Adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome is caused by an ACTH-producing tumor, as is the case with Cushing’s disease and ectopic ACTH syndrome (EAS). Diagnosis and differential diagnosis of Cushing’s disease from EAS in ACTH-dependent Cushing’s syndrome are thus challenging problems in clinical endocrinology. The diagnostic criteria for Cushing’s disease in Japan, established by the working group of the Japan Ministry of Health, Labour and Welfare, were originally reported in 2003 and revised in 2007 and 2010. In addition, criteria for subclinical Cushing’s disease were established in Japan in 2010. In this review, we evaluate the usefulness and accuracy of the most recent diagnostic criteria. Previous data suggest that as an initial test of Cushing’s syndrome, 0.5 mg dexamethasone is more sensitive than 1 mg in the overnight dexamethasone suppression test (DST). Here, we recommend 0.5 mg plus a plasma cortisol cut-off level of 3 μg/dL as a suitable low-dose overnight DST for screening of all cases of ACTH-dependent Cushing’s syndrome in Japan. Recently, standardization of cortisol measurements by the ID-LC/MS/MS method using seven assay kits with standard plasma material containing synthetic hydrocortisone-d4 was carried out in Japan. The resulting relative standard deviation was within 10%. The cut-off value remains valid even after standardization of plasma cortisol measurements. Although the recent diagnostic criteria achieve higher diagnostic specificity, care should be taken since data for Cushing’s disease partially overlaps with some cases of EAS. Overall, therefore, this review suggests that the accuracy of each diagnostic test should be considered.

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Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an “in vitro contraction model” using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (_˜12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our “in vitro contraction model”.

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Substitution therapy of glucocorticoid is a major part of the treatment for 21-OHD (21-hydroxylase deficiency). However, the therapy causes two major adverse effects, impairment of linear growth and obesity, so that collecting precise growth data is essential for optimizing the therapy. We longitudinally evaluated the linear growth and the body composition of Japanese 21-OHD patients during childhood. For the present study, we chose 16 patients (eight of each sex) who were diagnosed during the newborn period, and continuously observed them in our institute until they were at least 15 years old. All patients were treated according to the guidelines from The Japanese Society for Pediatric Endocrinology. The final height standard deviation score (Ht-SDS) of all the patients was -1.18 ± 0.85 SD, and no significant differences were observed between males and females or between the simple virilizing form and the salt wasting form. As previously reported, in spite of nearly normal height at the onset of puberty, the pubertal height gains were severely impaired, resulting in reduced final heights. Body composition of the patients was evaluated with BMI-SDS. Our longitudinal data showed that BMI was increased up to +1.23 SD in males and up to +1.75 SD in females, and that adiposity rebound was precipitated. Our study should alert physicians to the risk of metabolic syndrome and provide a framework for further studies of metabolic syndrome in 21-OHD patients.

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Various patterns of calcification have been detected in benign and malignant thyroid nodules on ultrasonography (US). Microcalcification has been found to be highly associated with papillary thyroid carcinoma. However, other patterns of calcification have unclear clinical significance. The aim of this study was to evaluate which pattern of calcification could be predictive of malignancy. A total of 1,431 thyroid nodules of 1,078 patients who received preoperative ultrasound examinations and subsequently underwent thyroidectomy were retrospectively reviewed. The types of calcification were defined as follows: microcalcification, annular-like peripheral calcification, crescent-like peripheral calcification, intranodular coarse calcification, and calcified spot. Of these 1,431 nodules, 1,305 (91.1%) were thyroid carcinomas and 126 (8.9%) were benign nodules. Calcifications were detected in 38.6% (552/1,431) of all nodules. Calcifications were found in 40.2% (524/1,305) of malignant nodules and 22.2% (28/126) of benign nodules. Of the 524 malignant nodules with calcification, microcalcification was the most common pattern, occurring in 42.9% (225/524), and annular type was the least common pattern, occurring in 5.9% (31/524). Among the calcification types, only microcalcification and intranodular had a significant association with malignancy (p = 0.001, 0.035), with OR values of 3.5 (95% CI, 1.6-7.7) and 2.4 (95% CI, 1.1-5.6). Though using the patterns of calcification alone for predicting malignancy had limitation, microcalcification and intranodular calcification were significantly associated with malignancy.

