Differentiation of human adipose tissue-derived mesenchymal stromal cells into steroidogenic cells by adenovirus-mediated overexpression of NR5A1 and implantation into adrenal insufficient mice.

BACKGROUND AIMS: Cell therapy for adrenal insufficiency is a potential method for physiological glucocorticoid and mineralocorticoid replacement. We have previously shown that mouse mesenchymal stromal cells (MSCs) differentiated into steroidogenic cells by the viral vector-mediated overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), an essential regulator of steroidogenesis, and their implantation extended the survival of bilateral adrenalectomized (bADX) mice. METHODS: In this study, we examined the capability of NR5A1-induced steroidogenic cells prepared from human adipose tissue-derived MSCs (MSC [AT]) and the therapeutic effect of the implantation of human NR5A1-induced steroidogenic cells into immunodeficient bADX mice. RESULTS: Human NR5A1-induced steroidogenic cells secreted adrenal and gonadal steroids and exhibited responsiveness to adrenocorticotropic hormone and angiotensin II in vitro. In vivo, the survival time of bADX mice implanted with NR5A1-induced steroidogenic cells was significantly prolonged compared with that of bADX mice implanted with control MSC (AT). Serum cortisol levels, which indicate hormone secretion from the graft, were detected in bADX mice implanted with steroidogenic cells. CONCLUSIONS: This is the first report to demonstrate steroid replacement by the implantation of steroid-producing cells derived from human MSC (AT). These results indicate the potential of human MSC (AT) to be a source of steroid hormone-producing cells.

A simple questionnaire for the detection of testosterone deficiency in men with late-onset hypogonadism.

The Aging Males' Symptoms (AMS) score, developed to screen for late-onset hypogonadism (LOH), contains 17 questions regarding mental, physical, and sexual parameters. In the Japanese guidelines, a free testosterone (FT) <8.5 pg/mL is recommended for testosterone treatment. However, previous studies have shown no correlation between total AMS scores and testosterone concentration. We aimed to develop a better questionnaire for the detection of testosterone deficiency in men, for the diagnosis of LOH. In 234 Japanese men, aged 40-64 years, we analyzed the relationships of AMS with serum total testosterone (TT), FT, calculated FT (cFT), and calculated bioavailable testosterone (cBT), and identified useful questions for the detection of testosterone deficiency. Four scores, a decrease in muscular strength, a decrease in ability to perform sexually or the frequency, a decrease in the number of morning erections, and a decrease in sexual desire/libido, were negatively associated with two or more of the above four testosterone parameters, and the sum of these four scores (named the selective score) correlated with TT and cFT, independent of age. Statistical analysis revealed an association between insulin resistance and testosterone deficiency, and a higher selective score in smokers than non-smokers. Cubic function model analysis and logistic regression analysis revealed that selective scores ≥10 corresponded with the testosterone concentrations recommended for the diagnosis of LOH, including FT <8.5 pg/mL, independent of age, insulin resistance, and smoking. Thus, the selective score represents a simple and useful means for screening of testosterone deficiency in Japanese men, as an indicator of LOH.

Combined treatment with glucagon-like peptide-1 receptor agonist exendin-4 and metformin attenuates breast cancer growth.

