The Insertion Allele of the ACE Gene I/D Polymorphism
A Candidate Gene for Insulin Resistance?
Background The insertion/deletion (ID) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with increased coronary heart disease (CHD), although the mechanism of this association is not apparent. We tested the hypothesis that the deletion allele of the ACE gene is associated with insulin resistance.
Methods and Results We related ACE genotype to components of the insulin-resistance syndrome in 103 non–insulin-dependent diabetic (NIDDM) and 533 nondiabetic white subjects. NIDDM subjects with the DD genotype had significantly lower levels of specific insulin (DD 38.6, ID 57.1, and II 87.4 pmol · L−1 by ANOVA, P=.011). Non–insulin-treated subjects with the DD genotype had increased insulin sensitivity by HOMA % (DD 56.4%, II 29.4%, P=.027) and lower levels of des 31,32 proinsulin (DD 3.3, II 7.6 pmol · L−1, P=.012) compared with II subjects. There were no differences in prevalence of CHD or levels of blood pressure, serum lipids, or plasminogen activator inhibitor–1 (PAI-1) activity between the three ACE genotypes. In nondiabetic subjects there were no differences in insulin sensitivity, levels of insulin-like molecules, blood pressure, PAI-1, serum lipids, or CHD prevalence between the three ACE genotypes.
Conclusions We conclude that increased cardiovascular risk of the DD genotype is not mediated through insulin resistance or abnormalities in fibrinolysis. Conversely, we report an increased sensitivity in NIDDM subjects with the ACE DD genotype.
A polymorphism in the ACE gene has been described consisting of an insertion or deletion (I/D) of a 287-bp fragment in intron 16. Individuals homozygous for the deletion allele have plasma ACE levels about twice as high as those homozygous for the insertion allele.1 The ACE (I/D) polymorphism has been identified as a risk factor for CHD in both nondiabetic and diabetic subjects.2 3 It has been postulated that the association of the deletion polymorphism with CHD could be mediated through an effect on insulin resistance.3 ACE inhibitor therapy is associated with improved insulin sensitivity in most studies,4 but the observation that low-dose infusions of AngII increase insulin sensitivity5 may suggest that this effect of ACE inhibitors is mediated through an action on bradykinin. It is unclear from these observations what effect the deletion allele may have on insulin resistance.
The aim of our study, therefore, was to test the hypothesis that the ACE gene DD polymorphism is associated with increased insulin resistance. We have related insulin sensitivity and the insulin-resistance syndrome variables, including PAI-1, to ACE genotype in NIDDM and nondiabetic subjects.
Subjects and Study Design
White NIDDM subjects (n=103; 54 men, 49 women) attending the outpatient clinic at the Whittington Hospital were studied. Subjects were diagnosed according to WHO criteria and had been allocated to different hypoglycemic therapies as part of two separate studies,6 7 both of which involved maximizing improvement in glycemic control. At the time of study they were on the following treatments: insulin (n=20), sulphonylurea (n=43), sulphonylurea plus metformin (n=16), metformin (n=8), and diet alone (n=16).
Characteristics of the nondiabetic population have been previously described.8 In brief, 959 nondiabetic white subjects aged 40 to 75 years, randomly selected from a north London general practice list, were investigated. Of these subjects, 533 had samples collected for genotyping and were representative of the total study population. The study was approved by the Ethical Committee of Islington Health Authority.
Subjects attended after an overnight fast. Weight and height were recorded and BMI (kg · m−2) was calculated. Blood pressure in diabetic subjects was taken with an automatic sphygmomanometer (A & D Company Ltd) and in nondiabetic subjects with a random zero sphygmomanometer (Hawksley and Sons Ltd); a mean of two readings was used. A resting ECG was recorded and coded according to Minnesota criteria.9 “Major CHD” was defined as Minnesota-coded ECG changes 1.1, 1.2, or 7.1. “All CHD” includes subjects with major CHD or Minnesota-coded ECG changes 1.3, 4.1-4.4, and 5.1-5.3.
Plasma glucose was assayed with glucose oxidase reagent (Beckman). PAI-1 activity was measured by using commercial kits (diabetic subjects: Kabi Vitrum; nondiabetic subjects: Biopool Spectrolyse pL).10 Specific insulin, intact proinsulin, and des 31,32 proinsulin were assayed by in-house two-site immunometric assays.11 12 Serum lipids were determined by enzymatic colorimetric methods, and LDL was calculated by the Friedewald formula.13
Insulin sensitivity was estimated with the HOMA model,14 employing fasting specific insulin and glucose concentrations. The computer program used was developed from the original HOMA formula (J.C. Levy, personal communication). Values are expressed as a percentage of a normal population with 100% being taken as normal and correlate well with insulin sensitivity measured using a euglycemic clamp (r=.88, P<.0001).14 In insulin-treated subjects the physiological relation between glucose concentrations and insulin secretion no longer applies, and the HOMA model cannot be used.
Genomic DNA was amplified as previously described using the polymerase chain reaction with primers flanking the polymorphic region.1 Polymerase chain reaction products of the two alleles of 490 and 190 bp were separated on 1.5% agarose gels and visualized by ethidium bromide staining. All genotyping was assessed independently by two individuals.
