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Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Although acromegaly has been reported in patients with Neurofibromatosis type 1 (NF1), these cases have not been associated with growth hormone (GH)-producing somatotroph adenoma, but with optic pathway glioma. A 68 year-old Japanese woman, who had been clinically diagnosed with NF1, was referred to our hospital due to a thyroid tumor and hypercalcemia. Acromegaly was suspected due to her facial features, and subsequent examinations revealed the presence of GH excess with a pituitary tumor, leading to the diagnosis of acromegaly. Histological and immunohistochemical analysis demonstrated an eosinophilic pituitary adenoma with diffuse positivity for GH, indicating typical somatotroph adenoma. In addition, her thyroid tumor was diagnosed histologically as follicular thyroid carcinoma (FTC) with primary hyperparathyroidism (PHPT). To investigate the pathogenesis of this untypical multiple endocrine tumor case of NF1, genetic analysis was performed using peripheral leukocytes and tissue of resected tumors. A heterozygous novel germline nonsense mutation (p.Arg1534*) in exon 35 of the NF1 gene was detected from peripheral leukocytes, which results in a truncated protein lacking the critical domain for GTPase activity, strongly suggesting its causal role in NF1. The loss of heterozygosity (LOH) in exon 35 of the NF1 gene was not detected in the somatotroph adenoma, parathyroid adenoma, and FTC. Although any mutations of the following genes; MEN1, CDKN1B, and PAX8-PPARγ were not detected, a heterozygous GNAS R201C mutation was detected in the somatotroph adenoma. To our knowledge, this is the first rare MEN1-like case of genetically diagnosed NF1 complicated with acromegaly caused by a somatotroph adenoma.

Context: Anti-pituitary-specific transcriptional factor-1 (anti-PIT-1) antibody syndrome is characterized by acquired and specific deficiencies in growth hormone, prolactin, and thyroid-stimulating hormone. Although PIT-1-reactive cytotoxic T lymphocytes (CTLs) have been speculated to recognize anterior pituitary cells and to cause the injury in the pathogenesis of the syndrome, it remains unclear whether endogenous PIT-1 protein is processed through the proteolytic pathway and presented as an antigen on anterior pituitary cells., Objective: To examine how PIT-1 protein is processed and whether its epitope is presented by major histocompatibility complex (MHC)/HLA class I on anterior pituitary cells., Materials and Methods: Immunofluorescence staining and proximity ligation assay (PLA) were performed using anti-PIT-1 antibody and patients' sera on PIT-1-expressing cell line GH3 cells and human induced pluripotent stem cell (iPSC)-derived pituitary tissues., Results: PIT-1 was colocalized with MHC class I molecules, calnexin, and GM130 in the cytosol. PLA results showed that PIT-1 epitope was presented by MHC/HLA class I molecules on the cell surface of GH3 cells and iPSC-derived pituitary cells. The number of PIT-1/HLA complexes on the cell surface of pituitary cells in the patient was comparable with that in the control subject., Conclusions: Our data indicate that PIT-1 protein is processed in the antigen presentation pathway and that its epitopes are presented by in MHC/HLA class I on anterior pituitary cells, supporting the hypothesis that PIT-1-reactive CTLs caused the cell-specific damage. It is also suggested that number of epitope presentation was not associated with the pathogenesis of anti-PIT-1 antibody syndrome., (Copyright © 2019 Endocrine Society.)

Purpose: To evaluate the advantages of intraprocedural CT during adrenal venous sampling (AVS) to confirm accurate catheterization of the right adrenal vein (RAV)., Materials and Methods: This single-institution study included 106 patients (mean age 52.4 years; range 28-74 years) with primary aldosteronism who performed contrast-enhanced CT (CECT) before AVS following AVS between January 2011 and March 2018. After catheterization of the RAV under fluoroscopic guidance, unenhanced CT images were obtained to confirm catheter position on unified CT angiography system. Catheter repositioning was performed when the catheter was inaccurately positioned. Venography findings were classified into two groups: (1) presumably cannulated in the RAV (presumed RAV group) and (2) obscured visualization of the RAV because of collateral vessels (obscured RAV group). Success rates of AVS were compared using Fisher's exact test., Results: The overall success of AVS was achieved in 104 patients (98.1%). Catheter was deviated into the IVC during intraprocedural CT in four patients. Fourteen patients (14.0%) required catheter repositioning by intraprocedural CT images, and accurate catheterization in the RAV was eventually accomplished. The success rate of AVS was significantly higher in the presumed RAV group (90.1% [73/81]) than that in the obscured RAV group (68.4% [13/19]) (p = 0.024). If intraprocedural CT was not acquired during AVS, the success rate of AVS would have been significantly lower (84.9% [90/106]) compared with that use of intraprocedural CT (98.1% [104/106]) (p < 0.001)., Conclusions: Intraprocedural unenhanced CT by referring to the preprocedural CECT before AVS enables the confirmation of accurate catheterization of the RAV., Level of Evidence: Level 4, case series.

Purpose: IgG4-related disease involves various organs including the pituitary and pancreas. The prevalence of IgG4-related hypophysitis is relatively rare compared with IgG4-related pancreatitis (autoimmune pancreatitis). Although several cases demonstrating both autoimmune pancreatitis and hypophysitis have been reported, the prevalence of IgG4-related hypophysitis in patients with autoimmune pancreatitis remains unknown. This study aimed at screening for IgG4-related hypophysitis to accurately determine its prevalence in patients with autoimmune pancreatitis., Methods: In this cohort study, we screened IgG4-related hypophysitis via pituitary magnetic resonance imaging (MRI) and endocrinological examination in 27 patients who were undergoing follow-up for autoimmune pancreatitis at Kobe University Hospital between 2014 and 2018., Results: Among 27 patients with autoimmune pancreatitis, 5 patients exhibited morphological abnormalities in the pituitary (18.5%). Among them, one patient (3.7%) met the criteria for hypophysitis with an enlarged pituitary and stalk concomitant with hypopituitarism. After glucocorticoid treatment, the enlarged pituitary shrank and became empty sella during the clinical course. Four patients (14.8%) revealed empty sella without obvious pituitary dysfunction. Four of 5 patients with morphological pituitary abnormalities showed multiple organ involvement in addition to pancreatic and pituitary involvement. Accordingly, multiple organ involvement was more prevalent in patients with morphological pituitary abnormalities (80%) compared to those without (48%)., Conclusions: Although a large-scale study is necessary to validate these results, these data suggest that the prevalence of hypophysitis in patients with autoimmune pancreatitis may be underestimated. Based on our findings, we recommend screening for hypophysitis, especially in patients with multiple organ involvement.

Purpose: In isolated adrenocorticoropic hormone (ACTH) deficiency (IAD), autoimmunity against corticotrophs has been suggested; however, the pathogenesis remains largely unknown. Large cell neuroendocrine carcinoma (LCNEC) of the lung is a pulmonary tumor of high-grade malignant neuroendocrine tumor and it reportedly caused paraneoplastic syndrome by autoimmunity in several cases., Methods: A 42-year-old woman with isolated adrenocorticotropic (ACTH) hormone deficiency (IAD) was diagnosed with large cell neuroendocrine carcinoma (LCNEC) 3 years after being diagnosed with IAD. We hypothesized that the LCNEC played a causal role in the development of IAD as a paraneoplastic syndrome and analyzed the autoimmunity. We also analyzed another case of ectopic ACTH syndrome to prove this hypothesis., Results: The LCNEC tissue revealed an ectopic ACTH expression and lymphocyte infiltration. Interestingly, autoantibody against the proopiomelanocortin (POMC) protein was detected in the peripheral blood. Although, patient's serum did not show any effects on cell viability, proliferation, nor pomc expression in a corticotroph cell line, AtT20 cells, patient's lymphocytes in the peripheral blood specifically reacted toward POMC protein, indicating a presence of cytotoxic T lymphocytes (CTLs). In addition, the analysis of another case of ectopic ACTH syndrome showed lymphocyte infiltration not only in the metastatic liver tumors but also in the pituitary. Moreover, most CD8-positive cells resided adjacent to corticotrophs., Conclusions: These data indicate that the ectopic ACTH expression in the tumor evoked the autoimmunity to corticotrophs and caused IAD as a form of paraneoplastic syndrome.

