Gitelman syndrome with a novel frameshift variant in SLC12A3 gene accompanied by chronic kidney disease and type 2 diabetes mellitus.

Gitelman syndrome is an autosomal recessive genetic disease caused by pathogenic variants in SLC12A3 resulting in the loss of function of the Na-Cl co-transporter (NCC) in the distal tubules. Hypokalemia and diuretic effects can cause secondary type 2 diabetes and renal function decline. Here, we present the case of a 49-year-old male patient with chronic persistent treatment-resistant hypokalemia for the past 13 years who had been receiving treatment for type 2 diabetes mellitus for 6 years. He was referred to our department due to the presence of urinary protein, impaired renal function, high renin activity, and hyperaldosteronism. Laboratory test results showed hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. Using next-generation and Sanger sequencing, we identified a novel stop-gain variant (NM_000339.3:c.137del [p.His47fs]) and a missense variant (NM_000339.3:c.2927C > T [p.Ser976Phe]) in the SLC12A3 gene. This novel pathogenic variant was located at the intracellular N-terminus of the NCC. Based on these findings, the patient was diagnosed with Gitelman syndrome. The use of next-generation sequencing facilitated the exclusion of diseases with similar clinical symptoms.

Plasma aldosterone level within the normal range is less associated with cardiovascular and cerebrovascular risk in primary aldosteronism.

BACKGROUND: Previous studies showed higher risk of cardiovascular and cerebrovascular (CCV) events in primary aldosteronism compared with essential hypertension, but the patients of these studies were limited to primary aldosteronism patients with high plasma aldosterone concentration (PAC). The introduction of the aldosterone-renin ratio as the screening test for primary aldosteronism led to the recognition of primary aldosteronism patients with normal PAC (nPA). However, there is no information on the risk of primary aldosteronism including nPA. METHOD: In this retrospectively and cross-sectional study, the clinical features and CCV event risk of primary aldosteronism at diagnosis including nPA were investigated and compared with essential hypertension. The study included 292 consecutive primary aldosteronism patients and 498 essential hypertension outpatients. All primary aldosteronism patients were diagnosed by autonomous aldosterone secretion using confirmatory tests, and then divided into nPA (n = 130) and primary aldosteronism patients with high PAC (hPA: n = 162) using a PAC cutoff level of less than 443 pmol/l (16 ng/dl), representing the normal upper limit of PAC. RESULTS: nPA patients were significantly older at diagnosis of primary aldosteronism and at onset of hypertension compared with hPA patients. They had milder hypokalemia and easier-to-control blood pressure. The results suggested that nPA could be considered a mild type of primary aldosteronism but not an early-stage hPA. Moreover, the risk of all CCV events in nPA was significantly lower than that in hPA (odds ratio 0.42, 95% confidence interval 0.18-0.90, P < 0.05) and not significantly higher than that in essential hypertension (odds ratio 0.95, 95% confidence interval 0.43-1.94, P = 0.899). CONCLUSION: This study suggests that aggressive diagnostic workout for nPA is less effective to prevent CCV events.

Clinical and endocrinological characteristics of adrenal incidentaloma in Osaka region, Japan.

The aim of this study was to investigate the clinical and endocrinological characteristics of adrenal incidentalomas in Osaka region, Japan. The study was a multicenter retrospective analysis of 150 patients with adrenal incidentalomas who underwent radiographic and endocrine evaluations between 2005 and 2013. Most adrenal incidentalomas were discovered by computed tomography (77.0%) and the rest were identified by abdominal ultrasonography (14.6%), magnetic resonance imaging (4.2%), or positron emission tomography (4.2%). Adrenal incidentalomas were more frequently localized on the left side than on the right. The average diameter of tumors was 21 ± 11 mm. On endocrinological evaluation, 14 patients were diagnosed with primary aldosteronism (9.3%), 10 with subclinical Cushing's syndrome (6.7%), 7 with pheochromocytoma (4.7%), 7 with Cushing's syndrome (4.7%), 2 with both subclinical Cushing's syndrome and primary aldosteronism (1.3%), and 110 with non-functioning tumors (73.3%). Patients with functioning tumors were significantly younger and had larger tumor diameters than those with non-functioning tumors. Except for hypertension, complications were comparable between patients with functioning and non-functioning tumors, including the presence of glucose intolerance, cardiovascular disease, and dyslipidemia. In conclusion, a higher prevalence of primary aldosteronism was observed compared with a previous report. Complications were comparable between patients with functioning and non-functioning tumors, including the frequencies of glucose intolerance, cardiovascular disease, and dyslipidemia. Long-term follow-up is required in patients with non-functioning tumors because the frequency of complications, such as glucose intolerance, cardiovascular disease, and dyslipidemia, was equal to that in patients with functioning tumors.

