PAPER Polymorphisms in the insulin response element of ... - Nature

International Journal of Obesity (2001) 25, 1012±1017 ß 2001 Nature Publishing Group All rights reserved 0307±0565/01 $15.00 www.nature.com/ijo

PAPER Polymorphisms in the insulin response element of APOC-III gene promoter in¯uence the correlation between insulin and triglycerides or triglyceride-rich lipoproteins in humans J Dallongeville1,2*, A Meirhaeghe1, D Cottel1, J-C Fruchart2,3, P Amouyel1 and N Helbecque1 1

Service d'EpideÂmiologie et de SanteÂ Publique et INSERM U-508, Institut Pasteur de Lille, Lille, France; 2DeÂpartement d'atheÂroscleÂrose, Institut Pasteur de Lille, Lille, France; and 3INSERM U-325; Institut Pasteur de Lille, Lille, France

OBJECTIVE: To assess whether the 7455 and 7482 mutations in APOC-III gene insulin response element affect the relationships between plasma insulin and triglyceride-rich lipoprotein levels. DESIGN: Population-based studies. SUBJECTS: The population sample was composed of 983 subjects (485 men and 498 women), aged between 35 and 65 y, randomly sampled from the electoral rolls in Northern France and strati®ed on gender and 10 y age groups. MEASUREMENTS: Plasma triglyceride, apolipoprotein C-III, apoB, LpC-III:B and LpE:B lipoprotein particles and insulin levels were measured. Two polymorphisms in APOC-III gene insulin response element (T?C at 7455 and=or C?T at 7482) were determined. RESULTS: Plasma insulin was positively correlated to triglyceride levels (P < 0.0001), apo C-III (P < 0.003), LpC-III:B (P < 0.0001), apoB (P < 0.0001) and LpE:B (P < 0.0001). This association differed signi®cantly according to APOC-III insulin response element polymorphisms. The relationship between insulin and LpC-III:B (P < 0.02) or apoB (P < 0.02) was greater in women bearing the C allele of 7455 than the T allele. Similarly, the relationship between insulin and LpC-III:B (P < 0.02) or LpE:B (P < 0.05) was greater in women bearing the T allele of 7482 than the C allele. There was no evidence for any effect in men. CONCLUSION: These results suggest that the relationship between plasma insulin and triglyceride-rich lipoprotein levels is partly in¯uenced by polymorphisms in APOC-III insulin response element. International Journal of Obesity (2001) 25, 1012 ± 1017 Keywords: triglycerides; apoC-III; insulin; lipoproteins

Introduction

Several studies have demonstrated a positive correlation between plasma insulin and triglyceride (TG) levels.1 The mechanism by which insulin increases triglyceride levels is complex, affecting very low density lipoprotein (VLDL)-apolipoprotein B2 and VLDL-TG production,3 fatty acid ¯uxes4 and lipase activities.5,6 In addition to these effects, insulin

*Correspondence: J Dallongeville, Service d'EpideÂmiologie et de SanteÂ Publique et INSERM U-508, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59019 Lille Cedex, France. E-mail: [email protected] Received 16 March 2000; revised 22 August 2000; accepted 19 September 2000

also decreases apolipoprotein (apo) C-III gene expression in diabetic rats7 and in cultured hepatocytes.8 apoC-III is a 96 amino-acid transferable apolipoprotein whose function is not fully known. In vitro evidence9 and transgenic animal experiments10,11 suggest that apoC-III is an important regulator of VLDL clearance. High levels of APOC-III have been demonstrated to inhibit triglyceride-rich lipoproteins (TRL) lipolysis and=or TRL cellular uptake. A number of polymorphic sites have been described in the promoter region of APOC-III gene.12 Two such sites are located in an insulin response element (IRE) at 7455 and 7482 from the transcription initiation site of APOC-III gene. In vitro, insulin decreases APOC-III gene expression. Mutations in the IRE at 7455 (T?C) and 7482 (C?T) result in

Polymorphisms in IRE of APOC-III J Dallongeville et al

the loss of APOC-III gene down-regulation by insulin.8 In the present study we tested the hypothesis that APOC-III IRE polymorphisms in¯uence the relationship between plasma insulin and TRL levels. This hypothesis was plausible because (1) insulin down regulates APOC-III gene expression, (2) mutations at the 7455 and 7482 loci impair insulinmediated regulation of apoC-III gene expression, and (3) apoC-III in¯uences TRL clearance.

