Chronic administration of an angiotensin II receptor antagonist resets the hypothalamic–pituitary–adrenal (HPA) axis and improves the affect of patients with diabetes mellitus type 2: Preliminary results

Abstract

Diabetes mellitus type 2 (DM type 2) is associated with depressive symptomatology and intermittent hyperfunction of the hypothalamic–pituitary–adrenal (HPA) axis. DM type 2 is also accompanied by increased tissue levels of angiotensin II (Ang II), which stimulates the HPA axis through the Ang II type 1 receptors (AT1). We investigated the effect of candesartan, an angiotensin receptor blocker (ARB) that crosses the blood brain barrier, on the activity of the HPA axis and on the affect of 17 patients with DM type 2, aged 40–65 years, who were treated with 4 mg/day candesartan per os for at least 3 months. Before and after candesartan administration, a corticotropin-releasing hormone (CRH) stimulation test and psychological tests were performed. In response to hCRH, time-integrated secretion of ACTH was not altered by candesartan administration, however, the cortisol response was decreased significantly compared to baseline (mean ± SEM, 2327 ± 148.3 vs. 1943 ± 131.9 μg/dl, P = 0.005) suggesting reduced sensitivity of the adrenals to ACTH. In parallel, there was a significant improvement in interpersonal sensitivity (0.91 ± 0.16 vs. 0.70 ± 0.15, P = 0.027) and depression scores (0.96 ± 0.15 vs. 0.71 ± 0.10, P = 0.026). We suggest that candesartan resets the HPA axis of patients with DM type 2 and improves their affect.

• Angiotensin receptor blocker
• cortisol
• diabetes mellitus type 2
• HPA axis
• depression

Introduction

Angiotensin II (Ang II), the final effector of the renin–angiotensin system (RAS) (Lavoie and Sigmund 2003) plays major roles in fluid and electrolyte homeostasis and the regulation of the hypothalamic–pituitary–adrenal (HPA) axis and the autonomic nervous system (Phillips 1987; Saavedra 1992; Phillips and Sumners 1998). Ang II exerts its effects through angitotensin receptors 1 and 2 (AT1 and AT2 receptors). These G protein-coupled receptor subtypes are present in various proportions in Ang II target tissues. In the brain, AT1 and AT2 are widely distributed in structures that are both outside and inside the blood brain barrier (Landas et al. 1980; Tsutsumi and Saavedra 1991A,B,C). Ang II modulates CRH neurons directly in the rat hypothalamus, through AT1 receptors, which are located in the perikarya of the periventricular and parvicellular divisions of the paraventricular nucleus (Aguilera et al. 1995; Latchford and Ferguson 2005). In rodents and primates, AT1 receptors have also been described on the pituitary corticotrophs (Hauger et al. 1982; Lenkei et al. 1999; Latchford and Ferguson 2005; Pawlikowski 2006), with Ang II directly stimulating ACTH secretion in vitro (Gaillard et al. 1981; Ganong 1993). The in vivo effect of Ang II on ACTH secretion is probably mediated primarily by CRH, since an increase in plasma ACTH requires relatively high doses of peripherally administered Ang II, while intracerebroventricular injection of low doses (1 ng) of this hormone suffice to elicit large increases in plasma ACTH concentration (Spinedi and Negro-Vilar 1983; Dessi-Fulgheri et al.1985; Spinedi and Rodriguez 1986; Murakami and Ganong 1987; Ganong 1993; Volpi et al. 1996; Coiro et al. 1998). Furthermore, Ang II stimulates the formation and release of vasopressin in the paraventricular nucleus (Phillips 1987; Veltmar et al. 1992; Lenkei et al. 1995). Vasopressin secreted into the hypophysial portal circulation synergizes with CRH, enhancing the secretion of ACTH (Gillies et al. 1982; Liu et al. 1983). Ang II also has direct effects on the adrenal gland, via the splanchnic nerves, stimulating the secretion of cortisol from the zona fasciculate (Rabano et al. 2004), of aldosterone from the zona glomerulosa (Aguilera 1993) and of catecholamines from the adrenal medulla (Livett et al. 1990). Furthermore, there are intrinsic RAS systems in the adrenal zona glomerulosa and medulla through which locally produced Ang II mediates the release of aldosterone and the catecholamines (Plunkett et al. 1985; Deschepper et al. 1986; Livett and Marley 1993; Phillips et al. 1993). In the rat adrenal medulla, AT1 and AT2 receptors regulate norepinephrine and epinephrine formation and release (Jezova et al. 2003). In addition, centrally administered Ang II increases the transcription rate of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine formation in the locus coeruleus (Seltzer et al. 2004).