High efficient and cost-effective screening method for diabetic cardiovascular risk.

BACKGROUND: The vascular complications of outpatients with diabetes at ordinary hospitals vary. Ischemic heart disease is barely predictable after treatment using previously reported therapeutic indices. We developed a simple and noninvasive screening method to evaluate the possibility of ischemic heart disease in patients with diabetes. METHODS: Five years of clinical data from 337 outpatients (196 males and 141 females) with diabetes were analyzed. Twenty-three males and 14 females had ischemic heart disease. We examined the possibility of predicting ischemic heart disease after analyzing this population. The analyzed laboratory data included the following: minimum value of right or left ankle-brachial indices (ABI), maximum value of right or left pulse wave velocities (PWV), aortic calcification diagnosed on plain chest radiographs, plaque score (PS), maximum value of intima media thickness at the cervical artery (IMT), electrocardiographic (ECG) ischemic changes (including ST-T changes or abnormal Q waves, which were re-examined by a cardiologist), HbA1c, low-density lipoprotein cholesterol (LDL-C), uric acid (UA), urine albumin, age, sex, disease duration, and body mass index. All data were subjected to multivariate logistic regression analyses. RESULTS: The presence of ECG ischemic changes, aortic calcification, minimum ABI, maximum IMT, LDL-C, and UA were evaluated in multivariate logistic regression analysis with the onset of ischemic heart disease. The receiver operating characteristic curve indicated an area under the curve of 0.879 (0.820 - 0.938; P = 0.00). CONCLUSIONS: Ischemic heart disease could be predicted in patients with diabetes using a combination of results from conventional physical and laboratory tests.

Effects of pitavastatin on serum lipids and high sensitivity C-reactive protein in type 2 diabetic patients.

AIM: Previous studies have been inconsistent results about the effects of statins on serum triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and high sensitivity C-reactive protein (hsCRP) levels. We therefore investigated the effects of pitavastatin on serum lipid profiles and hsCRP levels in patients with type 2 diabetes mellitus. METHODS: The study population was 65 Japanese type 2 diabetic patients who had been administered 2 mg daily of pitavastatin and completed a 6-month follow-up. Serum lipids and hsCRP were measured before and after treatment for 1, 3, and 6 months. RESULTS: Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and TG had significantly reduced after 1 month and remained reduced for 6 months, while HDL-C levels had significantly increased after 1 month and remained at the higher level for 6 months. Baseline median levels of hsCRP were 0.49 mg/L and showed a significant reduction to 0.37 mg/L at 6 months' treatment (p<0.001). Six-month changes in hsCRP levels were not associated with those in TC, LDL-C, HDL-C or TG. CONCLUSION: Pitavastatin improved serum lipid profiles and reduced serum hsCRP levels in type 2 diabetic patients with relatively low inflammation. The effect on hsCRP was not related to the effects on serum lipid profiles.

Combination of type 2 diabetes and malnutrition worsened by anastomotic stenosis and pancreas atrophy following resection of pancreas head.

A 77-year-old Japanese man was admitted due to hypoglycemia induced by small amount of insulin. He was diagnosed type 2 diabetes in 1978 and the pancreatic cancer in 1993. Resection of the pancreas head and duodenum was performed. Subsequently, anastomotic stenosis appeared to induce appetite loss. His flavor for carbohydrate-rich food accelerated protein malnutrition. Fatty liver and pancreas atrophy were diagnosed in 1999. After he was diagnosed as secondary kwashiorkor, nasal feeding of protein-rich food improved his fatty liver as well as his general condition rapidly. Anastomotic stenosis and pancreas atrophy contributed to a combination of type 2 diabetes and kwashiorkor.

