Food-dependent Cushing’s syndrome due to unilateral adrenocortical adenoma with cortisol secretion without ACTH elevation detected in peripheral blood by the CRH test: a case report

Abstract

We report an extremely rare case of a 61-year old woman with food-dependent Cushing’s syndrome (FDC) due to unilateral adrenocortical adenoma (UAA) with cortisol (CORT) secretion without ACTH elevation detected in peripheral blood by the CRH test. She was on oral medications for hypertension and depression, and presented weight gain, general fatigue, muscle weakness, and hypokalemia. Despite the fact that the diurnal variation of ACTH was always suppressed, a diurnal variation in CORT was observed, in the form of low levels in the early morning and high levels in the afternoon. An increase in CORT was shown in a 75 g-oral glucose tolerance test (OGTT) and in a mixed meal tolerance test, but no change in CORT levels was seen in intravenous glucose tolerance tests. Elevated CORT levels were observed in response to intravenous injection of CRH, although ACTH levels were always below the measured sensitivity. Laparoscopic left adrenalectomy was performed, which resulted in postoperative improvement in potassium and ACTH levels and disappearance of the CORT secretory response in the OGTT. Clear expression of glucose-dependent insulinotropic polypeptide receptor (GIPR), CRH and CRH receptor 2 (CRHR2) were confirmed in the surgically-resected UAA specimen by molecular and immunohistochemical analyses, suggesting the involvement of not only GIPR, but also CRH and CRHR2 in FDC.

PRIMARY CUSHING’S SYNDROME results from chronic hypercortisolemic exposure of glucocorticoid and mineralocorticoid receptors due to ACTH-independent cortisol (CORT) overproduction associated with adrenocortical tumors, bilateral macronodular adrenal hyperplasia (BMAH) or primary pigmented nodular adrenal dysplasia [1]. Ectopic expression and orthotopic activation of various G protein-coupled hormone receptors (GPCRs), especially in BMAH and unilateral adrenocortical adenoma (UAA), have also been shown to produce cortisol [1, 2]. Since the 1987 report by Hamet et al. [3] of food-dependent Cushing’s syndrome (FDC) due to ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR), normally expressed in pancreatic β cells, adipocytes, osteoblasts, etc., there have been a total of 38 reported cases of FDC, with 25 cases of BMAH and 13 cases of UAA [4-22], although their exact prevalence is still unknown.

CRH is a major regulator of the hypothalamus-pituitary-adrenal (HPA) axis, and is released from the hypothalamus in response to stress, causing the anterior pituitary to secrete ACTH into the blood. It exerts its effects on many peripheral tissues as an important mediator of autonomic, behavioral and immune responses [23]. CRH exerts its effects in most tissues by binding to the CRH receptor (CRHR), a member of the seven-transmembrane GPCR superfamily, which induces a change in receptor structure and activation of heterotrimeric G proteins, followed by an increase in cyclic AMP (cAMP) levels via activation of adenylate cyclase [24]. Two types of CRHRs have been cloned, CRHR1 and CRHR2 [24], and mRNAs encoding them have been reported to be overexpressed 6-fold in adrenocortical adenomas (AAs) and 10- to 60-fold in CORT-producing AAs [23].

Here, we report a rare case of a 61-year old woman with FDC due to UAA with CORT secretion without ACTH elevation detected in peripheral blood by the CRH test.

Case Presentation

The patient was a 61-year-old woman with no special medical or family history. At the age of 51 years, she was diagnosed with hypertension and was started on antihypertensive medication. She had also been depressed since that time. At the age of 57 years, she began to notice weight gain, fatigue and muscle weakness. She was referred to our cardiology department with the chief complaint of respiratory distress at the age of 61 years. Although there was no evidence of ischemic heart disease, arrhythmia or congestive heart failure, she was noted to have treatment-resistant hypertension (148/84 mmHg) and hypokalemia (2.9 mmol/L), and a 25 mm-sized tumor was observed in the left adrenal gland on CT, which led her to our department for endocrinological examination. At the time of her first visit to our department, she was receiving spironolactone 75 mg, nifedipine 80 mg, bisoprolol fumarate 5 mg, ethyl icosapentate 1,800 mg, mosapride citrate 15 mg, duloxetine hydrochloride 60 mg, etizolam 0.5 mg, triazolam 0.25 mg and mirtazapine 15 mg daily for the treatment of hypertension and depression, and lorazepam 0.5 mg was used as required during episodes of increased anxiety.

