Atherosclerosis and Lipoproteins |
From the Service dEpidémiologie et de Santé Publique, INSERM U.508 (A.M., D.C., P.A., N.H.) and INSERM U.325 (G.M., J.A.), Institut Pasteur de Lille, Lille, France; Division of Medical Genetics (M.N., S.D.), University of Washington, Seattle; and Centre Hospitalier et Universitaire de Lille (P.A.), Lille, France.
Correspondence to Prof Philippe Amouyel, Service dEpidémiologie et de Santé Publique, INSERM U.508, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, 59019 Lille Cedex, France. E-mail Philippe.Amouyel{at}pasteur-lille.fr
| Abstract |
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Key Words: fatty acid transport proteins fatty acid binding proteins polymorphism association studies fatty acids
| Introduction |
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| Methods |
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Laboratory Methods
Glucose was measured by the glucose oxidase method (DuPont
Dimension). Plasma insulin was measured by radioimmunoassay
(BiInsuline, ERIA Pasteur). Plasma total cholesterol and
triglyceride levels were measured enzymatically (DuPont
Dimension).
SSCP Analysis
SSCP analysis was performed with the primers listed in
Table 1
. The 582-bp fragment
comprising exons 8 to 10 was digested by Sau3AI into 3
fragments before SSCP analysis. Briefly, polymerase chain
reactions (PCRs) contained 100 ng genomic DNA, 62.5 µmol/L of
each dNTP, 10 pmol of each primer, 2.5 mmol/L
MgCl2, 0.25 U Taq polymerase, and 0.1
µL
-[32P]dCTP (3000 Ci/mmol, 10 mCi/mL) in
a volume of 10 µL. Each sample was electrophoresed at 2 different gel
temperatures: 4°C or room temperature. After electrophoresis, gels
were transferred to Whatman 3MM paper and dried.
Autoradiography with Kodak BIO-MAX MS film was
performed at -70°C. PCR products that yielded aberrantly
migrating band patterns were subjected to automated dideoxy sequence
analysis.
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Genetic Analysis
Genomic DNA was prepared from white blood cells by a "salting
out" procedure.10 DNA amplification was performed by use
of PCR.11 The primers that were used to amplify the
mutated intronic sequence were derived from the genomic sequence of the
FATP1 locus and are listed in Table 1
. The PCR
conditions were as follows: 94°C for 1 minute, 56°C for 1 minute,
and 72°C for 1 minute for 30 cycles and a final extension at 72°C
for 10 minutes with 1.5 mmol/L MgCl2 and 5%
dimethyl sulfoxide. The intron 8 A/G polymorphism was detected by
using allele-specific oligonucleotide hybridization
with the following primers: 5'-TCCCCACACCCTGCCT-3' for the A allele
and 5'-TCCCCACGCCCTGCCT-3' for the G allele
(polymorphism underlined). Membranes were hybridized at 48°C for
1 hour, washed twice in 1x SSC and 10% SDS buffer for 5 minutes, then
washed in 0.5x SSC and 10% SDS buffer for 5 minutes, and finally
washed in 0.5x SSC and 10% SDS buffer at 49°C for 3 minutes.
Statistical Analysis
Complete results were obtained for 1144 subjects. The data were
analyzed by use of SAS statistical software (release 6.12, SAS
Institute Inc). We considered the statistical significance to be at the
0.05 level. Because of skewness, triglycerides, glucose,
and insulin data were logarithmically transformed to achieve normal
distributions. Statistical tests were carried out on the transformed
values. The Pearson
2 test was used to compare
genotypic distributions and allelic frequencies between groups. When
necessary, the Fisher test of exact probability (2x2 table) was used
instead of the
2 test. Obesity markers and
plasma lipid, lipoprotein, or apolipoprotein levels were compared
between genotypes by an ANCOVA that used a general linear model
(GLM procedure, type III test).
