In Vivo Relationship Between Insulin and Endothelin Role Of

In vivo relationship between insulin and endothelin role of insulin-resistance ´ G Andronico, M Mangano, L Ferrara, D Lamanna, G Mule and G Cerasola ` Cattedra di Medicina Interna, Divisione di Medicina Interna, Nefrologia ed Ipertensione, Universita degli Studi di Palermo, Italy

Since endothelin production is stimulated in vitro by related with the glucose:insulin ratio that has been con- insulin, we performed this study to evaluate in vivo the sidered as an insulin-sensitivity index and positively relationships between endothelin and insulin plasma with the insulin:C-peptide ratio as hepatic insulin-resist- levels during a glucose load. ance index. The relationship between basal endothelin We studied 28 subjects; 17 with normal glucose toler- and insulin values was also found in each glucose tolerance (NGT) and 11 with impaired glucose tolerance ance group. At 120 min after the glucose load, mean (IGT). Ten of the subjects in this study were normotens- plasma values of endothelin were significantly higher ive and 18 with mild to moderate hypertension. Age, sex (6.66 ± 1.31 vs 4.17 ± 0.61 pmol/L); moreover, the per and body mass index were comparable among the cent increase of endothelin at 120 min was positively groups. After a 2-week period of washout they related to the per cent increase of insulin. Between the underwent an oral glucose tolerance test; blood was normotensive and hypertensive groups there were no drawn at 0 (basal), 90 and 120 min after the load for significant differences in studied endothelin para- determination of glucose, insulin, C-peptide of insulin meters. and endothelin-1 and -2. Our results appear to confirm that, even in vivo, insu- Basal endothelin in all the subjects under study was lin modulates circulating endothelin levels. correlated with basal insulin; moreover it was negatively

Keywords: endothelin; insulin; insulin-resistance; oral glucose load

Introduction logical increase of plasma insulin that can be obtained after a glucose load. Endothelin is a powerful vasoconstrictor peptide produced by endothelial cells in response to stimuli that cause vascular derangement;1,2 moreover, when Patients and methods injected intravenously, it causes a long term We studied 28 (16 male and 12 female) consecutive increase of arterial pressure.3,4 Genes for three dis- patients from outward clinic for diagnosis and treat- tinct endothelins (endothelin-1, endothelin-2 and ment of endocrine and metabolic hypertensive dis- endothelin-3) have been described,5 but human ease. All gave their informed consent and the study endothelial cells appear to produce exclusively was approved by the Ethic Committee of Internal endothelin-1 which derives from the 203-amino- Medicine Institute of our University. acid preproendothelin and 92-aminoacid big endo- Seventeen were with normal glucose tolerance thelin; thereafter it is converted to the final product (NGT) and 11 with impaired glucose tolerance (IGT) endothelin-1 by the endothelin converting enzyme.6 as defined by national Diabetes Data Group cri- Two different receptors (A and B) for endothelins teria.12,13 Ten of the subjects under study were have been found in mammalian cells, but only the normotensive and 18 with mild to moderate hyper- A receptor appears to mediate the vasoconstrictor tension (Table 1). Secondary hypertension in these effect of these peptides, through the stimulation of patients was excluded by means of clinical examin- phospholipase C which leads to the formation of ation and the determination of urinary aldosterone inositol 1,4,5 triphosphate and diacylglycerol.7 excretion, plasma renin activity and plasma catheco- Insulin, in vitro has a stimulatory effect on the lamines. endothelin-1 production8 and because increased After at least 2 weeks of washout from drugs, they values of this vasoconstrictor peptide have been underwent a standard oral glucose (75 g) tolerance found in diabetic patients9–11 it has been considered test; venous blood was drawn at 0, 90 and 120 min to play a role in the development of hypertension after the load for glucose, immunoreactive insulin, that frequently complicates diabetes mellitus. C-peptide of insulin and endothelin-1 and -2 deter- The aim of this work is to verify in vivo the minations. changes of plasma endothelin following the physio- Glucose was measured by the glucose-oxidase method; insulin and C-peptide of insulin by double- antibody radioimmunoassays (Incstar Corporation, Correspondence: G Andronico, via Campolo 92-90145 Palermo, Italy Stillwater, MN, USA); endothelin after ODS extrac- Received 11 June 1996; revised 5 November 1996; accepted 21 tion and purification by a radioimmunoassay November 1996 (Amersham International plc, Bucks, UK); used anti- Insulin, insulin-resistance and endothelin G Andronico et al 64 Table 1 Characteristics of the patients under study

