

The goat-immunopurified apoA-IV antibody conjugated to peroxidase was diluted in PBS-BSA at 1:5000, and 100 µl was added to each well. The plate was sealed and incubated at 37°C for 2 h. After this incubation, the plate was washed 5 times with PBST. The substrate used for the enzymatic color reaction is ortho-phenylene diamine (OPD) and H₂O₂ in 0.1 M citrate buffer. This solution was added to each well (100 µl) and incubated for 30 min at room temperature, then it was read at 410 nm on a microtiter plate reader (Dynatech MR 600).

Statistical analysis
Several variables were calculated to characterize the postprandial responses of plasma triglycerides, large TRL, and small TRL to the test meal. The area under the curve (AUC) is defined as the area between the plasma concentration versus time curve and a line drawn parallel to the horizontal axis through the 0 h concentration. This area was calculated by a computer program using the trapezoidal rule. Other variables were the normalized peak concentration, which is the average of the peak and the second highest concentration above baseline, and the peak time, which was the average of the time to peak concentration and the time to the second highest concentration. Data were tested for statistical significance between genotypes by analysis of variance (ANOVA) and Kruskal-Wallis test, and between genotypes and time by ANOVA for repeated measures. In this analysis we studied the statistical effects of the genotype alone, independent of the time in the postprandial study (represented as P1), the effect of time alone or changes in the parameter after ingesting fatty food over the entire lipemic period (represented as P2), and the effect of the interaction of both factors, genotype and time, indicative of the magnitude of the postprandial response in each group of subjects with a different genotype (represented as P3). When statistical significance was found, Tukey's post hoc comparison test was used to identify group differences. A probability value less than 0.05 was considered significant. Stepwise multiple regression analyses were carried out using the normalized peak of plasma triglycerides, small TRL-triglycerides, large TRL apoB-100 AUC, and large TRL apoAI-V AUC as dependent variables, and age, BMI, apoA-IV genotypes, basal cholesterol, HDL cholesterol and triglyceride values as independent variables. Discrete variables were divided into classes for analysis. All data presented in text and tables are expressed as mean ± SD.

	RESULTS

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The baseline characteristics of the study subjects are shown in Table 1. No significant differences for any of the variables analyzed were detected between A-IV-347Ser/+ (S/+, n = 14) and A-IV-Thr/Thr (T/T, n = 36) subjects.

Triglyceride in plasma, large TRL and small TRL responses after the fat load test are shown in Figure 1. Significant time effects were found for plasma triglyceride (P < 0.001), large TRL-TG (P < 0.001) and small TRL-TG (P < 0.001), showing that the triglyceride levels increased during the postprandial period in plasma and both TRL fractions. In both groups plasma triglyceride levels remained significantly elevated over baseline during the first 8 hours, and returned to baseline by the 11th h (Figure 1A). Triglycerides in large-TRL (Figure 1B) were significantly higher than baseline in T/T subjects during the study period and returned to baseline values by 11 h in S/+ subjects. Significant elevations over baseline were noted for triglycerides in the small-TRL (Figure 1C), during the first 6 h of the postprandial period for T/T subjects, and only during the first 4 h in S/+ subjects. Subjects with the apoA-IV S/+ genotype showed a lower postprandial response in plasma triglycerides (P < 0.025), large TRL triglyceride (P < 0.02), and small TRL triglyceride (P < 0.007) than subjects with the T/T genotype as demonstrated by ANOVA for repeated measures (genotype by time interaction) (Figure 1). In addition, a significant genotype effect was observed in the small TRL-TG fraction with T/T subjects showing significantly higher postprandial levels (P < 0.019) than S/+ subjects. Furthermore, both the maximum peak (P < 0.042) and the normalized peak (P < 0.047) of the small TRL-TG were significantly smaller in the Ser/+ subjects than in subjects homozygous for the T allele ( Table 2).

View larger version (23K): [in this window] [in a new window]   Figure 1. Line plots of postprandial plasma triglyceride (A), large TRL triglyceride (B), and small TRL triglyceride (C) response in apoA-IV347 Thr/Thr subjects (solid line, ) and apoA-IV347 Ser/+ subjects (dotted line, ) (Ser/+ indicates Ser/Ser or Ser/Thr). For each group, the levels at each time point were averaged and expressed as mean ± SEM. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measured. * Tukey's test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

No significant genotype effects were observed in total cholesterol, HDL-cholesterol, LDL-cholesterol, apoB, apoA-I, large TRL-retinyl palmitate (RP), large TRL-cholesterol, small TRL-RP, and small TRL-cholesterol postprandial response ( Table 3).