The patient was 156.3 cm tall, weighed 70.3 kg, had a body mass index of 28.8 kg/m², body temperature of 36.1°C, blood pressure of 148/84 mmHg, and pulse rate of 88 beats/min with regular rhythm. She showed no cognitive dysfunctions, and had no pigmentation of the skin and oral mucosa. Her cardiopulmonary examination was normal. She had no abnormal abdominal and neurological findings or skeletal abnormalities. She had exaggerated facial roundness, central obesity, a dorsocervical fat pad, and multiple subcutaneous hemorrhages on both upper and lower extremities. There were no reddish-purple striae or edema in her lower legs bilaterally. She was a non-drinker and non-smoker. Suspecting Cushing’s syndrome, we performed laboratory tests and imaging studies to confirm the diagnosis and identify the cause. Her serum levels of potassium, total protein and albumin were inappropriately low (Table 1). Serum lactate dehydrogenase, low density lipoprotein-cholesterol, triglycerides, sodium and glycosylated hemoglobin (HbA1c) levels were all elevated (Table 1). ACTH levels were suppressed to <1.5 pg/mL throughout the day. Dehydroepiandrosterone sulphate (DHEA-S) was in the low normal range (47 μg/dL) and urinary CORT was elevated (124 μg/day) (Table 1). Dexamethasone 1 mg did not suppress serum CORT levels to <5 μg/dL (Table 1). On the other hand, her fasting serum CORT level at 8:00 a.m. was as low as 3.84 μg/mL, but increased during the day to 19.9 μg/mL at 16:00 and 14.0 μg/mL at 23:00. In addition, elevated CORT levels (from 4.6 to 16.2 μg/dL) were observed in response to intravenous injection of 100 μg of human CRH, although ACTH levels were always below measurement sensitivity (<1.5 pg/mL) (Table 2). Both computed tomography (CT) and magnetic resonance imaging (MRI) displayed a tumor 25 mm in size, presumed to be an adenoma, on the left adrenal gland. No tumor was found in the right adrenal gland (Fig. 1A, B). A circular shaped area of highly abnormal tracer uptake was seen at the site of the left adrenal tumor, with diminished tracer accumulation in the right adrenal gland on ¹³¹iodine (I)-adosterol scintigraphy (Fig. 1C). Blood glucose levels on a 75 g-oral glucose tolerance test (OGTT) were maintained above 200 mg/dL from 30 min to 120 min after loading, indicating a diabetic-type blood glucose transition (Fig. 2A). Blood glucose transition in the mixed meal tolerance test (MMTT) and intravenous glucose tolerance test (IVGTT) peaked at 30 min after the glucose load (≥200 mg/dL) and decreased thereafter, but showed a tendency to rise again 120 min after the load only in the MMTT (Fig. 2A). In addition, insulin changes in OGTT and MMTT peaked at 30 min after loading (≥100 mg/dL), decreased thereafter, and showed a tendency to rise again after 120 min (Fig. 2B). On the other hand, the peak value of insulin in the IVGTT was <30 μU/mL (Fig. 2B). Furthermore, an increase in CORT was shown in the OGTT and MMTT, but no change in CORT was seen in the IVGTT (Fig. 2C). A diagnosis of FDC was made based on the fact that CORT secretion was only observed when nutrients were administered orally via the gastrointestinal tract, and not following their intravenous administration.

