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Original Studies |
Cardiovascular Diseases Unit, Clinical Pathology Laboratory (E.G.), Molecular Pathology Laboratory (N.P.), and Department of Medicina Sperimentale e Clinica G. Salvatore, University of Catanzaro, Magna Graecia, 88100 Catanzaro, Italy
Address all correspondence and requests for reprints to: Francesco Perticone, M.D., Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Policlinico Mater Domini, Via Tommaso Campanella, 88100 Catanzaro, Italy. E-mail: perticone{at}unicz.it
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Abstract |
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Introduction |
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Essential hypertension is frequently associated with IR and hyperinsulinemia, such as the compensatory response to decreased sensitivity to insulin-stimulated glucose uptake in peripheral tissues, particularly skeletal muscle (12, 13). Nevertheless, the mechanism responsible for the development of IR in hypertension and other common insulin-resistant states remains still unknown (14, 15, 16, 17). Moreover, the fact that IR is found among normotensive relatives of hypertensive patients implies that the predisposition to IR might be inherited (17).
Recently, the association between the I allele of the ACE gene and IR has been reported in glucose-tolerant and normotensive African-Americans (18), in noninsulin-treated noninsulin-dependent diabetes mellitus (NIDDM) patients, but not in nondiabetic subjects (19) or hypertensive patients. Furthermore, it has been reported that ACE inhibitors administration improve the insulin sensitivity in normal subjects (20), NIDDM patients (21), and nondiabetic hypertensive patients (22). The effect of the treatment with ACE inhibitors on insulin sensitivity (23) suggests the possibility of a relationship between RAS, the system of the bradykinins, and insulin action, making the gene coding for ACE a candidate for the genetic basis of IR. These findings support the idea that the ACE gene is related to a predisposition to IR, but results remain controversial.
Thus, we designed the present study to evaluate the hypothesis that polymorphism of the ACE gene is associated with the IR in a group of never-treated hypertensive patients compared with that in a normotensive control group.
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Materials and Methods |
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Hypertensive group. Two hundred consecutive out-patients at Catanzaro University Hospital (114 men and 86 women; mean age, 45.5 ± 4.7 yr) with well documented essential hypertension were recruited for the study. All patients were white, and their families had been living in Calabria (South Italy) for at least 2 generations. All participants underwent physical examination and review of their medical histories before entering the trial. Causes of secondary hypertension were excluded by appropriate clinical and biochemical examinations. None of the patients had a history of diabetes. At the first eligibility visit, none of the participants had been treated.
The local ethical committee approved the protocol study, and all participants gave written informed consent for all procedures.
Control group. The study included 96 normotensive subjects (54 men and 42 women; mean age, 44.0 ± 4.7 yr) recruited among those who had a healthy examination in our Catanzaro University Hospital. Age and gender were similar in hypertensive patients and the normotensive controls. Clinical history, physical examination, and laboratory analysis determined normality. The clinic systolic and diastolic BP in normotensive subjects was below 140/90 mm Hg.
Anthropometric measurements
A trained examiner collected measurements, according to a standardized protocol, of height, weight, and circumferences. Body weight was measured with a balance scale with the subjects in light clothing and without shoes; height was measured conventionally. Body mass index (BMI) was calculated according to the formula: weight in kilograms divided by the square of the height in meters. Waist and hip circumferences were measured using a steel tape with the subjects standing. Fat distribution was assessed by measurement of the waist to hip ratio (WHR), according to the Italian Consensus Conference on Obesity (24).
BP measurements
Clinical BP was measured using a mercury sphygmomanometer with the patients supine after 5 min of rest. A minimum of three BP readings were taken on three separate occasions at least 2 weeks apart. Patients with a clinical BP of 160 mm Hg or greater systolic and/or 95 mm Hg or greater diastolic were defined as hypertensive.
Ambulatory BP monitoring was obtained using an A&D TM-2421 recorder (Takeda, Japan). Recordings were taken every 10 min during the day (from 0700–2300 h) and every 20 min during the night (from 2300–0700 h). We considered the cut-off point for hypertension BP values of 140/90 mm Hg or more.
