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Clinical Chemistry 51: 864-871, 2005. First published March 11, 2005; 10.1373/clinchem.2004.044347
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2005;51:864-871.)
© 2005 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Additive Effects of the PPAR{gamma}, APOE, and FABP-2 Genes in Increasing Daylong Triglycerides of Normolipidemic Women to Concentrations Comparable to Those in Men

Josep Ribalta1,a, Constantijn J.M. Halkes2, Juliana Salazar1, Lluís Masana1 and Manuel Castro Cabezas2,3

1 School of Medicine, Universitat Rovira i Virgili, Reus, Spain.
2 Departments of Internal Medicine and Endocrinology, University Medical Center Utrecht, Utrecht, The Netherlands.
3 St. Franciscus Gasthuis, Rotterdam, The Netherlands.

aAddress correspondence to this author at: Unitat de Recerca de Lípids i Arteriosclerosi, Facultat de Medicina, Universitat Rovira i Virgili, Sant Llorenç, 21, 43201 Reus, Spain. Fax 34-977-759322; e-mail josep.ribalta{at}urv.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Fasting and postprandial triglyceride (TG) concentrations vary considerably among individuals. TG metabolism is more efficient in women than in men, which may partly explain why females are protected against atherosclerosis. Our aim was to identify gender-specific genetic influences on fasting and postprandial TG concentrations under typical living conditions in healthy, lean, normolipidemic women.

Methods: We studied 40 women and 48 men. Diurnal capillary TG profiles were calculated as the integrated area under the capillary TG curve averaged over 3 days. Genotypes of the FABP-2, HL, LPL, APOE, and PPAR{gamma} genes and the APOC-III, APOC-III/A-IV intergenic region were determined.

Results: Three genes (FABP-2, APOE, and PPAR{gamma}) had a significant additive effect only in women. Mean TG concentrations were fourfold higher in women carriers of the PPAR{gamma} wild-type allele (P = 0.044), threefold higher in carriers of the rare FABP-2 allele (P = 0.006), and fivefold higher in carriers of the E2 allele of the APOE gene (P = 0.037) than in noncarriers. None of these effects was observed in men. The presence of two or more of these adverse alleles increased TG concentrations in a dose-dependent manner. Women carriers of three adverse alleles had postprandial TG values comparable to those for men.

Conclusions: An adverse combination of common alleles of the FABP-2, APOE, and PPAR{gamma} genes in women increases their TG concentrations to values comparable to those seen in men. Although this influence is not appreciable when studying fasting plasma TGs, it becomes apparent with use of a more sensitive index such as measurements made throughout the day.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The plasma triglyceride (TG)1 concentration is a cardiovascular risk factor. Epidemiologic studies have shown a direct relationship between plasma TG concentrations and the risk of coronary heart disease in both men and women (1)(2). Women have lower fasting and postprandial plasma TG concentrations and better control of TG metabolism (3), and this may partly explain their lower risk of cardiovascular disease. Compared with men, lean women have better clearance of VLDL TGs (4) and much higher lipoprotein lipase (LPL) activity in both skeletal muscle and subcutaneous adipose tissue (5)(6).

The molecular mechanisms underlying these differences are not clear, but they are most likely related to gene variability and gender-specific differences in gene regulation. Many genes are involved in the regulation of exogenous and endogenous TGs (7)(8). Some of the best described gene products act during intestinal absorption of dietary fat [fatty acid-binding protein-2 (FABP-2)], the hydrolysis of TG-rich lipoproteins [apolipoprotein (apo) C-III, LPL, or hepatic lipase (HL)], the storage of excess free fatty acids [peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})], or the removal of remnant lipoproteins (apo E).

Most of the time, humans are in a postprandial state, and although there is a strong correlation between fasting and postprandial TG concentrations, it is conceivable that the latter better reflects TG metabolism. Among the different methodologies used to assess postprandial TG concentrations, namely, oral fat tolerance tests and serial measurements of capillary TG concentrations thought the day, it is of note that the latter reflects TG changes in typical living conditions (3). Serial daylong TG profiles reveal important metabolic disturbances in certain groups of patients (9)(10)(11)(12)(13), and intraindividual variability is far smaller for daylong TG profiles than for fasting plasma TG values (10)(14).