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Helicobacter pylori infection, which is a common cause of atrophic gastritis, has been reported to represent a causal factor increasing the vascular damage and consequent albuminuria. On the other hand, decreased serum pepsinogen (PG) I/II ratio can be used to assess gastric mucosal atrophy. To the best of our knowledge, there are no studies investigating the correlation between PG I/II ratio and diabetic nephropathy. Therefore, we investigated a relationship between PG I/II ratio and degree of urinary albumin excretion (UAE) in patients with type 2 diabetes. We evaluated relationships between PG I/II ratio and degree of UAE or estimated glomerular filtration rate as well as various factors, including age, body mass index, blood pressure, hemoglobin A1c, serum lipid concentrations, uric acid or C-reactive protein in 333 consecutive patients with type 2 diabetes. PG I/II ratio correlated positively with logarithm of UAE in all patients (r = 0.174, P = 0.0016) and in patients without Helicobacter pylori infection (r = 0.352, P < 0.0001). Multiple regression analysis identified that PG I/II ratio correlated independently with logarithm of UAE in all patients (β= 0.264, P = 0.0005) and in patients without Helicobacter pylori infection (β= 0.295, P = 0.0022). These data suggest that serum PG I/II ratio is correlated with diabetic nephropathy.

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The use of aromatase inhibitor (AI) in postmenopausal women with hormone receptor (HR)-positive early breast cancer (EBC) produces a deleterious effect on bone mass and an increase in fractures. Several studies using intravenous bisphosphonates have shown effective management of AI-induced bone loss. To determine whether a lower dosage in oral form combined with calcitriol can effectively manage AI-induced bone loss, we performed a randomized, double-blind, prospective, placebo-controlled 24-week trial with a combination of alendronate and 0.5-μg of calcitriol daily to HR-positive EBC patients. A total of 98 Korean postmenopausal women with HR-positive EBC who received daily AI, calcium and vitamin D were randomly assigned to 5-mg of alendronate and 0.5-μg of calcitriol (Maxmarvil^®) or placebo groups. The bone mineral density (BMD) and turnover markers were measured. At week 24, the difference in lumbar BMD between the groups was 3.0% (p < 0.005). The increase in C-telopeptide after 24 weeks was significantly less in the Maxmarvil group compared to that in the placebo group (35.2 ± 17.5% vs. 109.8 ± 28.6%, p < 0.05). Our study demonstrates that a combination of 5-mg alendronate and 0.5-μg calcitriol is effective to prevent bone loss due to AI in Korean postmenopausal women with EBC.

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Insulin glulisine (Glu) is a rapidly-acting insulin analog with a faster onset of action than the other insulin analogs of its class, which are insulin aspart (Asp) and insulin lispro (Lisp). While insulin Glu is usually injected just before meals, postprandial injection may help to avoid unexpected postprandial hypoglycemia or hyperglycemia by adjusting the insulin dosage according to food intake. However, the effect of postprandial insulin Glu on the glucose profile has not been evaluated. The aim of this study was to compare daily glucose excursion by continuous glucose monitoring (CGM) between multiple daily doses of preprandial insulin Asp or postprandial insulin Glu. In a randomized cross-over trial, we performed CGM to evaluate the 48-hour glucose profile during treatment with the same dosage of insulin Asp just before each meal in 12 hospitalized patients with type 2 diabetes. Patients also received the same dosage of long-acting insulin glargine at bedtime. The average glucose level, standard deviation of the glucose level, mean amplitude of glucose excursion, and daily glucose profile did not differ between preprandial Asp and postprandial Glu. The incidence of hypoglycemic episodes (glucose level<70 mg/dL with or without symptoms) and the area under the curve of glucose<70 mg/dL also did not differ between the two insulin regimens. Multiple daily injections of preprandial Asp and postprandial Glu achieved the same daily glucose excursion profile. Postprandial injection of Glu may provide greater flexibility for patients who require insulin therapy.