In diabetes mellitus type 2, (DM type 2) the circulating RAS is usually normal or suppressed, while tissue Ang II level is increased (Henriksen et al. 2001; Kersaw and Flier 2004; Shinozaki et al. 2004; Giacchetti et al. 2005). This increased tissue Ang II induces oxidative stress, endothelial damage and disease pathology including vasoconstriction, thrombosis, inflammation and vascular remodeling. Furthermore, insulin resistance is associated with upregulation of the AT1 receptor and an increase in oxygen free radicals in endothelial tissue caused by activation of NAD(P)H oxidase. Treatment with an AT1 receptor blocker (ARB) normalizes oxidase activity and improves endothelial function (McFarlane et al. 2003; Giacchetti et al. 2005; Zanchetti and Elmfeldt 2006). DM type 2 is a chronic disease associated with protracted metabolic dyshomeostasis of different degrees. Patients with this disorder have intermittent hyperfunction of their HPA axis expressed as non-suppression of plasma cortisol by dexamethasone, an increased 24 h urinary free cortisol (UFC) excretion rate and/or an abnormal CRH stimulation test (Roy et al. 1993; Tsigos et al. 1993). Subtle abnormalities in cortisol action may be one of the missing links among factors contributing to development of the metabolic syndrome and its manifestations in patients with obesity, hypertension, coronary heart disease, hyperlipidemia and DM type 2 (Andrews et al. 2002). Also it is known that cortisol administration impairs cholinergic vasodilation (Mangos et al. 2000) and suppresses nitric oxide (NO) release by down-regulating endothelial NO synthase proteins (eNOs) (Rogers et al. 2002). Interestingly, chronic CRH hypersecretion and/or mild hypercortisolism also characterize chronic anxiety (Tsigos and Chrousos 2002; Contoreggi et al. 2004) and major depression (Tsigos and Chrousos 1994; Habib et al. 2001; Makino et al. 2002; Tsigos and Chrousos 2002), while in DM type 2 there is an increased prevalence of both of these disorders (Wells et al. 1989; Thomas et al. 2003; Charmandari et al. 2005).

We hypothesized that an ARB that crosses the blood brain barrier could reset the HPA axis by inhibiting both peripheral and central AT1 receptors in disorders such as DM type 2 characterized by mild intermittent hyperactivity of this axis and mild affective symptomatology (Nishimura et al. 2000; Gohlke et al. 2002). To test the ability of candesartan to normalize the activity of the HPA axis in patients with DM type 2 to decrease their affective symptomatology, and improve arterial wall properties, we administered candesartan at a low dose and examined the patients and their HPA axis function before and after at least 3 months of candesartan administration.

Materials and methods

Patients

We studied 17 patients, 6 women and 11 men, aged 40–65 years (mean ± SEM, 53.9 ± 2.44 years) with DM type 2 of less than 5 years duration, who first were diagnosed and regularly followed at Diabetes Clinics of the Athens University Medical School. The diagnosis of DM type 2 was based on the criteria established by the World Health Organization in 1996 and modified by the American Diabetes Association in 1997 and 2003 (American Diabetes Association Standards of medical care in diabetes 2006).

Patients were included in the study provided that: (a) their DM type 2 was controlled by diet and/or oral antidiabetic medication, and (b) they had no clinical or biochemical evidence of retinopathy, nephropathy, or neuropathy. Patients that were receiving antidiabetic medication(s) continued their treatment. Patients were excluded if they had concurrent hypertension, an allergic, inflammatory or autoimmune disorder, or were pregnant or lactating. In addition, patients were excluded if they were receiving synthetic glucocorticoids, non-steroidal anti-inflammatory drugs, or psychotropic medication. Five of the 6 women included were menopausal and received no hormone therapy, while one was pre-menopausal not on oral contraceptives.

The patients' hemoglobin (Hb) A1c and albumin to creatinine ratio in a spot urine sample was < 7% (mean ± SEM, 6.5 ± 0.26%), with < 30 μg/mg creatinine (mean ± SEM, 18.0 ± 0.84 μg/mg), respectively (American Diabetes Association Standards of medical care in diabetes 2006).