Table 1 Laboratory findings

Peripheral blood		WBC	5,020/μL
		Neu	62.1%
		Lym	28.1%
		Baso	0.6%
		Eosin	0.8%
		Mono	8.4%
Biochemistry		AST	39 IU/L
		ALT	22 IU/L
		LDH	273 IU/L	[124–222]
		ALP	116 IU/L
		γ-GTP	82 IU/L	[9–32]
		Alb	3.72 g/dL	[4.10–5.10]
		Na	147 mmol/L	[138–145]
		K	3.3 mmol/L	[3.6–4.8]
		Cl	109 mmol/L
		Ca	9 mg/dL
		IP	3.5 mg/dL
		LDL-C	161 mg/dL	[65–163]
		HDL-C	68 mg/dL
		TG	326 mg/dL	[30–117]
		BUN	9.1 mg/dL
		Cr	0.82 mg/dL
		eGFR	54 mL/min/1.73m2	[60<]
		UA	6.9 mg/dL
		FBG	105 mg/dL
		HbA1c	6.5%	[4.9–6.0]
Endocrinology		DHEA-S	47 μg/dL
		U-CORT	124 μg/day	[11.2–80.3]
		U-MN	0.04 μg/mg·Cr
		U-NMN	0.55 μg/mg·Cr
	(After 1 mg DST)	ACTH	<1.5 pg/mL
		CORT	6.11 μg/dL	[<5.0]
	(After 8 mg DST)	ACTH	<1.5 pg/mL
		CORT	4.40 μg/dL
	(Daily fluctuation)	ACTH (06:00)	<1.5 pg/mL	[7.2–63.3]
		ACTH (16:00)	<1.5 ng/mL
		ACTH (23:00)	<1.5 pg/mL
		CORT (06:00)	3.8 μg/dL	[7.1–19.6]
		CORT (16:00)	19.9 μg/dL
		CORT (23:00)	14.0 μg/dL

The reference ranges are shown in square brackets. WBC, white blood cells; Neu, neutrophils; Lym, lymphocytes; Baso, basophils; Eosin, eosinophils; Mono, monocytes; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; γ-GTP, γ-glutamyltransferase; Alb, albumin; Na, sodium; K, potassium; Cl, chloride; Ca, calcium; IP, inorganic phosphorus; LDL-C, low-density lipoprotein-cholesterol; HDL-C, high-density lipoprotein-C; TG, triglycerides; BUN, blood urea nitrogen; Cr, creatinine; eGFR, estimated glomerular filtration rate; UA, uric acid; FBG, fasting blood glucose; HbA_1c, glycosylated hemoglobin; ACTH, adrenocorticotropic hormone; CORT, cortisol; DHEA-S, dehydroepiandrosterone sulphate; U-CORT, urinary-cortisol; U-MN, urinary-metanephrine; U-NMN, urinary-normetanephrine; DST, dexamethasone suppression test.

Table 2 CRH test results

	0	30	60	90	min
ACTH	<1.5	<1.5	<1.5	<1.5	pg/mL
CORT	4.6	12.2	16.2	11.2	μg/dL

100 μg of human CRH was injected intravenously. CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone; CORT, cortisol.

Fig. 1

Imaging for the adrenocortical adenoma

The arrowheads highlight an approximately 25 mm tumor in the left adrenal gland on computed tomography (A) and magnetic resonance imaging (B). The arrow indicates high accumulation in the left adrenal gland tumor on ¹³¹iodine (I)-adosterol scintigraphy (C), while accumulation in the right adrenal gland was suppressed and unclear.

Fig. 2

Changes in blood glucose, insulin and cortisol levels in the OGTT, MMTT and IVGTT

A shows changes in blood glucose levels, B shows changes in insulin levels, and C shows changes in cortisol levels. The closed circles, open circles and closed triangles represent the OGTT, MMTT and IVGTT, respectively. OGTT, 75 g-oral glucose tolerance test; MMTT, mixed meal tolerance test; IVGTT, intravenous glucose tolerance test.