ApoB-48 and B-100 in large and small TRL were analyzed. There were no significant genotype effects in the B-48 levels in the large and small TRL (Table 3). However, subjects with the apoA-IV S/+ genotype showed higher postprandial levels of large TRL apoB-100 (P < 0.041) than subjects with the T/T genotype as demonstrated by ANOVA for repeated measures (genotype effect) ( Figure 2A). There were no significant differences in small TRL apoB-100 levels between the two groups of subjects (Figure 2B). In addition, subjects with the apoA-IV T/T genotype have a trend toward higher triglyceride/apoB-100 ratio in large and small TRL fractions than S/+ subjects (Figure 2C and Figure D), although no significant differences attributable to genotype could be demonstrated by ANOVA for repeated measures.

View larger version (23K): [in this window] [in a new window]   Figure 2. Line plots of postprandial large TRL apoB-100 (A), small TRL apoB-100 (B), large TRL triglyceride/apoB-100 ratio (C), and small TRL triglyceride/apoB-100 ratio (D) response in apoA-IV347 Thr/Thr subjects (solid line, ) and apoA-IV347 Ser/+ subjects (dotted line, ) (Ser/+ indicates Ser/Ser or Ser/Thr). For each group, the levels at each time point were averaged and expressed as mean ± SEM. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measured. *Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

The postprandial response of apoA-IV levels in large TRL was significantly higher in individuals with the apoA-IV S/+ genotype than in the T/T subjects (P < 0.006; ANOVA) ( Figure 3A). However, there were no significant differences in the apoA-IV levels in the small TRL fractions between both groups of individuals (Figure 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3. Line plots of postprandial large TRL apoA-IV (A) and small TRL apoA-IV (B) response in apoA-IV347 Thr/Thr subjects (solid line, ) and apoA-IV347 Ser/+ subjects (dotted line, ) (Ser/+ indicates Ser/Ser or Ser/Thr). For each group, the levels at each time point were averaged and expressed as mean ± SEM. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measured. *Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

In the multiple regression analyses ( Table 4) the 347Ser/Thr polymorphism at the apoA-IV gene was a significant (P = 0.019) predictor of variability in large TRL apoA-IV postprandial response in our study population, accounting for 18.8% of the variance. In addition, the 347Ser mutation was a significant predictor of variability in large TRL apoB-100 postprandial response, accounting for 21% of the variance. The apoA-IV 347Ser/Thr polymorphism was also a significant predictor of variability in normalized peak of plasma TG (P = 0.027) and small TRL-TG (P = 0.032), accounting for 10.5% and 11.5% of the variance, respectively.

	DISCUSSION

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The results presented in this study demonstrate that carriers of the 347 Ser mutation in the apoA-IV gene show a reduced postprandial response of triglyceride-rich lipoproteins of intestinal origin, and higher levels of apoA-IV in these particles compared with AIV347-Thr/Thr subjects.

Interindividual variability in postprandial lipid transport after a standard meal exceeds that observed in the fasting state and is influenced by numerous environmental and genetic factors affecting the synthesis and catabolism of TRL originating from the liver and intestine. Thus, decreased postprandial lipemia has been shown in apoA-I Milano carriers (42). Other common variants at the apoB (15) and apoE gene loci have also been shown to affect the absorption or clearance of dietary fats (14) (43) (44), with E2 individuals having delayed clearance and with E4 individuals having faster clearance as shown by retinyl palmitate concentrations in plasma and the non-chylomicron fraction associated with a higher LDL-C response to changes in dietary fat. In order to remove the confusing effect of variability associated with the apoE gene locus, this study was carried out on subjects homozygous for the apoE3 allele. Moreover, there were also no differences in other factors that could influence postprandial lipemic response such as age, BMI, alcohol and tobacco consumption between carriers of the AIV-347Ser allele and homozygous for the AIV-347Thr allele.

ApoA-IV is a major constituent of the triglyceride-rich particles of intestinal origin. ApoA-IV is involved in the absorption of exogenous fats (23) (45) and its synthesis is stimulated by the absorption of dietary triglycerides (46) (47). Zaiou et al. (48) found that much of the variability in apoA-IV concentration is due to genetic factors. Mutations of the apoA-IV gene bring about a different lipid response to diet. The 360His mutation has been associated with a hyporesponse to changes in dietary fat and cholesterol (49) (50). Moreover, we have shown that the 347Ser mutation of the apoA-IV gene is associated with a greater increase in total cholesterol, LDL-C and apoB levels after the consumption of diets rich in saturated fats (33). We, therefore, decided to study whether this effect was associated with changes in the postprandial lipoprotein metabolism.

