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.

Effects of alogliptin on the ratio of glycated albumin to HbA1c in patients with type 2 diabetes mellitus.

The ratio of glycated albumin (GA) to HbA1c (the GA/HbA1c ratio) has been used as a glycemic control indicator that reflects postprandial plasma glucose levels or glycemic variability. In this study, we investigated the effects of alogliptin, a DPP-4 inhibitor, on the GA/HbA1c ratio in patients with type 2 diabetes mellitus. Thirty-eight patients with type 2 diabetes mellitus whose glycemic control was stable were enrolled, and alogliptin (12.5 or 25 mg/day) was then administered to them for 24 weeks. HbA1c and GA levels both significantly decreased after 24 weeks (P < 0.0001), whereas the GA/HbA1c ratio did not (P = 0.129). No correlation was observed between the change in the GA/HbA1c ratio (the ΔGA/HbA1c ratio) and HbA1c or GA level before the administration of alogliptin; however, a negative correlation was found between the ΔGA/HbA1c ratio and the GA/HbA1c ratio before the administration of alogliptin (R = -0.322, P = 0.049). Although the GA/HbA1c ratio in the low-value group (<2.80) was not significantly affected by the administration of alogliptin, that in the high-value group (≥2.80) significantly decreased (P = 0.008). The administration of alogliptin significantly decreased the GA/HbA1c ratio in the high-value group after 24 weeks. Alogliptin may be more useful for patients with high postprandial plasma glucose levels than in those with low postplandial plasma glucose levels.

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.

Degree of discrepancy between HbA1c and glycemia in variant hemoglobin is smaller when HbA1c is measured by new-type Arkray HPLC compared with old-type HPLC.

OBJECTIVES: Although variant hemoglobin mainly demonstrates inappropriate HbA1c values measured by high-performance liquid chromatography (HPLC), these values differ depending on the HPLC model. In the 1990s, old-type HPLC models were replaced with new-type HPLC models which could separate stable HbA1c from labile HbA1c and modified hemoglobin. This study compared HbA1c values in subjects with variant hemoglobin measured using old-type Arkray HPLC (HA-8150) and new-type Arkray HPLC (HA-8160 or HA-8180). DESIGN AND METHODS: This study included non-diabetic subjects with apparently low HbA1c values who had variant hemoglobins due to a ß-chain heterozygous mutation. HbA1c was measured by old-type HPLC in 28 subjects with 12 variant hemoglobins (group 1) and new-type HPLC in six subjects with four variant hemoglobins (group 2). When HbA1c was measured by HPLC (HPLC-HbA1c), HbA1c measured by immunoassay (IA-HbA1c) and glycated albumin (GA) were also measured. RESULTS: IA-HbA1c and GA did not significantly differ between both groups. However, HPLC-HbA1c in group 2 was significantly higher than that in group 1 (group 1: 2.9 ± 0.7% vs. group 2: 3.7 ± 0.2%, P = 0.006). CONCLUSIONS: When HbA1c in subjects with variant hemoglobin is measured by new-type Arkray HPLC, the degree of discrepancy between HbA1c and glycemia is smaller compared with that measured by old-type HPLC.

Estimation of HbA1c response to sitagliptin by change in glycated albumin level for 2 weeks.

UNLABELLED: Aims/Introduction: Since glycated albumin (GA) reflects shorter-term (about 2 weeks) control of plasma glucose levels compared with HbA1c, GA is thought to be a useful glycemic control indicator for the early period following commencement of the treatment of diabetes. In this study, we attempted to estimate HbA1c using the change in GA level before and after the first 2 weeks (ΔGA2w) of administration of sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor. MATERIALS AND METHODS: The study included 28 patients with type 2 diabetes who were administered sitagliptin at a dose of 50 mg/day for 12 weeks. RESULTS: At 2 weeks after administration of sitagliptin, GA markedly decreased, while HbA1c had only slightly decreased. A significant positive correlation was observed between the ΔGA2w and the change in HbA1c before and after the first 12 weeks of administration of sitagliptin (ΔHbA1c12w) (R = 0.793, P < 0.0001). The latter was about 0.6 times the former. The estimated HbA1c after 12 weeks of therapy was calculated by adding ΔGA2w × 0.6 to the baseline HbA1c. A significant positive correlation was observed between the estimated HbA1c and the measured HbA1c after 12 weeks (R = 0.735, P < 0.0001) and both were similar levels. CONCLUSIONS: HbA1c in the first 12 weeks after administration of sitagliptin could be estimated from the formula using the ΔGA2w. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00167.x, 2011).

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.

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.

Efficacy and adverse effects of nateglinide in early type 2 diabetes. Comparison with voglibose in a cross-over study.