The objective of the present study was to identify gender-specific genetic influences on fasting and postprandial TG concentrations under typical living conditions in lean, normolipidemic men and women. By studying common single-nucleotide polymorphisms in the genes listed above, we have been able to identify three genes, FABP-2, APOE, and PPAR{gamma}, that have an additive effect on daylong TG concentrations only in women and cause their TG values to resemble those of men.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
Healthy men and women from The Netherlands between the ages of 20 and 55 years were recruited by advertisement. All participants were recruited by the University Medical Center Utrecht. Exclusion criteria were age <20 or >55 years, a body mass index (BMI) >30 kg/m2, smoking, alcohol intake >2 units/day, renal and liver diseases, fasting plasma glucose >6.1 mmol/L, current use of lipid-lowering medication, a family history of premature myocardial infarction (at <55 years of age), and in females, the use of oral estrogens. Women were classified as postmenopausal if they had been amenorrheic for at least the 12 months before inclusion, if they had serum follicle-stimulating hormone concentrations >35 IU/L, and if they were not using estrogen replacement therapy.

Blood pressure and the waist-to-hip ratio were measured on the morning of inclusion in the study, and venous blood was drawn for analyses. Participants were required to fast overnight for at least 12 h and to abstain from alcohol use for at least 48 h before the morning of inclusion.

assessment of capillary blood tg concentrations
Participants performed self-testing of capillary blood TGs with a TG-specific point-of-care testing device (Accutrend GCT®; Roche Diagnostics) (3)(15). They received instructions at the outpatient clinic on how to conduct the self-assessment at home and were instructed to wash and dry their hands thoroughly before each measurement. A drop of blood (30 µL) from the finger was obtained with a lancet, squeezed on the TG test strip, and introduced into the TG analyzer. The analyzer measures TG concentrations by dry chemistry (capillary blood) or colorimetry (plasma). In the case of insufficient blood sample on the test strip, the participants were advised to repeat the measurement. Each was instructed in the whole procedure by the same investigator. The range of measured TG concentrations was 0.80–6.86 mmol/L, with CVs of 3.3–5.3% (15)(16). The correlation coefficient for capillary TG measurements with the TG strip analyzer compared with conventional laboratory plasma measurements by enzymatic methods was 0.94 (3)(12), and the daylong capillary TG profiles correlated with postprandial lipemia as assessed by standardized oral fat loading tests (12). Daylong triglyceridemia, estimated by six measurements a day, was not different from the hourly measurements, suggesting that these six time points are representative of the daylong study period (17).

Participants were instructed to measure their capillary blood TGs on three different days (preferably Mondays, Wednesdays, and Fridays to avoid the weekend) at the following time points: fasting, 3 h before and after lunch, 3 h before and after dinner, and on retiring to bed for the night. The 3-h postprandial measurements were performed exactly 3 h after meals regardless of the intake of snacks. The participants recorded all results in a diary. They were requested to refrain from any unusual physical activity and to maintain their usual activities, such as cycling to work. Females with a regular menstrual cycle were asked to perform the TG measurements during the second week of the cycle.

When one or more measurements were missed during a day, the data for that particular day were not used for constructing the "averaged" daylong TG profile. In 16 participants, mean diurnal capillary TG profiles were based on two instead of three days because of missing TG values for one day. When the value recorded was at the lowest detection limit and was described as "LOW" by the analyzer, the value 0.80 mmol/L was used in calculating daylong triglyceridemia. The averaged daylong TG profile was used for all statistical analyses.

laboratory analyses
Lipids, apolipoproteins, insulin, and glucose were measured in the plasma obtained from the fasting blood sample.

Plasma cholesterol and TGs were measured in duplicate by colorimetric assays with the CHOD-PAP and GPO-PAP reagents (Roche), respectively. HDL-cholesterol was measured according to Gidez et al. (18). Plasma apo B was measured by nephelometry using anti-apo B monoclonal antibodies (OSAN 14/15; Behring Diagnostics NV). Plasma apo AI was measured by nephelometry using anti-apo AI monoclonal antibodies (OUED 14/15; Behring Diagnostics NV). Glucose was measured by glucose oxidase dry chemistry (Vitros GLU slides) and colorimetry. Insulin was measured by competitive RIA with polyclonal antibodies. Plasma free fatty acids were measured by an enzymatic colorimetric method (Wako Chemicals GmbH and Neuss) (19).

genotyping
DNA was isolated from 10-mL EDTA-blood samples by standard procedures. DNA was amplified in a 25-µL reaction volume containing 1.25 mM deoxynucleotide triphosphates, 100 nM each primer, and 1.5 mM MgCl.