Carriers of the A-IV-347Ser allele present a smaller postprandial lipemic response both in chylomicrons and in their remnants. These effects are associated with elevated apoA-IV levels in chylomicrons in these subjects and these findings could be related to several factors. In the first place, the differences in postprandial lipemic response could be due to changes in the absorption of dietary fat and cholesterol as previously shown in association with apoE polymorphism. However, recently it has been demonstrated that the 360His mutation of the apoA-IV gene does not cause changes in the degree of absorption of dietary cholesterol (51). It is, therefore, unlikely that the 347Ser mutation, which is located very near to the previous mutation, could be associated with changes in the degree of absorption of dietary fat and cholesterol. Secondly, it is also possible that the differences in postprandial lipoprotein metabolism observed in this study are the result of a faster clearance of triglyceride-rich particles of intestinal origin. The higher apoA-IV levels observed in the large TRL supports this concept since the apoA-IV secreted in the chylomicrons could regulate the activation of the LPL and, therefore, their hydrolysis. LPL acts in two ways, first by promoting hydrolysis of large TRL triglycerides and second by facilitating hepatic uptake of these particles (52). Goldberg et al. (53) reported that in the presence of HDL, apoA-IV facilitates apoC-II catalysis of LPL by increasing transfer of apoC-II from HDL to substrate particles. Thus, the higher concentrations of apoA-IV on triglyceride-rich particles in the apoA-IV Ser/+ subjects would seem to indicate that less apoC-II has been transferred from HDL. This observation could be a consequence of faster triglyceride clearance, leaving smaller particles, as suggested by the trend toward lower TG/apoB-100 ratios in both TRL fractions observed in apoA-IV Ser/+ subjects. This faster hydrolysis of the chylomicrons by LPL could also favor hepatic uptake of the remnants, by the binding action of this enzyme to the hepatocyte. The immediate consequence would be increased hepatic uptake of cholesterol within the chylomicron remnants of dietary origin, which would bring about an increase in hepatic cholesterol content and a decrease in hepatic expression of LDL receptors. These events result in a greater increase in LDL-C levels in response to a high fat diet as we observed in previous studies in carriers of the mutation. Alternatively, the increased levels of apoB-100 in TRL observed in carriers of the mutation could be due to greater VLDL synthesis by the liver caused by an increase in the amount of fatty acids resulting from accelerated hydrolysis of chylomicrons and uptake of remnants which occurs in these subjects. Both phenomena could explain the increased response in LDL-C levels observed previously in carriers of the A-IV 347Ser mutation after consumption of a saturated fat rich diet (33).

At present, we do not know the mechanisms by which this mutation can cause an increase in apoA-IV levels in TRL. However, this could be due to an increase in the degree of intestinal synthesis of this apolipoprotein or changes in the degree of distribution between its different lipoprotein particles. In the first place, it is not very likely that a mutation that produces changes in the primary structure of the protein and is not located in regions which regulate expression of the apoA-IV gene could change the rate of apoA-IV production. Thus, the fact that differences were not observed in the total plasma levels of apoA-IV suggests that this phenomenon could be caused by a different distribution of the apoA-IV synthesized in the intestine. Thus, apoA-IV 347Ser can regulate the affinity of this apolipoprotein for the TRL so that it remains there for a longer time before being released into the free fraction of the plasma apolipoprotein, therefore increasing the possibility of it being exchanged with the apoC-II of the HDL. In addition, it should be noted that although an effect of apoA-IV 347Ser on apoA-IV lipid affinity is a plausible mechanism by which apoA-IV 347Ser affects the intravascular metabolism of triglyceride-rich lipoproteins, the effect of the Ser347 substitution on the structure or function of apoA-IV is unknown at present.

In accordance with these findings, the 347Ser mutation of the apoA-IV gene causes higher levels of apoA-IV to be transported in chylomicrons resulting in the rapid hydrolysis and hepatic uptake of these particles of intestinal origin. This would lead to two important effects which could explain the greater increase in LDL-C observed in these subjects after a high fat diet: an increase in the intrahepatic cholesterol pool, which would result in decreased expression of LDL receptors, and an increase in the production of VLDL, from which LDL are derived.

	ACKNOWLEDGMENTS

This work was supported by a grant from the Consejeria de Agricultura y Pesca, Consejeria de Educacion y Ciencia, Junta de Andalucia and the Spanish Ministry of Health (FIS 94/1547, 93/0746, 95/1144, 96/1540, 98/1531) (to J. L-M.), Fundación Cultural Hospital Reina Sofia-Cajasur and HL54776 (to J. M. O.) the National Institutes of Health, Bethesda, MD.

Manuscript received December 18, 1997; and in revised form May 27, 1998; and in revised form August 24, 1998.

Abbreviations: HDL, high density lipoproteins; LDL; low density lipoproteins; TRL, triglyceride-rich lipoproteins; RP, retinyl palmitate; PCR, polymerase chain reaction; AUC, area under the curve; TG, triglyceride

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TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

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