Aberrant cortisol regulations in bilateral macronodular adrenal
hyperplasia: a frequent finding in a prospective study of 32
patients with overt or subclinical Cushing’s syndrome
Rossella Libe ´1,8, Joe ¨l Coste2,7, Laurence Guignat3, Fre ´de ´rique Tissier1,2,4, Herve ´ Lefebvre9, Gae ¨lle Barrande10,
Christiane Ajzenberg11, Igor Tauveron12, Eric Clauser2,5, Bertrand Dousset2,6, Xavier Bertagna1,2,3,8,
Je ´ro ˆme Bertherat1,2,3and Lionel Groussin1,2
1Department of Endocrinology, Metabolism and Cancer, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 567, Centre National de la
Recherche Scientifique Unite ´ Mixte de Recherche 8104, Institut Cochin, 75014 Paris, France,2Universite ´ Paris Descartes, 75006 Paris, France,
3Department of Endocrinology, Center for Rare Adrenal Diseases, Assistance Publique des Ho ˆpitaux de Paris, Ho ˆpital Cochin, 75014 Paris, France,
4Department of Pathology, Assistance Publique des Ho ˆpitaux de Paris, Ho ˆpital Cochin, 75014 Paris, France,5Service d’Hormonologie, Assistance Publique
des Ho ˆpitaux de Paris, Ho ˆpital Cochin, 75014 Paris, France,6Digestive and Endocrine Surgery Unit, Assistance Publique des Ho ˆpitaux de Paris, Ho ˆpital
Cochin, 75014 Paris, France,7Biostatistics Unit, Assistance Publique des Ho ˆpitaux de Paris, Ho ˆpital Cochin, 75014 Paris, France,8INCa Comete
Network, France,9Department of Endocrinology, University Hospital of Rouen, Institute for Biomedical Research, University of Rouen, 76031 Rouen,
France,10Service d’Endocrinologie, CHR d’Orle ´ans, 45032 Orle ´ans, France,11Service de Me ´decine Interne et Endocrinologie, Ho ˆpital Henri Mondor,
94000 Cre ´teil, France,12Service d’Endocrinologie et Maladies Me ´taboliques, CHU de Clermont-Ferrand, 63003 Clermont-Ferrand, France
(Correspondence should be addressed to L Groussinwhois nowat Service des MaladiesEndocrinienneset Me ´taboliques,Ho ˆpitalCochin, 27ruedu Faubourg
Saint-Jacques, 75014 Paris, France; Email: email@example.com)
Context: ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare and heterogeneous
condition characterized by abnormal steroid production. Cortisol secretion can be regulated by
aberrant hormone receptors.
Objective: A large series of patients with AIMAH were evaluated to provide information on the
prevalence and profile of aberrant regulations, in relation with the functional status.
Design and patients: Thirty-two consecutive patients with AIMAH were prospectively studied: 10 had a
Cushing’s syndrome (CS), and 22 had a subclinical CS (SCS).
Methods: A baseline endocrine evaluation was followed by an in vivo protocol in search of aberrant
cortisol responses (seven provocative tests). An acute inhibition test with the somatostatin analog
octreotide was also performed.
Results: At least one aberrant cortisol response was identified in 28 of 32 (87%) patients. The overall
prevalence of aberrant responses was independent of the functional status. Responses to the upright
posture and to metoclopramide were frequently observed (67 and 56% respectively). A glucagon
response was frequently observed in the SCS group (58%). A cortisol inhibition by octreotide
was specifically found in the three CS patients who positively responded to the mixed meal, and was
observed also in 12 of 13 (92%) patients with SCS.
Conclusions: Cortisol responses indicative of aberrant receptor expression were highly prevalent in
AIMAH. Thorough phenotyping of AIMAH may help uncover the underlying pathophysiology.
European Journal of Endocrinology 163 129–138
ACTH-independent macronodular adrenal hyperplasia
(AIMAH) is a rare form of benign bilateral adrenocor-
tical tumor. Nowadays, the most frequent clinical
presentation is that of bilateral adrenal incidentalomas.
The initial endocrine evaluation usually demonstrates
subtle abnormalities of cortisol secretion, most often an
abnormal response to the suppression tests, whereas
urinary cortisol is still normal, and the clinical features
are modest, thereby defining the concept of subclinical
Cushing’s syndrome (SCS). AIMAH might also be
revealed in the diagnostic workup of an overt Cushing’s
syndrome (CS). AIMAH is a heterogeneous condition,
the pathophysiology of which is incompletely under-
stood (1). Without a final histological examination, the
diagnosis of AIMAH is based on the clinical phenotype
because of the lack of a specific diagnostic marker.
In the last few years, several studies have shown that
the steroidogenesis in AIMAH is dependent on non-
ACTH factors: it is regulated by a variety of non-ACTH
circulating factors as the result of aberrant expression
of various hormone receptors at the membrane of
tumoral adrenocortical cells. Cortisol secretion is
therefore ‘controlled’ by ‘ectopic’ receptors such as
those for glucose-dependent insulinotropic peptide
European Journal of Endocrinology (2010) 163 129–138 ISSN 0804-4643
q 2010 European Society of Endocrinology DOI: 10.1530/EJE-10-0195
Online version via www.eje-online.org
(GIP), catecholamine, vasopressin (V2- and V3-vaso-
pressin receptors), serotonin (5-HT7 receptor), and
angiotensin II (AT1 receptor) (2–7). The role of ‘eutopic’
receptors such as those for vasopressin (V1-vasopressin
(LH/hCG-R), serotonin (5-HT4 receptor), and leptin is
also established (8–11).
