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Original Studies |
Division of Endocrinology and Departments of Medicine (N.N., P.H., J.T., J.-M.B., A.L.) and Pathology (L.G.), Research Center, Hôtel-Dieu, Centre Hospitalier de l’Université de Montréal, 3850 Saint-Urbain, Montreal, Canada H2W 1T8
Address all correspondence and requests for reprints to: André Lacroix, M.D., Division of Endocrinology, Research Center, Hôtel-Dieu, Centre Hospitalier de l’Université de Montréal, 3850 St. Urbain Street, Montreal, Quebec, Canada H2W 1T8. E-mail: lacroixa{at}ere.umontreal.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanisms regulating tissue-specific expression of GIPR are still unknown, as are those leading to its increased adrenal expression. An acquired somatic mutation inducing the overexpression of GIPR may be responsible for the clonal expansion resulting in a single GIP-dependent cortisol-secreting adenoma; in the case of GIP-dependent bilateral macronodular adrenal hyperplasia, where all adrenal cells exhibit hyperplasia, the mutation must have occurred before the early stages of adrenal cortex embryogenesis. It is unclear, however, whether the ectopic expression of the GIPR precedes and is responsible for the adrenal overgrowth in addition to the regulation of cortisol secretion or whether the GIPR overexpression is secondary to dedifferentiation during a proliferative process caused by another pathophysiology. We now report a patient with GIP-dependent Cushing’s syndrome with asynchronous development of bilateral nodular hyperplasia in whom there is evidence that the adrenal overexpression of the GIPR was present at the stage of hyperplasia as well as in larger nodules.
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Case Report |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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Initial investigation had included elevated free urinary cortisol level of 1077 nmol/day (normal range, 90–330 nmol/day); morning fasting plasma cortisol was 279 nmol/L. Morning plasma ACTH was decreased at 0.3 pmol/L (normal range, 2–12 pmol/L). Serum levels were: dehydroepiandrosterone sulfate (DHEAS), 1.5 µmol/L (normal range, 0.9–11.6); testosterone, 0.4 nmol/L (normal range, <2.9); FSH, 3.6 U/L; LH, 0.4 U/L; and PRL, 10 µg/L. Blood electrolytes and fasting and postprandial glucose were normal, whereas low density lipoprotein cholesterol and triglycerides were elevated at 5.78 and 2.99 mmol/L, respectively. An abdominal computed tomography scan revealed that the right adrenal was the site of two 2.5- to 3-cm nodules; the left adrenal was of normal morphology, except for the presence of a 0.8 x 0.6-cm postero-superior nodule. An iodocholesterol scan performed without dexamethasone suppression showed uptake of the tracer only in the right adrenal.
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Materials and Methods |
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The study protocols were approved by the institutional review committee, and written informed consent was obtained from the patient. Medications were discontinued for at least 1 week before conducting the evaluation. Studies were performed after an overnight fast in the supine position for 60 min before testing. An iv dexamethasone suppression test (1 mg/h from 1100–1500 h) was performed as described previously (1), where the patient remains fasting until the end of the infusion of dexamethasone. The protocol to screen for potential adrenal ectopic receptors included serial measurements at 30- to 60-min intervals during 2–3 h of plasma ACTH, cortisol, aldosterone, 17-hydroxyprogesterone, free testosterone, DHEAS, and estradiol during the course of the various tests, which were performed sequentially over the course of several days. Tests included the administration of 100 µg GnRH, iv (Factrel, Wyeth-Ayerst Laboratories, Inc., Saint-Laurent, Canada); 200 µg TRH, iv (Relefact, Hoechst-Roussel, Montreal, Canada); 10 IU arginine vasopressin, im (Pitressin, Parke-Davis, Scarborough, Canada); 1 mg glucagon, iv (Eli Lily Canada, Inc., Scarborough, Canada); 0.2 U/kg regular human insulin, iv (Humulin, Eli Lily Canada, Inc.); 10 mg cisapride, orally (Prepulsid, Janssen Pharmaceuticals, Mississauga, Canada); and 250 µg ACTH-(1–24), iv (Cortrosyn, Organon Canada, Scarborough, Canada). Other tests included a standard mixed meal and a posture test performed by a 2-h supine position, followed by a 2-h ambulation period.