PCR amplifications and genotype determinations were conducted as follows:

FABP-2 (Ala54Thr).
The primers were as follows: forward, 5'-ACAGGTGGTAATATAGTGAAAAG-3'; reverse, 5'-TACCCTGAGTTCAGTTCCGTC-3'. The thermal cycling conditions were denaturation at 94 °C for 4 min and 33 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 3 min. All 180-bp fragments were digested overnight with HhaI restriction enzyme, and the digestion products were resolved by 4% agarose gel electrophoresis.

HL (C–480T).
The primers were as follows: forward, 5'-AAGAAGTGTGTTTACTCTAGGATCA-3'; reverse, 5'-GGTGGCTTCACGTGGCTGCCTAAG-3'. The thermal cycling conditions were denaturation at 94 °C for 4 min and 33 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 3 min. All 299-bp PCR products were digested overnight with NlaIII restriction enzyme at 37 °C, and the digestion products were resolved by 3% agarose gel electrophoresis.

APOE.
The primers were as follows: forward, 5'-ACAGAATTCGCCCCGGCCTGGTACAC-3'; reverse, 5'-TAAGCTTGGGCACGGCTGTCCAAGGA-3'. The thermal cycling conditions were denaturation at 94 °C for 4 min and 33 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 3 min. Digestion was with HhaI restriction enzyme, and the digestion products were resolved by 2% agarose gel electrophoresis.

APOCIII-AIV intergenic region (T–2854G).
A restriction enzyme site was "forced" for the T–2854G polymorphism by incorporation of a single base change into one of the primers used in the PCR reaction. The primers were as follows: forward, 5'-CAACAGGAGCTGTCCTTCAGTTCTGCC-3'; reverse, 5'-GGTCAGTCCAGAGGTCAGAGTCAGGAGGAG-3' (the underlined base indicates the forced restriction site).

After an initial denaturation of 5 min, denaturation was at 95 °C for 1 min, annealing was at 58 °C for 1 min, and extension was at 72 °C for 30 s for 35 cycles. PCR products were digested overnight with 2 U of Alw26I (MBI) at 37 °C, and the digestion products were resolved by 2% agarose gel electrophoresis.

APOC-III (IRE).
The primers were 5'-GGTGCTGGGAGGGGCGGTGAGAGCTCAGCC-3' (forward) and 5'-CCCTCCACCAGCCCCAAGCCCGGAACACAG-3' (reverse). A two-step PCR was used with a combined annealing and extension step of 72 °C for 1 min. The PCR product was digested with 2 U of BstNI restriction enzyme (NEB) for 4 h at 60 °C, and the digestion products were resolved by 2% agarose gel electrophoresis.

LPL (N291S).
The primers were as follows: forward, 5'-GCCGAGATACAATCTTGGTG-3'; reverse, 5'-TTGCTGCTTCTTTTGGCTCTGACTTTTGT-3' (wild type) and 5'-GAGTCTTCAGGTACATTGGGCTGCTTCTTTTGGCTCTGACTTGAC-3' (mutant).

The thermal cycling procedure for the PCR was as follows: denaturation for 4 min at 94 °C, followed by 33 cycles of denaturation for 60 s at 94 °C, annealing for 60 s at 52 °C, and extension for 3 min at 72 °C. This PCR yields several products: homozygous wild type (two 234-bp fragments), heterozygous (a 234- and a 254-bp fragment), and homozygous mutant (two 254-bp fragments). The possible outcomes are homozygous wild type (234 bp), heterozygous (234 and 254 bp), and homozygous mutant (254 bp).

PPAR{gamma} (Pro12Ala).
The primers were 5'-GCCAATTCAAGCCCAGTC-3' (forward) and 5'-GATATGTTTGCAGACAGTGTATCAGTGAAGGAATCGCTTTCCG-3' (reverse; the underlined base indicates the forced restriction site). The reverse primer forces a restriction site for BstUI when the polymorphism is present. PCR conditions were as follows: 1 cycle of 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension of 72 °C for 7 min.

