Abstract

Introduction

Adrenal masses incidentally disclosed on computed tomography (CT) scans, ‘adrenal incidentalomas’ are common. Prevalence increases during life such that they are found in ten percent of the population aged 70 years or more [1], [2]. Between five to twenty percent of these are associated with mild cortisol excess without the classical external features typically associated with Cushing's syndrome [3], often termed ‘subclinical Cushing's syndrome’, or ‘subclinical hypercortisolism’ [4]. One of cortisol's major functions is the regulation of glucose metabolism: it promotes gluconeogenesis by activation of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver [5], [6]; inhibits insulin release in the pancreas [7]; decreases insulin sensitivity in skeletal muscle and adipose tissue [8]. Consequently, an important complication of cortisol excess is insulin resistance (IR) [9], a major independent marker of cardiovascular risk [10], and this is reflected by the fact that over 60% of individuals with adrenal incidentalomas and low grade cortisol secretion have IR, impaired glucose tolerance or diabetes mellitus compared to age, sex and BMI-matched controls [11]. However, as IR is common in the general population it is unknown whether the IR in patients with adrenal incidentalomas is secondary to the low level cortisol secretion or other factors.

The GR antagonist mifepristone (11-[4-(Dimethylamino)phenyl]-17-hydroxy-17-[1-propynyl]-[11ß,17ß]-estra-4,9-dien-3-one) has been shown to improve glucose tolerance in patients with overt Cushing's syndrome [12]. We hypothesized that insulin resistance in individuals with adrenal incidentalomas and low-grade cortisol excess without the classical external features typically associated with Cushing's syndrome might also be improved by short-term GR blockade if in these individuals IR is driven by cortisol excess. Our findings showed, for the first time, that even short-term GR blockade in these individuals improved insulin resistance. These data could inform the design of a much awaited prospective interventional randomized controlled study aimed to identify whether such an approach could be used as a prediction tool to stratify an affected individual to surgery, medical therapy or observation [13].

Methods

Study Design & End-points

The flow chart in Figure 1, as recommended by the TREND statement, describes the different phases and design of the study. One patient who was approached and screened did not meet the entry criteria, whilst one other declined to participate. Study visits occurred weekly up to week 4 and procedures at each visit are shown in Figure 2. The baseline glucose tolerance test was performed on the first day of treatment (treatment start) before the first dose of mifepristone whilst the baseline 24-hour ambulatory BP monitoring was performed between the first visit (screening) and second visit (treatment start). During each visit renal function, 0900 h and 2300 h salivary cortisol (representing free cortisol) and 0900 h plasma ACTH and serum cortisol (total cortisol) levels were assessed to confirm compliance and efficacy of GR blockade. All interventions were performed by the clinical research team on the Facility. Since mifepristone blocks the GR the expectation was a rise in plasma ACTH and serum and salivary cortisol by activation of the hypothalamo-pituitary-adrenal (HPA) axis, but with blockade of GR-mediated activity. Saliva samples were obtained using the Salivette tubes (cotton swab with citric acid preparation; Sarstedt, Numbrecht). Subjects were instructed to refrain from brushing their teeth, smoking, eating or drinking anything for at least 60 min prior to sampling. To provide a saliva sample, patients were asked to chew on the cotton swab for 1–2 min and then place it back in the plastic container. The bone formation marker serum osteocalcin was measured in a 0900 h fasting sample, and the second morning urine sample was used to measure the bone resorption marker urine N-telopeptide crosslinks of type 1 collagen (NTX). Full clinical assessment including resting BP, weight and temperature was made at each visit - monitoring for symptoms and signs for adrenal insufficiency can only be performed on a clinical, and not biochemical, basis when on mifepristone. The oral dose of mifepristone was kept the same at 200 mg twice a day at 0900 h and 2100 h, one hour before any food, throughout the study. Although once daily dosing might have sufficed, twice daily dosing was chosen in an effort to maintain as constant a GR blockade effect as possible.

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Figure 1

Flow chart indicating the different phases of the study to week four as recommended by the TREND statement [14].

Analysis performed at week four as all six patients had insulin resistance and blood pressure measurements suitable for analysis.

