Q J Med 2001; 94: 321-326
© 2001 Association of Physicians
Activation of the endothelin system in insulin resistance
From the University of Edinburgh, Department of Medical Sciences, Western General Hospital, Edinburgh, and 1 University of Glasgow, Department of General Practice, Glasgow, UK
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Summary |
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Endothelin-1, released from the vascular endothelium after cleavage from big endothelin-1, is a potent paracrine vasoconstrictor peptide. Small studies suggest that circulating levels of endothelin-1 are elevated in subjects with cardiovascular risk factors. Big endothelin-1 levels may better reflect endothelin-1 generation. We examined relationships between plasma endothelin-1, plasma big endothelin-1, and predisposition to hypertension or other cardiovascular risk factors associated with insulin resistance in a large group of healthy young men. We recruited 96 healthy men aged 24–33 years from a cohort of 864 young men and women in whom predisposition to hypertension had been defined on the basis of their own blood pressure and the blood pressures of their parents. They attended after an overnight fast for measurement of blood pressure, anthropometry, and plasma lipids, insulin, glucose, endothelin-1 and big endothelin-1. Plasma endothelin-1 and big endothelin-1 levels did not correlate with blood pressure (r=0.09, -0.002 respectively) and were not influenced by parental blood pressure. Higher plasma endothelin-1 levels were associated with higher body mass index (r=0.29, p<0.005), and higher plasma insulin (r=0.21, p<0.05). Higher plasma big endothelin-1 levels were associated with insulin resistance, as assessed by the Homeostasis Model of Assessment resistance index (r=0.30, p<0.005). Endothelin-1 levels are not related to blood pressure, but are higher in healthy young men with insulin resistance and obesity.
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Introduction |
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Endothelin-1 (ET-1) is a potent paracrine vasoconstrictor peptide synthesized by the vascular endothelium.1 It is cleaved from its inactive precursor big endothelin-1 (big ET-1) and secreted predominantly abluminally.2 It has a short plasma half-life of 2–5 min3 because it is quickly cleared from the circulation by binding to endothelin receptors, followed by enzymic degradation.4 Plasma concentrations are therefore a crude reflection of active peptide levels at the interface between endothelium and target tissue. Big ET-1 is present at higher concentrations in plasma than ET-1 and it is cleared more slowly;5 measurement of this peptide may more accurately reflect ET-1 synthesis.6,7 Nevertheless, plasma ET-1 levels correlate with the severity of disease in patients with established atheroma8 and renal failure.3 Higher big ET-1 levels predict poor prognosis in chronic heart failure.9 Moreover, a role for ET-1 has been suggested in the pathogenesis of essential hypertension, and elevated levels have been found in some hypertensive animal models10 and patients,11 although this is not consistent.12–14 In small case-control studies, plasma ET-1 has also been found to be elevated in patients with the metabolic syndrome15,16 or type 2 diabetes mellitus,17,18 leading to speculation that increased ET-1 secretion may play a central role in the development of vascular complications in diabetes mellitus.17–19 No study has yet examined the circulating concentrations of big ET-1 in subjects with any of these cardiovascular risk factors.
Against this background, we investigated whether plasma ET-1 and big ET-1 concentrations had any relationship to predisposition to hypertension or to insulin resistance in a large group of healthy young men.
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Methods |
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These studies were approved by our local Research Ethics Committee, and written informed consent was obtained from all participants. The investigation conformed with the principles outlined in the Declaration of Helsinki. Subjects were selected from a cohort which has been described elsewhere.20,21 In brief, blood pressure was measured in 603 married couples in 1979, and in 864 of their offspring, then aged 16–24 years, in 1986. Age-adjusted Z-scores were used to define tertiles for both offspring and mean parental blood pressures (see Figure 1
). Offspring for whom both their own blood pressure and the mean blood pressure of their parents were outside the middle tertile were identified as belonging to one of four ‘corners’. Subgroups of offspring randomly selected from these corners have participated in previous investigations which have identified correlates of the inherited predisposition to high blood pressure.20–28. As this was a population of young adults, only men were studied, in order to eliminate the effects of oral contraceptives and the menstrual cycle.
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We studied 96 Caucasian male offspring drawn at random from the four corners (Table 1
). No subject was taking vasoactive medication or aspirin. Each provided a 30 ml venous blood sample obtained at 0930 h after overnight fast and 30 min supine. The blood was taken on ice, spun at 4 °C at 4000 rpm for 20 min and frozen at -80 °C until analysis. Weight and height were recorded. Blood pressure was then recorded in the right arm, with the subject resting supine, using a Hawksley random-zero sphygmomanometer.
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Plasma ET-1 and big ET-1 were extracted from plasma, after ethyl acetate extraction to remove lipids, with >90% efficiency using acetic acid29 and measured by radioimmunoassay (RIA) (modified Peninsula assay). The ET-1 assay has 100% reactivity with ET-1, 11% cross-reactivity with big ET-1, and zero cross reactivity with endothelin-2 or endothelin-3. The big ET-1 assay has 100% reactivity with big ET-1 and zero cross-reactivity with ET-1. Intra- and inter-assay coefficients of variation were 6.3% and 7.2%, respectively, for both ET-1 and big ET-1. Insulin was measured by RIA.30 Glucose and lipids were measured by autoanalyser. Homeostasis Model Assessment (HOMA) indices for ß-cell function and insulin resistance were calculated from fasting insulin and glucose concentrations as previously described.31
Statistics
Normal distribution of baseline data and cardiovascular risk factors was assessed using Lilliefor's test of normality. Data on plasma triglyceride, insulin levels, and HOMA resistance index were log-transformed to obtain normal distribution. Examination of the influence of offspring and parental blood pressure was done by two-way ANOVA, in which blood pressure was coded as low (i.e. in lowest tertile of age-adjusted blood pressure) or high (i.e. in highest tertile of age-adjusted blood pressure) (Figure 1
). Correlations were identified by simple linear regression. Multiple regression was used to correct for confounding effects from plasma creatinine, because renal clearance of ET-1, proportionate to GFR, is a major determinant of ET-1 levels and a correlate in simple regression (i.e. the ET-1 corrected for creatinine is more likely to reflect production independently of clearance).