Methods

Subjects The population sample was composed of 1195 subjects (601 men and 594 women), living in the urban community of Lille (Northern France), aged between 35 and 65 y, who were randomly sampled from the electoral rolls, and strati®ed on gender and 10 y age groups. This sample was recruited within the framework of the WHO-MONICA (World Health Organization Ð Monitoring of Trends and Determinants of Cardiovascular Disease) project. A questionnaire was completed, which included details on personal medical history, drug intake and lifestyle variables such as cigarette smoking, physical activity and alcohol consumption. Body mass index (BMI), waist-to-hip ratio and blood pressure were also recorded according to the procedure of the MONICA project.13 The present data were analyzed on a sample of this population (n 983) not treated for diabetes and dyslipidemia. The study was approved by the Ethics Committee of the Centre Hospitalier et Universitaire de Lille. Each individual signed an informed consent prior to the investigation. Laboratory methods A 20 ml fasting blood sample was drawn on disodium EDTA and centrifuged within 4 h. Lipid and lipoprotein levels were measured on fresh samples in a central laboratory at Purpan Hospital Biochemical Laboratory (Toulouse). Plasma TG were measured enzymatically (Boehringer Mannheim, Mannheim, Germany). Apolipoprotein B, C-III, LpC-III:B and LpE:B were quanti®ed as previously described.14 Genomic DNA was prepared from white blood cells by a `salting out' procedure.15 The promoter 7455 T?C mutation corresponds to the suppression of a FokI restriction site. The promoter 7482 C?T mutation corresponds to the suppression of a MspI restriction site. Both mutations were detected using the following procedure: PCR ampli®cations16 were 0 carried out using the primers 5 -GGCTGTGAGAGCT0 0 CAGCCCT-3 (sense strand) and 5 -TCACACTGGAATTT0 CAGGCC-3 (antisense) and annealing was at 58 C. After enzymatic digestion, the 7455 and 7482 genotypes were detected using agarose gel electrophoresis. For technical reasons some genotypes were missing (22 in the 7455 group and 24 in the 7482 group respectively). Statistical analysis The data were analyzed using the SAS statistical software (release 6.12, SAS Institute Inc., Cary, NC). Because of skewness, TG, APOC-III, LpC-III:B, LpE:B and insulin were log

transformed to approximate normal distribution. Statistical tests were carried out on the transformed values. Univariate regression equations and coef®cients were used to assess the association between insulin and lipid variable levels. The general linear model (GLM) procedure was used to evaluate the impact of genotype on insulin levels. The GLM was also performed to account for confounding variables. The in¯uence of IRE polymorphisms on the relationship between plasma insulin and lipid levels was assessed in a multivariate model using the interaction term between genotype and insulin levels as independent variable.

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Results

Plasma insulin concentration was positively correlated to TG (r 0.28; P < 0.0001), apoC-III (r 0.10; P < 0.003), LpC-III:B (r 0.18; P < 0.0001), apoB (r 0.19; P < 0.0001, Figure 1) and LpE:B (r 0.22; P < 0.0001) levels. In multivariate analysis these associations remained statistically signi®cant independently of confounding variables (age, gender, BMI, alcohol consumption and number of cigarettes smoked daily) suggesting that insulin has a direct in¯uence on TG, apoC-III, LpC-III:B, apoB and LpE:B levels. Earlier studies from our laboratory have shown that the impact of APOC-III polymorphisms on lipoprotein particles is in¯uenced by gender.17 Thus, the analysis of the in¯uence of the 7455 and 7482 polymorphisms on biological variables was carried out in men and women separately. There was no evidence for any statistically signi®cant difference in insulin levels between subjects bearing the 7455 or 7482 wild-type or mutated allele of APOC-III gene (Table 1). In order to test whether the relationship between insulin and lipid variables was dependent on the APOC-III IRE polymorphisms a multivariate analysis was carried out with lipid variables as independent variables and with insulin, APOC-III IRE polymorphisms and their product as dependent variables. In women, there was a statistically signi®cant interaction between insulin and APOC-III 7455 polymorphism for LpC-III:B (P < 0.02) and for apoB (P < 0.05) suggesting that the in¯uence on insulin on LpC-III:B and apoB levels was different in women bearing the common allele or the mutated allele. Similarly, there was a statistically signi®cant interaction between insulin and APOC-III 7482 polymorphism for LpC-III:B (P < 0.03) and LpE:B (P < 0.05). The slope of the regression line between plasma insulin and lipid variables was higher in women bearing the mutated alleles than in those with the wild-type allele (Figures 2 and 3). In men, there was no evidence for any statistically signi®cant interaction between insulin and the 7455 and 7482 APOC-III polymorphisms for any variable.