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Figure 2

Study Design showing investigations and intervention performed on six patients from baseline to four weeks.

One subject was withdrawn after end of second week. ¹variables measured at baseline and end of study. OGTT: Oral Glucose Tolerance Test - insulin & glucose at 0, 15, 30, 60, 90, 120 min, F: cortisol, ACTH: Adrenocorticotropic Hormone, U & E: urea, electrolytes and creatinine, LFT: Liver Function Tests, FBC: Full Blood Count, TFT: Thyroid Function Tests.

To establish a normal 95% reference range for IR we analyzed data from the Helsinki Policemen study, a 22 year follow up investigating what level of IR, as reflected by insulin AUC on an OGTT, predicted cardiovascular events. By taking the mean±2SD of the values of those followed for 22 years (n=755) and in whom no cardiovascular event was observed, we define the upper end of the 95% reference range of insulin AUC associated with no cardiovascular risk as 63420 pmol/l.min (26940±2SD pmol/l.min) [10]. In addition we took values <26940 pmol/l.min to represent definite normalization of AUC and only a decrease in insulin AUC greater than 7237 pmol/l.min, during a 2-hour OGTT, was considered significant as this exceeds biological variation [19]. The insulin AUC was appropriate for our group as in patients with fasting insulin levels less than 200 pmol/l the insulin AUC is reproducible [19]. We chose insulin AUC on OGTT as our primary estimate of IR as this allowed direct comparison to the seminal Helsinki heart study data examining the long term effects of IR on cardiovascular risk. Resting and 24-hour ambulatory blood pressure as measured using standard British Hypertension Society approved monitors were other primary end points.

Secondary end points were the means for fasting glucose, fasting insulin, the homeostasis model assessment of IR and beta cell function (HOMA-IR and HOMA-%beta) [20], and the Insulin Sensitivity Index (ISI) – Matsuda [21], as calculated from an oral glucose tolerance test. The difference in fasting lipids and bone turnover markers at study end were also measured.

Results

Baseline Characteristics

Demographic data for each subject and means (±95%CI) are shown in Table 1. Baseline characteristics were similar to the target population of interest [24], [25].

Table 1

Demographic data – Subject Baseline Characteristics.

Subject	1	2	3	4	5	6	Mean (±95% CI)
Gender	M	F	F	F	M	M
Age (yrs) (range)	72	58	74	56	64	75	67 (58–75)
Mean Weight (kg)	86.0	75.6	70.9	109.0	101.5	110.0	92.2 (74.3–110.0)
Mean BMI (kgm2)	29.1	31.1	24.5	43.7	32.0	39.9	33.4 (26.9–40.5)
Serum Cortisol post ONDST (nmol/l)	67.9	104.0	83.9	88.8	59.9	73.9	79.7 (63.2–96.3)
Serum Cortisol post LDDST (nmol/l)	80.0	54.1	85.0	105.1	80.0	61.0	77.5 (58.2–96.8)
0900 h salivary cortisol (nmol/l)	4.7	6.8	5.2	3.4	5.3	3.4	4.8 (3.4–6.2)
2300 h salivary cortisol (nmol/l)	0.5	0.6	0.9	3.0	2.1	0.5	1.3 (0.2–2.4)
ACTH (pmol/l)	1.9	1.1	2.0	4.1	1.1	1.2	1.9 (0.9–2.9)
Side of adenoma	R	R	L	R	R	R
Size of adenoma (cm)	3.8	2.5	3.6	3.5	2.7	2.0	3.02 (2.3–3.8)
24-hour blood pressure	136/91	143/75	149/86	150/86	138/81	147/77
Fasting glucose (mmol/l)	4.8	6.3	4.7	4.9	5.1	6.5	5.4 (4.6–6.2)
Fasting insulin (pmol/l)	44.4	124.2	34.0	177.1	69.2	86.8	76.4 (39.9–146.4)1

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ONDST: Overnight dexamethasone suppression test; LDDST: 48-hour 2 mg low dose dexamethasone suppression test; R=Right, L=Left;

¹Geometric mean.