Background: Most of circulating IGF-I is derived from the liver and circulating IGF-I levels are decreased in several pathological conditions, such as liver cirrhosis, uncontrolled diabetes, renal failure, and malnutrition. However, it has not fully been elucidated which factors modify IGF-I level in a physiological condition., Objective: To identify the factors which are associated with circulating IGF-I levels., Design: Cross-sectional study., Methods: This study included 428 subjects who undertook health check-up. Subjects diagnosed with non-alcoholic fatty liver disease (NAFLD) by ultrasonography were analyzed separately. Univariate and multivariate regression analyses were conducted to identify the factors associated with circulating IGF-I levels., Results: Regression analyses revealed that serum albumin levels, total-bilirubin levels, calcium levels, and HOMA-IR were positively correlated with IGF-I levels. Serum transaminase levels and habitual drinking (ethanol intake >20 g/day) were negatively correlated with serum IGF-I levels. Although serum IGF-I standard deviation scores (SDS) in subjects with and without NAFLD were comparable, after adjusting confounding factors clarified by multivariate regression analysis, IGF-I SDS negatively correlated with the presence of NAFLD., Conclusion: In this study, we demonstrated that serum bilirubin and calcium levels are correlated with serum IGF-I levels. Although further study is necessary, these data suggest a presence of interaction between GH-IGF-I axis and bilirubin and calcium metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Measuring salivary cortisol is both convenient and non-invasive for patients; however, its usefulness as a marker for monitoring medical therapy has not yet been established. The aim of this study was to assess the utility of multiple salivary cortisol measurements in patients with Cushing's syndrome (CS) during medical therapy. Six patients with CS (three with cortisol-secreting adrenocortical adenoma and three with ACTH-secreting pituitary adenoma) were recruited. Samples for morning serum cortisol, urinary free cortisol (UFC), and multiple salivary cortisol levels were collected before and during metyrapone treatment. The area under the curve (AUC) and mean value (MV) of daily salivary cortisol levels were calculated. In five out of six patients, UFC were normalized; however, multiple salivary cortisol measurements revealed an impaired diurnal cortisol rhythm in these patients. To verify the usefulness of multiple salivary cortisol measurements, we performed a prospective case study of a patient in whom the excess secretion of cortisol was not controlled (UFC 211 μg/day) with 2,250 mg/day in four divided doses of metyrapone. Multiple measurements of salivary cortisol revealed that cortisol levels elevated before the next administration. Accordingly, we shortened the interval by increasing the number of administration from four to five times per day, with a slight increment of daily dose of 2,500 mg. These optimizations resulted in a drastic improvement of diurnal pattern as well as UFC level (101 μg/day). Changes in both the MV and AUC of salivary cortisol levels were more correlated with those in UFC levels (Correlation coefficient 0.75, p  = 0.007, and 0.70, p  = 0.017) than those in the morning serum cortisol levels (0.42, p  = 0.200), indicating that multiple salivary cortisol measurements reflect more precisely the excess secretion of cortisol. Our preliminary data suggest that multiple salivary cortisol measurements can be a useful tool to visualize the diurnal cortisol rhythm and to determine the dose and timing of metyrapone during the treatment in patients with CS.

Purpose: Acromegaly is a disease associated with an increased risk for several kinds of neoplasms including colon and thyroid cancer. Although the association between acromegaly and pancreatic neoplasms has not been elucidated, it has recently been reported that GNAS gene mutations were found in 58% of intraductal papillary mucinous neoplasms (IPMNs), which are representative pancreatic cystic lesions, suggesting a link between IPMNs and acromegaly. To assess the prevalence of pancreatic cystic lesions in patients with acromegaly, we performed a retrospective cross-sectional single institute study., Methods: Thirty consecutive acromegalic patients (20 females and 10 males; mean age, 60.9 ± 11.9 years) who underwent abdominal contrast-enhanced computed tomography or magnetic resonance imaging between 2007 and 2015 at Kobe University Hospital were recruited. We also analyzed the relationship between presence of pancreatic cystic lesions and somatic GNAS mutations in pituitary tumors., Results: Seventeen of 30 (56.7%) patients studied had pancreatic cystic lesions. Nine of 17 patients (52.9%) were diagnosed with IPMNs based on imaging findings. These results suggest that the prevalence of IPMNs may be higher in acromegalic patients in acromegalic patients than historically observed in control patients (up to 13.5%). In patients with pancreatic cystic lesions, the mean patient age was higher and the duration of disease was longer than in those without pancreatic cystic lesions (67.0 ± 2.3 vs. 53.0 ± 2.7 years, p < 0.001, 15.5 ± 2.4 vs. 7.3 ± 2.8 years, p = 0.04). There were no differences in serum growth hormone levels or insulin-like growth factor standard deviation scores between these two groups (21.3 ± 6.4 vs. 23.0 ± 7.4 ng/ml, p = 0.86, 6.6 ± 0.5 vs. 8.0 ± 0.6, p = 0.70). Neither the presence of somatic GNAS mutation in a pituitary tumor nor low signal intensity of the tumor in T2 weighted magnetic resonance imaging was associated with the presence of pancreatic cystic lesions., Conclusions: These data demonstrate that old or long-suffering patients with acromegaly have a higher prevalence of pancreatic cystic lesions. Moreover, the prevalence of pancreatic cystic lesions may be increased in acromegalic patients.

Context: Pheochromocytoma and paraganglioma are catecholamine-producing tumors that often impair glucose tolerance. The effects of these tumors on insulin sensitivity and insulin secretion in patients have remained unclear, however., Objective: To characterize the influence of pheochromocytoma or paraganglioma on glucose tolerance, we comprehensively analyzed various parameters related to insulin secretion or insulin sensitivity in patients with these tumors., Design: Hyperglycemic and hyperinsulinemic-euglycemic clamps, as well as an oral glucose tolerance test (OGTT), were performed in patients before and after tumor excision., Setting: Patients underwent metabolic analyses on admission to Kobe University Hospital., Patients: Eleven patients with pheochromocytoma and two with paraganglioma were examined., Intervention: None., Main Outcome Measures: We evaluated various parameters related to insulin secretion or insulin sensitivity as determined by an OGTT and by hyperglycemic and hyperinsulinemic-euglycemic clamp analyses., Results: Surgical treatment of the tumor reduced urinary catecholamine excretion and improved glucose tolerance. The insulinogenic index, but not total insulin secretion, measured during the OGTT as well as the first phase, but not the second phase, of insulin secretion during the hyperglycemic clamp were improved after surgery. The insulin sensitivity index determined during the hyperinsulinemic-euglycemic clamp remained unchanged after surgery., Conclusion: These results suggest pheochromocytoma and paraganglioma impair glucose tolerance primarily through impairment of insulin secretion-in particular, that of the early phase of the insulin secretory response. A prospective study with more patients is warranted to further confirm these results., (Copyright © 2017 Endocrine Society)

Anti-PIT-1 antibody syndrome has recently been reported and characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with autoimmunity to a pituitary specific transcription factor PIT-1, which plays an essential role in GH-, PRL-, and TSH-producing cells. Although circulating anti-PIT-1 antibody and PIT-1-reactive cytotoxic T cells (CTLs) were detected in the patients, the pathophysiology and precise mechanisms for the autoimmunity remain unclarified. During the follow up, thymoma was diagnosed in all 3 cases with anti-PIT-1 antibody syndrome. Immunohistochemical analysis revealed that PIT-1 was strongly expressed in neoplastic cortical thymic epithelial cells. Importantly, after thymectomy, the titer of anti-PIT-1 antibody decreased and reactivity of CTLs toward PIT-1 diminished. These data strongly suggest that the aberrant expression of PIT-1 in the thymoma plays a causal role in the development of this syndrome. Thus, we define that this syndrome is a novel thymoma-associated autoimmune disease.