Protocol

On the first visit, a complete medical history was obtained, while a physical examination, screening laboratory examinations (a complete blood count and standard chemistry) and an electrocardiogram were performed. To exclude retinopathy, patients underwent eye fundoscopy. Nephropathy was excluded by plasma measurements of creatinine and urea and spot urine measurements of the albumin-to-creatinine ratio (as above; American Diabetes Association Standards of medical care in diabetes 2006). Neuropathy was excluded both by a neurological examination and the use of a vibrameter for the assessment of sensory perception thresholds.

Patients received 4 mg/day candesartan per os for at least 3 months. To ensure compliance with candesartan administration, patients were asked to note down the daily taking of the medicine. During this time, and throughout the study, patients receiving medication(s) for diabetes type 2 continued their standard treatment. Before and at the end of candesartan administration, patients underwent standard anthropometric measurements (body mass index, BMI; height, waist to hip ratio), laboratory examinations (glucose, HbA1c, urea, creatinine, lipids, C-reactive protein (CRP), serum amyloid A protein (SAA), serum insulin, cortisol-binding globulin (CBG), albumin and creatinine in the urine free cortisol (UFC); estimation of indices of insulin resistance and sensitivity by homeostasis model assessment (HOMA) and quantitative insulin sensitive check index (QUICKI), respectively, endocrine (CRH test), psychological tests [symptom checklist-90-Revised (SCL-90-R), state-trait-anxiety-inventory (STAI), Zung Depression scale, Whiteley index, Leyton obsessional inventory and visual analogue scales] and peripheral hemodynamic measurements [flow-mediated dilation of the brachial artery (FMD), Carotid–femoral pulse wave velocity (PWV), heart rate, systolic and diastolic blood pressure).

Methods

Anthropometry

Height in centimeters was measured on a portable stadiometer calibrated with a machine meter rod, and weight in kilograms was measured with an electronic scale. Body mass index (BMI) was calculated by dividing body weight in kilogram by height in meters squared. Waist to hip circumference ratio was also obtained.

Biochemical tests

Blood samples were drawn in the morning (0800 h) from an antecubital vein after at least 8 h of fasting and before taking any medication(s). Peripheral venous blood samples (20 ml) were drawn into plastic syringes under sterile conditions and transferred immediately to appropriate tubes for measurements of glucose, HbA1c, urea, creatinine, SAA, CRP, lipids, insulin and CBG. UFC was determined in 24 h urine collections, while albumin and creatinine were also determined in urine spot collections for ratio calculation. All measurements were performed before and after chronic candesartan administration.

HOMA index

Insulin resistance was estimated by the HOMA index by employing the following formula: [Glucose (mmol/l) × Insulin (mIU/ml)/22.5] (Hanley et al. 2002).

Quicki Index

Insulin sensitivity was defined using the quantitative insulin sensitivity check index by employing the following formula: 1/(log₁₀ [fasting Glucose] + log₁₀ [fasting Insulin] (Katz et al. 2000).

CRH stimulation test

This was performed for the evaluation of HPA axis function. At 1600 h, patients were placed at bed rest for the duration of the procedure, and an i.v. catheter was inserted 40 min before CRH administration. At 1700 h, human (h)CRH was administered at a dose 1 μg/kg as an i.v. bolus over 2 min. Blood for ACTH determinations was drawn and placed in pre-chilled EDTA containing tubes 15 and 1 min before and 5, 15, 30, 60, 90 and 120 min after administration of hCRH and placed on ice. Blood for cortisol determinations was also drawn and placed in appropriate tubes, at the same time-points. Plasma and serum were separated in a refrigerated centrifuge within 3 h of withdrawal, stored in polystyrene tubes and frozen at − 20°C until assayed for ACTH and cortisol.