IR evaluation
To exclude diabetes in unaware patients we performed an oral glucose tolerance test with subjects seated between 0800–0900 h after overnight fasting for at least 12 h. Fasting glucose and insulin values were averaged from the values obtained 15 and 5 min before administration of a 75-g glucose solution. Four additional blood samples were collected at 30-min intervals. Blood was analyzed for glucose using the glucose oxidase method, and insulin levels were measured using standard RIA techniques.
We estimated the IR using the homeostasis model assessment (HOMAIR) from the fasting glucose and insulin concentrations (25, 26). HOMAIR has been commonly used in clinical studies, and recently, it has been employed in a population-based study (27). Finally, it is necessary to remark that in the original report in the HOMA model (25) IR was highly correlated (r = 0.88) with IR measured by euglycemic clamp.
Detection of ACE polymorphism and ACE activity
The ACE genotypes were determined in duplicate by PCR by using the primers and methods described by Rigat and co-workers (28). The genotype was verified using an insertion-specific primer, according to the method described by Shanmugam and co-workers (29). For further details, see Ref. 30 .
ACE activity was evaluated with the ACE kinetic method from Bohlmann Laboratoires AG (Allschwil, Switzerland), standardized with the Bohlmann ACE colorimetric kit according to the method previously described (31, 32).
Statistical analysis
ANOVA of clinical and biological data was performed, and
differences between means were compared using unpaired Student’s
t test as appropriate. Because it is common knowledge that
gender may influence biological measures of health, analysis of the
genotype-phenotype relationships was performed separately for males and
females. Allele frequencies were estimated by the gene-counting method,
and Hardy-Weinberg’s equilibrium was verified by a
2 test. The possible correlation between ACE
activity and HOMAIR was tested by Pearson’s
coefficient. Multiple linear regression was used to compare
quantitative data on HOMAIR among hypertensive
patients and normotensive subjects. ACE activity, age, gender, BMI,
WHR, systolic and diastolic BP were considered independent variables.
In the analysis we included only ACE activity because it is an ACE
genotype function. Thus, we considered ACE activity as the fittest
variable to avoid possible colinearity. The possible interaction
between hypertension and ACE gene polymorphism on
HOMAIR was tested by a multivariate ANOVA,
including hypertension status and ACE genotype as independent
variables. Significant differences were assumed to be present at
P < 0.05. All group data are reported as the mean
± SD.
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Results |
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Demographic, clinic, humoral, and hemodynamic characteristics of
both hypertensive patients and normotensive subjects are summarized in
Table 1
. The hypertensive and
normotensive groups were matched for age, gender, and BMI. The WHRs of
two groups were also similar, indicating the same central fat
distribution.
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Clinical and biochemistry data in normotensive subjects vs. hypertensives
The clinical and ambulatory BP values were 128/79 ± 6/5 and 120/70 ± 6/5 mm Hg in normotensive subjects, and they were 157/98 ± 12/9 and 148/92 ± 10/8 mm Hg in hypertensive patients (P < 0.0001).
Fasting plasma glucose levels were slightly, but significantly,
increased in hypertensive patients compared with those in normotensive
controls (5.00 ± 0.32 vs. 4.79 ± 0.33 mmol/L;
P < 0.0001; Fig. 1A).
Moreover, hypertensive patients had higher fasting insulin levels
(12.3 ± 4.7 vs. 4.9 ± 1.5 µU/mL;
P < 0.0001; Fig. 1B) and a higher significant
HOMAIR than normotensive subjects (2.7 ±
1.1 vs. 1.1 ± 0.3; P < 0.0001; Fig. 1C). These observations suggest that hypertensive patients are more
insulin resistant than normotensive subjects. A slight, but
significant, difference was observed in total cholesterol, low density
lipoprotein cholesterol, and triglycerides (Table 1
).
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When we tested the possible interaction between ACE activity and
HOMAIR, we detected in hypertensive patients a
positive significant correlation (r = 0.679; P <
0.0001) between the two variables, as reported in Fig. 2. No significant correlation was found
in normotensive subjects (r = 0.020; P =
0.629).
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There were no differences in the ACE genotype distribution and
allele frequencies between hypertensive patients and control subjects.