Cancer is a major cause of death in patients with diabetes. Incretin therapy has received much attention because of its tissue-protective effects. We have previously reported an anti-breast cancer effect of glucagon-like peptide-1 receptor agonist exendin-4 (Ex-4). An anti-cancer effect of metformin is well recognized. Therefore, we examined the effect of combined treatment with Ex-4 and metformin in breast cancer cells. In human breast cancer cell lines MCF-7, MDA-MB-231, and KPL-1, 0.1-10 mM metformin significantly reduced the cell number in growth curve analysis in a dose-dependent manner. Furthermore, combined treatment with 0.1 mM metformin and 10 nM Ex-4 additively attenuated the growth curve progression of breast cancer cells. In a bromodeoxyuridine (BrdU) assay, Ex-4 or metformin significantly decreased breast cancer cell proliferation and further reduction of BrdU incorporation was observed by combined treatment with Ex-4 and metformin, which suggested that Ex-4 and metformin additively decreased DNA synthesis in breast cancer cells. Although apoptotic cells were not observed among Ex-4-treated breast cancer cells, apoptotic cells were clearly detected among metformin-treated breast cancer cells by apoptosis assays. Furthermore, metformin decreased BCL-2 expression in MCF-7 cells. In vivo experiments using a xenograft model showed that Ex-4 and metformin significantly decreased the breast tumor weight and Ki67-positive proliferative cancer cells, and metformin reduced the serum insulin level in mice. These data suggested that Ex-4 and metformin attenuated cell proliferation and metformin induced apoptosis in breast cancer cells. Combined treatment of Ex-4 and metformin may be an optional therapy to inhibit breast cancer progression.

Inhibition of NR5A1 Phosphorylation Alleviates a Transcriptional Suppression Defect Caused by a Novel NR0B1 Mutation.

Context: Mutations in the NR0B1 gene, also well-known as the DAX1 gene, are known to cause congenital adrenal hypoplasia associated with hypogonadotropic hypogonadism. The abnormal NR0B1 protein fails to suppress the transcription of promoters of steroidogenic enzymes, which are also targets of NR5A1 protein, also well-known as Ad4BP/SF-1 protein. Since NR5A1 and NR0B1 have antagonistic effects on steroidogenesis, the loss of function due to NR0B1 mutations may be compensated by inducing loss of function of NR5A1 protein. Patient: A middle-aged man was diagnosed with congenital adrenal hypoplasia associated with hypogonadotropic hypogonadism and genetic analysis revealed him to have a novel NR0B1 mutation, c.1222C>T(p.Gln408Ter). Methods: NR0B1 activity was evaluated in CLK1/4 inhibitor-treated 293T cells via immunoblotting and luciferase assays of the STAR promoter. Results: TG003 treatment suppressed NR5A1 protein function to compensate for the mutant NR0B1 showing inhibited suppression of transcription. Immunoblotting analyses showed that the phosphorylation status of NR5A1 at Ser203 was attenuated by the CLK1/4 inhibitor. Conclusion: The specific reduction of NR5A1 phosphorylation by a CLK1/4 inhibitor may alleviate developmental defects in patients with NR0B1 mutations.

Comparison and commutability study between standardized liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and chemiluminescent enzyme immunoassay for aldosterone measurement in blood.

A commutability confirmation test for the blood aldosterone measurement was performed on liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) as a designated comparison method (DCM) and four chemiluminescent enzyme immunoassay (CLEIA) measurement procedures based on metrological traceability. A conventional radioimmunoassay (RIA) and two measurement procedures of CLEIA which obtains RIA equivalent values were also compared. The relationship between the DCM value and the CLEIA value with respect to 120 pg/mL of the RIA value, which is the screening criterion of primary aldosteronism (PA) was clarified. For the correlation test, 75 samples of patient serum and plasma were used. Regression analysis revealed that the standardized LC-MS/MS and four CLEIA measurement procedures were in good agreement. This is the effect of measurement specificity and calibration using by certified reference material (CRM). The median of the LC-MS/MS corresponding to 120 pg/mL of RIA was 48.5 pg/mL. In the mean of standardized four CLEIA values corresponding to the 48.5 pg/mL of LC-MS/MS value was 47.51 pg/mL and the standard deviation (SD) was 2.93 pg/mL. However, the correlation between the RIA value and the RIA equivalent of the two measurement procedures by CLEIA differed depending on the measurement procedure. This is due to the influence of RIA measurement performance. Standardized CLEIA measurements are suitable for routine measurement procedure. When converting the LC-MS/MS equivalent value by the standardized CLEIA to the conventional RIA value, it is necessary to use the conversion formula.

Sodium-glucose cotransporter 2 inhibitor canagliflozin attenuates lung cancer cell proliferation in vitro.