Pituitary-specific transcription factor 1 (PIT-1; POU domain, class 1, transcription factor 1 (POU1F1)) is an essential transcription factor for the differentiation of somatotrophs, lactotrophs, and thyrotrophs, and for the expression of growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH). Mutations in PIT-1 cause congenital defects in GH and PRL secretion and severe TSH insufficiency. Anti-PIT-1 antibody syndrome, firstly reported in 2011, is characterized by acquired GH, PRL, and TSH deficiencies without PIT-1 mutation and is associated with the presence of the circulating antibody against PIT-1 protein as a marker. Various autoantibodies are detected with multiple endocrine organopathies in this syndrome; therefore, it meets the criteria of autoimmune polyglandular syndrome. Mechanistically, cytotoxic T lymphocytes specifically reacting with PIT-1 protein play an important role in the development of this syndrome., (© 2017 S. Karger AG, Basel.)

Most of acromegaly is caused by a sporadic somatotropinoma and a couple of novel gene mutations responsible for somatotropinoma have recently been reported. To determine the cause of sporadic somatotropinoma in Japanese patients, we analyzed 61 consecutive Japanese patients with somatotropinoma without apparent family history. Comprehensive genetic analysis revealed that 31 patients harbored guanine nucleotide-binding protein, alpha stimulating (GNAS) mutations (50.8%) and three patients harbored aryl hydrocarbon receptor interacting protein (AIP) mutations (4.9%). No patients had G protein-coupled receptor 101 (GPR101) mutations. The patients in this cohort study were categorized into three groups of AIP, GNAS, and others and compared the clinical characteristics. The AIP group exhibited significantly younger age at diagnosis, larger tumor, and higher nadir GH during oral glucose tolerance test. In all patients with AIP mutation, macro- and invasive tumor was detected and repetitive surgery or postoperative medical therapy was needed. One case showed a refractory response to postoperative somatostatin analogue (SSA) but after the addition of cabergoline as combined therapy, serum IGF-I levels were controlled. The other case showed a modest response to SSA and the switching to cabergoline monotherapy was also effective. These data suggest that although resistance to SSA has been reported in patients with AIP mutations, the response to dopamine agonist (DA) may be retained. In conclusion, the cause of sporadic somatotropinoma in Japanese patients was comparable with the previous reports in Caucasians, patients with AIP mutations showed unique clinical characteristics, and DA may be a therapeutic option for patients with AIP mutations.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) and cirrhosis determines patient prognosis; however, effective treatment for fibrosis has not been established. Oxidative stress and inflammation activate hepatic stellate cells (HSCs) and promote fibrosis. In contrast, cellular senescence inhibits HSCs' activity and limits fibrosis. The aim of this study was to explore the effect of IGF-I on NASH and cirrhotic models and to clarify the underlying mechanisms. We demonstrate that IGF-I significantly ameliorated steatosis, inflammation, and fibrosis in a NASH model, methionine-choline-deficient diet-fed db/db mice and ameliorated fibrosis in cirrhotic model, dimethylnitrosamine-treated mice. As the underlying mechanisms, IGF-I improved oxidative stress and mitochondrial function in the liver. In addition, IGF-I receptor was strongly expressed in HSCs and IGF-I induced cellular senescence in HSCs in vitro and in vivo. Furthermore, in mice lacking the key senescence regulator p53, IGF-I did not induce cellular senescence in HSCs or show any effects on fibrosis. Taken together, these results indicate that IGF-I induces senescence of HSCs, inactivates these cells and limits fibrosis in a p53-dependent manner and that IGF-I may be applied to treat NASH and cirrhosis.

Objective: Although serum insulin like growth factor type 1 (IGF-I) levels are negatively correlated with hemoglobin A1c (HbA1c) in patients with type 1 diabetes, this correlation is controversial in patients with type 2 diabetes mellitus (T2DM) because of the influence of multiple factors including insulin secretion and obesity. The aim of this study was to evaluate the influence of T2DM on serum growth hormone (GH) and IGF-I levels in Japanese patients, who exhibited relatively low BMI compared with white patients in the previous studies., Design: We retrospectively analysed 315 consecutive Japanese hospitalized patients with T2DM. We analysed factors correlated with changes in serum IGF-I levels and those related to diabetes., Results: The median HbA1c was 8.7% (7.4-10.2) and the median body mass index (BMI) was 26.2kg/m(2) (23.1-29.7), which was relatively low compared with the previous studies. Overall, no correlations was found between serum GH or IGF-I levels and fasting plasma glucose (FPG) or HbA1c; however, when stratified by FPG and HbA1c levels, serum IGF-I levels were significantly lower in patients with FPG≥200mg/dL than in those with FPG<200mg/dL (p=0.039). In addition, serum IGF-I levels were significantly lower in patients with HbA1c≥12% than in those with HbA1c<12% (p=0.046). Multiple linear regression analysis revealed a positive correlation between fasting C-peptide levels and serum IGF-I levels (p=0.040), whereas no correlations was found for BMI, duration of T2DM, FPG levels, or HbA1c. Moreover, patients with improved HbA1c levels during the follow up period showed a significant increase in serum IGF-I levels., Conclusions: Serum IGF-I levels were significantly decreased in Japanese patients with uncontrolled T2DM, and impaired insulin secretion may be a mechanism underlying this effect. When diagnosing acromegaly in patients with uncontrolled diabetes, these factors should be taken into account., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective Various organs are known to be affected by the comorbidities of acromegaly. However, the involvement of renal structural comorbidities, such as cysts, has so far remained largely unknown. In this single-center study, we aimed to determine the prevalence and factors associated with simple renal cysts in Japanese patients with acromegaly. Methods A total of 71 consecutive patients with acromegaly were analyzed, who all underwent abdominal ultrasonography at diagnosis between 1986 and 2012 at Kobe University Hospital. Results Of these 71 patients, 23 (32.4%) exhibited simple renal cysts. Acromegalic patients with renal cysts tended to be significantly older, had a higher prevalence of smoking- and higher nadir growth hormone (GH) levels during the oral glucose tolerance test (OGTT) than did those without renal cysts. A multivariate logistic regression analysis showed age, smoking, and nadir GH to be independent factors associated with renal cysts. Interestingly, the number of renal cysts positively correlated with both the basal GH levels and nadir GH levels during OGTT (r=0.66, p<0.05 and r=0.70, p<0.05, respectively). In addition, the mean diameter of renal cysts positively correlated with the systolic blood pressure (r=0.84, p<0.005). Conclusion This is the first report to show the prevalence of simple renal cysts in patients with acromegaly. Elevated nadir GH levels during OGTT were found to be associated with an increased risk of simple renal cysts. Therefore, an excessive secretion of GH may be related to the development of renal cysts.