Data were analyzed using the Statistical Package for Social Sciences (SPSS). Data are presented as mean (SD) for normally distributed data and geometric mean (SD) for skewed data. Differences in distribution of ACE genotypes and alleles were analyzed with χ2 tests. The comparison of level of variables between the three genotypic groups were analyzed by ANOVA followed by unpaired t tests. The null hypothesis was that ACE genotype was without effect on insulin sensitivity, for which statistical significance was taken as P<.05. For comparison of the different components of the insulin-resistance syndrome and for unpaired t tests, more rigorous criteria for significance than P<.05 might be appropriate. For this reason, probability values for t tests quoted in tables and text are corrected for multiple comparisons using the Bonferroni correction.
Table 1⇓ shows the results in NIDDM subjects. The allele frequency was D:0.56, I:0.44. The genotype distributions in men and women were in Hardy-Weinberg equilibrium and all subjects were pooled for analysis. Due to the impact of age and obesity on insulin-resistance syndrome variables, all comparisons were adjusted for age and BMI. DD subjects had significantly lower concentrations of specific insulin than II subjects (DD 38.6, II 87.4 pmol · L−1, corrected P=.006 (Table 1⇓) (Fig 1⇓). Since insulin treatment precludes HOMA analysis and can also reduce concentrations of proinsulin-like molecules,15 analyses for these were repeated after exclusion of insulin-treated patients and correction for age and BMI. DD subjects had significantly higher insulin sensitivity (DD 56.4%, II 29.4%, corrected P=.027) and lower concentrations of des 31,32 proinsulin (DD 3.3, II 7.6 pmol · L−1, corrected P=.012) compared with II subjects (Fig 2⇓). There was no association of the I/D polymorphism of the ACE gene with PAI-1 activity, serum lipids, blood pressure, or prevalence of CHD (Table 1⇓).
Characteristics of the nondiabetic subjects are shown in Table 2⇓. The allele frequency was D:0.57, I:0.43; the genotype distributions in men and women were in Hardy-Weinberg equilibrium. All subjects were therefore pooled for further analysis. There was no significant difference in insulin sensitivity, levels of insulin-like molecules, or of insulin-resistance syndrome variables among the three ACE genotypes. In the subset of 97 subjects who had PAI-1 activity measured, there was again no difference in activity between the three genotypes studied. There was no association of ACE genotypes with prevalence of CHD (Table 2⇓).
We have found, using the HOMA model, a 132% increased insulin sensitivity in NIDDM subjects with the DD versus the II genotype. Moreover, after we excluded insulin-treated patients, II subjects had higher concentrations of both insulin and des 31,32 proinsulin, which we also associated with insulin resistance. AngII infusions in low doses have been shown to result in increased, rather than decreased, insulin sensitivity, perhaps related to the effects of AngII infusions on increasing muscle blood flow.5 A substantial component of insulin action on peripheral glucose uptake is mediated by the effects of insulin on muscle perfusion.16 Increased muscle blood flow through the action of AngII might act as a compensatory mechanism to prevent the rise in blood pressure that would otherwise occur in the presence of increased ACE activity. Nevertheless, the higher insulin sensitivity shown in DD subjects by using the HOMA model needs confirming with euglycemic hyperinsulinemic clamp studies. The lack of associations of ACE genotype with insulin sensitivity in nondiabetic subjects may imply an interaction between diabetes and the renin-angiotensin system in determining insulin sensitivity. It could, however, simply relate to the use of the HOMA model rather than a more precise measure of insulin action. Our study showed no association of the ACE gene I/D polymorphism with components of the insulin-resistance syndrome or with CHD prevalence in nondiabetic or NIDDM subjects. In vivo studies have shown that infusions of AngII increase plasma PAI-1 activity, an important inhibitor of fibrinolysis.17 AngII has also been shown to stimulate rat aortic smooth muscle cell PAI-1 mRNA,18 and treatment with ACE inhibitors lowers plasma PAI-1 activity.19 These studies suggest a possible link between the renin-angiotensin system and increased thrombotic risk. Our study, however, found no difference in plasma PAI-1 activity among the three ACE genotypes either in diabetic or nondiabetic subjects. It remains possible, nevertheless, that tissue or platelet PAI-1 levels may show a relation with ACE genotype.
In conclusion, our study showed that NIDDM subjects with the DD genotype had increased insulin sensitivity and lower concentrations of insulin and des 31,32 proinsulin. There was no relation between the ACE gene I/D polymorphism and components of the insulin-resistance syndrome. While the influence of ACE genotype on insulin sensitivity may be mediated through action on muscle blood flow, these effects are not able to explain associations of the ACE gene deletion allele with increased cardiovascular risk.
Selected Abbreviations and Acronyms
|ACE||=||angiotensin I converting enzyme|
|BMI||=||body mass index|
|CHD||=||coronary heart disease|
|HOMA||=||homeostasis model assessment|
|NIDDM||=||non–insulin-dependent diabetes mellitus|
|PAI-1||=||plasminogen activator inhibitor–1|
This study was supported by grants from the Medical Research Council, British Diabetic Association, and Wellcome Trust. Dr Panahloo was supported by a grant from Lilly Industries, Drs Humphries and Talmud by the British Heart Foundation (RG16). We would like to thank Dr J.C. Levy (Diabetes Research Laboratories, Oxford) for the homa program, L.A. Luong and E. Carstensen for technical assistance, and the patients and staff of the Goodinge Health Centre.
- Received August 16, 1995.
- Revision received October 3, 1995.
- Accepted October 4, 1995.
- Copyright © 1995 by American Heart Association
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