Assessment of endothelial function

This was measured as FMD of the brachial artery. All subjects were studied in the morning, having abstained from alcohol, caffeine, food and tobacco use for 8 h before the study as well as, before they had taken their medication(s). The patient rested supine in a quiet room for at least 30 min before endothelial function was assessed. The procedure was performed according to recently published guidelines. Optimal imaging of the right brachial artery was obtained and a resting scan was recorded using an Echo–Doppler ultrasound machine (Vivid 7, GE, Horton, Norway) and a 7–10 MHz transducer. Reactive hyperemia was then induced by inflation of a blood pressure cuff on the forearm for 5 min and subsequent deflation; the brachial artery was scanned continuously 30 s before and 90 s after cuff deflation. All images were recorded on super VHS videotape for analysis. Artery diameter measurements were made using electronic calipers from the anterior to the posterior mid-line. Flow-mediated dilation was calculated as the percent increase in arterial diameter during hyperemia as compared to the resting scan. At the end images were obtained 4 min after sublingual nitroglycerine (0.4 mg) for measurement of nitrate-induced, endothelium-independent vasodilation (NID). The inter- and intra-observer variability for brachial diameter measurements in our laboratory is 0.1 ± 0.12 and 0.08 ± 0.19 mm respectively, while FMD variability measured on two different days was 1.1 ± 1% (Corretti et al. 2002).

PWV for measurement of arterial stiffness

All patients were examined in the morning hours and after a 10 min rest period. Patients did not receive their medications on the day of examination. PWV was measured automatically using Complior apparatus (Artech Medical France). PWV determination is based on the simultaneous recording of pulse waves in the common carotid and femoral arteries by two transducers, and calculated as the distance separating the two transducers divided by the time delay between the onset (foot) of the two recorded waves (Protogerou et al. 2006).

Peripheral hemodynamics measurements—brachial blood pressure and heart rate

Brachial systolic blood pressure, diastolic blood pressure and heart rate were recorded from the right arm using an automated sphygmomanometer (after 15 min of supine rest in a quiet room).

Psychometring testing

A battery of self-administered questionnaires was given to all patients. The following tests were employed:

1. Symptom checklist-90 revised (SCL-90-R). This provides 9 sub-scores and a total score. It is a general psychopathology test, as describes somatization, obsessive-compulsive symptoms, interpersonal sensitivity, depression, anxiety, aggression, phobic anxiety, paranoid ideation and psychoticism (Derogatis 1983).

2. State-trait-anxiety-inventory (STAI). This questionnaire assesses anxiety and provides separate scores for “state” (X1) and “trait” (X2) Anxiety (Spielberger et al. 1970).

3. Zung. Depression scale. This provides a total score of depressive symptomatology, focused on the bodily manifestations of depression (Zung 1965).

4. Whiteley index. This provides a total estimation of hypochondriacal manifestations (Pilowski 1967).

5. Leyton obsessional inventory (trait portion). This provides a total score of obsessional personality traits (no-symptoms) (Cooper 1970).

6. Visual analogue scales. This provides a subjective evaluation of the individual across a line connecting two opposite aspects of a notion (Mottola 1993).

The study was approved by the Bioethics Committee of the Laikon and Aretaieion University Hospitals, as well as by the Greek National Drug Administration (EOF). All patients gave informed consent of participation.

Assays

Glucose, total cholesterol (Chol), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), urea and creatinine as well as urine albumin and creatinine were measured using the Siemens Advia 1650 Clinical Chemistry System (Siemens Medical Solutions, Erlangen, Germany). Cholesterol bound to low density lipoprotein (LDL) was estimated by the Friedewald equation. Internal quality control for lipids was carried out according to the laboratory manual of the Lipid Research Clinics Programme. Whole blood HbA1c levels were measured with cation exchange HPLC (HA8121 HPLC system, Arkray Inc., Kyoto, Japan).

The concentrations of SAA and CRP in serum were measured by particle-enhanced immunonephelometric assays (BN ProSpec nephelometer, Dade Behring, Liederbach, Germany).

The serum concentration of insulin was measured by the automated chemiluminescence system ACS: 180 (Siemens Medical Solutions, Erlangen, Germany). Assays for serum cortisol concentrations were performed on previously frozen serum with the same system as for insulin. UFC was also measured with this system. Conversion of the results for the direct urine sample provided by the system in μg/dl, to μg/24 h, was made using the following equation: direct urinary cortisol (μg/24 h) = assay result (μg/dl) × 10 × V, where V = volume of urine in liters excreted per 24 h. Normal values for UFC measured by the ACS 180, are 28.5–213.7 μg/24 h.

Serum levels of cortisol-binding globulin (CBG) were measured by radioimmunoassay “RIA” (BioSourse Europe SA, Belgium). The analytical sensitivity (lower detection limit) was 0.25 μg/ml.