The frequency of D allele in hypertensives (0.667) was similar to the
frequency observed in the control group (0.641), as was the
distribution of each genotype: DD, 41.0% (n = 82)
vs. 37.5% (n = 36); ID, 51.5% (n = 103)
vs. 53.5% (n = 51); and II, 7.5% (n = 15)
vs. 9.4% (n = 9). The allelic frequencies were similar
to previously published data from the same area (11, 30).
Genotype distributions in both hypertensive (2
= 0.042; P = 0.837) and normotensive
(2 = 0.045; P = 0.833) groups
were in Hardy-Weinberg equilibrium.
Relationship between genotype and HOMAIR in normotensives
Table 2 compares characteristics of
the 3 genotypes among 54 male and 42 female normotensive subjects.
There were no significant differences in age, BMI, WHR, and clinical
systolic and diastolic BP among the 3 genotypes for both sexes. In
addition, the mean levels of fasting glucose, insulin, and
HOMAIR among the 3 genotypes for both male and
female normotensive subjects were not statistically significant. Thus,
the 3 normotensive ACE genotypes did not have significant
HOMAIR for males and females. ACE activity was
higher in the DD genotype than in ID or II groups for both males and
females. However, this difference was statistically significant only in
males.
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No significant differences among the three hypertensive genotypes
were found in BP values, BMI, WHR, total cholesterol, low density
lipoprotein cholesterol, and triglycerides for males and females (Table 3). Similarly, no significant differences
were observed in fasting glucose values among the three genotypes
(5.03 ± 0.35 mmol/L in DD, 4.96 ± 0.30 mmol/L in ID, and
5.02 ± 0.30 in II genotype; P = 0.322; Fig. 1A). However, patients homozygous for the short allele (DD) had
significantly (P < 0.0001, by ANOVA) higher fasting
plasma insulin levels (16.3 ± 3.3 µU/mL; Fig. 1B) and higher
HOMAIR (3.6 ± 0.8; Fig. 1C) compared with
those with the ID genotype (fasting insulin, 9.4 ± 3.1 µU/mL;
HOMAIR, 2.1 ± 0.7) and the II group
(insulin, 8.3 ± 2.8 µU/mL; HOMAIR,
1.9 ± 0.7). Thus, our data indicate that hypertensive patients
with the double D allele are more insulin resistant than those with the
I allele. A stepwise multiple linear regression was then performed to
determine the relative contribution of each variable to
HOMAIR. The multiple r for the model was 0.701,
accounting for a total of 49.2% of this variation (Table 4). It is necessary to remark that ACE
activity is strongly related to ACE genotype; therefore, according to
the fit model, only ACE activity was evaluated as the independent
determinant of HOMAIR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The important component of insulin action on peripheral glucose uptake is mediated by its effect on muscle perfusion (14). In fact, the insulin-mediated increase in skeletal muscle blood flow is blunted in the insulin-resistant states of diabetes, obesity, and essential hypertension (14, 15, 16, 17), suggesting that reduced skeletal muscle perfusion may contribute to the IR of these patients. Because the insulin-resistant state is frequently present in hypertensive patients, the possibility has been raised that IR causes essential hypertension; however, not all hypertensive patients have IR, and hypertension does not occur in all patients with hyperinsulinemia. On the other hand, racial factors may be important as well, because high BP and fasting plasma insulin levels are significantly related in whites, but not in blacks or Pima Indians (34).
Although the mechanisms responsible for the association between IR and high BP have not been clarified, some of these for the hemodynamic effects of insulin have been postulated: sympathetic nervous system activity (35), increase in renal sodium reabsorption (36), and promotion of the growth of vascular smooth muscle cells (37). Besides, in physiological conditions insulin potentiates acetylcholine-mediated vasodilatation (38), which seems to be mediated by nitric oxide production, presumably of endothelial origin (38, 39, 40). Conversely, several reports demonstrate that endothelium-dependent vasodilatation is impaired in hypertensive patients (41, 42, 43); thus, this endothelial dysfunction may be responsible for a failure of insulin-induced vasodilatation in hypertensive patients. Therefore, the possible influence of ACE gene polymorphism on IR may be mediated through an action on muscle blood flow. Recently, we reported that deletion polymorphism in the ACE gene was associated with an impairment of endothelium-dependent vasodilation in a group of newly discovered never-treated hypertensive patients. In particular, patients homozygous for deletion (DD) were characterized by significantly less endothelium-dependent vasodilation than patients who were homozygous for insertion (II) and heterozygous (ID) (43). These data are consistent with a greater ACE activity, as documented in our DD hypertensive patients, that induces a decrease in blood flow and a lower peripheral glucose uptake.
