CASE REPORT. Abstract. Introduction. Misaki Miyata 1,2, Masanori Yoshida 1,2, Junji Shinoda 1, Hironobu Sasano 3 and Yutaka Oiso 4

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1 CASE REPORT A Marked Difference in the Vasopressin Responsiveness Between the Adrenal Glands in a Patient with Adrenocorticotropin-independent Macronodular Adrenal Hyperplasia Misaki Miyata 1,2, Masanori Yoshida 1,2, Junji Shinoda 1, Hironobu Sasano 3 and Yutaka Oiso 4 Abstract We herein present the case of a 53-year-old patient with adrenocorticotropin-independent macronodular adrenocortical hyperplasia (AIMAH), which is a rare form of Cushing syndrome. He had hypercortisolemia and bilateral macronodular adrenal glands with a left side predominance. The administration of vasopressin significantly increased the plasma cortisol level (1.9-fold). Following left adrenalectomy, the patient s hypercortisolemia significantly improved and vasopressin responsiveness was lost, suggesting that the responsiveness originated from the resected left adrenal gland. The marked difference in vasopressin responsiveness between the adrenals corresponded with their asymmetrical size and function. Evaluating the differences in the vasopressin sensitivity may therefore be helpful for understanding the progression of AIMAH. Key words: adrenocorticotropin-independent macroadrenal hyperplasia, Cushing syndrome, vasopressin receptor, vasopressin responsiveness (Intern Med 52: , 2013) () Introduction Adrenocorticotropin (ACTH)-independent macronodular adrenocortical hyperplasia (AIMAH) is a rare disorder characterized by the macronodular enlargement of the bilateral adrenal glands with autonomous cortisol hypersecretion. AI- MAH accounts for less than 1% of cases of Cushing syndrome (CS) (1). secretion in AIMAH patients is mediated by aberrant membrane receptors for vasopressin, gastric inhibitory polypeptide (GIP), catecholamine, serotonin and luteinizing hormone (LH)/human chorionic gonadotropin (hcg), which are expressed in adrenocortical cells (2-9). Chronic stimulation of these receptors also plays an important role in the development of AIMAH. Asymmetrical development of bilateral adrenal lesions is often observed in AIMAH patients. In addition, the adrenal glands differ in their respective ability to synthesize cortisol. This unbalanced size and function of the adrenals may originate from the differential responsiveness of the glands to the ligands listed above. Although there are many studies regarding aberrant receptors and cortisol hypersecretion in AIMAH patients, the variable effects of ligands acting on the two adrenals in the same AIMAH patient have not been completely elucidated. We herein describe the case of an AIMAH patient with a marked difference in the responsiveness to vasopressin between the adrenals. Biochemical assays All blood samples were obtained in the morning after an overnight fast and after a 30-minute rest in the supine position. The levels of hormones were measured using commercial radioimmunoassay or enzymatic immunoassay kits. The normal ranges of the hormones are shown in Table. Dexamethasone was given at either 1-mg or 8-mg doses at 23: 00 hours for an overnight dexamethasone suppression test. Department of Endocrinology, Toyota Memorial Hospital, Japan, Department of Endocrinology and Diabetes, Nagoya Ekisaikai Hospital, Japan, Department of Pathology, Tohoku University School of Medicine, Japan and Department of Endocrinology and Diabetes, Nagoya University School of Medicine, Japan Received for publication August 28, 2012; Accepted for publication January 22, 2013 Correspondence to Dr. Masanori Yoshida, myavp@zm.commufa.jp, yoshidaz@ekisai.or.jp 1073

2 Table. Hormone Values Obtained on Admission A reference range ACTH pg/ml g/dl Growth Hormone 0.05 ng/ml IGF ng/ml Vasopressin 6.1 pg/ml LH 3.45 miu/ml FSH 2.2 miu/ml Prolactin 9.9 ng/ml TSH 0.64 IU/mL Free T pg/ml Free T ng/dl Plasma renin activity 1.0 ng/ml/hr Aldosterone 64.5 pg/ml Adrenaline 29 pg/ml < 170 Noradrenaline 231 pg/ml Dopamine 14 pg/ml < 30 DHEA-S 16 g/dl B reference range 17-KS 10.7 mg/day OHCS 24.3 mg/day Urinary free cortisol 508 g/day Aldosterone 4.7 g/day 0 10 Adrenaline 7.3 g/day 1 14 Noradrenaline 82.2 g/day Dopamine g/day Metanephline 0.17 mg/day Normetanephline 0.12 mg/day A, Basal hormone values obtained in the morning. B, 24-h urinary hormone values. IGF-1: insulin-like growth factor 1, FSH: follicle stimulating hormone, TSH: thyroid stimulating hormone, T3: triiodothyronine, T4: thyroxine, DHEA-S: dehydroepiandrosterone sulfate, 17-KS: 17-ketosteroid, 17-OHCS: 17-hydroxycorticosteroid The plasma ACTH and cortisol values were measured the next morning. To investigate the relationship between cortisol secretion and aberrant hormone receptors, the plasma cortisol values were measured at the suggested times following the administration of 0.2 U of