The possible outcomes were homozygous wild type (270 bp), heterozygous (270, 227, and 43 bp), and homozygous mutant (227 and 43 bp).

statistical analyses
Data are presented as the mean (SD). Differences between groups obtained with unadjusted data were tested with the Student t-test. ANOVA was performed to compare the means of all variables between genotypes, using adjusted data (covariates; see below). In the case of TG concentrations, calculations were performed on log-transformed values although nontransformed concentrations are shown in Table 1 and the figures. Daylong capillary blood TG profiles were calculated as the mean integrated area under the curve (AUC) and are reported as h · mmol/L. Incremental daylong triglyceridemia was calculated as the change in AUC for capillary TGs after adjustment for fasting capillary TG concentrations. In the original report of the daylong triglyceridemia assessment, Castro Cabezas et al. (3) indicated that it was influenced mainly (i.e., explaining 72% of the observed variation) by fasting capillary TG concentrations, gender, systolic blood pressure, and mean daily energy intake. Incremental daylong triglyceridemia was best described by the variables gender, mean daily protein intake, and systolic blood pressure (i.e., explaining 42% of the observed variation). We therefore used these variables as the covariates in ANOVA analyses comparing genotypes with the AUCs for capillary TGs or incremental daylong triglyceridemia. SPSS (Ver. 11.0) was used for all statistical analyses, and AUCs were calculated with GraphPad Prism (Ver. 3.0). A two-sided P value <0.05 was considered statistically significant.


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  		Table 1. Anthropometric and biochemical data for the individuals (n = 88) recruited into the study.1


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
general characteristics of the participants
Forty young, lean, normolipidemic women were included in this study. For comparison purposes, we studied a group of 48 men with comparable characteristics (Table 1Up ) and for whom the same data had been obtained. Anthropometric and biochemical data showed that waist-to-hip ratio and plasma TG, cholesterol, apo B, and free fatty acid concentrations were significantly higher in men, whereas HDL-cholesterol and plasma glucose concentrations were significantly increased in women (Table 1Up ).

daylong tg profiles
The daylong TG profiles were compared between the men and the women. Because fasting TG concentrations largely determine the postprandial response, the variable incremental daylong triglyceridemia, which is calculated as the AUC for capillary TGs adjusted for fasting TG concentration, was the preferred variable for assessment of the influence of genetic variants on postprandial TG concentrations.

Using the appropriate adjustment (3), we observed that, relative to women, men had significantly higher mean (SD) AUCs for capillary TGs [23.68 (6.78) vs 15.80 (4.04) mmol/L; P <0.0001; Fig. 1 ] and incremental daylong triglyceridemia [6.96 (4.86) vs 1.30 (3.33) h · mmol/L; P <0.0001]. Except for the fasting values, capillary blood TG values were significantly higher in men than in women at all time points (Fig. 1 ).



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  		Figure 1. Capillary TG concentrations measured at six fixed times of the day.

<lunch, 3 h before lunch; >lunch, 3 h after lunch; <dinner, 3 h before dinner; >dinner, 3 h after dinner.

genotypes
We assessed nine common single-nucleotide polymorphisms of relevant genes affecting lipid metabolism to identify genetic determinants of TG metabolism in women. Some of the genes explored, such as HL, LPL, and the APOC-III, APOC-III/A-IV intergenic region, did not show any influence on daylong triglyceridemia in women (data not shown).

FABP-2 (ALA54THR)
The frequency of the rare Thr54 allele in women (0.28) was not different from the frequency in men (0.24). Neither plasma nor capillary TG concentrations were different between the genotypes. Women carriers of the Thr54 allele had a higher AUC for incremental daylong triglyceridemia [2.93 (2.46) h · mmol/L] than carriers of the wild-type allele [–0.10 (3.46) h · mmol/L; P = 0.006]. Such an effect was not observed in men [wild-type vs rare, 7.10 (5.13) vs 6.77 (4.59) h · mmol/L; Fig. 2 ].



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  		Figure 2. Concentrations of capillary TGs adjusted for fasting capillary TGs (TGc-dAUC).

WT, wild-type Ala54 allele; rare, rare Thr54 allele.