The importance of both circulating and tissue ACE generation of angiotensin II has been demonstrated in experimental as well as in human studies. Thus, because ACE levels in humans are affected by the ACE I/D polymorphism (3, 4), it is plausible that elevated ACE levels might lead to chronically elevated circulating and/or tissue angiotensin II levels (44, 45, 46) that activate a number of intracellular pathways involved in insulin action (47). In addition, angiotensin II-mediated activation of protein kinase C via receptor-linked formation of diacylglycerol represents another mechanism by which angiotensin II might affect insulin action (48). Finally, it has been recently demonstrated that angiotensin II impairs insulin stimulation of insulin receptor substrate 1 tyrosine phosphorylation and coupling of the insulin receptor pathway to phosphatidylinositol 3-kinase (49). This interaction between angiotensin II and the insulin signaling system in the vasculature suggests that activation of the RAS affects the regulation of vascular physiology, leading to an increase in IR. Thus, the clinical data showing the efficacy of either ACE inhibitors (20, 21, 22) or angiotensin II receptor blockers (50) in improving insulin sensitivity in different disease populations, including hypertensives, may be explained in terms of these mechanisms.
Previous reports on the association between I/D polymorphism of the ACE gene and IR in some resistant states are controversial (8, 11, 18, 19, 51, 52, 53). In particular, Katsuya et al. (51) found an increased insulin sensitivity in the nondiabetic DD genotype using the insulin suppression test, but this difference may be due to a lower BMI detected in the DD genotype than in the ID or II genotype. Similarly, our results disagree with conclusions recently reported by Panahloo (18) and Chiu (17). In fact, both researchers reported that the I allele is associated with IR in NIDDM patients and in glucose-tolerant and normotensive African-Americans. This discordance is, in our opinion, only apparent because the study groups are not comparable; in the first study, the association of the ACE genotype with IR was found only in treated NIDDM patients, not in normal subjects; in the second study only African-Americans subjects without a prior history of diabetes or hypertension were examined.
Our findings are consistent with the association of D polymorphism and a higher degree of IR observed in hypertensive patients. The lack of correlation of ACE activity with IR in the normotensive control group may imply a true interaction between the RAS and hypertension in determining IR. Therefore, as ACE activity is associated with hypertension, and hypertension is associated with IR, it follows that association of ACE activity with IR is probably reflective of its association with hypertension and is not a direct association. This point is supported by the lack of an association among ACE levels, genotypes, and IR in normotensive subjects.
On the other hand, it is still possible to interpret our results based upon a possible linkage between the ACE gene, located on chromosome 17, and another still unidentified gene, modulating the expression of factors affecting insulin sensitivity in hypertensive patients. This hypothesis could explain the association of IR to different ACE gene alleles in different populations (11, 18, 19).
The implications of this study derive from the data showing that plasma and tissue ACE activity are elevated in subjects homozygous for the short allele (DD) of the ACE gene. This suggests that elevated ACE activity contributes to the development of atherosclerosis with an increased risk for CAD and subsequent myocardial infarction. On the other hand, it is well established that IR is an independent risk factor for CAD, and it is has been recently demonstrated that it is also linked to endothelial dysfunction. Therefore, on the basis of these findings it is possible to affirm that ACE/DD hypertensive patients have an increased risk for coronary atherosclerosis and cardiovascular complications. Thus, it appears reasonable, at least in these high risk patients, to use drugs acting on both the regulation of hypertension and the improvement of IR.
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Acknowledgments |
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Footnotes |
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Received January 20, 2000.
Revised July 31, 2000.
Accepted September 21, 2000.
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