© 1995 American Heart Association, Inc.
Articles |
Relations Between Deletion Polymorphism of the Angiotensin-Converting Enzyme Gene and Insulin Resistance, Glucose Intolerance, Hyperinsulinemia, and Dyslipidemia
From the Falk Cardiovascular Research Center, Divisions of Cardiovascular Medicine (T.K., M.H., G.K., R.E.P., V.J.D.) and Gerontology, Endocrinology, and Metabolism (Y.-D.I.C., G.M.R.), Department of Medicine, Stanford University School of Medicine and Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Palo Alto, Calif.
Correspondence to Gerald M. Reaven, MD, Department of Veterans Affairs Medical Center, GRECC 182B, 3801 Miranda Ave, Palo Alto, CA 94304.
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Abstract |
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Abstract Recent reports have shown that the frequency of the homozygous deletion genotype (DD) of the angiotensin-converting enzyme (ACE) gene is highly associated with myocardial infarction and cardiomyopathy, particularly in those considered to be at low risk for coronary heart disease (CHD) on the basis of their apoB or LDL cholesterol concentrations. The present study was initiated to extend this inquiry by exploring the possibility that the ACE/DD genotype might be associated with risk factors not evaluated in the initial reports. Consequently, we determined the ACE genotype in 181 subjects, 124 with normal glucose tolerance and 57 with non–insulin-dependent-diabetes mellitus (NIDDM), and compared various aspects of glucose, insulin, and lipoprotein metabolism in the three ACE genotypes. In general, normal subjects with the DD genotype had a lower body mass index, were more insulin sensitive (as assessed by the insulin suppression test), and had lower plasma glucose and insulin responses to oral glucose. In addition, plasma triglyceride and cholesterol concentrations were lowest and HDL cholesterol concentrations highest in the DD group. However, the only statistically significant differences were between the ID and DD groups; the latter had lower values for body mass index, was more insulin sensitive, and had a lower plasma insulin response to oral glucose. Similar but insignificant trends were noted in the patients with NIDDM. The present results show that subjects with the ACE/DD genotype are not at increased risk for CHD because of insulin resistance, relative hyperglycemia and hyperinsulinemia, or a dyslipidemia characterized by a high triglyceride and low HDL cholesterol concentration. Indeed, the risk for CHD from these variables seems to be decreased in subjects with the ACE/DD genotype.
Key Words: insertion/deletion polymorphism • coronary heart disease risk factors • steady-state plasma glucose • insulin response to glucose • lipoproteins
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Introduction |
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A recent study has reported1 that a specific polymorphism of the angiotensin-converting enzyme (ACE) gene increases risk for myocardial infarction (MI) in middle-aged men. Specifically, it was found that the homozygote deletion polymorphism at intron 16, termed the DD genotype, occurred more frequently than either the heterozygote ID or homozygote II genotypes in patients surviving an MI. Of particular interest was the observation in this initial report of the ECTIM (Etude Cas-Témoins sur l'Infarctus du Myocarde) study, a large case-control study of MI in Europe, that the relative risk of MI due to the ACE/DD genotype more than doubled in the subset of the study population defined as being at low risk for MI on the basis of an apoB concentration <125 mg/dL and a body mass index (BMI) <26 kg/m2. Essentially similar results were seen when total cholesterol or LDL cholesterol was used to define the group at "low risk" for MI. The conclusion drawn from these observations was that "the ACE/DD genotype is a new potent risk factor for MI in middle-aged men, particularly in subjects considered to be at low risk." Obviously, this generalization depends to a considerable extent on the status of the metabolic factors considered to affect the risk of coronary heart disease (CHD). For example, other factors known to increase risk of CHD include a low HDL cholesterol concentration, glucose intolerance, resistance to insulin-mediated glucose disposal and compensatory hyperinsulinemia, hypertriglyceridemia, and high blood pressure.2 Although BMI, apoB and LDL cholesterol were examined in the ECTIM study, many of the other risk variables were not evaluated. It is certainly conceivable that the risk of the ACE/DD genotype is associated with the coexistence of these other variables known to increase the risks of CHD. The study described here, which was initiated to test this alternative possibility, examined the relations between ACE gene polymorphism and multiple risk factors for CHD in a population of volunteers.