PPAR{gamma} (PRO12ALA)
The frequencies of the rare Ala12 allele were 0.13 and 0.11 in women and men, respectively. Neither plasma nor capillary TG concentrations were different between genotypes.

Women carriers of the rare Ala12 allele had lower AUCs for incremental daylong triglyceridemia [0.39 (1.96) h · mmol/L] than those with the wild-type genotype [1.60 (3.64) h · mmol/L; P = 0.037]. Again, this effect was absent in men [wild-type vs rare, 7.01 (4.82) vs 6.76 (5.28) h · mmol/L; Fig. 3 ]. In relation to other biochemical variables, fasting plasma glucose was significantly lower in Ala12 females [4.90 (0.70) mmol/L] than in noncarriers [5.53 (0.58) mmol/L; P = 0.006].



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  		Figure 3. Concentrations of capillary TGs adjusted for fasting capillary TGs (TGc-dAUC).

WT, wild-type Pro12 allele; rare, rare Ala12 allele.

APOE ({epsilon}2, {epsilon}3, {epsilon}4)
The allele frequencies of the {epsilon}2, {epsilon}3, and {epsilon}4 alleles in men were 0.06, 0.83, and 0.16, respectively, and in women were 0.08, 0.74, and 0.18, respectively. Neither plasma nor capillary TG concentrations were different between genotypes.

The AUC for incremental daylong triglyceridemia was significantly higher [4.54 (2.63) h · mmol/L; P = 0.037] in women carriers of the {epsilon}2 allele than in carriers of the {epsilon}3 [0.82 (3.45) h · mmol/L] and {epsilon}4 alleles [0.81 (2.24) h · mmol/L]. The effect was not observed in men (Fig. 4 ).



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  		Figure 4. Association of increased incremental daylong triglyceridemia (dTGc-AUC) with APOE genotype in women (left) and men (right).

additive effects of genes
The results indicate that each of the alleles FABP-2 Thr54, PPAR{gamma} Pro12, and APOE {epsilon}2 has a TG-increasing potential that affects daylong TG metabolism, specifically in women. To explore whether their influence may be additive, we compared daylong TG profiles in women carriers of none, one, two, or three of the "adverse" alleles. The AUCs for incremental daylong triglyceridemia, expressed as h · mmol/L, were 1.31 (3.44), –0.71 (3.69), 2.73 (1.90), and 5.27 (3.42), respectively, and increased significantly with the number of alleles (R = 0.401; P = 0.009), particularly when two or more TG-increasing alleles were present (Fig. 5 ). This effect was not observed with fasting plasma TG concentrations. Analysis of the data for men indicated that although women carriers of none, one, or two alleles had AUCs for incremental daylong triglyceridemia that were significantly lower than the AUCs for men [7.00 (4.80) h · mmol/L], those females carrying three alleles had daylong TG profiles comparable to those of men (Fig. 5 ). No allele seemed predominant because representation of the different TG-increasing alleles was comparable in each of the categories.



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  		Figure 5. Association of increased incremental daylong triglyceridemia (dTGc-AUC) with the FABP-2 Thr54, APOE {epsilon}2, and PPAR{gamma} Pro12 alleles in comparison with the other alleles in the study population.

(Left), additive effect of alleles in women compared with men. (Right), lack of additive effect of alleles in men. NS, not significant.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that an adverse combination of common alleles of the FABP-2, APOE, and PPAR{gamma} genes in women increases their daylong TG concentrations to values comparable to those in men. Our results also show that although this influence is not appreciable when fasting plasma TGs are used to assess TG metabolism, it becomes apparent when a more sensitive indicator, such as daylong TG concentrations, is used.

We based our strategy on two aspects. One was the recruitment of a "normal" population. Our volunteers were in their 30s, had a BMI <23 kg/m2, and had a normal lipid profile. In this group, therefore, the metabolic challenge to genes was reduced to the minimum, and great genetic influences were not expected to be present. We hypothesized that, conversely, this would have the advantage that in this population, any gene or combination of genes influencing TG metabolism could be considered relevant. We complemented this approach with what we believe is an appropriate method to assess TG metabolism in free-living persons, i.e., the determination of daylong capillary TG concentrations. It allows assessment of TG concentrations during the day, in contrast to the acute response to a nonphysiologic oral fat load in the oral fat tolerance test.