To study the effects of carbohydrates, proteins, or lipids on cortisol secretion, the patient sequentially received orally at 3-h intervals 75 g oral glucose, an isocaloric protein-rich meal, or a lipid-rich meal as described previously (1). On a different day, 25 g glucose were administered iv, and 3 h later, 100 µg octreotide (Sandostatin, Novartis, Pointe Claire, Canada) were administered sc 60 min before repeating an oral 75-g glucose challenge; plasma levels of cortisol, ACTH, GIP, and insulin were determined at regular intervals during these tests. Human GIP (Bachem, Torrance, CA) was prepared and infused at a rate of 0.6 µg/kg·min during the administration of 150 cc/h 10% glucose as described previously (1); to maximize the response, endogenous levels of GIP were suppressed by the sc administration of 100 µg octreotide 90 min before starting the infusion of human (h) GIP. Glucagon-like peptide-1 (GLP-1; Bachem) was provided by Dr. John Dupre (London, Canada) as 50 µg/mL in 0.1% human serum albumin and was infused at a rate of 0.75 pmol/kg·min also under 10% glucose, as described previously (12); the GLP-1 infusion was not preceded by the administration of octreotide.
Assays
Plasma and urinary cortisol and plasma estradiol were measured by immunofluorometric assay (Bayer Immuno I System, Tarytown, NY), ACTH by immunoradiometric assay (Allegro, Nichols Institute Diagnostics , San Juan Capistrano, CA), and plasma GIP (Peninsula Laboratories, Inc. Belmont, CA) and other steroid hormones by commercial RIA kits.
RNA preparation and GIPR RT-PCR
Total RNA was extracted from adrenals by the guanidium-phenol chloroform method (13). First strand cDNA synthesis was carried out with 2 µg total RNA and random primers (hexamers) by using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Burlington, Canada) as recommended by the manufacturer. In control reactions, reverse transcriptase was omitted to ensure that the PCR amplification did not result from contaminating genomic DNA. The PCR reaction contained 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L of each deoxy-NTP, 10 pmol each of sense and antisense primers specific for the human GIP receptor (GenBank U39231), one fifth of the RT reaction, 2.5 U Taq DNA polymerase, and 5% formamide. The amplification was achieved with 35 cycles (94 C, 30 s; 49 C, 30 s; 72 C, 30 s) with a pair of primers specific for hGIPR [5'-TGCTAGCCCTGCTCATCTTGA-3' (513–533) and 5'-ACACGGGGATCCCGCCCCCTA-3' (1453–1474)]. The PCR products were separated on agarose gel. The RNA samples were also amplified (94 C, 30 s; 51 C, 30 s; 72 C, 30 s) with a pair of primers specific for the human ß-actin cDNA (5'-GATTCCTATGTGGGCGA-3' and 5'-GATTCCTATGTGGGCGA- 3').
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Results |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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); the iv administration of ACTH-(1–24)
also resulted in a stimulation of plasma cortisol (180%), whereas the
upright posture test, TRH, GnRH, glucagon, insulin-induced
hypoglycemia, arginine vasopressin, and cisapride were
without effect (Table 1). Plasma cortisol increased in response to
75 g oral glucose (202%) and a lipid-rich meal (183%), but not
after a protein-rich meal (95%) or 25 g glucose, iv (93%); the
response to oral glucose was decreased by pretreatment with 100 µg
octreotide, sc (Fig. 1). Plasma cortisol
elevations followed and were positively correlated with plasma GIP
levels during these various tests (r = 0.95; P =
0.0001). Cortisol levels were stimulated by the infusion of 0.6
µg/kg·h hGIP (225%), but not by 0.75 pmol/kg·min
GLP-1 (88%) (Fig. 2);
plasma insulin levels increased from 138 to a peak value of 344 pmol/L
after 60 min of GLP-1 infusion. The plasma levels of GIP
reached during the hGIP infusion (1000 ng/L) were similar to those
produced by the 75-g oral glucose test (1202 ng/L). The infusion of GIP
in vivo induced an increase in plasma levels of
17-hydroxyprogesterone and free testosterone, but not of aldosterone,
DHEAS, or estradiol (Table 2); plasma
aldosterone increased after upright posture (46 to 234 pmol/L) or
cisapride administration (53 to 524 pmol/L).