Hypothalamo-pituitary-adrenal axis analysis

Mifepristone blocked glucocorticoid action as evidenced by activation of the HPA axis. Baseline mean serum cortisol level was 372.5 nmol/l (95%CI 309.0–435.9 nmol/l), this increased progressively in each subject, such that at one week levels had increased significantly (1065 nmol/l 95% CI 636.0–1495.4 nmol/l; p=0.03) and further increased by 4 weeks (1551 nmol/l (95%CI 615–2486 nmol/l; p=0.03). Baseline mean plasma ACTH was low at 1.9 pmol/l (95%CI 0.7–3.1 pmol/l) whilst plasma ACTH levels at 4 weeks were 13.6 pmol/l (95%CI 4.4–22.9 pmol/l; p=0.03) (Figure 3a). Baseline mean salivary cortisol levels at 0900 h and 2300 h were 4.8 nmol/l (95%CI 3.4–6.2 nmol/l) and 1.3 nmol/l (95%CI 0.2–2.4 nmol/l), respectively. There was a significant increase in salivary cortisol over 4 weeks at 0900 h (99.3 nmol/l; p=0.03) and 2300 h (9.4 nmol/l; p=0.03) (Figure 3b).

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Figure 3

a: Graph showing mean ±SEM 0900 h serum cortisol and ACTH levels at baseline and 4 weeks.

Activation of the HPA axis, that is a rise in serum cortisol and ACTH, is already evident one week after starting mifepristone. b: Graph showing mean ±SEM 0900 h and 2300 h salivary cortisol levels at baseline and 4 weeks. Circadian rhythm of cortisol is maintained evidenced by high levels at 0900 h and low levels at 2300 h, but amplified by the use of mifepristone 200 mg twice per day.

Changes in insulin resistance

As a group there were significant reductions in IR (lnHOMA-IR) and improvements in sensitivity (lnMatsuda index) compared to baseline following mifepristone treatment (Table 2). Five out of six individuals showed a reduction in insulin AUC (Figure 4). In subjects 2 and 5 there was a clinically significant reduction in insulin AUC from above the reference range into the upper half of the normal reference range. In subjects 1 and 6, insulin AUC fell by approximately 50% from the upper end of the reference range to the lower half of the reference range (<26940 pmol/l.min): patient 1: 51001 pmol/l.min to 25742 pmol/l.min; patient 6: 55972 pmol/l/min to 23668 pmol/l.min. In subject 3 insulin AUC fell, but this was within the lower half of the reference range at baseline. Subject 4 was highly insulin resistant and did not show any improvement in IR.

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Figure 4

Graphs showing the changes in insulin AUC pre (red circle) and post (black square) mifepristone.

The upper white area represents insulin AUC levels associated with increased risk for cardiovascular (CV) events. The grey shaded area represents the insulin AUC upper and lower 95% reference range (not associated with cardiovascular events). Mean: 26940 pmol/l.min; 2SD: 63420 pmol/l.min. Subjects 2 and 5 show a clinically significant improvement in their insulin AUC post mifepristone (dotted arrow). Subjects 1 and 6 both show a reduction in insulin AUC from the upper to the lower 95% reference range (solid arrow). Patient 4 remains at CV risk post mifepristone, indicating no clinical benefit. The insulin AUC is plotted on the y axis as a logarithmic scale to base 4.

Table 2

Baseline and 4 week insulin sensitivity and glucose tolerance data after the use of mifepristone in 6 patients.

Variable (means ±95% CI)	Baseline	4 weeks	p value
Fasting glucose (mmol/l)	5.4 (4.6–6.2)	5.6 (5.0–6.1)	0.34
2-hour glucose (mmol/l)	10.2 (8.0–12.5)	11.4 (8.7–14.1)	0.21
AUC glucose (mmol/l.min)	1176 (1013–1339)	1187 (988–1384)	0.60
Geom Fasting insulin (pmol/l))	76.4 (39.9–146.4)	53.3 (25.8–109.9)	0.03
Geom 2-hour insulin (pmol/l)	628.5 (258.4–1528.3)	420.5 (159.4–1109.5)	0.20
Geom AUC insulin (pmol/l.min)	61973 (30889–124337)	40083 (16020–100289)	0.03
lnHOMA-IR	1.0 (0.2–1.7)	0.6 (−0.1–1.4)	0.03
lnHOMA-%β	4.8 (4.2–5.4)	4.3 (3.7–5.0)	0.03
lnMatsuda	1.2 (0.4–1.9)	1.6 (0.7–2.4)	0.03

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Repeated measures effect analysed by non-parametric Wilcoxon's signed rank test.