i.v. vasopressin, 2.5 μg of s.c. desmopressin, 500 μg of i.v. thyrotropin releasing hormone (TRH), 100 μg of i.v. luteinizing hormone-releasing hormone (LH-RH) or 75 g of oral glucose. In these provocation tests, a positive response was defined as an increase of more than 1.5-fold above the baseline following the administration of the stimulus (10, 11). Histology Tissue samples obtained during surgery were fixed in a 10% formalin solution and embedded in paraffin. The sections were stained with hematoxylin-eosin (HE). Immunohistochemical staining of steroidogenic enzymes, including 3β-hydroxysteroid dehydrogenase (3βHSD) and 17αhydroxylase (P-45017α), was performed with formalin-fixed, paraffin-embedded sections. The antibodies used have all been described previously (12). Case Report A 53-year-old man was referred to Toyota Memorial Hospital on suspicion of CS by a general practitioner. He had been treated at a local clinic for hypertension, diabetes, hyperlipidemia and vasospastic angina. He had been taking 10 mg of amlodipine, 50 mg of spironolactone, 80 mg of valsartan, 3 mg of glimepiride and 2 mg of pitavastatin. He was 170 cm tall and weighed 78 kg and had typical features of CS such as a moon face, central obesity, a buffalo hump and leg edema. His blood pressure was 120/91 mmhg, and the levels of HbA1c, low-density lipoprotein (LDL) cholesterol and triglycerides were 8.6%, 154 mg/dl and 266 mg/ dl, respectively. The patient s endocrinological data are shown in Table. One week before the endocrine study was conducted, amlodipine, spironolactone and valsartan were changed to 40 mg of nifedipine CR. The morning plasma cortisol and 24-h urinary free cortisol (UFC) levels were significantly high. A circadian variation of cortisol was absent, and the plasma ACTH levels were suppressed throughout the day (Fig. 1A). A high dose (8 mg) of overnight dexamethasone administration failed to suppress the morning cortisol level. Abdominal computed tomography (CT) and magnetic resonance imaging (MRI) revealed bilateral macronodular adrenal glands 131 (right, mm; left, mm) (Fig. 2A-D). I- adosterol scintigram demonstrated bilateral adrenal uptake that was predominant on the left side, thus indicating that the extent of cortisol production was greater in the left adrenal gland (Fig. 2E). The i.v. administration of 0.2 U of vasopressin, but not s.c. injection of 2.5 μg of desmopressin, increased cortisol secretion 1.9-fold above the baseline. This exceeded the criterion value for a positive response (>1.5- fold) (10, 11), suggesting that this effect was caused by either the V1a or V1b receptor, not the V2 receptor (Fig. 1B). On the other hand, the plasma cortisol level failed to rise following the administration of oral glucose, LH-RH or TRH (Fig. 1B), suggesting that GIP, LH/hCG and TRH receptors were not associated with the cortisol overproduction observed in this patient. These observations were consistent with a diagnosis of vasopressin-responsive AIMAH. Laparoscopic left adrenalectomy was performed. AIMAH was pathologically confirmed; the cut surface displayed multiple yellowish nodules (Fig. 3A). The nodules were composed of compact cells and clear cells (Fig. 3B). Immunoreactivity of P-45017α and 3βHSD was observed exclusively in the small compact cells and large clear cells, respectively (Fig. 3C, D) (12). Hydrocortisone was given as steroid cover for four days after surgery, and thereafter the patient did not receive any glucocorticoid replacement. The findings of diurnal variation in the plasma ACTH and cortisol levels and the overnight 8- mg dexamethasone suppression test are shown in Fig. 4A. The patient s UFC levels on two consecutive days were 10.7 and 40.1 μg/day. These observations indicated that the hypercortisolemia was significantly improved; however, the cortisol autonomy persisted. Notably, a vasopressin loading test performed 11 days after surgery revealed a blunted cortisol response (Fig. 4B, C), thus suggesting that there was an apparent laterality of vasopressin responsiveness between 1074

3 Intern Med 52: , 2013 A ACTH 6h h h h h 21.7 pg/ml Ǵg/dL 8 mg dexamethasone B Stimulant Vasopressin DDAVP Glucose LH-RH TRH ACTH ACTH 0 min 15 min 30 min 60 min 90 min 120 min Figure 1. Evaluation of the adrenal function. A, Diurnal variation of the plasma ACTH and cortisol levels and the results of an 8-mg dexamethasone suppression test. B, The responsiveness of the plasma cortisol levels to the administration of vasopressin, desmopressin, oral glucose, LH-RH and TRH. Figure 2. Radiological findings of AIMAH. A-C, CT scans. D, MRI. A T2-weighted coronal image is shown. The arrows indicate the bilateral adrenal lesions. E, 131I-adosterol scintigram performed on day 8 after isotope injection. the adrenal glands. Seven months after surgery, the patient had lost 16 kg and his metabolic parameters significantly improved; the