Cancer is a major cause of death in patients with type 2 diabetes mellitus (T2DM) and lung cancer is one of the most prevalent cancers in patients with T2DM. In the present study, we examined the anti-cancer effect of the Sodium-glucose cotransporter 2 (SGLT2) inhibitor, canagliflozin, using a lung cancer model. In lung cancer tissues from non-T2DM human subjects, SGLT2 was detected by immunohistochemistry. SGLT2 mRNA and protein were also detected in A549, H1975 and H520 lung cancer cell lines by RT-PCR and immunohistochemistry, respectively. Canagliflozin at 1-50 µM significantly suppressed the growth of A549 cells in a dose-dependent manner. In BrdU assays, canagliflozin attenuated the proliferation of A549 cells, but did not induce apoptosis. In cell cycle analysis, S phase entry was attenuated by canagliflozin in A549 cells. In in vivo experiments, a xenograft model of athymic mice implanted with A549 lung cancer cells was treated with low and high dose oral canagliflozin. Despite the results of the in vitro experiments, tumor weight was not decreased by canagliflozin. In addition, the serum insulin level, but not body weight or blood glucose level, was decreased by canagliflozin. The number of cells positive for Ki67 was slightly decreased by canagliflozin, but this was not statistically significant. In conclusion, SGLT2 is expressed in human lung cancer tissue and cell lines, and the SGLT2 inhibitor, canagliflozin, attenuated proliferation of A549 lung cancer cells by inhibiting cell cycle progression in vitro but not in vivo.

High-mobility group box 2 protein is essential for the early phase of adipogenesis.

Understanding of the mechanism of adipogenesis is essential for the control of obesity, which predisposes toward numerous health problems. High-mobility group box protein 2 (HMGB2) is a non-histone chromosomal protein that facilitates DNA replication, transcription, recombination, and repair. Here, we studied the role of HMGB2 in adipogenic differentiation. The expression of HMGB2 was measured at the mRNA and protein levels in cultured 3T3-L1 pre-adipocyte cells and during the process of adipogenic differentiation induced bya cocktail of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone. This increased in the early phase and decreased in the late phase of differentiation. However, 3T3-L1 pre-adipocyte cells did not differentiate into adipocytes after the knockdown of HMGB2 expression by small interfering RNA (siRNA). Similarly, mesenchymal stem cells (MSCs) isolated from Hmgb2-/- mice did not express peroxisome proliferator-activated receptor gamma (PPARγ) in response to the adipocyte differentiation cocktail and did not differentiate. Wnt/ß-catenin signaling is a negative regulator of adipogenic differentiation. We found that ß-catenin expression was downregulated during 3T3-L1 adipogenic differentiation, as expected, but not when endogenous HMBG2 expression was knocked down using siRNA. These results indicate that HMGB2 plays an essential role in the early phase of the differentiation of pre-adipocytes and MSCs, and probably interacts with other regulators, such as PPARγ and Wnt/ß-catenin signaling.

Pemafibrate, a PPAR alpha agonist, attenuates neointima formation after vascular injury in mice fed normal chow and a high-fat diet.

Recently, the prevention of cardiovascular events has become one of the most important aims of diabetes care. Peroxisome proliferator-activated receptor (PPAR) agonists have been reported to have vascular protective effects. Here, we examined whether pemafibrate, a selective PPAR alpha agonist, attenuated neointima formation after vascular injury and vascular smooth muscle cell (VSMC) proliferation. We performed endothelial denudation injury in mice treated with a high-fat diet (HFD) or normal chow. Orally administered pemafibrate significantly attenuated neointima formation after vascular injury in HFD and normal chow mice. Interestingly, pemafibrate increased the serum fibroblast growth factor 21 concentration and decreased serum insulin concentrations in HFD mice. In addition, body weight was slightly but significantly decreased by pemafibrate in HFD mice. Pemafibrate, but not bezafibrate, attenuated VSMC proliferation in vitro. The knockdown of PPAR alpha abolished the anti-VSMC proliferation effect of pemafibrate. BrdU assay results revealed that pemafibrate dose-dependently inhibited DNA synthesis in VSMCs. Flow cytometry analysis demonstrated that G1-to-S phase cell cycle transition was significantly inhibited by pemafibrate. Pemafibrate attenuated serum-induced cyclin D1 expression in VSMCs. However, apoptosis was not induced by pemafibrate as assessed by the TUNEL assay. Similar to the in vitro data, VSMC proliferation was also decreased by pemafibrate in mice. These data suggest that pemafibrate attenuates neointima formation after vascular injury and VSMC proliferation by inhibiting cell cycle progression.