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).
However at histology, diffuse hyperplasia was present in the adrenal
cortex, forming small micronodules outside of the two macronodules,
which were composed of an alternance of clear and acidophilic cells
(Fig. 3). The levels of GIPR mRNA in the adrenal and control tissues
were detected using RT-PCR amplification and ethidium bromide staining.
The expected size (980 bp) GIPR band was detected and overexpressed in
both of this patient’s right adrenal nodules (Fig. 4, lanes 2 and 3), whereas it was absent
in the control normal adult adrenal cortex (lane 5). Interestingly, a
GIPR band was also detectable in the patient’s hyperplastic adrenal
cortex adjacent to the macronodules (lane 4). The GIPR bands in this
patient’s macronodules were similar to those found in the previously
reported positive control (5, 11) with a documented GIP-dependent
adrenal adenoma (lane 1) or in normal pancreas (lane 7).
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). The
response of plasma cortisol to 75 g oral glucose was abolished
when the GIP stimulation was inhibited by pretreatment with 100 µg
octreotide, sc. One year after the surgery, the patient still required
replacement with 20 mg hydrocortisone daily because of the persistent
suppression of the hypothalamic-pituitary-adrenal axis; 24-h urinary
free cortisol levels are maintained in the normal range on this
medication. Signs and symptoms of Cushing’s syndrome have disappeared,
weight has decreased to 50.8 kg, and the patient is normotensive
without any other medication. On repeat abdominal computed tomography
scan, the left adrenal nodule now measures 0.8 x 0.9 cm.
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Discussion |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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GIP-dependent Cushing’s syndrome has been reported in a relatively small number of patients to date (1, 2, 3, 4, 5, 6, 7); however, this patient presented several features that render the case of particular interest. Previous patients with GIP-dependent Cushing’s syndrome had fasting plasma cortisol levels ranging from as low as 4–47 nmol/L (5), 68–140 nmol/L (7), 102–119 nmol/L (2), 121–200 nmol/L (6), and 146 nmol/L (5) to 160–193 nmol/L (1); the current patient had fasting plasma cortisol ranging between 279–480 nmol/L, which indicates that GIP-dependent Cushing’s syndrome should not be excluded without performing a test meal. It has been previously proposed that the suppression of ACTH coupled with the low levels of GIP in the fasting state were responsible for the decreased plasma cortisol levels, which can be accompanied by symptoms of relative cortisol insufficiency (1, 2). The various other tests performed were not able to identify another abnormal receptor that could have explained the relatively normal fasting levels of cortisol in this patient. We cannot rule out the existence of ectopic receptors for other hormones that our protocol would not have identified; alternatively, a proportion of cortisol production by the two large nodules may be autonomous and non-GIP dependent.
Food- or GIP-dependent Cushing’s syndrome was previously identified in patients with either bilateral large macronodular adrenal hyperplasia (1, 2, 3) or single unilateral adrenal adenoma (3, 4, 5, 6, 7). We were initially unclear whether this patient had two distinct adenomas in the right adrenal and a nonfunctional incidentaloma in the left adrenal, as the iodocholesterol uptake was restricted to the right adrenal. The macroscopic appearance of the right adrenal tended to support the first hypothesis; however, the histological findings clearly indicate the presence of macronodular adrenal hyperplasia. There was one preliminary report of the coexistence of a schwannoma, pigmented skin lesions in a patient with GIP-dependent bilateral nodular hyperplasia that contained lipofuscin (3); there were no similar characteristics reminiscent of the Carney complex (16) in our or other patients.
This study confirmed the increased expression of GIPR mRNA in the two
GIP-dependent macronodules, as reported previously in patients with
large bilateral adrenal hyperplasia or unilateral adenomas and
GIP-dependent Cushing’s syndrome (3, 5, 6, 7, 11); however, GIPR
overexpression was also detectable in this patient’s adrenal cortex
adjacent to the two larger nodules at a stage of relatively early
hyperplasia. This finding supports the possibility that this patient
has bilateral disease; the probable increased expression of GIPR in the
small left adrenal cortex and nodule would explain the GIP-dependent
cortisol production that was still present after right adrenalectomy.