Purpose: To develop a Japanese version of the acromegaly quality of life (QoL) questionnaire (AcroQoL) and investigate the factors associated with impaired QoL in patients with acromegaly., Methods: We developed a Japanese version of the AcroQoL by a forward-backward method and evaluated QoL in 38 patients with acromegaly who had been followed up at an outpatient clinic at Kobe University Hospital. Its reliability was examined with Cronbach's alpha and item-total correlations. Second examination was performed for concurrent validity by assessment of correlations with the Short Form-36 (SF-36) and longitudinal analysis of the AcroQoL in 25 patients., Results: Cronbach's alpha and item-total correlations showed a range of 0.76-0.93 and 0.20-0.84, respectively, and significant correlations were found between the AcroQoL and the SF-36. Younger age and a history of radiotherapy were associated with worse total score by the multivariate linear regression analysis (p = 0.020 and p = 0.042, respectively). Intriguingly, in the biochemically-controlled group after the exclusion of patients who received radiotherapy, patients who underwent surgery alone exhibited a higher psychological (75.0 vs. 65.7 %, p = 0.036) and appearance (64.3 vs. 53.6 %, p = 0.036) score than those who are treating with pharmaceutical therapy., Conclusions: The reliability of the Japanese version of the AcroQoL was satisfactory. Younger age and a history of radiotherapy were associated with lower QoL in patients with acromegaly. In biochemically-controlled acromegaly, patients who underwent surgery alone exhibited better QoL than those under pharmaceutical therapy.

Objective: Patients with acromegaly exhibit reduced life expectancy and increased prevalence of age-related diseases, such as diabetes, hypertension, and cardiovascular disease. However, the underlying mechanism has not been fully elucidated. Telomere shortening is reportedly associated with reduced life expectancy and increased prevalence of these age-related diseases., Methods: We measured telomere length in patients with acromegaly using quantitative PCR method. The effect of GH and IGF-I on telomere length and cellular senescence was examined in human skin fibroblasts., Results: Patients with acromegaly exhibited shorter telomere length than age-, sex-, smoking-, and diabetes-matched control patients with non-functioning pituitary adenoma (0.62 ± 0.23 vs. 0.75 ± 0.35, respectively, P = 0.047). In addition, telomere length in acromegaly was negatively correlated with the disease duration (R2 = 0.210, P = 0.003). In vitro analysis revealed that not GH but IGF-I induced telomere shortening in human skin fibroblasts. Furthermore, IGF-I-treated cells showed increased senescence-associated β-galactosidase activity and expression of p53 and p21 protein. IGF-I-treated cells reached the Hayflick limit earlier than GH- or vehicle-treated cells, indicating that IGF-I induces cellular senescence., Conclusion: Shortened telomeres in acromegaly and cellular senescence induced by IGF-I can explain, in part, the underlying mechanisms by which acromegaly exhibits an increased morbidity and mortality in association with the excess secretion of IGF-I.

Introduction: Immunoglobulin (Ig) G4-related hypophysitis is an emerging clinical entity, which is characterized by an elevated serum IgG4 concentration and infiltration of IgG4-positive plasma cells in the pituitary. Although some criteria for its diagnosis have been proposed, they have not been fully established. In particular, differential diagnosis from secondary chronic inflammation including granulomatosis with polyangiitis (GPA) is difficult in some cases. We describe central diabetes insipidus with pituitary swelling exhibiting infiltration of IgG4-positive cells., Patient: A 43-year-old woman in the remission stage of GPA presented with sudden-onset polyuria and polydipsia. Pituitary magnetic resonance imaging revealed swelling of the anterior and posterior pituitary and stalk, with heterogeneous gadolinium enhancement and disappearance of the high signal intensity of the posterior pituitary. Evaluation of biochemical markers for GPA suggested that the disease activity was well-controlled. Endocrinological examination revealed the presence of central diabetes insipidus and growth hormone deficiency. Pituitary biopsy specimen showed IgG4-positive cells, with a 43% IgG4(+)/IgG(+) ratio, which met the criteria for IgG4-related hypophysitis. However, substantial infiltration of polymorphonuclear neutrophils with giant cells was also noted, resulting in a final diagnosis of pituitary involvement of GPA., Conclusion: These results suggest that pituitary involvement of GPA should be taken into account for the differential diagnosis of IgG4-related hypophysitis.

Objective: Although it has been recommended that serum free thyroxine (FT4) levels should be targeted to middle-upper normal levels during levothyroxine (l-T4) replacement therapy in patients with central hypothyroidism (CeH), the rationale has not been clarified., Methods: A retrospective single-center study enrolled 116 patients with hypothyroidism (CeH, n=32; total thyroidectomy (Tx), n=22; primary hypothyroidism (PH), n=33; and control benign thyroid nodule (C), n=29). The patients had received L-T4 therapy at the Kobe University Hospital between 2003 and 2013. They were stratified according to serum FT4 level (≥ 1.10 or <1.10 ng/dl), and body temperature (BT), serum free triiodothyronine (FT3) levels, FT3/FT4 ratio, and lipid profiles were compared. The effect of GH replacement therapy on thyroid function was also analyzed., Results: FT3 levels and FT3/FT4 ratios were significantly lower in patients with CeH than in patients with PH (P<0.05) or C (P<0.05). In patients with FT4 <1.10 ng/dl, BT was significantly lower in patients with CeH (P=0.002) and Tx (P=0.005) than in patients with PH, whereas no differences were found in patients with FT4 ≥ 1.10 ng/dl. In patients with CeH, FT3 levels were higher in those with GH replacement therapy (P=0.018)., Conclusion: In CeH, patients with median-lower normal levels of serum FT4 exhibited lower serum FT3 levels and lower BT. These results support the target levels of serum FT4 as middle-upper normal levels during l-T4 replacement therapy in patients with CeH., (© 2015 European Society of Endocrinology.)

Purpose: Colorectal neoplasms are well known to be a complication in cases of acromegaly; however, data on the prevalence of colorectal neoplasms in Asian patients with acromegaly are limited. Further, the factors associated with colorectal neoplasms in cases of acromegaly are controversial. Therefore, we aimed to clarify the prevalence of and factors associated with colorectal neoplasms in Japanese patients with acromegaly in a single center., Methods: We analyzed consecutive 57 patients who had undergone full-length colonoscopy at the time of diagnosis at Kobe University Hospital between 1986 and 2012., Results: Of the 57 patients, 22 (38.6%), 18 (31.6%) and 3 (5.3%) patients were diagnosed with hyperplastic polyps, adenomas, and adenocarcinomas, respectively and the prevalence was significantly higher than in a historical control group, Chinese patients with irritable bowel syndrome (The odds ratio was 4.0, 8.7, and 17.5, respectively). The prevalence of adenocarcinomas was also significantly higher in these patients than in the general Japanese population (odds ratio 14.5). Patients with acromegaly who had colorectal neoplasms had longer disease duration than those without colorectal neoplasms. Of note, the area under the growth hormone (GH) concentration-time curve (GH AUC) during the oral glucose tolerance test was significantly higher in patients with adenocarcinomas than in those with no colonic lesion or those with hyperplastic polyps., Conclusion: Japanese patients with acromegaly exhibited an increased risk of colorectal neoplasms, especially colorectal adenocarcinomas. An increased GH AUC was associated with an increased risk for colon adenocarcinomas in patients with acromegaly.