A novel growth hormone receptor gene deletion mutation in a patient with primary growth hormone insensitivity syndrome (Laron syndrome).

OBJECTIVE: Growth hormone (GH) insensitivity syndrome (Laron syndrome) is known to be caused by genetic disorders of the GH-IGF-1 axis. Although many mutations in the GH receptor have been identified, there have been only a few reports of deletions of the GH receptor gene. DESIGN: A Japanese adult female patient with Laron syndrome was subjected to chromosome analysis with basic G-banding and also with a high accuracy technique. Each exon of the GH receptor gene was amplified by means of PCR. Since this patient was diagnosed with osteoporosis, the effects of alendronate on bone mineral density (BMD) were also examined. RESULTS: The chromosome analysis with the high accuracy technique demonstrated a large deletion of the short arm in one allele of chromosome 5 from p11 to p13.1 [46, XX, del (5) (p11-p13.1)]. PCR amplification of exons of the GH receptor gene showed that only exons 2 and 3 were amplified. Low-dose IGF-1 administration (30microg/kg body weight) failed to increase her BMD, whereas alendronate administration resulted in an increase associated with a decrease in urinary deoxypyridinoline (DPD) and serum osteocalcin concentrations. CONCLUSIONS: The GH receptor gene of the patient was shown to lack exons 4-10. To the best of our knowledge, this is the third case report of Laron syndrome with large GH receptor deletion. Alendronate was effective for the enhancement of BMD.

The thiazolidinedione drug troglitazone up-regulates nitric oxide synthase expression in vascular endothelial cells.

Endothelial dysfunction is a phenomenon often observed in diabetic patients, which is a cause for vascular complications of diabetes mellitus. Endothelium-derived nitric oxide (NO) is responsible for vasodilatation, and NO-dependent vasodilatation is diminished in diabetic patients. In the present study, we evaluated the effects of thiazolidinediones (TZDs), antidiabetic drugs known to improve insulin resistance and to have vasodilating properties, on endothelial NO synthase (eNOS) expression in cultured vascular endothelial cells. Human umbilical vein endothelial cells were treated with the TZDs troglitazone and pioglitazone, or the peroxisome proliferator-activated receptor (PPAR) gamma activator 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-dPGJ2). The expression of eNOS protein and its mRNA was determined by Western and Northern blot analyses, respectively. The effect of alpha-tocopherol that possesses structural similarity to troglitazone was also examined. Troglitazone up-regulated eNOS protein and its mRNA levels, whereas pioglitazone and 15-dPGJ2 failed to increase their levels. By contrast, alpha-tocopherol also increased in eNOS protein and mRNA. These results suggest that troglitazone up-regulates eNOS expression probably through its 6-hydroxychromanes structure but not activating PPARgamma.

Reduction of a pancreatic tumor after total removal of an ACTH secreting pituitary tumor: differential diagnosis of Cushing's syndrome.

Endocrinologic tests sometimes fail to distinguish adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma from ectopic ACTH-secreting tumor. The authors experienced a case of Cushing's disease associated with a pancreatic tumor. Venous sampling contributed to the final diagnosis of Cushing's disease in this complex case, while endocrinologic tests showed paradoxical results. A 54-year-old woman presented with Cushing's syndrome and pancreatic tumor. Magnetic resonance imaging (MRI) failed to reveal a pituitary tumor, but a gadolinium-enhanced tumor with cystic components was seen in the pancreatic tail. Results of conventional endocrinologic tests suggested ectopic ACTH syndrome, but venous sampling including cavernous sinus sampling indicated an ACTH-secreting pituitary adenoma. Transsphenoidal surgery revealed a pituitary microadenoma, and total removal of the tumor was achieved. Postoperative abdominal MRI revealed that the pancreatic tumor diminished gradually without treatment. Selective cavernous sinus sampling was useful for distinguishing ACTH-secreting pituitary adenoma from ectopic ACTH syndrome in this complex case. This was a rare case in which the pancreatic tumor diminished after total removal of the ACTH-secreting pituitary adenoma.