Plasma levels of ACTH were measured by the electrochemiluminescence immunoassay “ECLIA” using the Roche Elescys 2010 immunoassay analyzer (Roche Diagnostics Mannheim, Germany).

Intra- and inter-assay imprecision were less than 5% for all assays.

Statistical analyses

Values are expressed as mean ± SEM, as indicated. ACTH and cortisol measured during the CRH test performed before and after candesartan administration were compared using ANOVA repeated-measures. Dunnett's post-hoc test was applied. The total area under the curve (AUC) of plasma ACTH and serum cortisol concentrations vs. time during CRH tests was estimated using the trapezoid method, a numerical integration method. The AUC of the ACTH/cortisol ratios during CRH tests was also estimated. The AUCs of ACTH, cortisol and ACTH/cortisol during CRH tests performed before and after candesartan administration, were compared using paired Student t-test analysis. Biochemical (cholesterol, TG, HDL, LDL, HbA1c, glucose, CRP, SAA, insulin, CBG, UFC and the ratio albumin to creatinine of a spot urine sample) and clinical (body weight, BMI, waist/hip ratio) parameters, indices of insulin sensitivity (QUICKI) and resistance (HOMA) measurements of the hemodynamic tests and scores of psychometric tests before and after candesartan administration were compared with paired Student's t-test. Relationships between the psychometric test scores were investigated by calculation of Pearson's correlation coefficient. Statistical significance was set at P < 0.05.

To quantify the difference between two groups and taking into account the variability of the measurements of the study, we calculated effect size for the number of patients participating in this study.

Results

Anthropometric and biochemical characteristics

Body weight, BMI and the waist to hip ratio of the patients remained unchanged with candesartan administration (Table I).

Table I.  Anthropometric, clinical and biochemical characteristics before and after candesartan administration (BMI, body mass index; UFC, urine free cortisol; CBG, cortisol-binding globulin; Glu, glucose; HOMA, the homeostasis model assessment; HbA1c, glycosylated Hb A1c; QUICKI, quantitative insulin sensitivity check index; SEM, standard error of the mean).

	Before candesartan (n = 17)		After candesartan (n = 17)
	Mean	SEM	Mean	SEM
Height (m)	1.69	0.02
Weight (kg)	78.54	4.18	78.88	4.51
BMI (kg/m^2)	27.50	1.09	27.62	1.11
Waist/Hip ratio	0.946	0.945	0.021	0.011
Heart rate (pulses/min)	62.9	1.88	57.7	1.63
UFC (μg/24 h)	81.74	5.58	88.15	9.75
CBG (μg/ml)	55.87	7.52	50.46	6.39
Cholesterol (mg/dl)	175.0	7.60	179.4	9.70
LDL (mg/dl)	107.2	5.50	111.1	6.10
HDL (mg/dl)	43.40	1.70	43.70	1.90
Triglycerides (mg/dl)	121.7	14.50	126.2	17.50
Fasting insulin (mU/l)	13.15	3.68	9.63	2.00
Fasting Glu (mg/dl)	142.6	3.10	123.8	2.92
Albumin/creatine (μg/mg)	18	0.84	17.8	0.89
HbA1C (%)	6.5	0.26	6.42	0.31
HOMA	5.20	1.45	2.95	0.58
QUICKI	0.324	0.009	0.340	0.007

Twenty four-hour UFC, circulating CBG, cholesterol, HDL, LDL, TG, fasting serum insulin and fasting glucose concentrations, albumin to creatinine ratio of a spot urine sample, whole blood HbA1c levels and Quicki and HOMA indices did not change significantly between baseline and after candesartan administration (Table I). The effect sizes of the parameters weight, BMI, CRP, insulin, HOMA and QUICKI ranged between 0.30 and 0.60, whereas the remainder were below 0.2.