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Methods |
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This study was performed in 181 volunteers, 124 subjects with normal oral glucose tolerance and 57 patients with non–insulin-dependent diabetes mellitus (NIDDM) diagnosed on the basis of conventional criteria.3 None of these subjects had a history or clinical evidence of CHD. The protocol was approved by the Stanford University Committee for the Protection of Human Subjects, and written informed consent was obtained from all subjects. The volunteer subjects were instructed to maintain their usual diet and activities before they were admitted to the Stanford Medical Center General Clinical Research Center, and tests were performed in random order. All subjects were in good health, as determined by a medical history, physical examination, and routine screening laboratory analyses.
Blood was drawn after an overnight fast for measurement of plasma glucose,4 insulin,5 triglyceride (TG),6 and cholesterol7 concentrations. In addition, an aliquot of plasma was used to isolate VLDL, LDL, and HDL by sequential ultracentrifugation,8 and TG and cholesterol concentrations were measured on these three lipoprotein fractions. After the fasting blood samples were drawn, all subjects were given a 75-g oral glucose challenge, and blood was removed 30, 60, 120, and 180 minutes later for determination of plasma glucose and insulin concentrations. The total areas under the plasma glucose and insulin concentrations from 0 to 180 minutes were calculated and are referred to as the glucose (milligrams per deciliter per hour) and insulin (microunits per milliliter per hour) responses.
On another day, resistance to insulin-mediated glucose disposal was quantified by the insulin suppression test.9 In brief, intravenous catheters were placed in both of the subject's arms after an overnight fast. Blood was sampled from one arm for plasma glucose and insulin concentration, and the contralateral arm was used for the administration of test substance. Somatostatin was administered at 350 µg/h in a solution containing 1% autologous human plasma by Harvard infusion pump to suppress endogenous insulin secretion.9 Simultaneously, insulin and glucose were infused at 25 mU · m-2 · min-1 and 240 mg · m-2 · min-1, respectively. Blood was sampled hourly until 2 hours into the study, and then every 10 minutes at 150, 160, 170, and 180 minutes. Insulin concentrations typically plateau after 30 minutes, whereas glucose concentrations plateau after 120 minutes. The four glucose and insulin values obtained from 150 to 180 minutes were averaged and considered to represent the steady-state plasma glucose (SSPG) and insulin concentrations.
DNA was extracted from 200 µL whole blood by using a QUIAmp Blood Kit (QUIAGEN Inc.). The insertion/deletion polymorphism of the ACE gene was determined according to the method of Tiret et al.10 The sequences of the sense and antisense primers were 5'-CTG GAG ACC ACT CCC ATC CTT TCT-3' and 5'-GAT GTG GCC ATC ACA TTC GTC AGA T-3', respectively. Polymerase chain reaction (PCR) was performed in a final volume of 50 µL that contained 100 ng genomic DNA, 10 pmol of each primer, 250 µmol/L dNTP, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.4, and 1 U Taq DNA polymerase (Life Technologies). Amplification was performed by using the Omni Gene TR3 CM110 (Hybaid Ltd Inc.). Samples were denatured for 3 minutes at 94°C and then cycled 35 times through the following steps: 1 minute at 94°C, 1 minute at 58°C, and 2 minutes at 72°C. PCR products were electrophoresed in 1.5% agarose gel and were visualized directly with ethidium bromide staining. The insertion allele (I) was detected as a 490-bp band, and the deletion allele (D) was visualized as a 190-bp band.