Since the patient desired resection of the AA on her left adrenal gland, laparoscopic left adrenalectomy was performed. The size of the resected tumor was 26 × 25 × 20 mm, and the pathological diagnosis was benign adrenocortical adenoma. Postoperatively, her serum K levels improved compared to those before surgery. Furthermore, at 8 months postoperatively, her morning fasting ACTH level had recovered from less than measurement sensitivity to 7.4 pg/mL. The CORT secretory response in the OGTT also disappeared (Table 3).

Table 3 OGTT after tumor resection

	0	30	60	90	120	min
Glucose	115 (101)	212 (212)	154 (168)	199 (150)	196 (176)	mg/dL
Insulin	7.2 (16.0)	194.1 (165)	26.7 (27.0)	100.1 (17.9)	90.3 (56.0)	μU/mL
ACTH	21.4 (0.00)	21.1 (0.00)	20.9 (0.00)	17.2 (0.00)	16.4 (0.00)	pg/mL
CORT	8.53 (4.54)	8.78 (29.5)	6.97 (26.0)	6.24 (23.9)	5.62 (30.0)	μg/dL

OGTT, 75 g-oral glucose tolerance test; ACTH, adrenocorticotropic hormone; CORT, cortisol. The numbers in brackets indicate the respective preoperative values.

RT-PCR

In order to identify the expression of the CRH gene (CRH), CRHR1 gene (CRHR1), CRHR2 gene (CRHR2), GIPR gene (GIPR) and GLP-1R gene (GLP-1R), total RNA extraction from both, the patient’s resected adrenocortical adenoma tissue blocks (patient’s adenoma, PA) and non-functioning adrenocortical adenoma tissue blocks removed from a 54-year-old woman (control adenoma, CA) (Biovit, Detroit, USA), was performed. Next, we performed real-time polymerase chain reaction (RT-PCR) testing for the glyceraldehyde-3-phosphate dehydrogenase gene (GHPDH), CRH, CRHR1, CRHR2, GIPR and GLP-1R according to the manufacturers’ instructions. Differences in the average C_T values (ΔC_T value) for CRH, CRHR2, GIPR and GLP-1R were 15.5, 15.5, 2.8 and 15.9, respectively in PA, although CRHR1 was not detected (Table 4). Fold-changes in PA for CRH, CRHR2, GIPR and GLP-1R were 2.08, 7.26, 258.03 and 8.75, respectively (Table 4). These tests were conducted by the Technical Support Center of DNA Chip Research Institute, Inc., Tokyo, Japan.

Table 4 Average ΔC_T values and fold changes in CRH, CRHR1, CRHR2, GIPR and GLP-1R by RT-PCR

Sample Name	CRH		CRHR1		CRHR2		GIPR		GLP-1R
	Average ΔCT	Fold Change	Average ΔCT	Fold Change	Average ΔCT	Fold Change	Average ΔCT	Fold Change	Average ΔCT	Fold Change
PA	15.5	2.08	UD		15.5	7.26	2.8	258.03	15.9	8.75
CA	16.6		UD		18.4		10.8		19.1

PA, the patient’s resected adrenocortical adenoma tissue blocks; CA, non-functioning adrenocortical adenoma tissue blocks (control); CRH, corticotropin-releasing hormone gene; CRHR1, CRH receptor 1 gene; CRHR2, CRH receptor 2 gene; GIPR, Glucose-dependent insulinotropic polypeptide receptor gene; GLP-1R, glucagon-like peptide-1 receptor gene; RT-PCR, real-time polymerase chain reaction; C_T, threshold cycle; Average ΔC_T, Average C_T of CRH, CRHR1, CRHR2, GIPR or GLP-1R—Average C_T of GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene); Fold Change, gene expression ratio of each gene in PA relative to CA (control sample). These were calculated by ΔΔC_T power of 2. ΔΔC_T value, Average ΔC_T of PA—Average ΔC_T of control sample. Technical replicates; 2.