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Vitamin D is an important regulator of bone health. Previous studies examining the association between vitamin D deficiency and osteoporotic fractures have reported conflicting results. The relationship between vitamin D status and risk of vertebral fractures in diabetic patients is unknown. The objective of this study was to examine whether low serum 25-hydroxyvitamin D [25(OH)D] levels were associated with vertebral fractures in patients with type 2 diabetes. This cross-sectional study was conducted among 161 postmenopausal women and 180 men with type 2 diabetes. Serum concentrations of 25(OH)D were measured and the presence of vertebral fracture was assessed using lateral plain radiographs of the thoracolumbar spine. Women had lower 25(OH)D levels than men (31.3 ± 17.7 vs. 41.3 ± 26.5 ng/mL, p<0.001). Vertebral fractures were found in 16% of patients. Men with a serum 25(OH)D concentration greater than 30 ng/mL showed a lower prevalence of vertebral fractures compared to those with 20-29.9 ng/mL or those with less than 20 ng/mL (9.4% vs. 17.9% vs. 21.7%, p for trend=0.036). However, there was no significant association between vitamin D status and the prevalence of vertebral fractures in women (14.4% vs. 19.2% vs. 26.6%, p for trend=0.111). After adjusting for multiple confounding factors, men with a serum 25(OH)D concentration of less than 20 ng/mL were associated with an increased risk of vertebral fractures (OR 7.87; 95% CI 1.69-36.71), but not women. In conclusion, serum 25(OH)D levels below 20 ng/mL were associated with an increased vertebral fracture risk in men with type 2 diabetes.

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This study is aimed to explore the relationship between bone marrow characteristics and clinical prognosis of antithyroid drug (ATD) induced agranulocytosis. A retrospective study was conducted in the first affiliated hospital of the University of South China. A total of 33 hospitalized patients diagnosed with ATD-induced agranulocytosis were analyzed. The bone marrow characteristics were classified into two types. Type I was characterized by reduction or absence of granulocytic precursors and type II was recognized as hypercellular bone marrow with dysmaturity of granulocytic cells. Bone marrow of 20 cases (61%) were characterized with type I whereas 13 cases (39%) with type II. The median duration of neutrophil recovery and high-grade fever were 4.7 ± 1.0 days and 3.6 ± 2.5 days respectively for type II, compared to 8.0 ± 2.8 days and 8.6 ± 3.1 days for type I (p < 0.01 in both compared groups). However, there was no significant difference between the two types in terms of age, median duration of drug administration before the diagnosis of agranulocytosis, the amount of neutrophil count on admission and the total administration dose of granulocyte-colony stimulating factor (G-CSF) before bone marrow examination. Two cases of type I died of complications from infection. This study showed that the bone marrow characteristics of ATD-induced agranulocytosis could be classifed into two types. Also, the clinical prognosis was closely related to the bone marrow features. Type I is the dominant type which is usually associated with worse clinical prognosis compared to type II.

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Prostaglandins (PGs), whose synthesis is catalyzed by the rate-limiting enzyme cyclooxygenase (COX) including COX-1 and COX-2, are among the important mediators involved in the regulation of gonadotropin-releasing hormone (GnRH) secretion. However, the cellular origin of PGs remains obscure in terms of its relationship to GnRH neurons. The present study was therefore aimed to clarify the anatomical relationship between COX-1-producing microglia and GnRH neurons in the preoptic area (POA), and to examine possible influence of ovarian steroids. We performed a triple labeled immunofluorescent histochemistry of COX-1, CD11b (a specific marker for microglia) and GnRH in the POA of ovarian steroid-primed and non-primed ovariectomized rats. The result confirmed our previous study suggesting COX-1 immunoreactivity in the vicinity of, but not within, GnRH neurons in the POA. COX-1 around GnRH cells was entirely (100%) localized in cells containing CD11b regardless of steroid replacement in ovariectomized rats. These CD11b-immunoreactive cells had small cell bodies and highly branched fibers characteristic of ramified microglia. Three-dimensional reconstruction of confocal images revealed close proximity of some COX-1-containing microglia and GnRH neurons. These results showed selective and constitutive expression of COX-1 in ramified microglia in the vicinity of GnRH neurons, providing evidence for intercellular communication, mediated by PGs, from microglia to GnRH cells.

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We observed glucose levels >140 mg/dL measured at 30 minutes (min) during an oral glucose tolerance test (OGTT) in some obese patients. We aimed to investigate the significance of this finding by comparing lipid profiles, insulin resistance indices, and systemic inflammatory mediators between obese adolescents with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and elevated glucose levels at 30 min. The study involved 80 obese (body mass index >95^th percentile for age and sex) adolescents (48 female, 32 male) between 11 and 16 years of age. Depending on OGTT results, patients were divided into NGT and IGT groups. The third group was recruited from the NGT group as having glucose levels > 140 mg/dL at 30 minutes. Lipid profiles, [interleukin-6 (IL-6)], neopterin, and lipoprotein associated phospholipase A₂ (Lp-PLA₂)] were assessed. Neopterin and Lp-PLA₂ levels were significantly higher in obese adolescents with elevated glucose levels at 30 min. compared with those in both NGT and IGT groups (p=0.013, and 0.004, respectively). In these adolescents, IL-6 levels were significantly higher only than the NGT group (p=0.01). In logistic regression analysis, IL-6, neopterin and Lp-PLA₂ levels were detected to be related to high blood glucose levels at 30 min (OR 1.11, p=0.01; OR 9.03, p=0.013; OR 1.01, p=0.004 respectively). Obese adolescents with elevated glucose levels at 30 min. demonstrated higher inflammatory mediators levels, which were atherosclerotic indicators, than obese adolescents with NGT and IGT. These results suggest that glucose levels >140 mg/dL measured at 30 min during an OGTT may be a new disorder of glucose tolerance in obesity.

