Original Research

Obesity Research (2003) 11, 1502–1508; doi: 10.1038/oby.2003.201

Obesity Modulates the Association among APOE Genotype, Insulin, and Glucose in Men

Roberto Elosua*, Serkalem Demissie†, L. Adrienne Cupples†, James B. Meigs‡, Peter W.F. Wilson§, Ernst J. Schaefer*, Dolores Corella* and Jose M. Ordovas*

  1. *Nutrition and Genomics Laboratory and Lipid Metabolism Laboratory, Jean Mayer-U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  2. †Department of Biostatistics, Boston University School of Public Health and School of Medicine, Boston, Massachusetts
  3. ‡Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
  4. §The Framingham Heart Study, Framingham, Massachusetts

Correspondence: Jose M. Ordovas, Nutrition and Genomics Laboratory, JM-USDA-HNRCA at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail: jose.ordovas@tufts.edu

Received 3 March 2003; Revised  0000; Accepted 7 October 2003.

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Abstract

Objective: Obesity, insulin resistance, and apolipoprotein E (APOE) genotype have all been associated with coronary heart disease. We examined the interaction between obesity and APOE genotype in determining fasting insulin and glucose levels.

Research Methods and Procedures: From 1991 to 1995, 3799 subjects underwent a clinical examination and fasting insulin and glucose measurement. APOE genotypes were determined on 3500 participants. Participants taking oral hypoglycemic drugs or insulin preparations or with the rare APOE2/4 genotype were excluded. Finally, 2929 individuals were included in the present analysis.

Results: In men, we observed a statistically significant interaction between obesity and APOE genotype on insulin and glucose level (p = 0.003 and 0.008, respectively). Obese men with the APOE4 genotype presented with higher levels of insulin and glucose than obese men in the other genotype groups. No association between genotype and insulin or glucose in nonobese men was observed. Obesity was associated with higher insulin levels in the three APOE genotypes groups, whereas obesity was directly associated with glucose in those with the APOE4 genotype. In women, the effect of interaction between APOE genotype and obesity on fasting insulin and glucose was not statistically significant. Obesity was associated with higher levels of fasting insulin and glucose. APOE genotype was not associated with insulin or glucose.

Discussion: Obesity modulates the association between the APOE genotype and fasting insulin and glucose levels in men. Although weight control is important in all people, it may be especially important in APOE4 men to modify potentially elevated fasting insulin and glucose levels.

Keywords:

APOE genotype, insulin, glucose, interaction

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Introduction

Hyperinsulinemia has been associated independently with a higher risk of incident coronary heart disease (CHD)1 (1, 2, 3), primarily in men (4, 5). However, a direct relationship between insulin and CHD remains controversial because effects of insulin may be mediated by correlated risk factors, other comorbidities, and genetic factors (6, 7).

Apolipoprotein E (apoE) is a plasma protein modulating metabolism of plasmatic lipoproteins, particularly apoB-containing lipoproteins. The APOE locus is polymorphic, with three major alleles (epsilon2, epsilon3, and epsilon4) encoding the six most common isoforms. This genetic variation significantly affects plasma lipoproteins concentrations. The presence of the epsilon4 allele is associated with elevated low-density lipoprotein (LDL) cholesterol, whereas the presence of the epsilon2 allele is associated with decreased LDL cholesterol (LDL-C) (8). Moreover, epsilon2 and epsilon4 have also been found to be associated with elevations in plasma triglycerides (9) and/or higher cardiovascular disease risk (8, 10, 11). Conversely, the association between APOE genotype and insulin resistance and diabetes has been addressed in several studies, including our own (12), with negative results (13, 14). The aim of the current study was to determine whether the association between APOE genetic variation and fasting insulin and glucose levels is modulated by obesity.

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Research Methods and Procedures

Study Population

Participants in the Framingham Offspring Study, a long-term community-based prospective observational study of risk factors for cardiovascular diseases, were included in this study (15). From January 1991 through June 1995 (examination cycle 5), 3799 participants fasted overnight; each had standardized medical history and physical and laboratory examination. From these, 3500 participants had the APOE genotype determined. For these analyses, 118 participants were excluded for taking oral hypoglycemic drugs or insulin preparations, and 404 were excluded because no valid information was collected for variables of interest. We also excluded from the analysis 49 individuals with the rare APOE2/4 genotype, leaving 2929 individuals in the present analysis.

Laboratory Methods: Fasting Glucose and Insulin

Fasting plasma glucose was measured in fresh specimens with a hexokinase reagent kit (A-gen glucose test; Abbot, South Pasadena, CA). Glucose assays were run in duplicate; the intra-assay coefficient of variation was <3%. Fasting insulin was measured in EDTA plasma as total immunoreactive insulin (Coast-A-Court Insulin; Diagnostic Products, Los Angeles, CA) and calibrated to serum levels for reporting purposes. Cross-reactivity of this assay with pro-insulin at mid-curve was approx40%, the intra- and interassay coefficient of variation ranged from 5.0 to 10.0%, and the lower limit of sensitivity was 8 pM.

