Serum leptin levels in patients with primary hyperaldosteronism before ...

Journal of Human Hypertension (2002) 16, 41–45  2002 Nature Publishing Group All rights reserved 0950-9240/02 $25.00 www.nature.com/jhh

ORIGINAL ARTICLE

Serum leptin levels in patients with primary hyperaldosteronism before and after treatment: relationships to insulin sensitivity M Haluzı´k, G Sˇindelka, J Widimsky´ Jr, M Pra´zny´, T Zelinka and J Sˇkrha Third Department of Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic

Background: Leptin is a protein hormone produced predominantly by adipocytes that plays a role in food intake regulation and a series of other physiological processes including blood pressure regulation. Objectives: The aim of our study was to compare serum leptin levels in patients with primary hyperaldosteronism (PA) with those of healthy subjects and to explore the relationship of serum leptin levels and the parameters of insulin action in these patients before and after surgical or pharmacological treatment. Methods: Serum potassium, leptin, aldosterone, insulin levels and plasma renin activity were measured and hyperinsulinaemic euglycaemic clamp was performed in 11 patients with PA and 11 healthy age-, gender- and body mass index (BMI)-matched subjects. In eight of 11 patients the same measurements were repeated at least 6 months after surgical or pharmacological treatment. Results: The basal serum leptin levels in PA patients did not significantly differ from those of healthy subjects (mean ⴞ s.e.m. 8.4 ⴞ 1.9 vs 11.2 ⴞ 1.8 ng/ml, P ⴝ 0.30), although their insulin sensitivity was significantly impaired (PA patients vs control subjects: glucose dis-

posal rate in the last 20 min of clamp (M) 18.7 ⴞ 1.8 vs 30.6 ⴞ 3.3 ␮mol/kg/min, metabolic clearance rate of glucose (MCRg) 3.9 ⴞ 0.5 vs 7.2 ⴞ 1.1 ml/kg/min, P ⬍ 0.05). The surgical or pharmacological treatment of PA patients increased significantly their serum leptin levels (10.9 ⴞ 3.7 vs 8.4 ⴞ 1.9 ng/ml, P ⬍ 0.05) and simultaneously improved their insulin sensitivity. Basal serum leptin levels in both groups correlated positively with BMI and serum insulin levels. The inverse relationship between serum leptin levels and the insulin sensitivity parameters was found in both PA patients before treatment and healthy subjects. These relationships disappeared after treatment of PA patients except for those between serum leptin levels and MCRg. Conclusion: Basal serum leptin levels in untreated patients with PA do not significantly differ from those of healthy subjects, but increase significantly after surgical or pharmacological treatment. The increase in serum leptin levels is paradoxically accompanied by the improvement of insulin sensitivity in these patients. Journal of Human Hypertension (2002) 16, 41–45. DOI: 10.1038/sj/jhh/1001292

Keywords: leptin; primary hyperaldosteronism; insulin sensitivity; treatment

Introduction Leptin is a 16-kDa protein hormone produced predominantly by adipocytes identified in 1994 by ob gene cloning in mice.1 Its serum concentrations usually reflect the body fat content, ie they are higher in obese compared with lean subjects.2,3 Serum leptin levels represent the negative feedback loop from adipose tissue to the hypothalamus satiety centre. The increase of serum leptin levels suppresses the food intake in lean subjects. In obese subjects hyperlepti-

Correspondence: M Haluzı´k, MD, PhD, 3rd Department of Medicine, 1st Faculty of Medicine, Charles University, U nemocnice, 128 08, Praha 2, Czech Republic. E-mail: mhalu얀lf1.cuni.cz Received 6 June 2000; revised 30 July 2001; accepted 12 August 2001

naemia is unable to sufficiently suppress the food intake. The reason probably lies in the resistance to leptin effects, however the clear mechanism for this suggested leptin-resistance is currently unknown.4 Except for the action of leptin on the food intake regulation it also plays a role in the modulation of angiogenesis, haematopoiesis, and reproduction etc.5–7 Obesity is one of the well-known risk factors for arterial hypertension development.8 Because of the clear link between obesity and the increase of serum leptin levels the relationship of serum leptin levels and blood pressure regulation was studied by a series of authors. Leptin receptors were identified in the brain regions, which are also known to be important in cardiovascular control. Haynes et al9 demonstrated that intravenous leptin infusion increased sympathetic nerve activity to kidney,