AUC: Area under the curve HOMA - IR: Homeostatic Model Assessment of Insulin Resistance; HOMA - %β: Homeostatic Model Assessment of Beta Cell function.

Discussion

We have shown that short-term use of mifepristone can reduce IR in subjects with adrenal incidentalomas and low-grade autonomous cortisol secretion. To our knowledge, use of mifepristone in subjects with adrenal incidentalomas has never been tested. These data support recent findings in a study in patients with overt Cushing's syndrome where an improvement in insulin AUC and HOMA-IR was shown in the combined diabetes mellitus/impaired glucose tolerance and hypertension groups [12].

As a group we showed a significant reduction in IR and five of our subjects had a decrease in insulin AUC greater than 7237 pmol/l.min, exceeding biological variation. In addition, to establish whether GR blockade might also be associated with cardiovascular benefit, we defined our cut-off for insulin AUC from data published from the Helsinki Policemen Study, which investigated the relationship between baseline IR and the risk for cardiovascular disease over 22 years [10]. In that study, on an OGTT a mean (±SD) insulin AUC of 26940±18240 pmol/l.min was found in those without a future cardiovascular event. We used, therefore, the mean insulin AUC (±2SD) as our 95% reference range. Using this method we identified two subjects (numbers 2, 5) who had IR above this level but in whom IR decreased into the normal range with GR antagonism, consistent with a likely clinical benefit. Two other subjects (numbers 1, 6) experienced a reduction of their insulin AUC to the lower half of the reference range, whilst subject 3 had an insulin AUC value at baseline in the lower half of the reference range that improved further. Conversely, subject 4 has insulin resistance, which despite GR blockade, was not reversed. It may be that in some cases insulin resistance is more ‘fixed’ and less dependent on cortisol. We chose insulin AUC on OGTT as our primary estimate of IR as this allowed direct comparison to the seminal Helsinki heart study data examining the long term effects of IR on cardiovascular risk.

In addition to insulin AUC we used other estimates of insulin sensitivity. The Matsuda index of whole body insulin sensitivity, representing a composite of hepatic and peripheral tissues, and which considers insulin sensitivity in the basal state and after the ingestion of a glucose load, correlates strongly with estimates of insulin sensitivity from the ‘gold standard’ euglycemic insulin clamp (r=0.73; p<0.0001) [21]. Although HOMA, or even stronger, log-transformed HOMA indices, correlate well with euglycemic clamps, they measure fasting steady state glucose and insulin concentrations and assume that hepatic and peripheral insulin sensitivity are equivalent [20], [23]. Nonetheless all insulin sensitivity indices significantly improved. Our data give strong support to the notion that even mild cortisol excess may be deleterious on insulin sensitivity/resistance, and that short-term antagonism of GR may be one means of teasing out which subjects have potentially reversible IR.