miR-4463 regulates aromatase expression and activity for 17ß-estradiol synthesis in response to follicle-stimulating hormone.

OBJECTIVE: The aim of this study was to investigate microRNAs (miRNAs) related to follicle-stimulating hormone (FSH) responsiveness using miRNA microarrays and to identify their target genes to determine the molecular regulatory pathways involved in FSH signaling in KGN cells. METHODS: To change the cellular responsiveness to FSH, KGN cells were treated with FSH receptor (FSHR)-specific small interfering RNA (siRNA) followed by FSH. miRNA expression profiles were determined through miRNA microarray analysis. Potential target genes of selected miRNAs were predicted using bioinformatics tools, and their regulatory function was confirmed in KGN cells. RESULTS: We found that six miRNAs (miR-1261, miR-130a-3p, miR-329-3p, miR-185-5p, miR-144-5p and miR-4463) were differentially expressed after FSHR siRNA treatment in KGN cells. Through a bioinformatics analysis, we showed that these miRNAs were predicted to regulate a large number of genes, which we narrowed down to cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and estrogen receptor alpha (ESR1) as the main targets for miR-4463. Functional analysis revealed that miR-4463 is a regulatory factor for aromatase expression and function in KGN cells. CONCLUSION: In this study, we identified differentially expressed miRNAs related to FSH responsiveness. In particular, upregulation of miR-4463 expression by FSHR deficiency in human granulosa cells impaired 17ß-estradiol synthesis by targeting CYP19A1 and ESR1. Therefore, our data might provide novel candidates for molecular biomarkers for use in research into poor responders.

Efficacy and safety of a combination of an insulin secretagogue and a dipeptidyl peptidase-4 inhibitor in Japanese patients with type 2 diabetes mellitus; the repaglinide glucose oscillation study in Fukuoka (REGO-F).

ABSTRACT: Dipeptidyl peptidase-4 inhibitors (DPP-4Is) are one of the most frequently prescribed anti-diabetic agents in Japan, and they are often used in combination with insulin secretagogues, such as sulfonylureas and glinides. In the present study, we determined the efficacy and safety of the use of repaglinide or glimepiride, a sulfonylurea, in combination with a DPP-4I, in Japanese patients with type 2 diabetes mellitus (T2DM). This study was an investigator-initiated, open-label, randomized, multi-center prospective study. Patients with T2DM, which was inadequately controlled using a DPP-4I, were randomized to a repaglinide group or a glimepiride group and treated for 48 weeks. The primary outcomes were the reductions in glycated hemoglobin (HbA1c) and glucose oscillation, identified using continuous glucose monitoring, after 12 weeks. The secondary outcome was the change in carotid intima-media thickness (IMT), measured by ultrasonography, after 48 weeks. A total of 61 patients were recruited and analyzed in the study. Twelve weeks of treatment with 1.5 mg repaglinide or 1 mg glimepiride significantly reduced HbA1c, and a larger reduction in HbA1c occurred in the repaglinide group than the glimepiride group. Mean subcutaneous glucose concentration was significantly reduced in both groups, but the glucose oscillation did not decrease. Interestingly, the mean left IMT significantly increased in the glimepiride group, but not in the repaglinide group. More hypoglycemic events were observed in the glimepiride group. These data suggest that repaglinide reduces HbA1c more effectively than glimepiride when used in combination with a DPP-4I, and causes fewer hypoglycemic events. TRAIL REGISTRY: This study is registered with UMIN-CTR (UMIN000018321).