FABP-2, apo E, and PPAR{gamma} are key players in TG metabolism. FABP-2 is an intestinal protein involved in long-chain fatty acid absorption and metabolism. More than 25% of the population has a common variant, Ala54Thr, which is associated with rather negative effects, such as impaired glucose tolerance (20), altered lipoprotein profiles in response to diet (21), and altered postprandial triglyceridemia (22). Apo E is the strongest genetic determinant of lipoprotein metabolism. Apo E has three common isoforms, E2, E3, and E4, with frequencies in Caucasians of 0.08, 0.77, and 0.15, respectively. It has a consistent influence on plasma cholesterol concentrations, higher in {epsilon}4 and lower in {epsilon}2 than in {epsilon}3 individuals. The effect on TGs is less consistent, although {epsilon}2 and {epsilon}4 carriers tend to have increased TG concentrations (23)(24). PPAR{gamma} is a transcription factor involved in adipogenesis and the regulation of adipocyte gene expression, for example, relative to the storage and mobilization of TGs. More than 10% of the population carries the Pro12Ala variant, which is associated with a beneficial effect on BMI and insulin sensitivity (25). Globally, all three genes have a significant influence on the three steps of TG metabolism: absorption, transport, and storage.

The aim of this study was to investigate the genetic determinants of TG metabolism in women, and as a consequence, we focused on the three genes that showed a significant influence on postprandial TG concentrations. Although there is great variability of gene effects, the gender specificity reported here is in accordance with existing evidence.

In our study, women carriers of the Thr54 allele had higher daylong TG concentrations than noncarriers, but this was not observed among men. Agren et al. (22) reported an increased postprandial TG response in carriers of the Thr54/Thr54 genotype for the FABP-2 gene selected a priori, including 60% of females. However, when this polymorphism was analyzed in a much larger group of young males (n = 666), the effect on postprandial TG concentrations was not observed (17).

More contradictory are the results of apo E in relation to gender. Whereas the influence of apo E on total and LDL-cholesterol is typically more pronounced in men than in women, essentially because of hormonal regulation of the APOE gene (26), our results indicate that women carriers of the {epsilon}2 genotype have higher postprandial TG concentrations, this association not being significant among men. The most important factor to explain this may be that whereas the association between APOE genotype and plasma cholesterol seems invariant, the association with nonfasting TG concentrations seems a lot more context-dependent (27). This might indicate that in a young healthy population, the influence of the APOE gene is stronger in women than in men. Interestingly, in relation to vitamin A metabolism, which is tightly linked to postprandial TG concentrations, a recent report indicates that women carriers of the {epsilon}2 allele, but not men, have increased fasting vitamin A concentrations (28). Approximately 10% of the population carry the Ala12 variant of the PPAR{gamma} gene, providing effective protection against impaired glucose tolerance and diabetes (29). In our sample, only women benefited from this. In view of the critical role of PPAR{gamma} in adipocyte differentiation, the fact that women generally have a higher percentage of body fat than men (30) may help explain this observation.

Although abundant data on individual gene variants affecting lipid and lipoprotein metabolism are present in the literature, their usefulness for identifying individual profiles for cardiovascular risk is fairly limited. This is attributable to the small effect that a single gene has, which in addition can vary depending on factors such as gender, environmental stimuli, and other genes. Therefore, not taking into account, for example, gender influence or synergies between genes may lead to the false perception that in the general population only a given percentage of individuals are significantly influenced by genetic variants, whereas such influence is almost negligible in the rest. These data may in part explain the large interindividual variability in postprandial lipemia, whereas the differences in fasting TG concentrations are less pronounced. Genetic differences may explain why patients with coronary artery disease have increased postprandial lipemia despite the presence of fasting lipids within reference values or, conversely, that increased risk of coronary artery disease in genetically predisposed individuals might be caused by enhanced postprandial lipemia. The experimental characteristics of this study did not allow the inclusion of a large population; therefore, these results await confirmation with larger studies.