Western Blotting

Western blotting was performed using CRH/CRF rabbit polyclonal antibody (LSBio, Seattle, Washington, USA), CRHR2/CRF2 Receptor rabbit polyclonal antibody (LSBio), GLP-1R rabbit polyclonal antibody (Novus Biologicals, Littleton, Colorado, USA), GIPR rabbit polyclonal antibody (GeneTex Inc., Irvine, California, USA) and anti-GAPDH mouse monoclonal antibody (Bio-Rad Laboratories Inc., Hercules, California, USA) to identify the protein expression of CRH, CRHR2, GIPR and GLP-1R in PA and CA tissue. Bands were detected near 25–37 kDa (CRH) and near 50 kDa (CRHR2) in both the PA and CA tissues. Further, a band around 25 kDa (CRHR2) was identified in PA tissue, but not in CA tissue. In addition, the bands presumed to be GIPR and GLP-1R around 50 kDa showed stronger signals in PA than in CA. All of these immunoreaction procedures and evaluations were performed by GenoStaff Co., Ltd., Tokyo, Japan (Fig. 3).

Fig. 3

Western blotting analysis

CRH, corticotropin-releasing hormone; CRHR2, CRH receptor 2; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP-1R, glucagon-like peptide-1 receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Lane 1 indicates the patient’s resected adrenocortical adenoma tissue, and lane 2 indicates non-functioning adrenocortical adenoma tissue from another patient (control). Lane 2 showed no expression of GAPDH. The parts surrounded by the red dashed lines indicate bands detected around 25–37 kDa, 50 kDa, 50 kDa and 50 kDa in the left adrenal gland tissue, which were considered to correspond to CRH, CRHR2, GIPR and GLP-1R, respectively.

Immunohistochemical Staining

Immunohistochemical staining was performed on a 4% paraformaldehyde phosphate buffer solution-fixed tissue block from the resected PA. Coloration of both the vessels and the entire tissue was confirmed to be GIPR-positive (Fig. 4). Pathological processing and evaluation was performed by GenoStaff Co., Ltd.

Fig. 4

Immunohistochemical staining of GIPR in the patient’s resected adrenocortical adenoma

The lower left inset shows a negative control using normal rabbit immunoglobulin. The upper right inset shows a positive control of human pancreatic tissue stained using rabbit polyclonal anti-human GIPR antibodies. The inset at the lower right shows a weakly magnified photo of the patient’s resected adrenocortical adenoma. GIPR, glucose-dependent insulinotropic polypeptide receptor.

Discussion

Although the exact prevalence of FDC is unknown, a total of 38 cases have been reported worldwide [4-22]. GIPR is reportedly not expressed in normal adrenocortical tissue [7], although its ectopic expression has been shown to be a cause of FDC. Our patient had several characteristic physical findings of Cushing’s syndrome, such as full exaggerated facial roundness and central obesity, and, although there was no evidence of neutropenia, she manifested hypokalemia, hyperglycemia and dyslipidemia (Table 1). In addition, 24-h urinary CORT was elevated and CORT levels were not suppressed by either 1 mg and 8 mg dexamethasone (Table 1), a left adrenal adenoma was observed on CT and MRI (Fig. 1A, B), and strong accumulation of ¹³¹I-adosterol in the same tumor and suppressed uptake on the non-tumor side were also observed (Fig. 1C). Hence, a diagnosis of Cushing’s syndrome due to UAA was made. Furthermore, despite the fact that the diurnal variation of ACTH was always suppressed, a diurnal variation in CORT was observed, which was characterized by a low level at 6:00 and high levels at 16:00 and 23:00 (Table 1). Reznik Y et al. reported that plasma GIP concentrations are usually maximal after meal intake, at around 200 pmol/L, and are very low, at approximately 20 pmol/L, during fasting [5], and this low GIP level, coupled with suppression of ACTH, might be responsible for the specific finding of low early morning CORT levels. Since the findings in our patient were consistent with the characteristics of FDC, OGTT, MMTT and IVGTT were performed. Since CORT secretion was stimulated only by OGTT and MMTT (Fig. 2), the involvement of incretin was inferred, which was the basis for the diagnosis of FDC in this case.