Plasma ACTH and cortisol responses to hCRH test

The time-course of ACTH and cortisol responses to an i.v. bolus of hCRH are illustrated in Figure 1(A),(B). A statistically significant difference was found only at time-point +120 min for the ACTH response to hCRH (P = 0.017). At other time-points, the plasma ACTH response to hCRH was unchanged after at least 3 months of candesartan administration (Figure 1(A)). The time-integrated secretion AUC for ACTH did not differ at the two testing times (Figure 1(a)). Statistically significant differences were found in the cortisol response to hCRH before and after candesartan administration at time-points t = − 1 (P = 0.031), t = +5 (P = 0.021), t = +15 (P = 0.022), t = +30 (P = 0.034) and t = +90 min (P = 0.041) (Figure 1(B)). The time-integrated secretion AUC of cortisol after candesartan administration was significantly suppressed compared to baseline (P = 0.005; Figure 1(b)). The effect size for the time-course of ACTH responses to hCRH was greater for the time-points t = +5, +15, +30, +60 min (0.32–0.45). The effect size for the time-course of cortisol responses to hCRH was greater for the time-points t = − 1, +5, +15, +30, +60 and +90 min (0.42–0.75). The estimated effect size of the time-integrated secretion AUC of ACTH and cortisol were 0.24 and 0.91, respectively. The effect size for cortisol was particularly significant.

Figure 1 (A) Mean ± SEM plasama ACTH (pg/mL) response to hCRH in DM type 2 patients before (closed squares) and after (open squares) candesartan administration. Asterisks indicate statistically significant difference at the indicated time-point. P = 0.0017, n = 17. (a) Mean ± SEM AUC values of plasma ACTH in DM type 2 patients before (black bars) does not differ from that after (white bars) candesartan administration. (n = 17). (B) Mean ± SEM serum cortisol (μg/dl) response to hCRH in DM type 2 patients before (closed squares) and after (open squares) candesartan administration. Asterisks indicate statistically significant difference at the indicated time-point. − 1 min: P = 0.031; 5 min: P = 0.021; 15 min: P = 0.022; 30 min: P = 0.034; 90 min: P = 0.041; n = 17. (b) Mean ± SEM AUC values of serum cortisol in DM type 2 patients before (black bars) and after (white bars) candesartan administration. Asterisks indicate statistically significant difference at the indicated time-point. P = 0.005, n = 17. (C) Mean ± SEM plasma ACTH (expressed in pg/ml)/serum cortisol (expressed in μg/dl) ratios at the different time-points of the hCRH test in DM type 2 patients before (closed squares) and after (open squares) candesartan administration. Asterisks indicate statistically significant difference at the indicated time-point. − 1 min: P = 0.045; 5 min: P = 0.01; 90 min: P = 0.034; 120 min: P = 0.021; n = 17. (c) Mean ± SEM AUC values of plasma ACTH (expressed in pg/ml)/serum cortisol (expressed in μg/dl) ratios in DM type 2 patients before (black bars) and after (white bars) candesartan administration. Asterisks indicate statistically significant difference at the indicated time-point. P = 0.017, n = 17.

As an index of a change in adrenocortical sensitivity to ACTH the ratios of the circulating ACTH to cortisol concentrations were calculated at the different time-points after hCRH both before and after candesartan administration (Figure 1(C)). Statistically significant differences were found at the time-points t = − 1 (P = 0.045), t = +5 (P = 0.010), t = +90 (P = 0.034) and t = +120 min (P = 0.021). The time-integrated AUC of the ratios of the circulating ACTH to cortisol concentrations after hCRH was significantly increased compared to baseline after candesartan administration (P = 0.017; Figure 1(c)). The estimated effect size for the ratios of the circulating ACTH to cortisol concentrations, as well as of the time-integrated AUC of these ratios, calculated at the different time-points after hCRH, before and after candesartan administration, was greater for points t = − 15, − 1, +90, +120 min and above 0.4 for these time-points and 0.28 for the ratio of the AUCs, respectively.

Vascular endothelial function before and after candesartan administration

Systolic (124.20 ± 3.10 vs. 119.20 ± 4.60 mmHg) and diastolic (79.10 ± 2.60 vs. 74.60 ± 3.50 mmHg) blood pressure did not decrease significantly with candesartan. Candesartan significantly decreased heart rate (P = 0.01), but did not alter FMD (5.42 ± 1.12 vs. 4.95 ± 1.15) and arterial stiffness (10.27 ± 1.38 vs. 11.37 ± 1.86). The estimated effect sizes for diastolic blood pressure, heart rate at baseline and after hyperemia and nitrate-induced-hyperemia, were above 0.5.