A novel thiazolidinedione MCC-555 down-regulates tumor necrosis factor-alpha-induced expression of vascular cell adhesion molecule-1 in vascular endothelial cells.

Thiazolidinediones (TZDs) are anti-diabetic agents that enhance insulin sensitivity through activating peroxisome proliferator-activated receptor (PPAR) gamma. Besides their glucose-lowering effects, TZDs are shown to exhibit anti-inflammatory properties in vascular cells, although their precise molecular mechanisms are unknown. In the present study, we examined the effects of a novel TZD MCC-555, which has unique characteristics of ability to activate not only PPARgamma but also PPARalpha and PPARdelta on vascular cell adhesion molecule-1 (VCAM-1) expression in vascular endothelial cells (ECs). Human aortic ECs were treated with MCC-555, followed by stimulation with tumor necrosis factor (TNF)-alpha. Cell surface VCAM-1 protein expression and human monocytoid U937 cell adhesion to these cells were determined. MCC-555 efficiently inhibited TNF-alpha-stimulated VCAM-11expression and U937 cell adhesion. Transient transfection of bovine aortic ECs with a VCAM-1 promoter construct revealed that MCC-555 inhibited TNF-alpha-induced VCAM-1 promoter activity. Electrophoretic mobility-shift assay demonstrated that MCC-555 reduced the amount of nuclear factor-kappaB (NF-kappaB) bound to its recognition site on the VCAM-1 promoter. The considered PPARdelta activator GW501516 and the considered PPARalpha activator fenofibrate also inhibited TNF-alpha-induced VCAM-1 expression, whereas pioglitazone and rosiglitazone did not. These results indicate that MCC-555 is a strong TZD agent to inhibit the cytokine-induced VCAM-1 expression in vascular ECs. This effect is exerted probably through activation of PPARalpha and/or PPARdelta, rather than PPARgamma, mediating down-regulation of NF-kappaB activity.

Suppressor of cytokine signaling 1 suppresses muscle differentiation through modulation of IGF-I receptor signal transduction.

Suppressor of cytokine signaling (SOCS) 1 was initially identified as an intracellular negative feedback regulator of the JAK-STAT signal pathway. Recently, it has been suggested that SOCS1 affects signals of growth factors and hormones. One of them, SOCS1, is also known to be involved in auto-regulation of IRS-1-mediated signaling. However, the mechanism(s) of SOCS1 induction by insulin-like growth factor (IGF)-I and a role of SOCS1 on IGF-I receptor-mediated signaling are not clarified. Here, we investigate SOCS1 on muscle differentiation. We found that muscle differentiation was suppressed in SOCS1 stable transformant C2C12 myoblasts, while it was promoted in SOCS1-deficient myoblasts. Additionally, SOCS1 augmented MEK phosphorylation and reduced Akt phosphorylation induced by IGF-I. Then, SOCS1 stable transformant C2C12 myoblasts, infected with adenovirus bearing constitutively active Akt, have the ability to differentiate again. Collectively, these findings suggest that SOCS1 suppresses muscle differentiation through negative feedback regulation of IGF-I receptor-mediated signaling.

Regeneration of tubular complex is promoted by a free space.

OBJECTIVES: Regeneration of the pancreas is initiated by the tubular complexes that consist of a cluster of epithelia surrounded by the mesenchymal cells. They have the potential to become pancreatic lobes, but their growth stops before the complete regeneration of the organ. To elucidate the possibility that we could promote the regeneration of the pancreas, the potential for growth or differentiation of tubular complex was analyzed. METHODS: The intact lobes were growing around the silk knot after ligation of the pancreas in adult mice. To develop this reaction to a quantitative assay, tubular complexes were induced on the silk strings in the pancreas and were growing into a free space under the silicon cover. The proliferation and differentiation of new lobes with or without the space were analyzed. RESULTS: The number of tubular complexes, which express PDX-1, was increased 5.4 times by the space effect. The proliferating cell nuclear antigen labeling index of acinar cells was 1.7 times stimulated, but that of tubular complex was not changed. The amputated pancreas recovered 49.5% of the resected part under the silicon cover; however, it remained the same weight without the cover. CONCLUSION: The proliferation and differentiation of tubular complex are promoted by a free space.

