GERMLINE DELETION OF ARMC5 IN FAMILIAL PRIMARY MACRONODULAR ADRENAL HYPERPLASIA.

OBJECTIVE: Primary macronodular adrenal hyperplasia (PMAH) is considered a predominantly sporadic disease, but familial forms are well recognized. Genetic studies revealed germline mutations in the armadillo repeat containing 5 gene (ARMC5) in the majority of PMAH cases. Furthermore, somatic ARMC5 mutations, as different types of second-hit mutations and loss of heterozygosity have been reported in each adrenal nodule in PMAH. Here, we describe the involvement of ARMC5 alteration in a familial case of PMAH. METHODS: In our study, we performed clinical and genetic evaluations in a mother and her son with familial PMAH. To search for mutations and deletion of ARMC5, we used Sanger sequencing and droplet digital polymerase chain reaction (ddPCR), respectively. RESULTS: Both patients showed the same phenotype of subclinical Cushing syndrome, with mild excess of mineralocorticoids and vasopressin-responsive cortisol secretion. The ddPCR analysis demonstrated that both mother and son had germline deletions in exons 1 to 5 of the ARMC5 gene locus. Furthermore, Sanger sequencing of DNA from the right and left adrenal nodules as well as peripheral blood of the son revealed the presence of another germline, missense mutation in ARMC5 exon 3 (p.P347S). CONCLUSION: This is the first report demonstrating germline deletion of ARMC5 in familial PMAH. In addition to investigating mutations, germline and somatic deletions of ARMC5 could be examined by ddPCR, which permits rapid and accurate evaluation of the ARMC5 allelic status.

Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species.

We identified a p53 target gene, phosphate-activated mitochondrial glutaminase (GLS2), a key enzyme in conversion of glutamine to glutamate, and thereby a regulator of glutathione (GSH) synthesis and energy production. GLS2 expression is induced in response to DNA damage or oxidative stress in a p53-dependent manner, and p53 associates with the GLS2 promoter. Elevated GLS2 facilitates glutamine metabolism and lowers intracellular reactive oxygen species (ROS) levels, resulting in an overall decrease in DNA oxidation as determined by measurement of 8-OH-dG content in both normal and stressed cells. Further, siRNA down-regulation of either GLS2 or p53 compromises the GSH-dependent antioxidant system and increases intracellular ROS levels. High ROS levels following GLS2 knockdown also coincide with stimulation of p53-induced cell death. We propose that GLS2 control of intracellular ROS levels and the apoptotic response facilitates the ability of p53 to protect cells from accumulation of genomic damage and allows cells to survive after mild and repairable genotoxic stress. Indeed, overexpression of GLS2 reduces the growth of tumor cells and colony formation. Further, compared with normal tissue, GLS2 expression is reduced in liver tumors. Thus, our results provide evidence for a unique metabolic role for p53, linking glutamine metabolism, energy, and ROS homeostasis, which may contribute to p53 tumor suppressor function.

Safety and Efficacy of Ranibizumab and Luseogliflozin Combination Therapy in Patients with Diabetic Macular Edema: Protocol for a Multicenter, Open-Label Randomized Controlled Trial.

INTRODUCTION: Diabetic macular edema (DME) threatens daily life activities such as reading and driving and reduces the patients' quality-of-life. Recently, anti-vascular endothelial growth factor (VEGF) agents have become a first-line therapy in DME. However, therapy with anti-VEGF agents has several problems: repeated invasive injections are required; medical costs are high; and a certain proportion of patients with DME are resistant to treatment with anti-VEGF agents. While sodium-glucose co-transporter 2 (SGLT2) inhibitors have been widely used for the treatment of type 2 diabetes mellitus (T2DM), the effects of a combination therapy with anti-VEGF agent and SGLT2 inhibitor on DME are not yet known. METHODS: This study enrolls subjects with T2DM and DME, randomizes them into either a study agent treatment group (treated with ranibizumab as anti-VEGF agent and luseogliflozin as SGLT2 inhibitor) or a control group (treated with ranibizumab and glimepiride), and observes the subjects for 52 weeks after initiation of treatment. Planned outcomes: The primary endpoint is intergroup difference in the number of intravitreal anti-VEGF injections to the study eye from baseline to week 48. Secondary and exploratory endpoints include safety and ophthalmologic and internal medical clinical parameters. REGISTRATION: This study is registered at the University Hospital Medical Information Network Clinical Trial Registry (UMIN000033961) and Japan Registry of Clinical Trials (jRCTs031180210).