Laboratory Methods: APOE Genotype

Leukocyte DNA was extracted from 5 to 10 mL of whole blood, as previously described (16) and according to current guidelines (17). APOE genotyping was performed as described by Hixson and Vernier (18). A 244-bp sequence of the APOE gene including the two polymorphic sites was amplified by polymerase chain reaction in a DNA Thermal Cycler (PTC-100; MJ Research, Watertown, MA), using oligonucleotide primers F4 and F6 (18). Each reaction mixture was heated at 94 °C for 2 min, followed by 35 cycles of amplification (94 °C for 40 s, 62 °C for 30 s, and 72 °C for 1 min). The polymerase chain reaction products were digested with 5 U of Hha I, and the fragments were separated by electrophoresis on an 8% polyacrylamide non-denaturing gel. After electrophoresis, the gel was treated with ethidium bromide for 30 min, and DNA fragments were visualized by UV illumination.

Three APOE genotypes were defined: APOE2 for those subjects carrying the epsilon2/epsilon2 or epsilon2/epsilon3 genotypes, APOE3 for those carrying the epsilon3/epsilon3 genotype, and APOE4 for those carrying the epsilon3/epsilon4 or epsilon4/epsilon4 genotypes.

Other Variables

Height and weight were measured with the individual dressed in an examining gown and wearing no shoes. BMI was calculated as weight in kilograms divided by the square of height in meters (kilograms per meter squared). Obesity was defined as BMI greater than or equal to 30 kg/m2. Waist circumference was determined, and abdominal obesity was defined as a waist circumference higher than 102 cm in men and higher than 88 cm in women. Smoking status was based on the cigarette consumption in the year before the examination. Alcohol consumption and the use of beta-blockers, diuretics, and hormonal substitutive treatment (in women) were assessed by questionnaire. Dietary intake was estimated with the semiquantitative Willett food frequency questionnaire (19).

Ethical Issues

The study was approved by all the institutional review committees. All the subjects gave informed consent.

Statistical Analysis

chi2 tests to compare proportions across groups and ANOVA to compare means of continuous variables across groups were used. Analysis of covariance was used to evaluate interactions between APOE genotypes and obesity in determining fasting insulin and glucose. In this analysis, we adjusted for potential confounding variables and accounted for correlations due to familial relationships among the members of the study using Proc Mixed in SAS (SAS Institute Inc., Cary, NC). Scheffe adjustment was used to control for multiple comparisons. A two-tailed p value < 0.05 was considered as statistically significant. The SAS program was used for statistical analysis.

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Results

Characteristics of the study population are presented in Table 1. Framingham Offspring Study participants are primarily of mixed European white ethnicity. The relative frequencies of APOE genotypes are shown in Table 1. No significant differences in genotype or allele frequencies between men and women were observed.


The characteristics of participants according to APOE genotype and stratified by sex are presented in Table 2. An association between APOE genotype and insulin levels was observed in men but not in women. No other statistically significant differences were observed.


In men, the effects of interactions between APOE genotype and obesity on fasting insulin and glucose concentrations were statistically significant (p = 0.003 and 0.008, respectively). Among obese men, glucose was marginally significantly higher, and insulin was significantly higher in those with the APOE4 genotype compared with those in the APOE3 genotype group (p = 0.057 and 0.022) (Table 3). No association between APOE genotype and insulin or glucose in nonobese men was observed. Obesity was associated with higher fasting insulin in the three genotype groups and was associated with higher fasting glucose only in APOE4 men.


In contrast, we did not find similar patterns in women, where the interaction between APOE genotype and obesity on fasting insulin and glucose was not statistically significant. Obesity was associated with higher levels of fasting insulin and glucose independently of the APOE genotype. No association between the APOE genotype and insulin or glucose was observed.

Moreover, we examined the interaction between abdominal obesity and APOE genotype on insulin and glucose levels (Table 4). The data show similar trends to those observed for obesity. However, the interaction terms did not reach statistical significance at the level of p < 0.05.


A similar analysis was performed considering diabetes as the dependent variable. The 118 participants under hypoglycemic treatment (excluded for the previous analyses) and those with a fasting glucose level greater than or equal to126 mg/dL were considered diabetics. In men, the interaction between APOE genotype and obesity on diabetes was not statistically significant, although the p value was 0.069. The prevalence of diabetes among APOE2, APOE3, and APOE4 genotype groups was 13.8%, 14.4%, and 25.0%, respectively. In women, this interaction was not statistically significant (p = 0.999). Similar results were obtained when the cut-off point to define diabetes was 140 instead of 126 mg/dL.