Discussion

The goal of the present study was to assess whether mutations in the IRE of APOC-III gene in¯uence the relationship between plasma insulin and TG, apoC-III or TRL levels in International Journal of Obesity


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Figure 1 Relationship between plasma deciles of insulin levels and plasma triglyceride, APOC-III, LpC-III:B and apoB levels in the whole sample. Values are mean s.e.m.

Table 1

Association between APOC-III gene polymorphism and insulin levels Men

7455 Insulin (mU=ml) 7482 Insulin (mU=ml)

T=T (180) 12.1 9.3 C=C (263) 11.8 8.3

T=C (229) 11.3 8.7 T=C (183) 11.9 10.9

Women

P C=C (68) 13.0 11.9 T=T (30) 12.5 9.8

NS NS

T=T (182) 11.2 5.7 C=C (261) 11.4 5.9

T=C (245) 11.4 5.7 T=C (204) 11.0 5.2

P C=C (57) 11.1 5.9 T=T (30) 12.4 6.9

NS NS

P, multivariate analysis of variance adjusting for age, BMI, smoking and alcohol consumption. Analysis performed on logtransformed data.

Figure 2 Relationship between plasma deciles of insulin and LpC-III:B according to 7455 and 7482 APOC-III IRE genotypes in women. Values are mean s.e.m. P-values are for the interaction term between APOC-III IRE genotypes and insulin.

humans. The results indicate that the relationship between insulin and apoB-containing TRL differs in women but not in men bearing the common or the mutated allele of APOCIII IRE. This ®nding supports the concept that APOC-III IRE gene polymorphisms in¯uence the relationship between International Journal of Obesity

insulin and TRL levels and that this effect is dependent on gender. In the present study we analyzed the effect of mutations in the IRE of APOC-III gene on the relationship between plasma insulin and TG, apoC-III or TRL levels in men and


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Figure 3 Relationship between plasma deciles of insulin and LpE:B according to 7455 and 7482 APOC-III IRE genotypes in women. Values are mean s.e.m. P-values are for the interaction term between APOC-III IRE genotypes and insulin.

women separately. This strategy was based on the following reasons. First, the levels of TG, apoC-III, LpC-III:B and LpE:B are usually higher in men than in women, indicating that factors related to gender Ð either constitutive or environmental Ð in¯uence the levels of these variables. Second, previous reports have shown an in¯uence of gender on the relationship between APOC-III gene polymorphisms and plasma lipid variables.18 ± 24 Differences in lifestyle, diet, alcohol consumption or smoking habits between men and women or the in¯uence of sexual hormones on lipoprotein metabolism may explain these differences. Finally, a study from our laboratory has shown that the impact of APOC-III IRE polymorphism on apoB-containing particles was different in men and women, suggesting a different regulation of these variables in men and women.17 Plasma insulin concentration was positively correlated to plasma TG, apoC-III, apoB, LpC-III:B and LpE:B levels. The association persisted after adjustment on confounding variables (age, gender, BMI, alcohol consumption and number of cigarettes smoked daily). The relationship between insulin and LpC-III:B, apoB and LpE:B appeared to be in¯uenced by APOC-III IRE polymorphism in women but not in men. This effect reached the level of statistical signi®cance for LpC-III:B and was less consistent for apoB and LpE:B, suggesting a subtle effect. The slopes of the regression lines between insulin and LpE-III:B or apoB were signi®cantly greater in women bearing the 7455 mutated allele (`C') than in those bearing the wild-type allele (`T'). Similarly, the slopes of the regression lines between insulin and LpC-III:B or LpE:B were greater in women bearing the 7482 mutated allele (`T') than in those bearing the wild-type allele (`C') (Figures 2 and 3). LpC-III:B and LpE:B lipoprotein particles represent a mixture of TRL which associates apoC-III or apoE with apoB at their surface.25 ApoB-containing TRL are heterogeneous with respect to size, density, lipid composition and apolipoprotein content.26,27 ApoB may be associated with other apolipoprotein including apoC-III, C-II, C-I or E.26,27 Compared to LpE:B, LpC-III:B is enriched in TG and depleted in cholesterol. The presence of apoC-III at the surface of LpC-III:B also reduces the ability of this particle to bind cellular receptors in vitro.28,29 Plasma lipoprotein particle levels are sensitive to changes in their constituent apolipoprotein. For instance the