HbA1c, LDL cholesterol and triglyceride levels were 6.2%, 92 mg/ dl and 162 mg/dl, respectively, without taking any antidiabetic or hyperlipidemic drugs. The patient s blood pressure was well-controlled with 40 mg of nifedipine CR only. One year after unilateral adrenalectomy, a further vasopressin loading test was performed, which showed that cortisol responsiveness to vasopressin was apparently reduced after surgery (Fig. 4B, C). This result suggested that the stimulatory effects of vasopressin originated from the resected left adrenal lesion. The marked difference in vasopressin responsiveness between the adrenal glands corresponded with their asymmetrical size and function. The size of the remaining right adrenal grand did not increase for at least two years after surgery. Discussion Although the pathogenesis of AIMAH has been investigated from both molecular and clinical aspects, it is not yet fully understood. The pathogenesis of AIMAH is thought to be heterogeneous. Activation of camp/protein kinase A (PKA) signaling plays an important role in the formation of AIMAH (1); a somatic mutation of the Gsα subunit gene (GNAS) has been found in the adrenal nodules of some patients with AIMAH (13). In only a few limited cases, an ac- 1075

4 Figure 3. Histological findings of the resected adrenal gland. A, Macroscopic view. B, Hematoxylin and Eosin staining. The adrenal nodules were composed of compact cells and clear cells. C and D, Immunohistochemical stains. Exclusive immunoreactivity of P α in small compact cells (C) and 3βHSD in large clear cells (D) was observed. A 8 h 12 h 18 h 23 h 6 h ACTH pg/ml g/dl 8 mg dexamethasone B C (μg/dl) Before surgery After surgery Time (min) 0 min 15 min 30 min 60 min 90 min 120 min After 11 days ACTH After 1 year ACTH Figure 4. Evaluation of the adrenal function after surgery. A, Diurnal variation of the plasma ACTH and cortisol levels and the results of an 8-mg dexamethasone suppression test performed six days after surgery. B, The responsiveness of the plasma cortisol levels to vasopressin (0.2 U i.v.) before (circle with solid line), 11 days after (square with solid line) and one year after (triangle with dashed line) left adrenalectomy. C, Raw data for the vasopressin stimulation tests 11 days after and one year after surgery. 1076

5 tivating mutation of the melanocortin 2 receptor, which belongs to the G protein-coupled receptor superfamily, has been identified (14). On the other hand, various kinds of hormones, including vasopressin, serotonin, LH/hCG, GIP and angiotensin II, are known to induce excessive secretion of plasma cortisol via membrane receptors that are abnormally expressed in the adrenals. In particular, vasopressin stimulates cortisol secretion through eutopic V1a and/or ectopic V1b and V2 receptors in many cases of AIMAH (2-9). Antagonists of these receptors such as V1a receptor antagonists and β-blockers have been reported to suppress cortisol secretion (3, 15, 16). The long-term stimuli of these ligands are thought to be associated with adrenal enlargement as well as cortisol secretion in AIMAH patients. In the present study, vasopressin loading tests were performed three times in total (once before surgery and twice after surgery). The administration of vasopressin increased cortisol secretion before surgery. In contrast, this effect was lost following left adrenalectomy, indicating that the response to exogenous vasopressin was due to the resected left adrenal. When performing a vasopressin test, dexamethasone pretreatment is recommended to inhibit an ACTH increase through the V1b receptor in pituitary corticotroph cells (17). Although dexamethasone was not given in this case, the plasma ACTH level was completely suppressed in the first two vasopressin tests performed before and 11 days after surgery. Therefore, at least in both of these tests, the effects of ACTH could be ignored and vasopressin was thought to increase the plasma cortisol level through the V1a and/or V1b receptors in the adrenals before surgery. On the other hand, in the vasopressin test performed one year after surgery, a slight increase in the plasma ACTH level ( pg/ml) following vasopressin administration might have affected the cortisol response, which increased by approximately 25% above baseline. However, this rate of increase was below the criterion value for a positive response (10, 11). This responsiveness might thus have been lower if dexamethasone had been given. In any case, the vasopressin responsiveness was still apparently reduced one year after surgery. Therefore, the significant enhancing effects of vasopressin on cortisol secretion were thought to originate from the left adrenal lesion. In addition, the plasma and urinary cortisol levels markedly decreased after surgery, thus suggesting that the cortisol production capacity was greater in the left adrenal gland. This