Leptin in primary hyperaldosteronism M Haluzı´k et al

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hindlimb, the adrenal gland and brown adipose tissue in rats. Chronic leptin infusion treatment increased heart rate and blood pressure in normal rats.10 There are also several clinical studies showing the positive correlation between serum leptin levels and systolic or diastolic blood pressure11,12 or leptin and plasma renin activity.13 Primary aldosteronism (PA), the autonomous hypersecretion of aldosterone resulting in excessive sodium retention and potassium excretion, is usually accompanied by arterial hypertension.14 Moreover, a series of authors reported alterations of insulin sensitivity and/or release in patients with this disease, although the results of these studies are contradictory.14–17 Recently, Torpy et al18 demonstrated that serum leptin levels in patients with PA increased after surgical removal of aldosteroneproducing adenoma. The reason for this increase was not clarified by Torpy’s study. However, the authors suggested the possibility that decreased serum leptin levels in these patients are the result of alterations in insulin sensitivity and/or secretory capacity. There was no control group in Torpy’s study, thus the authors were not able to assess whether serum leptin levels in untreated patients with PA do differ from those of healthy subjects. Our study was performed to compare the serum leptin levels of patients with PA before and after treatment with those of healthy gender-, age- and body mass index (BMI)-matched subjects. We hypothesised that that the changes in serum leptin levels in patients with PA are the result of alterations in insulin sensitivity. To test this hypothesis the hyperinsulinaemic euglycaemic clamp was performed in all subjects at baseline and in the subset of the patients also after surgical or pharmacological treatment.

Materials and methods Patients Eleven patients (two females and nine males) with PA and 11 healthy gender-, age- and BMI-matched subjects were included into the study. Eight subjects with PA were examined for the second time at least 6 months after surgical or pharmacological treatment. Five patients with PA had aldosterone-producing adenoma and six had idiopathic hyperaldosteronism with bilateral adrenal hyperplasia. From eight patients examined both before and after the treatment five had aldosterone-producing adenoma and three had idiopathic hyperaldosteronism. All subjects included in the study had normal glucose tolerance confirmed by oral glucose tolerance test. None of the subjects suffered from acute infectious diseases and none was treated by medication known to affect food intake and/or appetite. The control subjects had normal blood pressure and no clinical or laboratory signs of PA. All subjects were informed about the purpose of Journal of Human Hypertension

the study and gave their informed consent to participate. The protocol of the study was approved by the local ethical committee. The diagnosis of PA was based on clinical finding of arterial hypertension accompanied by low serum potassium concentration, increased plasma aldosterone, low rein levels and increased aldosterone/ renin ratio. Hormonal tests (postural, captopril, dexamethasone) and topographical examinations were used for classification of the aldosterone-producing adenoma vs idiopathic hyperaldosteronism. The adrenal tumor was diagnosed by CT scans and ultrasonography. Casual blood pressure was measured by standard technique (Mercury sphygmomanometer). The antihypertensive therapy metoprolol (Vasocardin, Slovakofarma, Slovak Republic) or diltiazem (Diacordin, Leciva, Czech Republic) was withdrawn at least 3 weeks prior to examination. Five subjects with aldosterone-producing adenoma underwent unilateral adrenalectomy and the six patients with idiopathic hyperaldosteronism were treated by daily doses of 50–100 mg of spironolactone (Verospiron, Gedeon Richter, Hungary). Methods All subjects were measured and weighed and the blood pressure was measured in all of them. The hyperinsulinaemic euglycaemic clamp was performed in control subjects and in patients with PA at the time of diagnosis and the blood for hormonal determination, basal insulin, plasma glucose and potassium concentration was withdrawn before the beginning of the clamp. In eight subjects with PA the same examination was repeated at least 6 months after surgical removal of adenoma or pharmacological treatment. The hyperinsulinaemic euglycaemic clamp was performed as described previously.19 Briefly, flexible cannula was inserted into the forearm vein, at the same time the blood samples for blood glucose, potassium and hormonal levels determinations were obtained. Then the cannula was connected with the infusion module of a Biostator (GCSII, Elkhart, IN, USA) to administer the insulin solution (160 iU of Actrapid HMR, Novo Nordisk, in 500 ml 0.9% sodium saline solution), 40% glucose solution and wash-out sodium saline solution (0.9% w/v). At the same time 7.5% potassium chloride infusion diluted with physiological saline solution 1:4 was supplied by perfusor (Infusor Secura FT, B. Braun, Germany) to another channel of the cannula with a rate 0.1 ⫾ 0.05 ml/min to maintain the basal potassium levels. The rate of this infusion was adjusted to maintain the basal potassium levels stable. A doublelumen catheter was inserted into a contralateral arm for continuous blood glucose determination. A third cannula placed into a wrist vein was used to collect blood samples for biochemical measurements. Two blood samples for insulin determination were col-