All Normotensives Hypertensives NGT IGT

n (M,F) 16,12 5,5 11,7 10,7 6,5 Age years 45.9 ± 1.6 44.1 ± 2.5 46.9 ± 1.8 43.6 ± 2.0 49.5 ± 1.9 BMI kg/m2 30.4 ± 0.9 29.5 ± 1.1 30.9 ± 1.3 30.3 ± 2.8 30.5 ± 1.6 MAP mm Hg 111.7 ± 2.0 98.3 ± 1.3 119.2 ± 1.0*** 109.8 ± 3.0 114.7 ± 2.0

NGT: normal glucose tolerance; IGT: impaired glucose tolerance; BMI: body mass index; MAP: mean arterial pressure. *P Ͻ 0.05; **P Ͻ 0.02; ***P Ͻ 0.01 hypertensives vs normotensives or IGT vs NGT.

body crossreacts 100% with endothelin-1, 204% with endothelin-2, 0% with synthetic endothelin-3 and 37.9% with human big endothelin-1. Values are expressed as means ± s.e.m. The results were analysed by one-way analysis of variance with the Bonferroni post hoc correction. For data which appeared not to be normally distributed, the Fried- man non-parametric statistic was used to evaluate the effect of the glucose load. Multiple regression analysis was undertaken with basal endothelin as the dependent variable and blood pressure (BP), body mass index (BMI), blood glucose at 120 min after the glucose load, and basal insulin as independent variable. Rank Spearman’s correlation test was also used for the relationship between variables not normally distributed. A value of P Ͻ 0.05 was considered significant. Figure 2 Relationship between fasting (basal) glucose:insulin Results ratio and endothelin in all patients under study. On the multivariate regressions, only basal insulin was predictive for basal endothelin (logarithmic computed) (r = 0.37; P Ͻ 0.05; Figure 1); whereas BMI, blood glucose at 120 min after the oral glucose load and BP were not related to endothelin as an independent variable. Fasting glucose:insulin ratio and insulin:C-peptide ratio also showed a significant correlation with the basal endothelin (respectively r =−0.42; P Ͻ 0.03; Figure 2 and r = 0.40; P Ͻ 0.03; Figure 3). After the glucose load, endothelin mean plasma levels increased significantly in all the groups of patients (Table 2) except those with NGT.

Figure 3 Relationship between fasting (basal) insulin:C-peptide ratio and endothelin in all patients under study.

The per cent increase of endothelin 2 h after the glucose load was positively correlated with the per cent increase of insulin in the sub-group of hypertensive patients (rho: 0.50; P: 0.034), in the sub- group of patients with impaired glucose tolerance (rho: 0.65; P: 0.020) and when all the subjects under study were considered together (rho: 0.41; P: 0.015). No significant relationship could be found with the plasma glucose increase. Endothelin values were not significantly different Figure 1 Relationship between fasting (basal) insulin and endo- in hypertensive patients in comparison with normo- thelin in all patients under study. tensive ones and in subjects with IGT compared Insulin, insulin-resistance and endothelin G Andronico et al 65 with those with normal tolerance at any time of the responsive element that could be responsible for glucose load (Table 2). No differences in insulin and this stimulating effect of insulin.21 C-peptide parameters were seen between the groups Wolpert et al22 and Ferri et al8 have shown, how- except for the glucose:insulin ratio that was signifi- ever, that an acutely induced increase in circulating cantly lower in hypertensives in comparison with insulin is rapidly followed by an increase of plasma the normotensives (Table 2). endothelin values, suggesting that in this situation this peptide could also be released from cell storage Discussion or rapidly produced by endothelial cells. In the experimental design of these works, however, insu- Because of its powerful vasoconstrictor property an lin was injected in pharmacological amount and involvement of endothelin has been thought poss- obviously not in a physiological way, whereas we ible in the development of human hypertension; analysed the consequence of an usual insulin rise however only the minority of reports show that can be obtained by oral carbohydrates. increased circulating levels of endothelin in hyper- After the glucose load we did not measure the tensive patients,14,15 whereas more often normal variations of blood flow or of BP, so we do not know values of this peptide have been found.16–18 Our if any measurable hemodynamic effect is joined to results confirm no significant difference in plasma the observed endothelin increase. However, we endothelin levels between normotensive and hyper- know that a very low concentration of endothelin, tensive subjects, but our patients were, however, all even if is not able to produce vasoconstriction, still free from renal complications that could be a cause potentiates the response to other vasoconstrictor of the increased endothelin concentrations reported hormones23 and the median endothelin increase of by others. about 50% could well obtain these effects. In our study, moreover, significant differences in Fasting glucose:insulin ratio can be considered as endothelin values was not found between subjects an insulin-sensitivity index; when blood glucose is with NGT and IGT; it is noteworthy, however, that not abnormally increased, a lower ratio indicates none of our patients had overt diabetes. that in a basal state more insulin is necessary to Our results still show that in all the subjects under maintain optimal glucose metabolism.13 Similarly, study, basal plasma endothelin values are correlated the insulin:C-peptide ratio calculated from a periph- with fasting insulin levels and that the increase in eral venous blood sample can be an index of insulin insulin concentration following a standard glucose extraction so that a higher value indicates insulin load is accompanied by a correlated endothelin rise, resistance.24 particularly in hypertensives and in subjects with Among the subjects that underwent our study, as IGT. expected hypertensives25 had a lower basal glu- Insulin stimulated production of endothelin from cose:insulin ratio in comparison with normoten- endothelial cells has been observed in vitro8,19,20 and sives; between subjects with normal and IGT no sig- it seems to require de novo protein synthesis; pre- nificant difference was seen for this parameter, proendothelin-1 promoter contains an insulin- perhaps because the high percentage (52.9%) of hypertensives were in the NGT group.