FDC has been recognized in BMAH, which is presumably due to the acquisition of mutations during adrenal embryogenesis, as well as in UAA, presumably due to the expansion of single-cell clones that abnormally express GIPR [6]. The ectopic expression of GIPR in UAA has been suggested as being due to somatic mutations in the DNA segments that regulate their expression, and might also be related to abnormal mRNA splicing [9]. On the other hand, since GIP has been shown to stimulate DNA synthesis in tumor cells, it has been speculated that GIPR might also be involved in tumor cell development [9]. In a previous study, a somatic mutation in p. Ser45Cys of β-catenin/CTNNB1, a mediator in the WNT signaling pathway, was identified in one of the UAAs expressing the GIP receptor, which might contribute to tumor cell development [22]. Both the PA and CA showed expression of the GIPR gene and protein. However, the expression was clearly increased in PA compared to CA (Table 2 and Fig. 3). In PA, GLP-1R gene and protein expression was also elevated compared to in the CA (Table 2 and Fig. 3). Chabre O et al. reported that adrenal tumors stimulated to secrete CORT by any type of food intake in vivo were also stimulated by GIP, but not by GLP-1, in vitro [9]. GLP-1 has been shown to have no effect on cortisol production at concentrations of up to 10⁷ pmol/L [19], while total GLP-1 concentrations in normal subjects are only elevated up to 40 pmol/L after dietary intake [23]. Furthermore, in immunohistochemical staining, the entire PA was stained GIPR-positive (Fig. 4). Thus, in our case, overexpression of GIPR in the UAA was also considered to be the cause of FDC. However, a limitation of our study is that we did not examine GPCRs other than GIPR, GLP-1R and CRHR, nor did we search for genes involved in tumor growth.

In this case, the CRH test showed CORT secretion without ACTH elevation in peripheral blood (Table 2). In previous reports, cases in which either both ACTH and CORT were non-responsive [4, 6] or both these hormones were responsive [13, 14] to CRH administration have been observed, but this is the first report of a CORT response without an ACTH response in peripheral blood. It has been reported that the expression of mRNA encoding CRHR1 and CRHR2 is very high in tissue specimens rich in adrenocortical cells, with 6-fold overexpression in AA and 10- to 60-fold overexpression in CORT-producing AA [24], which supports the observations in our case. Although CRHR1 gene expression was not observed in both the PA and CA, CRHR2 gene and protein expression was observed in both, with stronger expression in PA compared to CA (Table 2 and Fig. 3). The expression of CRHR2 is surprising, since the expression of CRHR1, which is supposed to be highly expressed in the anterior pituitary, is instead expected [25]. Splice variants called α, β, and γ have been identified in CRHR2, which are speculated to contribute to its diversity in binding efficiency or affinity with CRH and CRH-related agonists, and to its ability to acquire multiple signaling pathway activation capabilities [26]. We speculate that a splice variant of CRHR2 might have triggered cortisol overproduction, mediated by the action of CRH, for which it has a low affinity, in our case. A truncated form of CRHR2α (CRHR2α-tr) has been identified in rat amygdala. CRHR2α-tr is a truncated CRHR (approximately 26 kDa) consisting of 236 amino acids, including the first three transmembrane domains and part of the fourth transmembrane domain of CRHR2α, and has been reported to bind to CRH with low affinity, but not to other ligands [27]. As seen in Fig. 3, in our case, a strong band was observed at around 26 kDa in the PA tissue, but not in CA tissue. We speculate that this protein, which suggests a truncated splicing variant of CRHR2, might have been involved in expression of the CRH effect. Furthermore, complex formation and functional interactions between different receptors have recently been reported in G protein-coupled receptors (adenosine A1 receptor and type-1 metabotropic glutamate receptor) [28]. Hence, we speculated that the CRHR2-tr-mediated signal might functionally share or amplify the cortisol-producing signal of GIPR, which could be the reason for the cortisol secretory response to the CRH stimulation test in this case. On the other hand, ectopic ACTH production in adrenocortical cells in Cushing’s syndrome has been reported in a few BMAH and UAA cases [1, 29, 30], and expression of propiomelanocortin mRNA and ACTH in BMAH has also been confirmed [31]. ACTH gradients in adrenal venous sampling have also been demonstrated in two patients with BMAH [31]. These reports led us to speculate that, in the present case, CRH might have stimulated cortisol secretion via paracrine secretion of ACTH locally in the AA. A limitation of our study is that we did not examine the expression of ACTH or urocortin, which are known ligands for CRHR2 [32], in PA, nor did we examine stimulation-secretion linkage in vitro (e.g., batch incubation or perfusion experiments) or perform adrenal venous sampling. Furthermore, we consider it a limitation that we did not measure ACTH by other highly sensitive assays or bioassays.