Autoimmunity against the pituitary has been observed in patients with hypophysitis. Although various autoantibodies against pituitary proteins have been reported, it is known that most of them are markers for the disease. Recently, a unique autoantibody against pituitary transcription factor PIT-1 (POU1F1) was detected in patients with an acquired combined pituitary hormone deficiency characterized by a specific defect in growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH). The antibody has been suggested to be involved in the pathogenesis because PIT-1 is an essential transcription factor that plays a role in the differentiation and maintenance of GH-, PRL-, and TSH-producing cells and mutations in the PIT-1 gene, resulting in a specific defect in these hormones. This syndrome was found to be a novel clinical entity; therefore, it was named 'anti-PIT-1 antibody syndrome'. Circulating anti-PIT-1 antibody and various autoantibodies were detected with multiple endocrine organopathy, meeting the definition of autoimmune polyglandular syndrome. Mechanistically, cytotoxic T lymphocytes that specifically react with PIT-1 protein play an important role in the development of this syndrome. In this review, we discuss the clinical aspects and pathophysiology of anti-PIT-1 antibody syndrome.

Various hypothalamic-pituitary diseases cause hypopituitarism. Inflammation related to autoimmunity also causes hypopituitarism. Hypophysitis is a representative disease caused by autoimmunity. Generally, anterior pituitary hormones are non-specifically impaired in this condition, but specific hormone defects have been reported in some cases. Anti-PIT-1 (pituitary-specific transcription factor 1) antibody syndrome is a novel clinical entity that presents an acquired combined pituitary hormone deficiency characterized by a specific defect in growth hormone, prolactin, and thyroid-stimulating hormone. Circulating anti-PIT-1 antibody along with various autoantibodies are detected with multiple endocrine organopathy, meeting the definition of autoimmune polyglandular syndrome. Mechanistically, cytotoxic T lymphocytes that specifically react with PIT-1 protein play an important role in the development of this syndrome.

The prevalence of acromegaly is estimated to be 8-24/100,000, but several recent studies suggest it is underestimated. In particular, acromegaly is considered more prevalent in patients with type 2 diabetes mellitus (T2DM) than in the normal population. This study aimed to evaluate the prevalence of acromegaly in hospitalized patients with T2DM. A total of 327 hospitalized patients with T2DM were recruited as subjects. If serum insulin-like growth factor 1 (IGF-1) levels were found to be elevated, random GH level was measured or oral glucose tolerance test (OGTT) was performed. Five patients with elevated serum IGF-1 levels and random GH level or inadequate suppression of GH in the OGTT underwent pituitary magnetic resonance imaging. Of those patients, pituitary adenoma was detected in 2 patients. These 2 patients were diagnosed with acromegaly, as they also exhibited mild acromegalic features. Intriguingly, both these patients exhibited severe macroangiopathy and an absence of microangiopathy. The prevalence of acromegaly in the hospitalized patients with T2DM in this study was therefore 0.6%, suggesting a higher prevalence than that predicted. Although a large-scale prospective study is required to clarify the precise prevalence of acromegaly in hospitalized patients with T2DM, the present study shows that it is useful to screen hospitalized patients with T2DM for acromegaly by measuring their serum IGF-1 level.

Objective: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are frequently observed in patients with adult growth hormone deficiency (AGHD) and short-term GH replacement therapy (GHRT) has reportedly been efficacious in NAFLD and NASH. The aim of this study was to investigate whether long-term GHRT is an effective treatment for the hepatic comorbidities in AGHD., Design: This is a retrospective observational study. We recruited 54 consecutive hypopituitary patients with AGHD. Among them, 31 patients who had received GHRT for more than 24 months were compared with 19 age- and sex-matched patients without GHRT. We also analyzed the long term effect of GHRT on 14 patients diagnosed with NASH by liver biopsy. In addition, we subdivided the GHRT group into GH-responder and GH-non-responder groups and analyzed the factors associated with the efficacy of the treatment., Results: For a period of 24 months, the significant reduction of serum liver enzyme levels and a fibrotic marker was observed in patients receiving GHRT compared with the control group. Furthermore, GHRT also improved liver enzyme levels in AGHD patients with NASH. The GH-non-responder group showed a higher proportion of patients who gained weight during the study period., Conclusions: These results indicate that GHRT is efficacious for improving serum liver enzyme levels for at least 24 months in patients with AGHD. To optimize this effect, it is important to avoid body weight gain during the treatment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Context: Anti-pituitary-specific transcriptional factor 1 (PIT-1) antibody syndrome is characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with circulating anti-PIT-1 antibodies. Although autoimmunity to PIT-1 has been suggested as a pathogenesis, the precise mechanism of the syndrome remains unclarified., Objective: To elucidate the involvement of antibody- or cell-mediated immunity in anti-PIT-1 antibody syndrome., Materials and Methods: To investigate a direct effect of anti-PIT-1 antibody on pituitary cells, cell proliferation, and cytotoxicity detection assays were performed using patient serum. Enzyme-linked immunospot (ELISpot) assay was performed to evaluate the involvement of PIT-1-reactive cytotoxic T lymphocytes (CTLs). An immunohistochemical analysis using anti-CD4 or anti-CD8 antibody was performed to examine tissue infiltration by CTLs., Results: Patient serum did not exhibit any inhibitory effect on cell proliferation and secretion of GH and PRL in GH3 cells. In addition, complement-dependent cytotoxicity was not detected in patient serum on GH3 cells or primary pituitary cells. The ELISpot assay revealed the presence of CTLs that specifically reacted to the recombinant PIT-1 protein in the patient's peripheral lymphocytes. CD8(+) cell infiltrations, which is the characteristic of CTLs, were observed in the pituitary gland, adrenal gland, stomach, thyroid gland, liver, and pancreas of the patient with anti-PIT-1 antibody syndrome., Conclusions: These results suggest that the anti-PIT-1 antibody is not a cause but a marker of anti-PIT-1 antibody syndrome, in which CTLs play a pivotal role in the pathogenesis.

A novel clinical entity related to autoimmune polygladular syndrome (APS) termed "anti-PIT-1 antibody syndrome" is characterized by a presence of circulating autoantibody against the pituitary-specific transcriptional factor-1 (PIT-1) with acquired specific defect in GH, PRL, and TSH. Although autoimmunity to PIT-1 has been suggested, the underlying mechanisms remain to be elucidated. Sialic acid acetylesterase (SIAE) plays a crucial role in regulating the threshold of autoantibody production of B-cells and the defective variants of SIAE are associated with an increased risk of various autoimmune diseases such as type 1 diabetes (T1DM). To explore the link between anti-PIT-1 antibody syndrome and SIAE, we analyzed SIAE gene in 3 patients with anti-PIT-1 antibody syndrome and 200 healthy control subjects, and compared the prevalence of single nucleotide polymorphisms. Intriguingly, we found A467V SIAE variants (c.1400C>T, rs7941523) in a heterozygous state in all the patients with anti-PIT-1 antibody syndrome, while we detected in 6 % of control subjects, in which the prevalence was significantly increased in the patients (P<0.0005). Considering the physiological function of SIAE and the clinical features of anti-PIT-1 antibody syndrome, present data imply a novel aspect of the pathogenesis in this disease.