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Results |
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Neither ET-1 (mean 2.04 pmol/l, SD 0.36) nor big ET-1(mean 27.0 pmol/l, SD 9.78) levels correlated with the blood pressures of the subjects or of their parents, although there was a trend for higher ET-1 concentrations in the offspring of parents with higher blood pressure (see legend to Figure 1
). Smoking status (72 smokers, 24 non-smokers) did not affect circulating levels of ET-1 (mean±SD 2.07±0.39 vs. 2.03±0.35, smokers vs. non-smokers) or big ET-1 (28.3±8.5 vs. 27.0±10.4). In the absence of a relationship between plasma ET-1 levels and blood pressure, and given the normal distribution of the other variables measured, we performed simple regression to identify relationships between ET-1, big ET-1 and other cardiovascular risk factors.
Plasma ET-1 levels were higher in men with higher body mass index, higher fasting plasma insulin, and higher fasting plasma triglycerides (Table 1), but were not associated with plasma total cholesterol or fasting plasma glucose. Plasma big ET-1 levels were higher in men with insulin resistance assessed by HOMA resistance index (HOMAR)31 (Figure 2) and with elevated triglycerides, but not in those with higher BMI. Plasma ET-1 and big ET-1 levels were higher in subjects with lower serum creatinine and higher creatinine clearance. All of these relationships remained statistically significant after adjustment for the effect of creatinine, except the relationship between ET-1 and fasting plasma insulin, for which statistical significance was borderline (r=0.21, p=0.06).
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Discussion |
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This study shows that plasma ET-1 levels correlate with several cardiovascular risk factors, and that big ET-1 levels correlate with insulin resistance in healthy young men. Blood pressure did not correlate with either plasma ET-1 levels or big ET-1 levels, although there was a trend for higher ET-1 concentrations in the offspring of parents with higher blood pressure. This negative finding is in accordance with most studies, which have found only slight increases or normal levels of circulating ET-1 in hypertensive patients.14,32 However, these data do not preclude a role for ET-1 in the pathophysiology of hypertension. Plasma ET-1 levels are a crude measure of locally active peptide, and we have previously shown that sensitivity to venoconstrictor effects of ET-1 is increased in hypertensive patients.33
In the absence of an association with hypertension, elevated plasma ET-1 was strongly associated with other cardiovascular risk factors, including fasting plasma insulin, triglyceride levels and body mass index. Previous studies have shown differences between plasma ET-1 levels in groups with and without established atheromatous disease,8 the metabolic syndrome34 or type 2 diabetes mellitus.35 Moreover, ET-1 levels have been shown to decline with weight loss in obese hypertensive and normotensive men.36 However, this study is the first to demonstrate a relationship between ET-1 levels and cardiovascular risk factors associated with insulin resistance in healthy young men. None of these men were diabetic, although fasting plasma glucose ranged from 3.0 to 6.5 mmol/l. The lack of relationships between ET-1 or big ET-1 and fasting plasma glucose suggests a mechanism related to insulin resistance rather than hyperglycaemia. Interestingly, there was no relationship between ET-1 and the strong cardiovascular risk factor of hypercholesterolaemia, which is less closely associated with insulin resistance.37
The mechanism of elevated plasma ET-1 in subjects with insulin resistance might relate to enhanced synthesis and release, or impaired clearance. ET-1 is cleared predominantly in the kidney by receptor-mediated clearance and enzymic degradation. We did not find that variation in renal function within the normal range (as measured by creatinine clearance) contributed to elevated ET-1 levels. However, ET-1 generation assessed by big ET-1 levels correlated directly with insulin resistance and elevated triglyceride levels, but not obesity. This is consistent with the observation in vitro and in vivo38 that hyperinsulinaemia stimulates ET-1 synthesis and elevates plasma levels. In contrast, obesity correlated with higher plasma ET-1 levels but not big ET-1 levels, suggesting that obesity may reduce clearance but not affect generation of ET-1. It is possible, therefore, that elevated ET-1 levels in insulin resistant young men are a direct consequence of hyperinsulinaemia. Alternatively, ET-1 secretion may be enhanced by loss of down-regulation of ET-1 synthesis by nitric oxide (NO).39 NO-mediated vasodilatation is impaired in subjects with insulin resistance in association with diabetes mellitus or hyperlipidaemia.40 Against impaired NO generation being solely responsible for enhanced ET-1 generation, however, is the lack of relationship between plasma cholesterol and ET-1 levels in this study, since hypercholesterolaemia is the most reproducible predictor of endothelial dysfunction. The mechanism of enhanced ET-1 synthesis in insulin resistance therefore requires further research.
In summary, elevated ET-1 may be another manifestation of ‘endothelial dysfunction’ in subjects with cardiovascular risk factors and insulin resistance and may contribute to subsequent progress to vascular disease.
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Acknowledgments |
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Supported by grants from the Scottish Office Home and Health Department and British Heart Foundation. We are grateful to Drs D.W. Holton and H.V. Edwards for assistance in recruiting subjects.
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Notes |
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Address correspondence to Dr B.R. Walker, Department of Medical Sciences, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU. e-mail: b.walker{at}ed.ac.uk
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References |
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