The finding of CRH gene expression in PA and CA is interesting. In humans, cortisol has been reported to stimulate placental CRH [33]. It is possible that a positive feedback system by CORT also exists in UAA, which might be involved in the overproduction of CORT. Furthermore, we speculated that autocrine-paracrine mechanisms in the UAA might be involved in the overproduction of CORT and tumor growth. Further studies on this topic are required in the future.

In conclusion, we report an extremely rare case of FDC due to UAA with CORT secretion without ACTH elevation detected in peripheral blood by the CRH test, in which clear expression of GIPR, CRH and CRHR2 were confirmed in the surgically-resected UAA specimen by molecular and immunohistochemical analysis. In addition, we found a relative increase in mRNA expression of these receptors in the PA compared to CA, suggesting the involvement of not only GIPR, but also CRH and CRHR2 in FDC.

Abbreviations

UAA, Unilateral adrenocortical adenoma; CORT, Cortisol; OGTT, 75 g-oral glucose tolerance test; GIPR, Glucose-dependent insulinotropic polypeptide receptor; CRHR, CRH receptor; BMAH, Bilateral macronodular adrenal hyperplasia; GPCRs, G protein-coupled hormone receptors; FDC, Food-dependent Cushing’s syndrome; GIP, Glucose-dependent insulinotropic polypeptide; GIPR, GIP receptor; HPA, Hypothalamus-pituitary-adrenal; AAs, Adrenocortical adenomas; HbA1c, Glycosylated hemoglobin; DHEA-S, Dehydroepiandrosterone sulphate; CT, Computed tomography; MRI, Magnetic resonance imaging; MMTT, Mixed meal tolerance test; IVGTT, Intravenous glucose tolerance test; CRH, CRH gene; CRHR1, CRHR1 gene; CRHR2, CRHR2 gene; GIPR, GIPR gene; GLP-1R, GLP-1R gene; GHPDH, Glyceraldehyde-3-phosphate dehydrogenase gene; PA, Patient’s resected adrenocortical adenoma tissue blocks; CA, Non-functioning adrenocortical adenoma tissue blocks; RT-PCR, Real-time polymerase chain reaction; ΔC_T value, Average C_T values; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase

Acknowledgements

We wish to thank GenoStaff Co., Ltd., Tokyo, Japan, for the Western blotting and immunohistochemical staining, and the Technical Support Center of DNA Chip Research Institute, Inc., Tokyo, Japan, for gene expression analysis by RT-PCR. We also thank the patient for her permission to publish this manuscript. Furthermore, we thank Forte Science Communications for their medical editing services.

Disclosures

Ethics approval and consent to participate

Performance of RT-PCR, Western blotting and immunohistochemical examinations on the excised tissue were approved by the clinical ethics review committee of Kagoshima Medical Center (Authorization number 21012, September 2, 2021). The patient gave written informed consent for evaluation of the tissue.

Consent for publication

Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Conflicts of interest

The authors declare no conflict of interest associated with this manuscript.

Contributions

M.M., N. Kori., N. Koji. and T.T. attended to the patient; M.M. and N. Kori. wrote the manuscript; Y.N. gave conceptual advice. N. Kori. supervised management of the case and contributed to writing and editing the manuscript. All authors have read and approved the final manuscript.

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