cAMP activation by PACAP/VIP stimulates IL-6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/vasoactive intestinal peptide (VIP) superfamily, stimulates cyclic AMP accumulation initiating a variety of biological processes such as: neurotropic actions, immune and pituitary function, learning and memory, catecholamine biosynthesis and regulation of cardiopulmonary function. Both osteoclasts and osteoblasts have been shown to express receptors for PACAP/VIP implicated in their role in bone metabolism. To further understand the role of PACAP/VIP family in controlling bone metabolism, we investigated differentiation model of MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria. Quantitative RT-PCR analysis demonstrated that MC3T3-E1 cells expressed only VPAC2 receptor and its expression was upregulated during osteoblastic differentiation, whereas VPAC1 and PAC1 receptors were not expressed. Consistent with expression of receptor subtype, both PACAP and VIP stimulate cAMP accumulation in a time- and dose-dependent manner with the similar potency in undifferentiated and differentiated cells, while Maxadilan, a specific agonist for PAC1-R, did not. Furthermore, downregulation of VPAC2-R by siRNA completely blocked cAMP response mediated by PACAP and VIP. Importantly, PACAP/VIP as well as forskolin markedly suppressed the induction of alkaline phosphatase mRNA upon differentiation and the pretreatment with 2',5'-dideoxyadenosine, a cAMP inhibitor, restored its inhibitory effect of PACAP. We also found that PACAP and VIP stimulated IL-6 release, a stimulator of bone resorption, and VPAC2-R silencing inhibited IL-6 production. Thus, PACAP/VIP can activate adenylate cyclase response and regulate IL-6 release through VPAC2 receptor with profound functional consequences for the inhibition of osteoblastic differentiation in MC3T3-E1 cells.

Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

Cushing Syndrome Due to ACTH-Secreting Pheochromocytoma, Aggravated by Glucocorticoid-Driven Positive-Feedback Loop.

CONTEXT: Pheochromocytoma is a catecholamine-producing tumor that originates from adrenal chromaffin cells and is capable of secreting various hormones, including ACTH. CASE DESCRIPTION: A 56-year-old female presented with Cushingoid appearance and diabetic ketoacidosis. Endocrinological examinations demonstrated ectopic ACTH production with hypercortisolemia and excess urinary cortisol accompanied by elevated plasma and urine catecholamines. Computed tomography revealed a large left adrenal tumor with bilateral adrenal enlargement. Metaiodobenzylguanidine scintigraphy revealed abnormal accumulation in the tumor, which was eventually diagnosed as pheochromocytoma with ectopic ACTH secretion with subsequent manifestation of Cushing's syndrome. Ectopic ACTH secretion and catecholamine production were blocked by metyrapone treatment, whereas dexamethasone paradoxically increased ACTH secretion. Left adrenalectomy resulted in complete remission of Cushing's syndrome and pheochromocytoma. IN VITRO STUDIES: Immunohistological analysis revealed that the tumor contained two functionally distinct chromaffin-like cell types. The majority of tumor cells stained positive for tyrosine hydroxylase (TH), whereas a minor population of ACTH-positive tumor cells was negative for TH. Furthermore, gene expression and in vitro functional analyses using primary tumor tissue cultures demonstrated that dexamethasone facilitated ACTH as well as catecholamine secretion with parallel induction of proopiomelanocortin (POMC), TH, and phenylethanolamine N-methyltransferase mRNA, supporting a glucocorticoid-dependent positive-feedback loop of ACTH secretion in vivo. DNA methylation analysis revealed that the POMC promoter of this tumor, particularly the E2F binding site, was hypomethylated. CONCLUSION: We present a case of ectopic ACTH syndrome associated with pheochromocytoma. ACTH up-regulation with paradoxical response to glucocorticoid, possibly through the hypomethylation of the POMC promoter, exacerbated the patient's condition.

Age, initial dose and dose increase are independent risk factors for symptomatic vertebral fractures in glucocorticoid-treated male patients.

OBJECTIVE: The incidence and risk factors for symptomatic vertebral fracture were analyzed in glucocorticoid-treated male patients. METHODS: This was an observational cohort study at Shimoshizu National Hospital in Japan. Analyzed were 161 male patients newly treated with high-dose glucocorticoid (≥20 mg/day prednisolone equivalent) (initial age: 53.5±16.9, initial glucocorticoid dose: 38.9±12.9 mg/day (0.66±0.23 mg/kg/day), follow-up time: 70.4±52.5 months) and 33 male patients with no glucocorticoid (initial age: 52.7±13.0, follow-up time: 76.4±62.7 months). The vertebral fracture was determined by x-rays. RESULTS: Symptomatic vertebral fractures occurred more frequently in the high-dose glucocorticoid group (21.1%) than in the no glucocorticoid group (3.0%). Using Cox model, the adjusted hazard ratio (HR) for the high-dose glucocorticoid group was 8.16 (95% confidence interval: 1.09-60.86) relative to the no glucocorticoid control group. In the high-dose glucocorticoid group, Kaplan-Meier analyses demonstrated that the incidence of fractures in the patients with glucocorticoid dose increase was significantly higher in comparison with those with no glucocorticoid dose increase. Cox model demonstrated that the risk was independently higher in every 10-year increment of initial age with HR 1.58 (1.18-2.13), in every 10 mg increment of initial dose of prednisolone with HR 2.03 (1.43-2.88), in every dose increase of glucocorticoid increase with HR 3.63 (2.04-6.46), and with each 1-gram decrease of cumulative dose of glucocorticoid with HR 0.88 (0.84-0.93). CONCLUSION: In male patients, high-dose glucocorticoid causes a significantly high prevalence of symptomatic vertebral fractures, and the independent risk factors are age, initial glucocorticoid dose, glucocorticoid dose increase, and decrease of cumulative glucocorticoid dose.