Development of a simple prediction model for adrenal crisis diagnosis.

To develop a prediction model for adrenal crisis (AC) diagnosis among individuals with adrenal insufficiency that relies on the values of routinely measured clinical parameters, for application in standard clinical practice. We retrospectively analysed data from five referral centres in Japan. Multivariate binary logistic regression was used to identify independent predictors of AC, and receiver operating characteristic curve analysis was used to determine their optimal cut-off points. The analysis included data from 54 patients with 90 AC events. Logistic regression revealed that serum sodium and C-reactive protein (CRP) levels were independent predictors of AC. Serum sodium levels < 137 mEq/L had a sensitivity of 71.1% and specificity of 95.6%. CRP levels > 1.3 mg/dL had a sensitivity of 84.4% and specificity of 94.9%. In combination, serum sodium levels < 137 mEq/L or CRP levels > 1.3 mg/dL for AC diagnosis had sensitivity and specificity values of 97.8% and 94.4%, respectively. The combined use of serum sodium and CRP levels had high sensitivity and specificity, and can be used for AC screening in standard clinical practice. The model can assist in identifying AC among high-risk individuals. A larger prospective study is needed to validate these results.

Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFß mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2C134W hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2C134W and the TGFß pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.

Low serum albumin, aspartate aminotransferase, and body mass are risk factors for frailty in elderly people with diabetes-a cross-sectional study.

BACKGROUND: Frailty is broadly characterized by vulnerability and decline in physical, mental and social activities and is more common in elderly patients with type 2 diabetes mellitus (T2DM). Frailty is closely associated with nutrition, muscle strength, inflammation, and hormones etc. In hormones, dehydroepiandrosterone sulfate (DHEA-S) and cortisol are suggested to be such candidates affecting frailty. Little investigation has been performed using a wider range of measures of frailty to clarify risk factors for frailty including the above two hormones. METHODS: We performed a cross-sectional study to investigate the risk factors for frailty in elderly T2DM patients (n = 148; ≥65 years), using a broad assessment, the clinical frailty scale. We compared parameters between the non-frail and frail groups using the unpaired t and Mann-Whitney U tests. The Jonckheere-Therpstra test was used to identify relationships with the severity of frailty, and risk factors were identified using binary regression analysis. RESULTS: Simple regression analysis identified a number of significant risk factors for frailty, including DHEAS < 70 µg/dL and cortisol/DHEA-S ratio ≥ 0.2. Multiple regression analysis showed that low albumin (< 4.0 g/dl) (odds ratio [OR] = 5.79, p < 0.001), low aspartate aminotransferase (AST) activity (< 25 IU/L) (OR = 4.34, p = 0.009), and low body mass (BM) (< 53 kg) (OR = 3.85, p = 0.012) were independent risk factors for frailty. A significant decrease in DHEA-S and a significant increase in the cortisol/DHEA-S ratio occurred alongside increases in the severity of frailty. DHEA-S concentration positively correlated with both serum albumin and BM. CONCLUSIONS: Hypoalbuminemia, low AST, and low BM are independent risk factors for frailty in elderly T2DM patients, strongly implying relative malnutrition in these frail patients. DHEA-S may be important for the maintenance of liver function and BM. A decrease in DHEA-S and an increase in the cortisol/DHEAS ratio may be involved in the mechanism of the effect of malnutrition in elderly T2DM patients.

Diabetes Mellitus Itself Increases Cardio-Cerebrovascular Risk and Renal Complications in Primary Aldosteronism.