Peroxisome proliferator-activated receptor alpha agonists increase nitric oxide synthase expression in vascular endothelial cells.

OBJECTIVE: There has been accumulating evidence demonstrating that activators for peroxisome proliferator-activated receptor alpha (PPARalpha) have antiinflammatory, antiatherogenic, and vasodilatory effects. We hypothesized that PPARalpha activators can modulate endothelial nitric oxide synthase (eNOS) expression and its activity in cultured vascular endothelial cells. METHODS AND RESULTS: Bovine aortic endothelial cells were treated with the PPARalpha activator fenofibrate. The amount of eNOS activity and the expression of eNOS protein and its mRNA were determined. Our data show that treatment with fenofibrate for 48 hours resulted in an increase in eNOS activity. Fenofibrate failed to increase eNOS activity within 1 hour. Fenofibrate also increased eNOS protein as well as its mRNA levels. RU486, which has been shown to antagonize PPARalpha action, inhibited the fenofibrate-induced upregulation of eNOS protein expression. WY14643 and bezafibrate also increased eNOS protein levels, whereas rosiglitazone did not. Transient transfection experiments using human eNOS promoter construct showed that fenofibrate failed to enhance eNOS promoter activity. Actinomycin D studies demonstrated that the half-life of eNOS mRNA increased with fenofibrate treatment. CONCLUSIONS: PPARalpha activators upregulate eNOS expression, mainly through mechanisms of stabilizing eNOS mRNA. This is a new observation to explain one of the mechanisms of PPARalpha-mediated cardiovascular protection.

New analytical method for pancreas and liver regeneration: normalization of streptozotocin-induced hyperglycemia by retrograde injection of insulin producing cells.

We established a new analytical system in which functioning cells were transplanted directly into the pancreas and liver. The retrograde transplantation of beta cell line, Min6 cells, into the streptozotocin-diabetic mice normalized plasma glucose and insulin levels. The injected cells were protected from pancreatic enzymes with enzyme inhibitor. Blood glucose decreased gradually over 10 days and the diabetic mice recovered weight at the same time. Intraperitoneal glucose tolerance test showed that the peak of plasma glucose of the transplanted mice was less than half that of the control. The insulin secretion of the transplanted mice was recovered and stimulated 4.6 times from the basal secretion. Histological analyses showed that the pancreas and liver were characterized by Min6 cell clusters dispersed throughout the organs. Min6 cells were detected near the pancreatic or bile ducts. It is suggested that the injected cells obstructed the peripheral ducts where they settled. The weight of pancreas and liver did not differ significantly in either Min6 transplanted or the control mice. The metabolic effects on the weights of these organs appeared the same in both groups. This is the first report that cells transplanted via ducts into the pancreas and liver performed their biological function. Our transplantation model makes possible the in vivo analysis of the regeneration machinery of the pancreas and liver.

Reduced IRS-2 and GLUT4 expression in PPARgamma2-induced adipocytes derived from C/EBPbeta and C/EBPdelta-deficient mouse embryonic fibroblasts.

In adipose tissue, the ability of cells to respond to insulin and to express genes such as those encoding fatty-acid-binding protein (422/aP2), lipoprotein lipase (LPL), adipsin and glucose transporter 4 (GLUT4) is acquired during their differentiation into mature adipocytes. It has been recognized that peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding proteins (C/EBPs) play critical roles in adipocyte differentiation. However, it remained uncertain whether PPARgamma or which C/EBP is involved in the acquisition of these characteristics. We introduced PPARgamma2 into C/EBPbeta/delta-double deficient mouse embryonic fibroblasts (MEFs), followed by stimulation with its ligands, in order to define the roles of C/EBPbeta and C/EBPdelta in phenotypic acquisition during adipocyte differentiation. This procedure resulted in differentiation of these MEFs into mature adipocytes morphologically similar to wild-type MEFs. However, the adipocytes derived from the C/EBPbeta/delta-deficient MEFs showed lower expression of GLUT4 and adipsin mRNA than those derived from wild-type MEFs, although aP2 and LPL mRNA levels were similar in both types. The C/EBPbeta/delta-deficient adipocytes also expressed lower amounts of insulin receptor substrate 2 (IRS-2) than the adipocytes derived from wild-type MEFs, whereas the amounts of insulin receptor and IRS-1 were similar. Finally, insulin-responsive 2-deoxyglucose uptake was lower in the C/EBPbeta/delta-deficient cells. It could thus be demonstrated that C/EBPbeta and C/EBPdelta are involved in the acquisition of IRS-2 and GLUT4 expression as well as in insulin-sensitive glucose uptake during adipocyte differentiation.