Table 2 Glucose, insulin, C-peptide of insulin and endothelin values in the patients under study before (basal) and at 90 and 120 min after standard (75 g) oral glucose load

All Normotensives Hypertensives NGT IGT

Glucose mmol/L basal (0 min) 4.97 ± 0.15 4.76 ± 0.30 5.09 ± 0.14 4.67 ± 0.18 5.44 ± 0.15 90 min 7.21 ± 0.29*** 6.49 ± 0.42*** 7.60 ± 0.35*** 6.29 ± 0.23*** 8.63 ± 0.33*** 120 min 5.88 ± 0.23*** 5.47 ± 0.42 6.11 ± 0.23*** 5.38 ± 0.23** 6.37 ± 0.36* Insulin pmol/L basal (0 min) 87.25 ± 10.05 65.08 ± 14.76 99.57 ± 12.39 73.82 ± 10.99 108.02 ± 17.35 90 min 656.00 ± 88.99*** 677.03 ± 187.61*** 644.32 ± 90.99*** 591.81 ± 115.59*** 755.20 ± 133.89*** 120 min 447.00 ± 59.82*** 349.71 ± 85.15*** 501.05 ± 77.26*** 398.51 ± 83.05*** 521.95 ± 76.66*** C-peptide nmol/L 0.87 ± 0.06 0.75 ± 0.10 0.94 ± 0.07 0.81 ± 0.07 0.97 ± 0.09 basal (0 min) 2.83 ± 0.23*** 2.76 ± 0.51*** 2.88 ± 0.22*** 2.80 ± 0.35*** 2.89 ± 0.23*** 90 min 2.22 ± 0.15*** 2.06 ± 0.31*** 2.32 ± 0.15*** 2.20 ± 0.23*** 2.26 ± 0.15*** 120 min Basal G:I ratio mmol/pmol 0.10 ± 0.02 0.16 ± 0.05 0.08 ± 0.01 0.13 ± 0.03 0.08 ± 0.02 Basal I:CP ratio pmol/nmol 98.74 ± 11.27 86.35 ± 22.32 105.63 ± 12.1 89.71 ± 14.37 112.7 ± 17.36 Endothelin pmol/L basal (0 min) 4.17 ± 0.60 4.37 ± 1.17 4.06 ± 0.68 4.37 ± 0.82 3.87 ± 0.88 90 min 6.07 ± 1.21** 8.00 ± 3.05 5.00 ± 0.72** 5.15 ± 0.77 7.51 ± 2.80* 120 min 6.65 ± 1.30** 7.93 ± 2.28* 5.95 ± 1.57 6.68 ± 1.68 6.63 ± 2.07*

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