CONTEXT: The prevalence of diabetes mellitus (DM) in patients with primary aldosteronism (PA) is higher than in those with essential hypertension and the general population. Although DM is a common major risk factor for cardio-cerebrovascular (CCV) diseases and renal complications, details of its effects in PA have not been demonstrated. OBJECTIVE: The aim of this study was to determine the effects of coexistent DM on the risk of CCV events and progression of renal complications in PA patients. DESIGN: A multi-institutional, cross-sectional study was conducted. PATIENTS AND METHODS: PA patients experienced between January 2006 and October 2016 and with available data of CCV events and DM were enrolled from the Japan PA registry of the Japan Primary Aldosteronism Study/Japan Rare Intractable Adrenal Diseases Study (n = 2524). CCV events and renal complications were compared between a DM group and a non-DM group by logistic and liner-regression analysis. RESULTS: DM significantly increased the odds ratio (OR) of CCV events (OR 1.59, 95% CI: 1.05-2.41) and that of proteinuria (OR 2.25, 95% CI: 1.59-3.16). DM correlated significantly with declines in estimated glomerular filtration rate (ß = .05, P = .02). CONCLUSIONS: This the first report to demonstrate the presence of DM as an independent risk factor for CCV events and renal complications, even in PA patients. Management of DM should be considered in addition to the specific treatment of PA.

Activation of overexpressed glucagon-like peptide-1 receptor attenuates prostate cancer growth by inhibiting cell cycle progression.

AIMS/INTRODUCTION: Incretin therapy is a common treatment for type 2 diabetes mellitus. We have previously reported an anti-prostate cancer effect of glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4. The attenuation of cell proliferation in the prostate cancer cell line was dependent on GLP-1R expression. Here, we examined the relationship between human prostate cancer severity and GLP-1R expression, as well as the effect of forced expression of GLP-1R using a lentiviral vector. MATERIALS AND METHODS: Prostate cancer tissues were extracted by prostatectomy and biopsy. GLP-1R was overexpressed in ALVA-41 cells using a lentiviral vector (ALVA-41-GLP-1R cells). GLP-1R expression was detected by immunohistochemistry and quantitative polymerase chain reaction. Cell proliferation was examined by growth curves and bromodeoxyuridine incorporation assays. Cell cycle distribution and regulators were examined by flow cytometry and western blotting. In vivo experiments were carried out using a xenografted model. RESULTS: GLP-1R expression levels were significantly inversely associated with the Gleason score of human prostate cancer tissues. Abundant GLP-1R expression and functions were confirmed in ALVA-41-GLP-1R cells. Exendin-4 significantly decreased ALVA-41-GLP-1R cell proliferation in a dose-dependent manner. DNA synthesis and G1-to-S phase transition were inhibited in ALVA-41-GLP-1R cells. SKP2 expression was decreased and p27Kip1 protein was subsequently increased in ALVA-41-GLP-1R cells treated with exendin-4. In vivo experiments carried out by implanting ALVA-41-GLP-1R cells showed that exendin-4 decreased prostate cancer growth by activation of GLP-1R overexpressed in ALVA41-GLP-1R cells. CONCLUSIONS: Forced expression of GLP-1R attenuates prostate cancer cell proliferation by inhibiting cell cycle progression in vitro and in vivo. Therefore, GLP-1R activation might be a potential therapy for prostate cancer.

Extension of Survival in Bilaterally Adrenalectomized Mice by Implantation of SF-1/Ad4BP-Induced Steroidogenic Cells.

Mesenchymal stroma/stem cells (MSCs) exist in adult tissues, such as adipose tissue and bone marrow, and differentiate into cells of multiple lineages. In previous studies, we found that MSCs differentiate into steroidogenic cells by forced expression of steroidogenic factor 1 (SF-1)/adrenal 4 binding protein (Ad4BP), the master regulator of steroidogenesis and differentiation of pituitary gonadotrophs, adrenal glands, and gonads. In this study, SF-1/Ad4BP-induced steroidogenic cells derived from mouse adipose tissue-derived MSCs (ADSCs) were implanted under the kidney capsule of bilateral adrenalectomized (bAdx) mice. bAdx mice did not survive after 7 days. However, 4 of 9 bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, 1 of 10 bAdx mice transplanted with control ADSCs, and bAdx mice transplanted with an adrenal gland survived for 30 days. Plasma corticosterone levels in bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells and control ADSCs were 5.41 ±â€…2.26 ng/mL (mean ±â€…SEM) and undetectable at 7 days after implantation, respectively. After removal of the kidney bearing the graft from the surviving mice at 30 days after implantation, plasma corticosterone was not detected in any of the samples. Immunohistochemical staining revealed SF-1/Ad4BP-positive cells under the capsule of the kidney. Although we performed an adrenocorticotropin (ACTH) loading test on bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, ACTH responsiveness was not observed. Implantation of steroidogenic cells derived from ADSCs into bAdx mice increased the basal plasma corticosterone level and extended the survival of bAdx mice, suggesting the capability of restoring steroidogenic cells by cell transplantation therapy for adrenal insufficiency.