Venous thromboembolism (VTE) is frequently associated with hypercortisolemia. This retrospective single-center study aimed to clarify the significance of plasma D-dimer levels for VTE screening in patients with subclinical or overt Cushing's syndrome (soCS). A total of 72 consecutive treatment-naïve patients with soCS diagnosed at Kobe University Hospital between 2002 and 2014 were enrolled. Patients with both lower extremity ultrasound and D-dimer measurement data (n = 19) were recruited in study 1 and divided into 2 groups, a deep vein thrombosis (DVT) (-) group (n = 12) and DVT (+) group (n = 7) for a comparison of the associated factors. The age and D-dimer levels were higher in the DVT (+) group than in the DVT (-) group (p = 0.04 and 0.02, respectively). A receiver operating characteristic analysis found that D-dimer level ≥2.6 μg/mL correlated with the presence of DVT (sensitivity, 100%; specificity, 91.7%). Next, patients with D-dimer measurement data (n = 36) were recruited in study 2 and divided into 2 groups according to D-dimer level: D-dimer (-), <1 μg/mL group (n = 23) and D-dimer (+), ≥1 μg/mL group (n = 13); the groups were compared with respect to various VTE-related risk factors. A logistic regression analysis revealed that elevated cortisol level after low-dose dexamethasone suppression was a significant risk factor for D-dimer elevation (OR = 1.21, p = 0.02). In conclusion, these data demonstrate that a D-dimer level ≥2.6 μg/mL is an indicator of DVT in treatment naïve patients with soCS and suggests that relatively high autonomous cortisol secretion may be associated with thrombus formation.

Objective: The prevalence and clinical characteristics of IgG4-related hypophysitis remain unclear due to the limited number of case reports. Therefore, in this study, we screened consecutive outpatients with hypopituitarism and/or diabetes insipidus (DI) to estimate its prevalence., Methods: A total of 170 consecutive outpatients with hypopituitarism and/or central DI were screened at Kobe University Hospital for detecting IgG4-related hypophysitis by pituitary magnetic resonance imaging, measuring serum IgG4 concentrations, assessing the involvement of other organs, and carrying out an immunohistochemical analysis to detect IgG4-positive cell infiltration., Results: Among the screened cases, 116 cases were excluded due to diagnosis of other causes such as tumors and congenital abnormalities. Additionally, 22 cases with isolated ACTH deficiency were analyzed and were found not to meet the criteria of IgG4-related hypophysitis. The remaining 32 cases were screened and seven were diagnosed with IgG4-related hypophysitis, of which three cases were diagnosed by analyzing pituitary specimens. IgG4-related hypophysitis was detected in 30% (seven of 23 patients) of hypophysitis cases and 4% of all hypopituitarism/DI cases. The mean age at the onset of IgG4-related hypophysitis was 61.8±8.8 years, and the serum IgG4 concentration was 191.1±78.3 mg/dl (normal values 5-105 mg/dl and values in IgG4-related disease (RD) ≥135 mg/dl). Pituitary gland and/or stalk swelling was observed in six patients, and an empty sella was observed in one patient. Multiple co-existing organ involvement was observed in four of the seven patients prior to the onset of IgG4-related hypophysitis., Conclusion: These data suggest that the prevalence of IgG4-related hypophysitis has been underestimated. We should also consider the possibility of the development of hypopituitarism/DI caused by IgG4-related hypophysitis during the clinical course of other IgG4-RDs.

Adaptation under fasting conditions is critical for survival in animals. Sirtuin 1 (SIRT1), a protein deacetylase, plays an essential role in adaptive metabolic and endocrine responses under fasting conditions by modifying the acetylation status of various proteins. Fasting induces growth hormone (GH) resistance in the liver, leading to decreased serum insulin-like growth factor-I (IGF-I) levels as an endocrine adaptation for malnutrition; however, the underlying mechanisms of this action remain to be fully elucidated. Here we report that in vivo knockdown of SIRT1 in the liver restored the fasting-induced decrease in serum IGF-I levels and enhanced the GH-dependent increase in IGF-I levels, indicating that SIRT1 negatively regulates GH-dependent IGF-I production in the liver. In vitro analysis using hepatocytes demonstrated that SIRT1 suppresses GH-dependent IGF-I expression, accompanied by decreased tyrosine phosphorylation on signal transducer and activator of transcription (STAT) 5. GST pull-down assays revealed that SIRT1 interacts directly with STAT5. When the lysine residues adjacent to the SH2 domain of STAT5 were mutated, STAT5 acetylation decreased concomitant with a decrease in its transcriptional activity. Knockdown of SIRT1 enhanced the acetylation and GH-induced tyrosine phosphorylation of STAT5, as well as the GH-induced interaction of the GH receptor with STAT5. These data indicate that SIRT1 negatively regulates GH-induced STAT5 phosphorylation and IGF-I production via deacetylation of STAT5 in the liver. In addition, our findings explain the underlying mechanisms of GH resistance under fasting conditions, which is a known element of endocrine adaptation during fasting.

PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Chemerin, a recently discovered adipocytokine plays an important role in obesity and obesity-associated metabolic complications. However, the role of chemerin in the pathogenesis of type 2 diabetes mellitus (T2DM) has not fully been elucidated. We compared the serum chemerin levels and metabolic parameters between 88 control subjects, 86 patients with metabolic syndrome (MS), and 147 patients with T2DM in a Japanese population and further analyzed their correlation. Enzyme-linked immunosorbent assay was used to measure the serum chemerin levels. The chemerin levels were significantly higher in male than in female control subjects (p < 0.005), with significant decreases in patients with T2DM compared with those with MS and control subjects (164.9 ± 6.3 ng/mL vs. 209.8 ± 7.7 and 218.7 ± 7.3 ng/mL; p < 0.0001 vs. p < 0.0001, respectively) but no significant differences in female subjects. The multiple regression analysis revealed that the chemerin levels negatively correlated with the fasting glucose and HbA1c levels in total and male subjects. In the patients with T2DM, the chemerin levels negatively correlated with fasting glucose and high-density lipoprotein cholesterol but positively correlated with body mass index (BMI), and total cholesterol and triglyceride levels. The negative correlation between the chemerin and fasting glucose levels remained significant after adjustment for age, sex, and BMI in the total and male subjects and those with T2DM. These results suggest the role of chemerin in sex dimorphism and a potential link between chemerin levels and T2DM pathogenesis in a Japanese population.

Although somatostatin analogues are effective medical therapy for acromegaly, the serum insulin-like growth factor-I (IGF-I) levels remain uncontrolled in 35% of patients. Combined therapy with octreotide LAR and cabergoline has been reported to normalize IGF-I levels in 42-56% of Caucasian patients with acromegaly. However, it remains to be clarified whether combination therapy is effective in Japanese patients and on tumor shrinkage. We conducted a retrospective study on combined therapy in patients with octreotide-resistant acromegaly. Ten patients with acromegaly who showed octreotide-resistance were enrolled in this study. Cabergoline was added in doses of 0.25-2.0mg/week. Serum GH and IGF-I levels and tumor volume were assessed before and after treatment, and factors correlated with effect of the combined therapy were analyzed. Although serum GH levels did not decrease, serum IGF-I levels significantly decreased by 20% after 6 months of combined therapy compared with baseline (p < 0.05). As a result, serum IGF-I levels normalized in 30% of the patients. Tumor volume after combined therapy also significantly decreased (p < 0.01). There were no correlations between the decrease of serum IGF-I levels during combined therapy and the response of GH in a bromocriptine test, random GH, IGF-I, and PRL levels, the tumor volume, and the expression of PRL and dopamine D2 receptor in the tumor. In conclusion, we demonstrated that the addition of cabergoline to octreotide LAR is a beneficial option in Japanese patients with octreotide-resistant acromegaly, irrespective of serum PRL levels and the response of GH levels in a bromocriptine test.