observation was consistent with the results of the 131 I-adosterol scintigram. Therefore, in the present case, a close relationship may have existed between the vasopressin responsiveness, the steroidogenic function and the size of the adrenals. The immunohistological examination performed in this case showed that 3βHSD was expressed only in the large clear cells, whereas P-45017α was seen predominantly in the small compact cells, consistent with the finding of a previous study (12). This differential expression pattern of steroidogenic enzymes results in inefficient cortisol synthesis in massive adrenal lesions in patients with AIMAH. In fact, the cortisol content per gram tissue obtained from AIMAH patients is lower than that obtained from patients with Cushing s adenoma or even normal subjects (18). Although bilateral adrenalectomy is a curative treatment for AIMAH, patients are required to receive life-long glucocorticoid replacement and are susceptible to adrenal insufficiency following surgery. Alternatively, unilateral adrenalectomy of the largest gland is proposed to be a safe and effective treatment for AIMAH, especially when a size difference between the adrenals is observed (19, 20). Lamas et al. reported four patients with AIMAH who are in long-term remission of CS following unilateral adrenalectomy (19). In a follow-up study of seven cases of AIMAH, for a median of 4.5 years after unilateral adrenalectomy, a significant improvement of CS in clinical and laboratory findings was observed in all but one patient (20). On the other hand, as shown in recent studies, the contralateral adrenal gland can become enlarged following unilateral adrenalectomy (21, 22). In the present case, providing follow-up is required, although the patient s clinical features markedly improved following left adrenalectomy. Although unilateral adrenalectomy of the largest adrenal gland is performed in some cases of AIMAH, the responsiveness of cortisol secretion from the remaining adrenal gland to ligands has been rarely evaluated after surgery. Therefore, the effects of secretagogues on each adrenal gland in the same AIMAH patient have not been fully elucidated. Albiger et al. examined the cortisol response before and after unilateral adrenalectomy in a food- and hcg/lhdependent AIMAH patient (11). In that case, the cortisol response disappeared in an oral glucose test but remained in an hcg test (50% above baseline) after surgery. However, similar studies have not yet been conducted in patients with vasopressin-responsive AIMAH. Therefore, it is unknown whether the marked difference in vasopressin responsiveness between the adrenals observed in the current study is common or specific to our patient. To clarify this point, further studies of more AIMAH patients are required. A size difference in the bilateral adrenals is often observed in AIMAH patients. In many cases, the degree of cortisol production in the largest gland is greater than that observed in the other, and there is a significant positive correlation between the volume of the adrenal glands and cortisol secretion in familiar AIMAH patients (8). To date, the causative factor(s) accounting for the differences in the size and function between the adrenals remain(s) unknown. Interestingly, Gicquel et al. found heterogeneous clonal patterns in the adrenal glands and even in nodules within the same gland (23). Therefore, the two adrenals in patients with AI- MAH may differ in many points, including the proliferative rate, the efficacy of steroidogenesis, the distribution patterns of aberrant receptors, and the responsiveness to secretagogues. In vasopressin-responsive AIMAH, which is the most common form of this disease, the difference in vasopressin responsiveness may result in differences in size and the cortisol secretion levels between the adrenals. In the pre- 1077

6 sent case, the marked improvement observed in the clinical and hormonal findings after left adrenalectomy was most likely due to the differing characteristics between the adrenals in addition to mass reduction. Evaluating the differences in vasopressin sensitivity may therefore help to further our understanding of the pathogenesis and progression of AIMAH. The authors state that they have no Conflict of Interest (COI). References 1. Lacroix A. ACTH-independent macronodular adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 23: , Bourdeau I, D Amour P, Hamet P, Boutin JM, Lacroix A. Aberrant membrane hormone receptors in incidentally discovered bilateral macronodular adrenal hyperplasia with subclinical Cushing s syndrome. J Clin Endocrinol Metab 86: , Daidoh H, Morita H, Hanafusa J, et al. In vivo and in vitro effects of AVP and V1a receptor antagonist on Cushing s syndrome due to ACTH-independent bilateral macronodular adrenocortical hyperplasia. 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