concentration of LpC-III:B is increased after ingestion of a fatty meal25 and in patients with hypertriglyceridemia.30,31 As mentioned above the combined in¯uence of insulin and APOC-III polymorphism appear to be rather weak. This may explain the lack of statistically signi®cant effect of these factors on apoC-III levels. In plasma, apoC-III is associated with VLDL and HDL. Studies in transgenic animal models expressing human apoC-III have shown that apoC-III preferentially associates with VLDL.10 Thus a modest increase in apoC-III should primarily result in an increase in LpC-III:B particles levels in women bearing the mutated allele. These clinical ®ndings are supported by biological data which have shown that the 7455 T?C and the 7482 C?T mutations result in the loss of insulin-mediated APOC-III gene down-regulation in vitro. In women bearing the wildtype allele, higher levels of insulin are associated with modest down-regulation of APOC-III gene expression and thus in unchanged levels of LpC-III:B as compared to women with lower levels of insulin. In contrast, in women bearing the mutated allele the in¯uence of insulin on LpC-III:B levels is not compensated by a down regulation of APOC-III gene, resulting in increased levels of LpC-III:B. Earlier reports have described inconsistent associations between APOC-III polymorphisms and lipid levels. Increased levels of TG and LDL-cholesterol were reported in subjects carrying the 7455 C APOC-III allele in some,32,33 but not all studies,34,35 suggesting a complex relationship. One possible explanation for this complexity might be the confounding contribution of insulin which was not assessed in these studies. Therefore, the impact of APOC-III gene polymorphisms on lipid variables appears to be more complex than initially evaluated. The results of the present study indicate that insulin and gender are important covariates of this relationship. In conclusion, the results of the present study indicate that insulin modulates TG-rich lipoprotein concentrations through an interaction with APOC-III gene promoter polymorphisms in women. Individuals with a functional IRE of APOC-III appear to be protected against the insulindependent increase of LpC-III:B, compared to individuals carrying the non-functional allele. This is to our knowledge the ®rst demonstration that insulin regulates TRL levels in International Journal of Obesity


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humans in combination with APOC-III gene promoter polymorphisms. However, further studies are warranted to extend these observations to other population samples.

Acknowledgements The authors wish to thank Mrs V Codron, Mr X Hermant, Mr Eric BaugeÂ for technical assistance. Mrs A Meirhaeghe was supported by a grant from the MinisteÁre de I'Enseignement SupeÂrieur et de la Recherche. The WHO-MONICA population study developed in the North of France was supported by unrestricted grants from the Conseil ReÂgional du NordPas de Calais, ONIVINS, Parke-Davies Laboratory, the Mutuelle GeÂneÂale de I'Education Nationale (MGEN), Groupe Fournier, the ReÂseau National de SanteÂ Publique, the Direction GeÂneÂrale de la SanteÂ, the Institut National de la SanteÂ Et de la Recherche MeÂdicale (INSERM), the Institut Pasteur de Lille and the UniteÂ d'Evaluation du Centre Hospitalier et Universitaire de Lille.

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International Journal of Obesity