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Discussion

Our previous analysis on this population did not reveal significant associations between variability at the APOE locus and insulin resistance (12). In view of the potential interactions between this locus and several environmental factors (20), we focused on the current analyses of interaction models, and we found statistically significant interactions between APOE genotype and obesity on insulin and glucose levels in men. Thus, male subjects carrying the epsilon4 allele had significantly higher fasting insulin and glucose concentrations only in the presence of obesity (BMI greater than or equal to 30 kg/m2).

Our findings are in partial agreement with those reported by Kataoka et al. (21) in Native Americans. In this population, with a high prevalence of diabetes and obesity, these authors observed higher levels of fasting insulin in nondiabetic women with the APOE3 and APOE4 genotype compared with those with the genotype epsilon2 (p = 0.03); in men, the same tendency was observed, although without reaching statistical significance. Valdez et al. (22) also observed that the APOE3 and APOE4 genotypes were associated with higher levels of fasting insulin and LDL-C in two different ethnic groups (Mexican Americans and non-Hispanic whites from San Antonio) where the prevalence of obesity is higher. This association has not been observed in other studies (13, 14). However, those studies did not examine the interaction between APOE genotype and obesity.

At this time, there are no clearly defined mechanisms to explain the modulation of the association between APOE genotype and insulin by obesity. One potential link has been established between lipid peroxidation and insulin resistance (23, 24, 25), suggesting that lipid peroxidation precedes insulin resistance (23, 25). Increased total and LDL-C (8, 26) and decreased LDL diameter (27, 28, 29) may accelerate lipid peroxidation and are associated with the APOE4 genotype and obesity. A speculative mechanism is that the coexistence of these environmental and genetic factors could promote lipid peroxidation leading to higher insulin level.

Alternatively, obesity and APOE4 are also associated with higher triglyceride concentrations (9), and interactions between obesity and APOE4 on plasma triglycerides have been reported (30). Patients at an early stage of insulin resistance already have lipoprotein alterations characterized by increased very-low-density lipoprotein, intermediate-density lipoprotein, and LDL apoB production rates (31) and show a lower lipolytic capacity (32). The interaction between APOE and obesity was observed in men but not in women. Notably, the association between the APOE4 genotype and cardiovascular disease (CVD) or CHD has been mainly reported in men but less consistently in women (8, 33). In particular, the presence of allele epsilon4 (8, 10, 11) has been associated with higher CVD risk; in the present study, the epsilon4 allele was associated with higher insulin and glucose levels in obese men. In addition, hyperinsulinemia has been associated with excess CVD risk in men but not in women (34). Moreover, the excess of CVD risk observed in men with the APOE4 has been confined to hyperinsulinemic men in the presence of glucose intolerance, obesity, or hypertension (34).

To our knowledge, the possible modulating effect of obesity on the relationship between APOE genotype or insulin and cardiovascular risk has not been documented previously. The present data seem to represent an obesity-genotype interaction on CVD risk factors and suggest the hypothesis that obesity and APOE genotype may interact in men to intensify insulin resistance or hyperinsulinemia, leading to increased CVD risk. Among women, the better lipid profile, with a higher concentration of HDL and greater size of LDL particles (35), may protect them from the deleterious combination of APOE4 and obesity on insulin levels.

The interaction between abdominal obesity and APOE genotype on insulin or glucose levels was not statistically significant in men or in women. These results suggest that, at least in this population, global obesity (defined as BMI >30 kg/m2) is a more sensitive variable than abdominal obesity for the detection of the interaction between APOE genotype and obesity on insulin and glucose levels.

In conclusion, obesity modulates the association between APOE genotype and fasting insulin and glucose levels in men. Obese men with the epsilon4 genotype are more hyperinsulinemic and hyperglycemic than obese men with the epsilon2 or epsilon3 genotypes, whereas nonobese men have lower glucose and insulin levels than obese men but have similar glucose and insulin levels across APOE genotypes. These data seem to represent a novel phenotype (obesity)-genotype interaction that affects CVD risk factor levels. Although weight control is important in all people to improve insulin sensitivity and reduce CVD risk, weight control may be especially important in APOE4 subjects to modify potentially elevated fasting insulin and glucose levels and lower consequent CVD risk.

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Notes

1 Nonstandard abbreviations: CHD, coronary heart disease; apo, apolipoprotein; LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; CVD, cardiovascular disease.

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Acknowledgments

This work was supported by NIH/National Heart, Lung, and Blood Institute Grant HL54776, NIH/National Heart, Lung, and Blood Institute Contract 1-38038, and by U.S. Department of Agriculture Research Service Contracts 53-K06-5-10 and 58-1950-9-001. R.E. is a fellow from the Fulbright-Generalitat de Cataluña program. J.B.M. is supported by an American Diabetes Association Career Development Award.

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