Results are expressed as mean±SEM. Statistical significance was
assessed by using ANOVA followed by Scheffe's multiple comparison
test. The 
value was preset at 5%.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The data in Table 1
compare the predicted frequency
of the three ACE genotypes as calculated by the Hardy-Weinberg
equation11 to that observed in the nondiabetic and
diabetic groups. The genotypic distribution of the ACE
genotypes in either the population at large or in the
nondiabetic and diabetic groups when considered separately was quite
close to the predicted outcome.
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Table 2 presents the actual values of the risk
factors for CHD as a function of the three ACE genotypes in
nondiabetic subjects. Subjects with the DD genotype were
generally less obese as estimated by BMI, had lower plasma glucose and
insulin responses to oral glucose, were more insulin sensitive (lower
SSPG concentrations), and had lower plasma TG, VLDL TG, cholesterol,
and LDL cholesterol concentrations. However, the only statistically
significant differences were between the ID and the DD groups, with DD
subjects having lower values for BMI, insulin response to glucose, and
SSPG concentration.
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Similar results for the NIDDM patients are shown in Table 3. Not surprisingly, these individuals were
hyperglycemic and insulin resistant (higher SSPG concentration)
compared with the nondiabetic subjects. Furthermore, they had higher
plasma TG and lower HDL cholesterol concentrations. However, the
tendency for the DD subjects to have lower values for CHD risk factors
was not seen, and even their BMI values were similar to those of the
other two groups. Thus, the three diabetic groups appeared quite
comparable regarding all measured variables.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study we examined the relations between the distribution of insertion/deletion polymorphisms in the ACE gene and a series of risk factors for CHD in 124 nondiabetic volunteers and 57 patients with NIDDM. This study was not designed to examine the validity of the predictive power of DD genotype for MI. It was initiated to see if any of the CHD risk factors were present to a greater or lesser degree in subjects with the DD genotype. Our results extend the observation of Cambien et al1 that patients with the DD genotype are neither obese nor hypercholesterolemic. Our data show that subjects with the DD genotype do not, as a group, exhibit resistance to insulin-mediated glucose uptake and/or hyperinsulinemia or demonstrate glucose intolerance, higher plasma TG and lower HDL cholesterol concentrations, or higher blood pressure. Indeed, subjects with the DD genotype tend to have lower values for all these variables than those with either the II or the ID genotype. Furthermore, the distribution of the II, ID, and DD genotypes was similar in the normal subjects and the NIDDM patients.
Since the publication of the work of Cambien et al,1 several reports have linked the ACE/DD genotype to cardiomyopathy as well as to CHD.11 12 13 14 15 However, the mechanism that associates ACE polymorphism and heart disease has not been identified. The current study was an attempt to evaluate the possibility that risk factors for CHD other than hypercholesterolemia, ie, insulin resistance, glucose intolerance, hyperinsulinemia, and high TG or low HDL cholesterol levels, might account for the association between the ACE/DD genotype and cardiac disease. Our results exclude that possibility. If the ACE/DD genotype is indeed an independent risk factor for CHD, the mechanism of increased risk is not due to the coexistence of traditional risk factors but rather due to other mechanisms, such as enhanced angiotensin cardiovascular effects.10 On the other hand, the association between the ACE/DD genotype and CHD may be due to the fact that individuals with the DD genotype have improved chances of surviving an MI compared with the II or ID subjects. Although this possibility can only be considered as speculative, it must be viewed in light of the evidence that subjects with the DD genotype tended to be less obese, more insulin sensitive, and with lower plasma glucose, insulin, and TG concentrations and higher HDL cholesterol levels. The fact that all these changes represent a more favorable risk-factor profile for CHD2 16 17 18 19 lends experimental support for this alternative hypothesis to explain the association between ACE/DD genotype and MI. Obviously, the answer to these questions can only come from future prospective studies.
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Acknowledgments |
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This work was supported by NIH grants HL-46631, HL-35252, HL-35610, HL-48638, HL-07708, HL-08506, and RR-00070 and the American Heart Association Bugher Foundation Center for Molecular Biology in the Cardiovascular System. Victor J. Dzau is the recipient of Merit Award HL-35610.