Psychological profile of the patients before and after candesartan administration

After candesartan administration, there was an improvement in the interpersonal sensitivity (0.91 ± 0.16 vs. 0.70 ± 0.15, P = 0.027) and depression (0.96 ± 0.15 vs. 0.71 ± 0.10, P = 0.026) scores obtained by the SCL-90-R. There were positive correlations between several of the psychometric scores reflecting psychological co-morbidities (data not shown). The effect sizes of the psychometric results from the psychological tests performed gave values above 0.4 for depression, hypochondriacal manifestations and obsessional traits.

No significant differences were found between male and female subjects, when the means of the main measures were compared.

Discussion

The patients with DM type 2 we studied were not clinically depressed, had no recent history of hospitalization for any reason and had no condition that might have affected their HPA axis activity in a major fashion. Furthermore, all DM type 2 patients had well-controlled, mild DM type 2, with a mean blood Hb A1c less than 7%. Hence, it was unsurprising that they had UFC values within the normal range. They showed a decreased cortisol response to a bolus infusion of hCRH after at least 3 months of candesartan administration, compared to the baseline response. This is consistent with previous findings indicating that dysregulation of the HPA axis may be present among patients with DM type 2 (Roy et al. 1993; Tsigos et al. 1993; Andrews et al. 2002), characterized by chronic intermittent mild hypercortisolism and a concomitantly altered HPA axis response to CRH and suggests that candesartan administration reset the HPA axis closer to normal. The unchanged baseline UFC excretion levels after candesartan administration indicates a properly functioning glucocorticoid negative feedback system before and after candesartan administration and a complete, time-integrated quantitative reset of the HPA axis. This is consistent with the unchanged plasma ACTH response to hCRH with candesartan administration, and the increased ACTH to cortisol response ratio, suggesting that candesartan decreased sensitivity of the adrenal cortex to ACTH. This is compatible with a chronically decreased central stimulation of the HPA axis and, presumably, a consequently smaller adrenal cortex (Bornstein et al. 1998; Bornstein and Chrousos 1999).

Endothelial function assessed by FMD did not change significantly after candesartan treatment. Hypercortisolemia leads to endothelial dysfunction, which is ameliorated by inhibition of cortisol production by metyrapone (Broadley et al. 2005, 2006), and candesartan at a dose of 8–16 mg daily improves endothelial function in postmenopausal women (Wassmann et al. 2006), and in patients with hypertension and non-insulin dependent DM (Rizzoni et al. 2005). The lack of FMD improvement in our study could be explained by the lower dose of candesartan employed (4 mg daily), which in turn had a mild but statistically non-significant hypotensive effect. PWV did not change with candesartan administration either, indicating no effect of low dose candesartan on arterial stiffness. The short period of treatment with candesartan could explain the lack of improvement in arterial stiffness. Sasamura et al. (2006) observed that PWV values decreased after 1 year of treatment with candesartan in hypertensive patients, which is in accordance with observations by Spoelstra-de Man et al. (2006) in hypertensive type 2 diabetic patients. In contrast, in the present study there was a significant decrease in heart rate, probably due to a decrease in sympathetic system activity that is in accordance with the hCRH test results showing a decrease in the responsiveness of the HPA axis. An increase in vagal tone was previously described after administration of losartan only in rats with myocardial infarction and heart failure, but not in control rats (Du et al. 1998). Ang II does not induce a decrease in vagal tone in normal subjects either (Sander-Jensen et al. 1988). Therefore, the decrease in heart rate observed in our study in patients with normal left ventricular function after administration of candesartan is unlikely to be due to increased vagal tone. Finally, in this study after chronic low dose candesartan administration there was a tendency for improvement of the affective psychopathology of patients with DM. Recently, Saavedra et al. (2005) showed that in rats long-term pretreatment with candesartan prevented the cortical CRH and benzodiazepine responses to isolation stress and was anxiolytic in the elevated plus-maze, whereas AT2 knockout mice exhibited anxiety behaviour (Okuyama et al. 1999). Furthermore, there is some preliminary clinical evidence for a possible role of Ang II in depression. Indeed, the angiotensin-converting enzyme (ACE) inhibitor captopril improved mood in hypertensive patients with depression, an effect that was not observed with other anti-hypertensive drugs (Zubenko and Nixon 1984; Deicken 1986; Germain and Chouinard 1988).

There is a large amount of evidence from preclinical studies that clearly shows the anti-stress effects of ARB in acute stress disorders (Saavedra and Pavel 2005). Armando et al. (2001) demonstrated that candesartan prevented HPA axis and sympathetic stimulation during isolation stress, while administration of an ARB protected against stress-induced gastric ulcers in rats (Bregonzio et al. 2003).