lected in the last 20 min of the clamp. After a 30min washout period, hyperinsulinaemic euglycaemic clamp was performed by Biostator (mode 7:1) for 120 min using the constant insulin infusion rate (1 mU/kg/min). Glucose solution (40% w/v) was sampled by Biostator to maintain blood glucose levels at basal value. During the clamp, blood glucose concentration was repeatedly determined by glucose analyser. The following characteristics of insulin action were calculated: glucose disposal rate as the amount of glucose supplied by Biostator to maintain blood glucose level during clamp (M, ␮mol/kg/min), the insulin sensitivity index, defined as ratio M to insulin concentration at the end of the clamp (M/I, ␮mol/kg/min per mU/l × 100), metabolic clearence rate of glucose expressed as a ratio of M to blood glucose concentration (MCRg, ml/kg/min). Assays Plasma glucose concentration was measured by glucose oxidase method by an automatic analyzer (ESAT 6660-2, Germany). Plasma insulin, aldosterone and renin concentrations were measured by commercial radioimmunoassay kits (Immunotech, Czech Republic). Serum leptin levels were determined by commercial double sandwich ELISA kit (Bio Vendor, Czech Republic). Serum potassium concentration was assessed by standard laboratory methods in the Department of Clinical Biochemistry of the University Hospital. Statistical analysis For the statistical analysis the SigmaStat software (Jandel Scientific, USA) was used. Results are expressed as means ⫾ standard error of means. Analysis of variance followed by Student–Newman– Keuls test was used for comparison of values of the control and patients group before and after treatment. Paired t-test was used to compare the values of patients group before and after treatment. The relationships between data were calculated by Pearson correlation test.

Results The basal systolic and diastolic blood pressure was significantly higher in patients with PA compared with the control group (Table 1). The serum potassium concentration, glucose disposal rate in the last 20 min of clamp and metabolic clearance rate of glucose were significantly lower in PA compared with the control group (Tables 1 and 2). Basal blood glucose, serum leptin and insulin concentrations in the PA group did not differ from those of the control group (Table 1). The treatment of PA significantly decreased serum aldosterone concentrations, increased serum potassium concentrations, plasma renin activity and

Table 1 Clinical and anthropometric characteristics of subjects Control group Number of subjects 11 Age (years) 47.4 ± 4.24 27.9 ± 1.24 BMI (kg/(m2)) Leptin (ng/ml) 11.22 ± 1.79 Potassium 4.1 ± 0.21 (mmol/l) Blood glucose 5.11 ± 0.18 (mmol/1) SBP (mm Hg) 128 ± 2.92 DBP (mm Hg) 85 ± 2.51 Aldosterone – (pg/ml) PRA (ng/ml/h) –

PA before treatment

43

PA after treatment

11 8 50.5 ± 4.01 48.4 ± 3.91 28.2 ± 1.50 27.36 ± 1.61 8.4 ± 1.9 10.88 ± 3.68** 3.18 ± 0.35* 4.0 ± 0.34** 5.3 ± 0.41

4.9 ± 0.22

163.2 ± 7.41* 131.9 ± 3.26** 105.9 ± 2.92* 85.1 ± 2.67** 361.4 ± 42.81 146.6 ± 47.40** 0.23 ± 0.048

0.89 ± 0.18**

Age, body mass index (BMI), serum leptin levels, serum potassium levels, blood glucose, systolic (SBP) and diastolic blood pressure (DPB), serum aldosterone and plasma renin activity (PRA) in control group (n = 11), patients with primary hyperaldosteronism (PA) before (n = 11) and after treatment (n = 8) Expressed as means ± standard error means. *Statistically significant difference from control group (P ⬍ 0.05, ANOVA, Student–Newman–Keuls test). **Statistically significant difference between PA patients before vs after treatment (P ⬍ 0.05, paired t-test).