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We reviewed 94 patients with Rathke’s cleft cyst (RCC) who were surgically treated at Nippon Medical School Hospital between December 1995 and July 2009 to clarify the effect of surgery on their endocrine function. In our statistical analysis we considered their age and sex, the cyst volume, and preoperative MRI findings. Using simple linear- and multiple regression analysis we evaluated the association between these factors and their preoperative hormone baseline levels. To assess pre- and postoperative anterior pituitary function we subjected the results of various hormone loading tests to the Wilcoxon rank sum test. Surgery improved headache and visual impairment in most patients and elevated PRL levels were significantly normalized after surgery (p = 0.004). However, pre- and postoperative anterior pituitary hormone loading tests revealed that the levels of GH, TSH, LH, and FSH were not improved significantly by surgery. Although the ACTH loading test showed postoperative improvement, the change was not statistically significant. We suggest that RCC patients with headache or visual impairment are good candidates for surgery. We also recommend that patients with hyperprolactinemia and those with ACTH deficiency whose MRI findings reveal low- intensity on T1WI and high-intensity on T2WI are likely to benefit from surgery. In contrast, RCC patients with other hormone dysfunctions do not appear to benefit from surgical intervention.

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Protein kinase A (PKA) regulatory subunit type Iα (RIα) is a major regulatory subunit that functions as an inhibitor of PKA kinase activity. We have previously demonstrated that elevated RIα expression is associated with diffuse-to-nodular transformation of hyperplasia in parathyroid glands of renal hyperparathyroidism. The aim of the current study was to determine whether or not RIα expression is increased in adenomas of primary hyperparathyroidism (PHPT), because monoclonal proliferation has been demonstrated in both adenomas and nodular hyperplasia. Surgical specimens comprising 22 adenomas and 11 normal glands, obtained from 22 patients with PHPT, were analyzed. Western blot and immunohistochemical analyses were employed to evaluate RIα expression. PKA activities were determined in several adenomas highly expressing RIα. RIα expression was also separately evaluated in chief and oxyphilic cells using the “Allred score” system. Expression of proliferating cell nuclear antigen (PCNA), a proliferation marker, was also immunohistochemically examined. Western blot analysis revealed that 5 out of 8 adenomas highly expressed RIα, compared with normal glands. PKA activity in adenomas was significantly less than in normal glands. Immunohistochemical analysis further demonstrated high expression of RIα in 20 out of 22 adenomas. In adenomas, the greater RIα expression and more PCNA positive cells were observed in both chief and oxyphilic cells. The present study suggested that high RIα expression could contribute to monoclonal proliferation of parathyroid cells by impairing the cAMP/PKA signaling pathway.

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Obesity is a major risk factor for sleep-disordered breathing (SDB). However, many Japanese subjects with diabetes are less obese despite compared with Caucasian. We evaluated the relationship between SDB and clinical characteristics other than obesity, especially in relation to cardiac autonomic neuropathy (CAN) in Japanese subjects with diabetes. The study included a total of 261 consecutive Japanese subjects with type 2 diabetes, including nonobese subjects defined as BMI <25 kg/m² for Japanese. SDB was screened by 4% oxygen desaturation index (ODI) level of 5 or more events per hour, which was measured by nocturnal pulse oximetry. CAN was examined with the variation of R-R intervals (CVRR). The SDB were found in 24.5% of total subjects and 16.3% of nonobese subjects with type 2 diabetes, respectively. The nonobese type 2 diabetes subjects with SDB had significantly lower coefficient of CVRR than those without SDB. Multiple regression analysis revealed that BMI and heart rate were significant independent factors for SDB in total subjects with type 2 diabetes, but CVRR was the only significant independent factor for SDB in nonobese subjects with type 2 diabetes. These findings suggest that the presence of SDB should be kept in mind in type 2 diabetic patients with abnormality in CVRR variation in electrocardiogram even though they are not obese.