Investigative protocols are used in vivo to identify
aberrant receptor expression (12, 13). The presence of
aberrant membrane hormone receptors has been
demonstrated in a relatively high proportion of
unilateral tumors, and in most patients with AIMAH
(1, 13–15). The presence of an abnormal response may
help distinguish between various causes of bilateral
adrenal hyperplasia. Identification of aberrant receptor
may also offer a specific pharmacological approach, or
target, to prevent tumor progression and control
abnormal steroidogenesis. In the case of food-dependent
CS, due to aberrant expression of the GIP receptor,
somatostatin analogs have been used to control cortisol
secretion (2, 16).
We prospectively evaluated the presence of aberrant
receptor responses in a large cohort of 32 consecutive
patients with bilateral macronodular adrenal hyperpla-
sia. The present study shows that, independent of the
functional status, aberrant receptors are highly
prevalent. In our series, a response to the standard
mixed meal was specific to patients with overt CS.
Interestingly, a cortisol response to provocative testing
with glucagon was observed in a large proportion of
patients with SCS. In addition, a significant cortisol
inhibition was obtained by the administration of a
somatostatin analog, especially in the SCS group.
Patients and methods
Thirty-two consecutive patients (23 women and 9 men)
with bilateral macronodular adrenal hyperplasia,
referred to our Endocrine Unit, have been investigated
between January 1999 and October 2008. Bilateral
macronodular hyperplasia was defined on the basis of
the computed tomographic appearance. Two radio-
logical aspects were encountered. In some patients,
adrenal glands were massively but smoothly enlarged
with a dominant nodule. In other patients, multiple
bilateral adrenocortical macronodules up to 5 cm in
diameter were presented. Normal, uninvolved adrenal
gland could not be identified. The main clinical
characteristics and initial endocrine investigations are
summarized in Table 1.
Baseline hormonal investigations were performed:
24-h urinary cortisol, diurnal variation in serum
cortisol, 1-mg overnight dexamethasone suppression
test, plasma aldosterone to renin concentration ratio
for patients with high blood pressure, testosterone,
dehydroepiandrosterone sulfate, estradiol, compound S,
desoxycorticosterone, baseline and post-corticotrophin
serum cortisol and 17-hydroxyprogesterone, and
plasma ACTH and cortisol levels before and after i.v.
administration of 1 mg/kg ovine CRH (oCRH). Assays
were performed as previously reported (17–19).
The diagnosis of CS was made on the basis of
elevated daily urinary free cortisol (O90 mg/day),
non-suppressibility of serum cortisol after 1-mg dexa-
methasone testing (O50 nmol/l) and suppressed
plasma ACTH (both basally and after administration
of 1 mg/kg of oCRH).
The definition of subclinical Cushing relies on an
abnormal 1-mg overnight dexamethasone suppression
test (serum cortisol level O50 nmol/l) and normal 24-h
In the 13 operated patients, AIMAH was histo-
logically proven. Multiple nodules or diffuse hyperplasia
were recognized with no residual normal adrenal
cortex. Non-nodular atrophic adrenal cortex could be
detected in some AIMAH.
The study was approved by and was performed
according to the recommendations of the Institutional
Review Board of Cochin Hospital. Written informed
consent was obtained from all the patients.
Provocative tests for the detection of aberrant
The protocol used to detect in vivo cortisol responses
indicative of aberrant adrenal receptors was modified
from that originally described by Lacroix et al. (12). Two
approaches were added in the present study: patients
were asked to provide salivary samples during the
provocative tests for the measurement of salivary
cortisol; an acute octreotide test was performed.
The investigation protocol was conducted over a 4-day
period, after discontinuing potential interfering medi-
cations at least 1 week before the studies. The strategy
consisted of either inducing transient modulation of
endogenous ligands for potentially aberrant adrenocor-
tical receptors or directly administering compounds
acting at these potential receptors, and secondarily
monitoring the steroidogenic responses (serum and
On day 1, a supine-to-upright posture test evaluated
the potential modulation by angiotensin-II, vasopressin,
catecholamines, endothelin, or natriuretic peptide. This
was followed by a standard mixed meal, assessing
adrenal receptors for GIP. Subsequently, i.v. injection of
250 mg ACTH 1–24 provided a reference for the adrenal
On day 2, administration of 100 mg GnRH i.v. was
used to assess the potential modulation of cortisol by
FSH, LH, or GnRH. A bolus injection of 200 mg TRH i.v.
was used to evaluate the adrenal presence of TSH,
prolactin, or TRH receptors. Subsequently, 10 mg of
metoclopramide (Primpe ´ran, Synthe ´labo Laboratories,
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Received 5 April 2010
Accepted 7 April 2010
R Libe ´ and others
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 163