| Results |
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We studied the effect of the A/G polymorphism on different
anthropometric and plasma lipid variables (Table 3
). No association was observed either
with anthropometric variables, such as body weight, BMI, and
waist-to-hip ratio, or with plasma insulin or glucose levels. However,
a significant association was observed between the intron 8 A/G
polymorphism and plasma triglyceride levels, which were
increased in the presence of the mutated allele, in an allelic
dose-dependent manner. Because our population was recruited to have the
same number subjects of each sex, we stratified the population
according to sex. The effect of the G/A polymorphism on plasma
triglyceride levels was observed mainly in women (11% in
AA subjects compared with GG subjects) and was
not statistically significant in men. No association was detected in
the obese subjects (BMI
30 kg/m2) or in NIDDM
subjects (data not shown).
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The association between the intron 8 G/A polymorphism and plasma lipid levels was further investigated by using multiple regression analysis in women (data not shown). The regression model was examined for plasma triglyceride and total cholesterol levels as dependent variables and for age, BMI, alcohol consumption, smoking status, and the polymorphism as independent variables. The model showed that BMI and alcohol consumption were the most significant determinants of triglyceride levels (P<0.001), with the G/A polymorphism contributing significantly to this model (P=0.03). Age, BMI, alcohol consumption, smoking status, and the intron 8 G/A polymorphism accounted for 29% of the plasma triglyceride level variance in women.
| Discussion |
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FATP1 is a 63-kDa plasma membrane protein mainly expressed in heart, testis, brain, and adipose tissue.3 Cell lines expressing FATP1 demonstrate a marked increase in the uptake of LCFA; this effect was much smaller for medium chain FAs (<C8), and no effect was observed for butyric acid (C4) for instance. It has been suggested that FATP1 could be a part, in association with other proteins such as FA translocase, of a multimeric complex that facilitates LCFA cellular uptake. Because this protein participates in LCFA cellular uptake, any defect in FATP1 affecting either the formation of the hypothetical complex mentioned above or FATP1 membrane anchoring or affecting the LCFA binding domain can inhibit this uptake, leading to an increase in plasma FA concentration.
It has been shown that an increase in plasma free FA levels stimulates VLDL production in humans.12 13 Free FAs are rapidly delivered to the liver, where they are processed to avoid accumulation of these substances. Processing includes ß-oxidation and reesterification, storage as triglycerides, and, ultimately, VLDL secretion.14 Such a mechanism could explain the observed association between the intronic A/G polymorphism in the FATP1 gene and an increase in plasma triglyceride levels (most triglycerides in plasma are carried in VLDLs). Although the triglyceride values still fell within the normal range, the observed association suggests an involvement of FATP1 in triglyceride metabolism. Because the FATP1 A/G polymorphism is located in the middle of intron 8, it is unlikely to be functional. This strongly suggests that the intronic A/G polymorphism may be in linkage disequilibrium, with an active mutation located, for instance, in the regulatory region of the FATP1 gene.
The association between the FATP1 A/G polymorphism and plasma triglyceride levels was mainly observed in women. This observation suggests that the regulation of plasma free FA levels and/or metabolism differs according to sex. This hypothesis has been confirmed by experiments in rodents: an increase of triacylglycerol synthesis was observed in isolated liver cells from female rats compared with male rats; concomitantly, FA oxidation was higher in male cells.15 16 Studies were also conducted in human lymphocytes and fibroblasts, in which highly significant differences were observed in FA esterification, oxidation, and FA composition of lipoproteins between male and female cells.17
In conclusion, the present study showed an association between an intronic polymorphism in the human FATP1 gene and an increase in plasma triglyceride levels, mainly in women. An increase of the same variable was observed when studying the Ala54Thr polymorphism of FABP2, an intestinal cytosolic FABP, in obese Finns.18 This last polymorphism has also been associated in nondiabetic Pima Indians with insulin resistance syndrome,19 whereas the FATP1 intron 8 polymorphism in the present study was not. We conclude from these results that FATP1 may be implicated in lipid metabolism, especially in women.
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| Acknowledgments |
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| Footnotes |
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Received July 13, 1999; accepted October 22, 1999.
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