In conclusion, we report that in healthy, lean, young women, the additive effects of the FABP-2, APOE, and PPAR{gamma} genes increase daylong capillary TG concentrations. Such an increase may have a biological significance because it increases TG concentrations to values comparable to those found in men, thus neutralizing part of the protection that women have against coronary artery disease as a result of their more effective TG metabolism. We also show that this genetic influence could be unnoticed if only fasting plasma TG concentrations were assessed and that, conversely, it becomes apparent when assessing postprandial daylong capillary TG concentrations.


   Acknowledgments
 
This article is dedicated to the memory of our friend, beloved colleague, and outstanding scientist, Willem Erkelens. We gratefully acknowledge the Accutrend GCT meters and accessories provided by Roche Diagnostics (Mannheim, Germany). Josep Ribalta is a researcher of the Ramón y Cajal Program from the Spanish Ministry of Science and Education. This work was supported by grants from the Instituto de Salud Carlos III, RCMN (C03/08), genetic hyperlipidemias (G03/181), and Mediterranean diet (G03/140) and SAF2001-02781 (Madrid, Spain).


   Footnotes
 
1 Nonstandard abbreviations: TG, triglyceride; LPL, lipoprotein lipase; FABP-2, fatty acid-binding protein-2; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; apo, apolipoprotein; HL, hepatic lipase; BMI, body mass index; and AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Castelli WP. Cholesterol and lipids in the risk of coronary heart disease—the Framingham Heart Study. Can J Cardiol 1988;4(Suppl):5A-10A.
  2. Cullen P. Evidence that triglycerides are an independent coronary heart disease risk factor. Am J Cardiol 2000;86:943-949.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Castro Cabezas M, Halkes CJ, Meijssen S, van Oostrom AJ, Erkelens DW. Diurnal triglyceride profiles: a novel approach to study triglyceride changes. Atherosclerosis 2001;155:219-228.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  4. Mittendorfer B, Patterson BW, Klein S. Effect of sex and obesity on basal VLDL-triacylglycerol kinetics. Am J Clin Nutr 2003;77:573-579.[Abstract/Free Full Text]
  5. Simoneau JA, Veerkamp JH, Turcotte LP, Kelley DE. Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss. FASEB J 1999;13:2051-2060.[Abstract/Free Full Text]
  6. Arner P, Lithell H, Wahrenberg H, Bronnegard M. Expression of lipoprotein lipase in different human subcutaneous adipose tissue regions. J Lipid Res 1991;32:423-429.[Abstract]
  7. Busch CP, Hegele RA. Variation of candidate genes in triglyceride metabolism. J Cardiovasc Risk 2000;7:309-315.[Web of Science][Medline] [Order article via Infotrieve]
  8. Ordovas JM. Genetics, postprandial lipemia and obesity. Nutr Metab Cardiovasc Dis 2001;11:118-133.[Medline] [Order article via Infotrieve]
  9. Van Wijk JPH, Halkes CJM, Erkelens DW, Castro Cabezas M. Fasting and daylong triglycerides in obesity with and without type 2 diabetes. Metabolism 2003;52:1043-1049.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Delawi D, Meijssen S, Castro Cabezas M. Intraindividual variation of fasting plasma lipids, apolipoproteins and postprandial lipemia in familial combined hyperlipidemia. Clin Chim Acta 2003;328:139-145.[Medline] [Order article via Infotrieve]
  11. Van Wijk JPH, Halkes CJ, De Jaegere PP, Plokker HW, Erkelens DW, Castro Cabezas M. Normalization of daytime triglyceridemia by simvastatin in fasting normotriglyceridemic patients with premature coronary sclerosis. Atherosclerosis 2003;171:109-116.[Medline] [Order article via Infotrieve]
  12. van Oostrom AJ, Castro Cabezas M, Ribalta J, Masana L, Twickler TB, Remijnse TA, et al. Diurnal triglyceride profiles in healthy normolipidemic male subjects are related to insulin sensitivity, body composition and diet. Eur J Clin Invest 2000;30:964-971.[Medline] [Order article via Infotrieve]
  13. Geluk CA, Halkes CJ, de Jaegere PP, Plokker TW, Castro Cabezas M. Daytime triglyceridemia in normocholesterolemic patients with premature atherosclerosis and in their first-degree relatives. Metabolism 2004;53:49-53.[Medline] [Order article via Infotrieve]