Received October 30, 1994; accepted March 15, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Cambien F, Porer O, Lecerf L, Evans A, Cambou J-P, Arveiler D, Luc G, Bard J-M, Bara L, Rivard S, Tiret L, Amouyel P, Alhenc-Gelas F, Soubrier F. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992;359:641-644.[Medline] [Order article via Infotrieve]
2. Reaven GM. Role of insulin resistance in human disease. Diabetes. 1988;37:1595-1607. [Abstract]
3. National Diabetes Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes. 1979;28:1039-1057. [Medline] [Order article via Infotrieve]
4. Kadish AH, Litle RL, Sternberg JC. A new and rapid method for determination of glucose by measurement of rate of oxygen consumption. Clin Chem. 1968;14:116-131. [Abstract]
5. Hales CN, Randle PJ. Immunoassay of insulin with insulin-antibody precipitate. Biochem J. 1963;88:137-146. [Medline] [Order article via Infotrieve]
6. Wahlefeld AW. Triglycerides: determination after enzymatic hydrolysis. In: Bergmeyer HU, ed. Method of Enzymatic Analysis. New York, NY: Academic Press; 1974:1831-1835.
7. Allain CA, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470-475. [Abstract]
8. Havel RJ, Eder HA, Bragdon JH. The distribution of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
9.
Shen DC, Shieh SM, Fuh MT, Wu DA, Chen Y-DI, Reaven
GM. Resistance to insulin-stimulated glucose uptake in patients
with hypertension. J Clin Endocrinol Metab. 1988;66:580-583.
10. Tiret L, Rigat B, Visvikis S, Breda C, Corvol P, Cambien F, Soubrier F. Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Genet. 1992;51:197-205. [Medline] [Order article via Infotrieve]
11. Hardy GH. Mendelian proportion in a mixed population. Science. 1988;28:49-50.
12. Tiret L, Kee F, Poirier O, Nicaud V, Lecerf L, Evans A, Cambou J-P, Arveiler D, Luc G, Amouyel P, Cambien F. Deletion polymorphism in angiotensin-converting enzyme associated with parental history of myocardial infarction. Lancet. 1993;341:991-992. [Medline] [Order article via Infotrieve]
13. Raynolds MV, Bristow MA, Bush EW, Abraham WT, Lowes BD, Zisman LS, Taft CS, Perryman MB. Angiotensin-converting enzyme DD genotype in patients with ischemic or idiopathic dilated cardiomyopathy. Lancet. 1993;342:1073-1075. [Medline] [Order article via Infotrieve]
14. Marian AJ, Yu QT, Workman A, Greeve G, Roberts A. Angiotensin-converting enzyme polymorphism in hypertrophic cardiomyopathy and sudden cardiac death. Lancet. 1993;342:1085-1086. [Medline] [Order article via Infotrieve]
15.
Schunkert H, Hense HW, Holmer SR, Stender M, Pertz S,
Keil U, Lorell BH, Riegger GA. Association between a deletion
polymorphism of the angiotensin converting-enzyme gene and left
ventricular hypertrophy. N Engl J Med. 1994;330:1634-1648.
16.
Castelli WP, Doyle JT, Gordon T, Hames CB, Hjortland
MC, Hulley SB, Kagan A, Zukel WJ. HDL cholesterol and other
lipids in coronary heart disease: the cooperative lipoprotein
phenotyping study. Circulation. 1977;55:767-772.
17. Pyörälä K. Relationship of glucose tolerance and plasma insulin to the incidence of coronary heart disease: results from two population studies in Finland. Diabetes Care. 1979;2:131-141. [Abstract]
18.
Austin MA. Plasma triglyceride and coronary
heart disease. Arterioscler Thromb. 1991;11:2-14.
19. Reaven GM. Are triglycerides important as a risk factor for coronary disease? Heart Dis Stroke. 1993;2:44-48.[Medline] [Order article via Infotrieve]
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