The previous sequencing of the GIPR cDNA indicated the existence of
spliced isoforms lacking exons 4 and 9 in the GIP-dependent or normal
adrenal tissues and the absence of receptor mutation in GIP-dependent
adrenals (6, 11); the presence of an isoform lacking exon 9 is not
detectable on the gel in Fig. 5 because the 61-bp difference is not
resolved, and the two bands appear as a single 980-bp band.
The molecular mechanisms regulating tissue-specific expression of GIPR are still unknown, as are those leading to its increased adrenal expression. The cloning and characterization of the 5'-promoter and 3'-regulatory regions of the GIPR gene and of their specific transcription factors will be necessary to elucidate this question. It is unclear whether the ectopic expression of the GIPR precedes and is responsible for the adrenal overgrowth in addition to the regulation of cortisol secretion or whether the GIPR expression is a secondary phenomenon occurring during the course of the adrenal proliferation resulting from another primary pathophysiology. The presence of abnormal GIPR expression at the stage of early hyperplasia found in this patient argues in favor of a primary role and suggests that its overexpression precedes the nodular formation and may thus be at least partly responsible for the proliferative process. Chabre et al. (6) recently demonstrated a stimulation of thymidine incorporation by GIP in adrenal cells from GIP-dependent Cushing’s syndrome, but not in normal cells. The steroidogenic secretory pattern suggests that in this case, the cells overexpressing the GIPR have a fasciculata phenotype, with a predominance of cortisol production without a significant stimulation of aldosterone or DHEAS production; different patterns were found previously in vasopressin- or catecholamine-dependent Cushing’s syndrome (14, 15).
The concept of alterations in G protein coupled-receptors and/or
postreceptor events leading to increased cAMP and proliferation is now
well established (17) and was well studied in somatotroph and thyroid
cells (18, 19, 20). Our hypothesis is that ectopic or abnormal expression
of a hormone receptor capable of being coupled to adenylyl cyclase
places the adrenal cells under the stimulation of a trophic factor that
is not under a regulatory negative feedback by glucocorticoids; this
constitutes an unregulated new trophic stimulus that leads to increased
function and possibly to a proliferative advantage. A recent study
indicates that the hormone-stimulated LH receptor can act as an
adrenocortical tumor promoter when ectopically expressed in the adrenal
cortex of mice transgenic for the inhibin 
-subunit promoter/simian
virus 40 T antigen fusion gene (21). It remains to be shown whether the
adrenocortical expression of an ectopic receptor without another
oncogenic event would be sufficient to induce adrenal overgrowth. The
asynchronous nature of nodule formation observed in this patient
suggests that the initial mutation is not uniformly distributed in all
adrenocortical cells, or that other secondary events are necessary to
generate within the hyperplastic cell population a clonal proliferation
of selected cells. Bilateral macronodular adrenal hyperplasia is
usually sporadic, but rare familial cases have been reported (22, 23);
we have not found any evidence of GIP-dependent stimulation of cortisol
production in three siblings of this patient.
The characterization of the pathophysiology of adrenal hyperplasias or tumors can eventually lead to diverse pharmacological therapies as alternatives to adrenalectomy. This has now been illustrated by the short term improvement of hypercortisolism with octreotide in GIP-dependent Cushing’s syndrome (2, 5) and by the long term control of ectopic ß-adrenergic receptors by propranolol (15). It would be beneficial for this and other patients with GIP-dependent Cushing’s syndrome to have access to an effective antagonist of the GIPR such as GIP-(7–30)-NH2 (24) to correct the hypercortisolism and possibly prevent the progression of adrenal cell proliferation.
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Acknowledgments |
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Footnotes |
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Received July 6, 1998.
Revised March 24, 1999.
Accepted May 3, 1999.
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References |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter/simian virus
40 T-antigen fusion gene. Mol Endocrinol 12:801–809.
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), or a lipid-rich meal (
); iv administration of 25 g
glucose (
); and oral administration of 75 g glucose 1 h
after the sc injection of 100 µg octreotide (
) in the patient with
food-dependent cortisol production. The first three tests were
performed consecutively at 3-h intervals on the same day; the last two
tests were also conducted sequentially at 3-h interval on the following
day.
) and in two sisters (