  14. Van Wijk JP, Van Oostrom AJ, Castro Cabezas M. Fasting and non-fasting triglycerides in healthy Dutch males and females. Clin Chim Acta 2003;337:49-57.[Medline] [Order article via Infotrieve]
  15. Moses RG, Calvert D, Storlien LH. Evaluation of the Accutrend GCT with respect to triglyceride monitoring. Diabetes Care 1996;19:1305-1306.[Web of Science][Medline] [Order article via Infotrieve]
  16. Luley C, Ronquist G, Reuter W, Paal V, Gottschling HD, Westphal S, et al. Point-of-care testing of triglycerides: evaluation of the Accutrend triglycerides system. Clin Chem 2000;46:287-291.[Free Full Text]
  17. Tahvanainen E, Molin M, Vainio S, Tiret L, Nicaud V, Farinaro E, et al. Intestinal fatty acid binding protein polymorphism at codon 54 is not associated with postprandial responses to fat and glucose tolerance tests in healthy young Europeans. Results from EARS II participants. Atherosclerosis 2000;152:317-325.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  18. Gidez LI, Miller GJ, Burstein M, Slagle S, Eder HA. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure. J Lipid Res 1982;23:1206-1223.[Abstract]
  19. Meijssen S, Castro Cabezas M, Ballieux CG, Derksen RJ, Bilecen S, Erkelens DW. Insulin mediated inhibition of hormone sensitive lipase in vivo can not be overruled by endogenous catecholamines in healthy subjects. J Clin Endocrinol Metab 2001;86:4193-4197.[Abstract/Free Full Text]
  20. Baier LJ, Sacchettini JC, Knowler WC, Eads J, Paolisso G, Tataranni PA, et al. An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance. J Clin Invest 1995;95:1281-1287.
  21. Hegele RA. A review of intestinal fatty acid binding protein gene variation and the plasma lipoprotein response to dietary components. Clin Biochem 1998;31:609-612.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  22. Agren JJ, Valve R, Vidgren H, Laakso M, Uusitupa M. Postprandial lipemic response is modified by the polymorphism at codon 54 of the fatty acid-binding protein 2 gene. Arterioscler Thromb Vasc Biol 1998;18:1606-1610.[Abstract/Free Full Text]
  23. Dallongeville J, Tiret L, Visvikis S, O’Reilly DS, Saava M, Tsitouris G, et al. Effect of apo E phenotype on plasma postprandial triglyceride levels in young male adults with and without a familial history of myocardial infarction: the EARS II study. European Atherosclerosis Research Study. Atherosclerosis 1999;145:381-388.[CrossRef][Medline] [Order article via Infotrieve]
  24. Reznik Y, Morello R, Pousse P, Mahoudeau J, Fradin S. The effect of age, body mass index, and fasting triglyceride level on postprandial lipemia is dependent on apolipoprotein E polymorphism in subjects with non-insulin-dependent diabetes mellitus. Metabolism 2002;51:1088-1092.[CrossRef][Medline] [Order article via Infotrieve]
  25. Altshuler D, Hirschhorn JN, Klannemark M, Lindgren CM, Vohl MC, Nemesh J, et al. The common PPAR{gamma} Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet 2000;26:76-80.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  26. Ordovas JM, Mooser V. The APOE locus and the pharmacogenetics of lipid response. Curr Opin Lipidol 2002;13:113-117.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  27. Frikke-Schmidt R, Nordestgaard BG, Agerholm-Larsen B, Schnohr P, Tybjaerg-Hansen A. Context-dependent and invariant associations between lipids, lipoproteins, and apolipoproteins and apolipoprotein E genotype. J Lipid Res 2000;41:1812-1822.[Abstract/Free Full Text]
  28. Gomez-Coronado D, Entrala A, Alvarez JJ, Ortega H, Olmos JM, Castro M, et al. Influence of apolipoprotein E polymorphism on plasma vitamin A and vitamin E levels. Eur J Clin Invest 2002;32:251-258.[CrossRef][Medline] [Order article via Infotrieve]
  29. Deeb SS, Fajas L, Nemoto M, Pihlajamaki J, Mykkanen L, Kuusisto J, et al. A Pro12Ala substitution in PPAR{gamma}2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet 1998;20:284-287.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  30. Blaak E. Gender differences in fat metabolism. Curr Opin Clin Nutr Metab Care 2001;4:499-502.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]




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