There remains an open question as to whether individuals who have been identified with adrenal incidentaloma should undergo any intervention. A number of studies have been carried out in individuals with cortisol secreting incidentalomas who have been subjected to adrenalectomy [26], [27] and improvements have been noted in weight, blood pressure, lipid profiles, fibrinogen levels, and glycemic control [11], [28], [29], [30], [31]. There is, however, no consensus on the criteria for the diagnosis of low-grade cortisol excess in patients with adrenal incidentalomas [13], [27] creating a dilemma on whom should be referred to surgery. Recently, tests of the HPA axis associated with known metabolic consequences of hypercortisolemia in these individuals have been suggested [32], but benefit from adrenal surgery has been demonstrated in subjects without ‘subclinical hypercortisolemia’ [26]. Whilst one interpretation is that this argues against a direct role for cortisol in their phenotype [27], an alternative interpretation is that the definition of ‘sub clinical’ is not correct, with some of those undergoing surgery being labeled as ‘normal’ in fact having clinically significant excess cortisol. Here, we have used dexamethasone suppression tests with a serum cortisol cut-off of 50 nmol/l. Diagnosis was supported by a low mean baseline 0900 h ACTH of 1.9 pmol/l. Whilst in a general population this strategy runs the risk of including too many false positives, in an individual with adrenal incidentalomas there is a considerable a priori likelihood that there is some abnormality of cortisol secretion, and thus a positive screening raises the post-test probability of hypercortisolemia to approximately 99% [18], [33]. It was for these reasons that we specifically focused on what would often be regarded as truly mild cases, to ask the question as to whether we would see any metabolic changes by antagonizing the GR without a pre-conceived definition ‘subclinical Cushing's/hypercortisolemia’; other than a diagnostic strategy that is in keeping with Endocrine Society guidelines [18]. Although we did not measure plasma dexamethasone levels there does not appear to be an advantage to using the 8 mg DST [34], and thus we feel that it is unlikely that our patients tested false positive.

As a group no significant changes were detected in the other metabolic markers investigated. Interestingly the two patients with clinically significant decreases in insulin AUC (patients 2 and 5) showed improvements in average 24-hour ambulatory blood pressure. Meta-analysis of several large prospective studies has shown that a 5 to 6 mm Hg decrease in diastolic blood pressure is associated with a 38% reduction in risk for stroke and a 16% reduction in coronary heart disease events [35], whilst a 10 mmHg reduction in systolic blood pressure is associated with a reduction in risk of stroke by one third [36]. Cortisol-induced hypertension is mediated by several mechanisms including an increased vascular responsiveness to catecholamines [37], elevated circulating catecholamine levels [38], and potentially, through a mineralocorticoid effect, when cortisol occupies the mineralocorticoid receptor if in excessive amounts consequent to HPA axis activation. The latter effect could explain why we did not see a significant change in BP in the group. The decrease in HDL although on first glance may seem non beneficial, in patients on mifepristone this has previously been shown to be associated with an increased efflux capacity of serum HDL on a per particle basis, hence no proportional impairment in HDL function [39].

As expected with glucocorticoid receptor antagonism the HPA axis was rapidly activated with serum and salivary cortisol and plasma ACTH levels rising appropriately. Hence, monitoring of changes in serum or salivary cortisol can be used to confirm subject compliance and drug efficacy. Throughout the study as indicated by the 0900 h/2300 h salivary cortisol ratio the physiological circadian rhythm of cortisol was maintained but the amplitude widened with maximal effects of mifepristone occurring in the morning. Limitations of our pilot study include the open design, a small sample and two patients were withdrawn after 2 and 4 weeks of mifepristone treatment, respectively. All patients received 400 mg per day, in two divided doses of 200 mg. The two patients who were withdrawn and who developed some symptoms of adrenal insufficiency were lighter, and hence had the highest relative dose, followed by highest HPA axis activation. Importantly, however, there was no hypotension, indicating the relative clinical safety of using mifepristone in this setting despite the vague symptoms in these individuals. These data reinforce the dose-dependent effect of mifepristone, with a dose of around 5 mg/kg/day being most appropriate. The nature of our study precludes generalization without further studies being performed.

In summary, we have shown that a short period of GR antagonism for up to four weeks in individuals with mild cortisol excess resulted in significant improvements in insulin sensitivity, and potentially clinically significant improvements in two out of six. These effects can now be examined in randomized controlled studies to establish whether GR blockade or cortisol-lowering strategies, are a suitable means for the individualized stratification of individuals with adrenal incidentaloma to medical or surgical intervention, or observation.

Supporting Information

Funding Statement

The study was funded in part by HRA Pharma grant number: R/123263. The funder had no role in data collection, analysis and decision to publish. The study design, and all ethical applications and associated administration, were conceived and written by JN-P and MD. The funder contributed to study design and the preparation of the manuscript.

References