RU486 antagonizes the inhibitory effect of peroxisome proliferator-activated receptor alpha on interleukin-6 production in vascular endothelial cells.

Peroxisome proliferator-acitivated receptor alpha (PPARalpha) is a member of nuclear receptor superfamily. Recent studies have shown that the activators for PPARalpha inhibit the expression of some inflammatory molecules in vascular endothelial cells (ECs) and vascular smooth muscle cells, indicating the anti-inflammatory roles of PPARalpha on vascular walls. In this investigation, we showed that RU486, already proved to be an active anti-glucocorticoid and anti-progesterone agent, blocked the inhibition of tumor necrosis factor (TNF)-alpha-stimulated interleukin-6 (IL-6) production by the PPARalpha activator fenofibrate in human umbilical vein ECs. Transient transfection of bovine aortic ECs with an IL-6 promoter construct demonstrated that RU486 blocked the inhibitory effect of fenofibrate on TNF-alpha-induced IL-6 promoter activity. By fluorescence microscopy, RU486 was found to prevent fenofibrate-induced nuclear translocation of PPARalpha. Thus, RU486 has an antagonizing effect on PPARalpha-mediated down-regulation of IL-6 in vascular ECs. This effect may be exerted by its interference with the nuclear translocation of PPARalpha.

PTHrP-producing tumor: squamous cell carcinoma of the liver accompanied by humoral hypercalcemia of malignancy, increased IL-6 and leukocytosis.

A 77-year-old man was admitted to our hospital showing symptoms of general fatigue and appetite loss. He had leukocytosis, thrombocytosis and hypercalcemia with elevated serum levels of parathyroid hormone related peptide (PTHrP) and interleukin-6 (IL-6). An increase in tumor markers SCC and CYFURA21-1 was observed. The liver contained a huge tumor, which was proved to be PTHrP producing squamous cell carcinoma by immuno-histochemical analysis. Since the tumor did not express IL-6, it was assumed to be induced by PTHrP in osteoblasts. This is the first report of PTHrP producing squamous cell carcinoma of the liver.

Akt1 and Akt2 differently regulate muscle creatine kinase and myogenin gene transcription in insulin-induced differentiation of C2C12 myoblasts.

Insulin and IGFs are potent inducers of skeletal muscle differentiation. Although PI3K is known to be involved in skeletal muscle differentiation, its downstream targets in this process are not clearly defined. We investigated the roles of Akt and mammalian target of rapamycin (mTOR) in skeletal muscle differentiation. LY294002, a pharmacological inhibitor of PI3K, and the immunosuppressant rapamycin inhibited insulin-induced differentiation of C2C12 myoblasts. LY294002 and rapamycin suppressed myosin heavy chain expression and myotube formation. Transient reporter assays showed that both inhibitors repress muscle creatine kinase (MCK) and myogenin gene transcription. Heterologous expression of Akt1/PKB(alpha) potently suppressed MCK gene transcription without affecting myogenin gene transcription, whereas heterologous expression of Akt2 increased myogenin and MCK gene transcription. Finally, overexpression of myogenin rescued the inhibitory effect of rapamycin on MCK gene transcription, whereas it failed to rescue the inhibitory effect of LY294002 and Akt1. These results suggest that insulin regulates myogenic differentiation chiefly at the level of myogenin gene transcription via PI3K and mTOR. PI3K activity, but not mTOR, may regulate transcriptional activity of myogenin. Our data also suggest that Akt1 and Akt2 play distinct roles in myogenic differentiation.