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Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare genetic disorder characterized by hypocalcemia and hyperphosphatemia due to imprinting defects in the maternally derived GNAS allele. Patients with PHP-Ib are usually identified by tetany, convulsions, and/or muscle cramps, whereas a substantial fraction of patients remain asymptomatic and are identified by familial studies. Although previous studies on patients with primary hypoparathyroidism have indicated that hypocalcemia can be associated with various neuromuscular abnormalities, such clinical features have been rarely described in patients with PHP-Ib. Here, we report a 12-year-old male patient with familial PHP-Ib and unique neuromuscular symptoms. The patient presented with general fatigue, steppage gait, and myalgia. Physical examinations revealed muscular weakness and atrophies in the lower legs, a shortening of the bilateral Achilles’ tendons and absence of deep tendon reflexes. Laboratory tests showed hypocalcemia, hyperphosphatemia, elevated serum intact PTH level, and impaired responses of urinary phosphate and cyclic AMP in an Ellsworth-Howard test, in addition to an elevated serum creatine kinase level. Clinical features of the patient were significantly improved after 1 month of treatment with alfacalcidol and calcium. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and subsequent PCR analyses identified a methylation defect at exon A/B of GNAS and a microdeletion involving exons 4-6 of the GNAS neighboring gene STX16 in the patient and in his asymptomatic brother. The results suggest that various neuromuscular features probably associated with hypocalcemia can be the first symptoms of PHP-Ib, and that MS-MLPA serves as a powerful tool for screening of GNAS abnormalities in patients with atypical manifestations.

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We examined the association of the RETN (resistin) -420 C>G polymorphism (rs1862513) with risk of diabetes mellitus (DM), considering lifestyle factors, in Japanese. Subjects were participants of J-MICC Study, where 2,651 participants aged 35-69 years provided their blood for genotyping and lifestyle data after informed consent. Odds ratio (OR) of DM for RETN-420 G/G genotype was estimated using unconditional logistic regression model. Statistically significant interaction on risk of DM was observed between RETN-420 G/G genotype and BMI<25 (OR for interaction = 0.12; P = 0.046), and when subjects with RETN-420 C/C+C/G and BMI ≥ 25 (n = 69 for DM and 544 for non-DM) were defined as the reference, the adjusted ORs for subjects with RETN-420 G/G genotype and BMI>25 (n = 10 for DM and 111 for non-DM), RETN-420 C/C+C/G and BMI<25 (n = 81 for DM and 1,605 for non-DM), and RETN-420 G/G and BMI<25 (n = 1 for DM and 230 for non-DM) were demonstrated to be 0.72 (95% confidence interval: 0.36-1.46), 0.40 (0.28-0.56) and 0.03 (0.005-0.25), respectively. The present study revealed the significant interaction of RETN-420 G/G genotype with lower BMI on the decreased risk of DM, but the direction was opposite to the reported ones in Japanese. We should be careful in interpretation of the present study results because of the limited sample sizes. Further investigation of this association as well as of the actual biological roles of RETN in the genesis of human metabolic disorders including DM will be required.

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Congenital central hypothyroidism (C-CH) is a rare disease known to be caused by mutations of the genes encoding TSH β or the TRH receptor gene, although the cause of the disease in a number of patients has not yet been clarified. Recently, mutations and deletions of the immunoglobulin superfamily member 1 (IGSF1) gene have been reported to be the cause of C-CH. Here we report a Japanese male patient with C-CH due to a novel IGSF1 mutation. He was detected by neonatal mass screening of simultaneous TSH and free T4 measurements and levothyroxine was initiated. At 6 years of age he underwent ¹²³ I scintigraphy after levothyroxine treatment had been discontinued for one month and his thyroid and pituitary function were evaluated. Since TSH and PRL responses after TRH stimulation were low, his diagnosis of C-CH was confirmed. During follow up, whereas onset of his puberty was delayed, his secondary sex characterization completed at 17 years old. In this patient we analyzed IGSF1 and TRHR. As results, we identified a novel insertion mutation in IGSF1 (c.3528-3529insC), resulting in a premature stop codon (p.Pro1082Trpfs39X). In conclusion, we identified a novel mutation of IGSF1 in a Japanese male patient with C-CH.

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