Chronic activation of the stress system includes changes in both the HPA axis and the sympathetic nervous system and a resetting of the entire system to greater activity (Tsigos and Chrousos 1994, 2002; Habib et al. 2001;). Ang II stimulates HPA axis via AT1 receptors regulating CRH release. Through the sympathetic system, brain Ang II increases heart rate and arterial blood pressure and (Phillips 1987; Reid 1992; Saavedra 1992; Phillips and Sumners 1998), via the splanchnic nerves and the intrinsic RAS in the adrenal zona glomerulosa and medulla (Plunkett et al. 1985; Deschepper et al. 1986; Livett and Marley 1993; Phillips et al. 1993) stimulates the adrenal medulla and, indirectly, the adrenal cortex (Livett et al. 1990; Ehrhart-Bornstein et al. 1995; Bornstein et al. 1998; Bornstein and Chrousos 1999; Jain et al. 2004). The adrenal zona fasciculata is thus stimulated both by circulating ACTH and the stress-activated medulla (Tsigos and Chrousos 1994; Bornstein et al. 1998; Bornstein and Chrousos 1999). A chronically activated stress system is associated with structural changes of the adrenals characterized by hypervascularization and cellular hypertrophy and hyperplasia of the cortices, a larger size and a greater responsiveness to ACTH (Bornstein et al. 1998; Bornstein and Chrousos 1999). Naturally the glucocorticoid negative feedback system is also activated, which exerts a restraining influence upon the stress system by inhibiting ACTH and central sympathetic activity (Tsigos and Chrousos 1994, 2002; Habib et al. 2001; Jain et al. 2004). In our subjects, candesartan exerted effects on both limbs of the stress system, resetting both the HPA axis and the sympathetic system at a lower level of activity.

Our results are in accordance with previous studies, examining decrease of HPA axis activity in association with other centrally acting drugs. Thus, chronic imipramine administration to healthy, non-depressed volunteers resulted in decreased ACTH and cortisol responses to ovine CRH and AVP without changes in indices of time-integrated HPA axis function, including UFC excretion (Michelson et al. 1997). This action of imipramine is potentially related to its therapeutic effects in melancholic depression, a disorder that is associated with hypothalamic hypersecretion of CRH. Similarly, alprazolam, a triazolobenzodiazepine, administered to jacketed, intravenously cannulated nonhuman primates and to normal humans suppressed HPA axis activity (Kalogeras et al. 1990). This most likely reflects suppression of the CRH neuron rather than the pituitary corticotroph, explaining its efficacy in the treatment of chronic anxiety.

Our results are also in accordance with previous studies suggesting that Ang II is an important stress hormone, implicated in the response to different stressors. Ang II contributes to the regulation of the sympathoadrenal and hormonal responses to stress, through stimulation of the brain and peripheral AT1 receptor. In rats, the effects of centrally administered Ang II on the sympathetic nervous system are mediated via an increase in oxidative stress in brain regions involved in the noradrenergic control of blood pressure (Campese et al. 2005). A two-week edaravone treatment significantly restored systolic blood pressure and lipid peroxidation to normal and enhanced the catalase and superoxide dismutase activity in diabetic rats suggests the involvement of hydroxyl radical stress in augmented responses of Ang II in diabetic animals (Saini et al. 2006). On the other hand, Ang II participates in the response to isolation stress (Armando et al. 2007). Furthermore, Ang II seems to regulate not only the acute (Armando et al. 2001) but also the chronic stress reaction (Uresin et al. 2004). In addition, blockade of the RAS by losartan significantly prevented the elevation of plasma glucose levels induced by chronic immobilization stress (Uresin et al. 2004). Furthermore, a large body of evidence suggests a possible link between Ang II-dependent end-organ damage and the advanced glycation end products in vivo (Bohlender et al. 2005).

In conclusion, candesartan administration for at least 3 months decreased central HPA axis activity and improved the affect of patients with DM type 2. We suggest that ARB at higher doses might be useful as antidepressants. We suggest that candesartan resets the HPA axis of patients with DM type 2 and improves their affect, possibly protecting these patients from long-term CRH hypersecretion.

Acknowledgements

We would like to acknowledge Anastasios Boutsiadis for the calculation of the effect sizes.