serum leptin concentrations (Tables 1 and 2). No changes in BMI, blood glucose concentration and serum insulin concentrations were found after treatment (Tables 1 and 2). The glucose disposal rate was significantly increased after treatment while the rest of the insulin action parameters studied did not change significantly (Table 2). The post-treatment systolic and diastolic blood pressure, serum potassium, leptin and insulin concentrations as well as glucose disposal rate in the last 20 min of clamp, insulin sensitivity index and metabolic clearance rate of glucose in patients with PA did not differ from the control group (Tables 1 and 2). Basal serum leptin concentrations in both the conTable 2 Insulin sensitivity parameters Control group Number of subjects 11 Insulin (mU/l) 20.7 ± 3.22 M (␮mol/kg/min) 30.6 ± 3.29 M/I (␮mol/kg/min 34.1 ± 6.31 per mU/l × 100) MCRg (ml/kg/min) 7.2 ± 1.06

PA before treatment

PA after treatment

11 18.61 ± 3.09 18.71 ± 1.82* 24.51 ± 2.96

8 17.72 ± 4.64 32.56 ± 4.64** 39.53 ± 7.09

3.92 ± 0.47*

5.93 ± 0.97

Serum insulin levels and insulin action characteristics. M, glucose disposal rate in the last 20 min of clamp; M/I, the insulin sensitivity index; MCRg, metabolic clearence rate of glucose) in control group (n = 11), patients with primary hyperaldosteronism (PA) before (n = 11) and after treatment (n = 8). Expressed as means ± standard error means. *Statistically significant difference from control group (P ⬍ 0.05, ANOVA, Student–Newman–Keuls test). **Statistically significant difference between PA patients before vs after treatment (P ⬍ 0.05, paired t-test). Journal of Human Hypertension


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Table 3 The relationships of serum leptin levels and the rest of parameters studied Control group

PA before treatment

PA after treatment

r = 0.76, P = 0.007

r = 0.75, P = 0.007

r = 0.68, P = 0.059

Potassium

NS

NS

NS

SBP

NS

NS

NS

DBP

NS

NS

r = 0.71, P = 0.049

Aldosterone

–

NS

NS

PRA

–

NS

NS

Insulin

r = 0.63, P = 0.036

r = 0.76, P = 0.006

r = 0.84, P = 0.008

GDR

r = −0.66, P = 0.027

r = −0.76, P = 0.007

NS

M/I

r = −0.64, P = 0.03

r = −0.71, P = 0.013

r = −0.75, P = 0.031

MCR

r = −0.73, P = 0.011

r = −0.70, P = 0.014

NS

BMI

The relationship of serum leptin levels with body mass index (BMI), serum potassium levels, systolic (SBP) and diastolic blood pressure (DBP), serum aldosterone levels, plasma renin activity (PRA), basal serum insulin levels, glucose disposal rate in the last 20 min of clamp (GDR), insulin sensitivity index (M/I), metabolic clearance rate of glucose. (MCRg) in control group (n = 11), patients with primary hyperaldosteronism (PA) before (n = 11) and after treatment (n = 8). r, correlation coeficient; P, significance levels; NS, nonsignificant (Pearson test).

trol and PA groups significantly positively correlated with BMI and basal serum insulin concentrations (Table 3). In both groups, the significant inverse relationship was found between basal serum leptin concentrations and glucose disposal rate in the last 20 min of clamp, the insulin sensitivity index and metabolic clearance rate of glucose respectively (Table 3). No significant relationships between serum leptin concentrations and the rest of the parameters studied were found at basal state in any of groups studied. Serum leptin concentrations in patients with PA after treatment positively correlated with serum insulin concentrations and diastolic blood pressure (Table 3). The correlation of serum leptin concentrations and BMI was on the borderline of statistical significance (P ⫽ 0.059). Serum leptin concentrations after treatment correlated negatively with insulin sensitivity index only, while the significant relationship of serum leptin concentrations with glucose disposal rate in the last 20 min of clamp and metabolic clearance rate of glucose respectively disappeared after treatment (Table 3). No additional significant relationships of serum leptin concenJournal of Human Hypertension

trations and the rest of the parameters studied were found in patients with PA after treatment.