Atrogin-1 and MuRF1, muscle-specific ubiquitin ligases, and autophagy play a role in protein degradation in muscles. We hypothesized that branched-chain amino acids (BCAAs) may decrease atrogin-1, MuRF1, and autophagy, and may have a protective effect on disuse muscle atrophy. To test this hypothesis, we selected hindlimb suspension (HS)-induced muscle atrophy as a model of disuse muscle atrophy because it is an established model to investigate the effects of decreased muscle activity. Sprague-Dawley male rats were assigned to 4 groups: control, HS (14 days), oral BCAA administration (600 mg/[kg day], 22.9% L-isoleucine, 45.8% L-leucine, and 27.6% L-valine), and HS and BCAA administration. After 14 days of the treatment, muscle weights and protein concentrations, cross-sectional area (CSA) of the muscle fibers, atrogin-1 and MuRF1 proteins, and microtubule-associated protein 1 light chain 3 II/I (ratio of LC3 II/I) were measured. Hindlimb suspension significantly reduced soleus muscle weight and CSA of the muscle fibers. Branched-chain amino acid administration partly but significantly reversed the HS-induced decrease in CSA. Hindlimb suspension increased atrogin-1 and MuRF1 proteins, which play a pivotal role in various muscle atrophies. Branched-chain amino acid attenuated the increase in atrogin-1 and MuRF1 in soleus muscles. Hindlimb suspension significantly increased the ratio of LC3 II/I, an indicator of autophagy, whereas BCAA did not attenuate the increase in the ratio of LC3 II/I. These results indicate the possibility that BCAA inhibits HS-induced muscle atrophy, at least in part, via the inhibition of the ubiquitin-proteasome pathway. Oral BCAA administration appears to have the potential to prevent disuse muscle atrophy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated β-galactosidase (SA-β-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the present study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, γH2AX, the increased levels of p53 and p21 proteins, and activated SA-β-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-β-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The progression to nonalcoholic steatohepatitis (NASH) from simple steatosis is associated with the mitochondrial dysfunction, enhanced oxidative stress, and inflammation. Recently, it has been reported that the prevalence of NAFLD (nonalcoholic fatty liver disease)/NASH is increased in patients with adult growth hormone deficiency (AGHD), suggesting that the deficiencies in GH and insulin-like growth factor (IGF-I) are involved in the development of NAFLD/NASH; however, the precise underlying mechanism remains to be elucidated. To clarify the mechanisms and the specific contribution of GH and IGF-I in these conditions, we examined the liver of a GH-deficient rat model, spontaneous dwarf rat (SDR) and the effect of GH and IGF-I administration. SDR showed steatosis and fibrosis in the liver in line with the phenotype observed in AGHD. Serum AST and ALT levels and triglyceride content in the liver were significantly increased in the SDR compared with the control. Intriguingly, the mitochondrial morphology in the SDR hepatocyte was impaired and the area was significantly decreased. Furthermore, oxidative stress in the SDR liver was enhanced. These changes were improved not only by GH but also by IGF-I administration, suggesting that GH-independent IGF-I action plays an essential role in the liver. In conclusion, we demonstrated that GH-deficient rat exhibits NASH and IGF-I plays an essential role to prevent the development of NASH. The improved mitochondrial function and reduced oxidative stress may contribute the effect of IGF-I in the liver., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Excessive oxidative stress plays a causal role in various diseases such as diabetes, hypertension, atherosclerosis, and heart failure. Acromegaly is a pathological condition associated with excess growth hormone (GH) and insulin-like growth factor-I (IGF-I) and a high prevalence of diabetes, hypertension, atherosclerosis, and heart failure; resulting in premature death. We hypothesized that these conditions may be associated with increased oxidative stress., Objective and Methods: We explored the oxidative stress levels in the serum and tissues of GH-transgenic rats as an animal model for acromegaly. We also measured the oxidative stress levels in the serum of patients with acromegaly and age-, sex-, and BMI-matched control subjects. We examined the effects of GH and IGF-I on reactive oxygen species (ROS) production in C2C12 myocytes., Results: The levels of an oxidative stress marker, serum thiobarbituric acid reactive substances (TBARS) were increased in the GH-transgenic rats. Further, tissue oxidative stress damage was enhanced in the cardiomyocytes and vascular smooth muscle cells in the aorta of the GH-transgenic rats. In addition, serum TBARS levels and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were increased in acromegaly in humans. IGF-I but not GH induced ROS production in C2C12 myocytes in vitro., Conclusions: These data indicate that the increased levels of IGF-I are associated with enhanced oxidative stress in rats and humans. In addition, increased ROS may play an important role in the complications and premature death in acromegaly., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.

Although various function of chemerin have been suggested, its physiological role remains to be elucidated. Here we show that chemerin-deficient mice are glucose intolerant irrespective of exhibiting reduced macrophage accumulation in adipose tissue. The glucose intolerance was mainly due to increased hepatic glucose production and impaired insulin secretion. Chemerin and its receptor ChemR23 were expressed in β-cell. Studies using isolated islets and perfused pancreas revealed impaired glucose-dependent insulin secretion (GSIS) in chemerin-deficient mice. Conversely, chemerin transgenic mice revealed enhanced GSIS and improved glucose tolerance. Expression of MafA, a pivotal transcriptional factor for β-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated β-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates β-cell function via maintaining MafA expression. These results indicate that chemerin regulates β-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner.

The pituitary-specific transcriptional factor-1 (PIT-1, also known as POU1F1), is an essential factor for multiple hormone-secreting cell types. A genetic defect in the PIT-1 gene results in congenital growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiency. Here, we investigated 3 cases of adult-onset combined GH, PRL, and TSH deficiencies and found that the endocrinological phenotype in each was linked to autoimmunity directed against the PIT-1 protein. We detected anti-PIT-1 antibody along with various autoantibodies in the patients' sera. An ELISA-based screening revealed that this antibody was highly specific to the disease and absent in control subjects. Immunohistochemical analysis revealed that PIT-1-, GH-, PRL-, and TSH-positive cells were absent in the pituitary of patient 2, who also had a range of autoimmune endocrinopathies. These clinical manifestations were compatible with the definition of autoimmune polyendocrine syndrome (APS). However, the main manifestations of APS-I--hypoparathyroidism and Candida infection--were not observed and the pituitary abnormalities were obviously different from the hypophysitis associated with APS. These data suggest that these patients define a unique "anti-PIT-1 antibody syndrome," related to APS.

Prop1 activates POU1F1 (Pit-1) gene expression, which in turn stimulates GH, PRL, TSHbeta and GHRH receptor gene expressions. Therefore the patients with Prop1 mutation show GH, PRL, and TSH deficiency. The mutation of Prop1 is a major abnormality causing combined pituitary hormone deficiency (CPHD). However, DNA-binding and activating functions of mutant Prop1 have not been examined fully because Prop1-binding elements (PBEs) in human POU1F1 gene were not identified until 2008. The aim of this study is to test DNA-binding and transcriptional activities of two mutant Prop1s (W194XProp1 and S156insTProp1, both of them were found in the patients with CPHD) whose mutation is located in putative transactivating domain but not in DNA-binding domain. W194XProp1 showed a marked DNA-binding to PBE as well as a consensus element of paired-like transcription factors (PRDQ9). Activating function for POU1F1 reporter genes expression was lost or decreased in W194XProp1 but still preserved for PRDQ9 reporter gene. S156insTProp1 did not bind PBE but bound PRDQ9. Consistent with the result, S156insTProp1 did not stimulate POU1F1 reporter gene but stimulated PRDQ9 reporter gene. These results support the inference that W194XProp1 is unable to increase POU1F1 gene expression by the defect of transactivating domain and that S156insTProp1 is unable to increase due to the loss of DNA-binding activity. DNA-binding domain that has been assumed is not sufficient to provide full DNA-binding activity of Prop1 and transactivating domain of Prop1 is likely to affect DNA binding to PBE., (2010. Published by Elsevier Ireland Ltd.)