Historical changes and between-facility differences in adrenal venous sampling for primary aldosteronism in Japan.

Primary aldosteronism (PA) is a common curable cause of hypertension. Adrenal venous sampling (AVS) is recommended for subtype diagnosis but is a difficult procedure. Recently, an increased prevalence of PA was reported, creating a greater demand for treatment of the condition in clinical facilities. The aim of the present study was to identify the historical changes over time and the differences between facilities in the success rate and subtype diagnosis of PA. The database of the PA registry developed by the Japan PA Study (JPAS) was used. A total of 2599 patients with PA who underwent AVS were evaluated. The overall success rate of AVS was 88%. The bilateral subtype was the dominant subtype, comprising 69% of cases. During the period 2004-2011 to 2011-2017, there were significant changes in the total number of AVS procedures (from 562 to 1732), ratio of ACTH administration with AVS (75 to 97%), success rate (79 to 90%), and proportion with bilateral subtype diagnosis (53 to 72%). There were also significant inter-facility differences in the number of AVS procedures (6 to 322), success rate (59 to 97%), and proportion with the bilateral subtype (44 to 86%). The principal enrolled department was Endocrinology (86%), and the ratio of unilateral PA was significantly higher in this department than in others (32% vs. 25%). In conclusion, the number of AVS procedures performed, the success rate, and the proportion with the bilateral subtype increased over time after normalizing the centre difference. Significant differences were observed between the centres.

SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation.

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 µM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

A nationwide survey of adrenal incidentalomas in Japan: the first report of clinical and epidemiological features.

The aim of this study was to reveal clear epidemiologic and clinical characteristics of incidentally discovered adrenal masses, termed adrenal incidentalomas (AIs), and to establish appropriate managemental and therapeutic regimens in Japan. This study had been originally carried out as a project of a research proposed on behalf of the Japanese Ministry of Health, Labour and Welfare, from 1999 to 2004. This nationwide multicenter study on AIs included 3,672 cases with clinically diagnosed AIs, involving 1,874 males and 1,738 females, with mean age 58.1 ± 13.0 years (mean ± SD). In the present study, we focused on the investigation of the real prevalence of various adrenal disorders with AI. The mean nodule size of AI based on computed tomography was 3.0 ± 2.0 cm. Compared to non-functioning adenomas (NFAs), tumor diameters were significantly larger in adrenocortical carcinomas (ACCs), pheochromocytomas, cortisol-producing adenomas (CPAs), myelolipomas, metastatic tumors, cysts, and ganglioneuromas (p < 0.01). Endocrinological evaluations demonstrated that 50.8% of total AIs were non-functioning adenomas, while 10.5%, including 3.6% with subclinical Cushing's syndrome, were reported as CPAs, 8.5% as pheochromocytomas, and 5.1% as aldosterone-producing adenomas. ACCs were accounted for 1.4% (50 cases) among our series of AIs. In conclusion, while almost 50 % of AIs are non-functional adenomas, we must be particularly careful as AIs include pheochromocytomas or adrenal carcinomas, because they may be asymptomatic. To our knowledge, this is the first and the largest investigation of AI, thus providing basic information for the establishment of clinical guidelines for the management of AI.