Discussion The aim of this study was to compare serum leptin levels in patients with PA with those of healthy subjects and to explore the relationship of insulin action parameters and serum leptin levels in patients with PA before and after surgical or pharmacological treatment. We have found that basal serum leptin levels in patients with PA do not significantly differ from those of healthy subjects, but increased significantly after surgical or pharmacological treatment. The insulin sensitivity was significantly impaired in patients with PA compared with healthy subjects and it was partially improved by surgical or pharmacological treatment. Leptin is a protein hormone produced predominantly by adipocytes involved primarily in food intake regulation.1 However a series of other actions in the human body including angiogenesis, haematopoiesis and possible significance in blood pressure regulation is also suggested.4–6,9,10 Recently Torpy et al18 reported that serum leptin levels in patients with PA caused by aldosteroneproducing adenoma increased significantly after surgical treatment. Our study is the first that compared serum leptin levels of patients with PA with age-, gender- and BMI-matched subjects. We failed to demonstrate a significant difference in basal serum leptin levels of these patients and those of healthy subjects. However, in agreement with Torpy’s results, we found a significant increase of serum leptin levels in these patients after treatment. Because body fat content was not measured in this study the possibility of alterations of this variable as the cause of increased serum leptin levels after the operation cannot be ruled out completely. However the lack of any significant change in BMI makes the variations in body fat content unlikely. We therefore suggest that the post-treatment increase in serum leptin levels is not explainable by body composition changes itself. Serum leptin levels in the case of stabilised nutritional status reflect the body fat content, thus they are increased in obese compared with lean subjects.2,3 However, in the case of dynamic changes of energy intake leptin levels seem to represent rather the ongoing triglyceride synthesis or breakdown in adipocytes than the body adiposity itself.20 Except for body fat content itself serum leptin levels are under the control of a series of other hormonal and metabolic factors among which insulin probably plays a pivotal role.21 The impairment of insulin secretion and/or sensitivity have been reported in patients with PA, however the reports concerning this topic are contradictory. Ishimori et al16 found increased insulin sensitivity in patients with PA. On the other hand our recent study17 and the study of


Shamiss et al14 demonstrated decreased insulin sensitivity in these patients. Here we have studied the relationships of serum leptin levels and insulin sensitivity parameters assessed by hyperinsulinaemic euglycaemic clamp in patients with PA and the age-, gender- and BMImatched group. We have demonstrated that patients with PA are insulin-resistant compared with healthy subjects and that the surgical or pharmacological treatment partially improves their insulin sensitivity. In basal state serum leptin levels correlated inversely with insulin sensitivity parameters in both PA patients and healthy subjects. Obesity with insulin resistance is in most cases accompanied by hyperleptinaemia.4 The drop of body weight usually leads to the improvement of insulin sensitivity together with the drop of serum leptin levels.4 In this study, however, the improvement of insulin sensitivity in patients with PA was accompanied by paradoxical increase of serum leptin levels. On the basis of these results we suppose that the changes in insulin sensitivity are unlikely to explain the alterations of serum leptin levels in patients with PA. The explanation for altered serum leptin levels in patients with PA thus could be still only speculative. The physiological significance of leptin in the normal regulation of blood pressure in humans is not definitely established so far. Thus, the possibility that the decrease of serum leptin levels could be the counterregulatory reaction of the human body to the longterm hypertension should be also taken into account. The direct effect of extremely increased serum aldosterone levels and/or decreased serum renin levels on leptin synthesis and/or release cannot be definitely excluded either. Moreover, the relationship between chronically decreased potassium levels and leptin synthesis should also be studied. Thus, further studies addressing these points are necessary to clarify the reason for altered serum leptin levels in patients with PA. In conclusion, our study failed to demonstrate the significant difference of serum leptin levels in patients with PA as compared with healthy subjects but further supported the fact that the treatment of PA increased serum leptin levels. The clarification of the precise mechanism and physiological relevance of altered serum leptin levels in PA is under the scope of our current studies.

Acknowledgements This study is supported by grants from IGA MH No. 5455-3, 5031-3, 6663-3, 6669-3 and Research Project of Ministry of Education No. J13/98:111100002-1. We thank Bio Vendor company for kindly providing ELISA kits for leptin measurements.

References

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