Objective: IGF-I is known to enhance insulin sensitivity in whole body mainly via the IGF-I receptors in muscles. However, the effect of IGF-I on the regulation of insulin sensitivity in the adipose tissue is yet unclear. Insulin sensitivity was found to be higher in the IGF-I receptor-deficient adipocytes than that in wild-type adipocytes, suggesting that IGF-I signaling induces insulin resistance in adipocytes. However, the underlying mechanism has not yet been elucidated. In addition, the effect of superphysiological levels of IGF-I, as is observed in patients with acromegaly, on insulin sensitivity remains unclear., Design: To clarify the role of IGF-I on insulin sensitivity in adipocytes, we determined insulin-induced glucose uptake and IRS-1 status in 3T3-L1 adipocytes treated with IGF-I. Since reactive oxygen species (ROS) are causally related to insulin resistance, we investigated the effect of IGF-I on ROS production to elucidate the molecular mechanism underlying insulin resistance., Results: Preincubation of the adipocytes with IGF-I attenuated insulin-dependent glucose uptake. Interestingly, we found that IGF-I significantly stimulated ROS production. Furthermore, preincubation of adipocytes with an antioxidant, N-acetyl-cysteine (NAC) restored the IGF-I-induced attenuation of insulin-dependent glucose uptake; this indicates that IGF-I induces insulin resistance via ROS. Serine phosphorylation of IRS-1 was strongly induced and the insulin-dependent tyrosine phosphorylation of IRS-1 was suppressed by preincubating the adipocytes with IGF-I. Further, NAC restored these changes induced by IGF-I on both serine and tyrosine phosphorylation of IRS-1., Conclusions: These data indicate that IGF-I inhibited insulin activity in the 3T3-L1 adipocytes via ROS production, which affects IRS-1 phosphorylation status., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We investigated the utility of branched-chain amino acids (BCAA) in dexamethasone-induced muscle atrophy. Dexamethasone (600 microg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone-induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross-sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin-1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin-1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone-induced conversion from microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy.

Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) and COX-2 are involved in cellular processes such as inflammation, apoptosis, and tumorigenesis. To address the relationship between COX-2 and NAG-1 expression, we investigated the expression of NAG-1 and COX-2 in normal and tumor tissue from human patients, Apc(Min/+) mice, and COX-2(-/-) mice. While COX-2 expression is highly induced in tumor tissue, NAG-1 expression is reduced. Furthermore, PGE(2) reduces NAG-1 while celebrex induces NAG-1 expression. The results suggest that a possible inverse relationship exists between the expression of NAG-1 and COX-2 in tumor formation of colon tissue.

Prophet of Pit-1 (Prop1) is a transcription factor that regulates Pit-1 gene expression. Because Pit-1 regulates the differentiation of pituitary cells and the expressions of GH, prolactin and TSHbeta genes, Prop1 mutation results in combined pituitary hormone deficiency in humans. However, Prop1-binding sites in human Pit-1 gene and the mechanism leading to combined pituitary hormone deficiency have remained unclear. In this study, we identified and analyzed Prop1-binding elements of the human Pit-1 gene. Prop1 stimulated the expression of the reporter plasmid containing Pit-1 gene from translation start site to -1340 dose dependently in GH3 cells. The activation by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells. Deletion analysis of Pit-1 gene showed that the Prop1-responsive elements were present within the -257-bp region. Within the -257-bp region, there are four elements similar to consensus sequence of paired-like transcription factors. Because Prop1 is a member of paired-like transcription factors, we assessed the elements. EMSA and transient transfection assay using the mutation of the elements revealed that the element from -63 to -53 (the proximal Prop1 binding element) was essential to Prop1-binding and Prop1-induced activation of Pit-1 reporter plasmid. A region at -8kb of human Pit-1 gene is similar to the distal region containing Prop1-binding elements in mouse Pit-1 gene. We showed the region functioned as an enhancer. Furthermore, chromatin immunoprecipitation assay showed that the proximal element could bind Prop1 in vivo cultured cells. Taken together, these findings indicated the novel functioning binding elements of Prop1 in human Pit-1 gene.

All forms of tobacco cause cardiovascular disease, and tobacco-related disease is a leading cause of death worldwide. Smoking oxidizes low-density lipoprotein (LDL) particles, and oxidized LDL particles are thought to play an early and critical role in atherosclerogenesis. Hyper-low-density lipoproteinemia is one of the risk factors for cardiovascular disease, but small, dense LDL particles have been associated with an increased risk for cardiovascular disease. Small, dense LDL correlates with some cardiovascular risk factors such as diabetes mellitus, hypertriglyceridemia, hypo-high-density lipoproteinemia, and hypertension. Although smoking is also a major risk factor for cardiovascular disease, the relationship between smoking and small, dense LDL particles has not been described previously. Our cross-sectional study examined this relationship in a population of 18 healthy young adult men (9 smokers and 9 never-smokers, aged 21-24 years) from the same college. Concentrations of blood lipids and the LDL migration index (LDL-MI) were examined. Although concentrations of blood lipids did not differ between smokers and never-smokers, the LDL-MI had a strong tendency to be lower in smokers. The LDL-MI is larger in the presence of a greater proportion of small, dense LDL particles. These results indicate that tobacco smoking is associated with a decrease in the proportion of small, dense LDL particles. Regardless of these surprising results, we do not recommend smoking, given that it is a major cause of cardiovascular disease.

Mitochondrial diabetes is characterized by diabetes and hearing loss in maternal transmission with a heteroplasmic A3243G mutation in the mitochondrial gene. In patients with the mutation, it has been reported that hepatic involvement is rarely observed. We demonstrated a case of hypertrophic cardiomyopathy and hepatic failure with mitochondrial diabetes. To clarify the pathogenesis we analyzed the mitochondrial ultrastructure in the myocytes, the reactive oxygen species (ROS) production in the liver and the status of heteroplasmy of the mitochondrial A3243G mutation in the organs involved. In cardiomyocytes and skeletal muscle, electron microscopic analysis demonstrated typical morphological mitochondrial abnormalities. Immunohistochemical analysis demonstrated enhanced ROS production associated with marked steatosis in the liver, which is often associated with mitochondrial dysfunction. Analysis of the A3243G mutation revealed a substantial ratio of heteroplasmy in these organs including the liver. The presence of steatosis and enhanced oxidative stress in the liver suggested that hepatic failure was associated with mitochondrial dysfunction.

For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region -197 to -160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation-Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.

Background/aims: Acromegaly is frequently accompanied with impaired glucose tolerance (IGT) and diabetes mellitus (DM). It remains unclear which factors determine the abnormal glucose tolerance status in acromegaly. In addition, diverse actions of GH and IGF-I in regulating glucose metabolism in acromegaly have not yet been well elucidated. The aim of this study was to investigate the factors associated with abnormal glucose tolerance in acromegaly., Subjects and Study Design: We conducted a retrospective cross-sectional study that included 48 patients with active acromegaly. The subjects were divided into two groups by the results of 75 g OGTT: normal glucose tolerance (NGT) group (n = 19) and IGT+DM group (n = 29)., Results: Systolic blood pressure (SBP) was significantly higher in the IGT+DM than in the NGT group. Homeostasis model assessment of beta-cell function (HOMA-beta) was significantly decreased in the IGT+DM group compared with the NGT group. Although serum GH or IGF-I levels were not different between the two groups, the IGF-I/GH ratio in the IGT+DM group was significantly lower than that in the NGT group., Conclusions: We have shown that a low serum IGF-I/GH ratio was associated with abnormal glucose tolerance in acromegaly. We propose that the IGF-I/GH ratio is a useful marker to understand the metabolic status in acromegaly., ((c) 2008 S. Karger AG, Basel)