An open-label prospective cross-over trial was performed to compare the efficacy and adverse effects of nateglinide with those of voglibose on Japanese early type 2 diabetes (who were oral hypoglycemic agent naïve and whose HbA(1C) levels were between 7.0 and 7.9% before treatment). Fourteen patients received 270 mg/day of nateglinide and 15 patients received 0.6 mg/day of voglibose. After 12 weeks of either therapy, the drugs were switched and treatment was continued for another 12 weeks. After 3-month treatment with each drug, HbA(1C) value decreased significantly (baseline HbA(1C) 7.24 +/- 0.42%, 6.70 +/- 0.47% with nateglinide: p<0.01, 6.93 +/- 0.62% with voglibose: p<0.05) but the difference in the effect between nateglinide and voglibose was not significant (p = 0.121). Symptoms related to hypoglycemia (e.g., increased appetite, palpitation, sweating, tremor) were scarcely observed with either voglibose or nateglinide treatments. Abdominal fullness/borborygmi was frequently reported, with variable severity, by patients on voglibose but this was absent or mild in those on nateglinide. After completion of both arms of the study, more patients favored nateglinide than voglibose. Our results suggest that nateglinide is an effective and safe drug in the treatment of early type 2 diabetes, similar to voglibose.

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.

Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor RORgamma.

The retinoid-related orphan receptor gamma (RORgamma) has been shown to function as a positive regulator of transcription in many cell lines. Transcriptional activation by nuclear receptors involves recruitment of co-activators that interact with receptors through their LXXLL motifs (NR box). In this study, we analyze the interaction of RORgamma with the co-activator SRC1 and use a series of LXXLL-containing peptides to probe for changes in the conformation of the co-activator interaction surface of the RORgamma LBD. We demonstrate that the H3-4/H12 co-activator interaction surface of RORgamma displays a selectivity for LXXLL peptides that is distinct from those of other nuclear receptors. LXXLL peptides that interacted with RORgamma efficiently antagonized RORgamma-mediated transcriptional activation. Mutations E502Q and Y500F in H12, and K334A, Q347A, and I348D in H3 and H4 of RORgamma, severely impact the recruitment of LXXLL peptides. The effects of these mutations are consistent with predictions made on the basis of the structure of the RORgamma(LBD) derived through homology modeling. These peptide antagonists provide a useful tool to analyze the conformation changes in the RORgamma(LBD) and to study RORgamma receptor signaling.

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.

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.

Identification of Glis1, a novel Gli-related, Kruppel-like zinc finger protein containing transactivation and repressor functions.

In this study, we describe the identification and characterization of a novel Krüppel-like protein named Gli-similar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krüppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca(2+)-dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development.

High incidence of T-cell lymphomas in mice deficient in the retinoid-related orphan receptor RORgamma.

Nuclear receptors are critical regulators of many physiological processes and have been shown to be involved in a variety of disease processes, including malignant neoplasms. Our laboratory is investigating the function of the retinoid-related orphan receptor gamma (RORgamma) and its possible role in disease. Studies of mice deficient in the expression of RORgamma demonstrated that this receptor plays a crucial role in the regulation of thymopoiesis and lymph node organogenesis. In this study, we show that changes in homeostasis in the thymus of RORgamma-/- mice are associated with a high incidence of T-cell lymphomas. Over 50% of the deficient mice of mixed genetic background die within the first 4 months as a result of thymic lymphomas. A high incidence of lymphomas was also observed in RORgamma-/- 129/SvEv mice. The lymphoblastic cells metastasized frequently to spleen and liver. No other tumor types were detected in any of RORgamma-/- mice that died during the course of the experiment, and none of the heterozygous mice developed thymic lymphomas. Lymphoma formation was associated with increased cellular proliferation and an increase in the number of apoptotic cells. When placed in culture, the RORgamma-/- lymphoblastic cells underwent accelerated "spontaneous" apoptosis at a rate similar to that of RORgamma-/- thymocytes. Upon prolonged culture, several lymphoblastic cell lines could be established. Analysis of the immunophenotype of the lymphoblastic cells showed that the CD4 and CD8 subpopulations varied substantially among different lymphomas. The established cell lines consisted mostly of CD44-CD25+CD4-CD8- cells. Our studies indicate that loss of RORgamma disturbs homeostasis in the thymus by enhancing apoptosis and cellular proliferation. The latter may enhance the probability of individual cells to acquire genetic alterations that make them escape negative selection and normal differentiation programs and as a consequence lead to increased susceptibility to the development of T-cell lymphoma.

Characterization of Glis2, a novel gene encoding a Gli-related, Krüppel-like transcription factor with transactivation and repressor functions. Roles in kidney development and neurogenesis.

In this study, we describe the characterization of a gene encoding a novel Krüppel-like protein, named Glis2. Glis2 encodes a relatively proline-rich, basic 55.8-kDa protein. Its five tandem Cys(2)-His(2) zinc finger motifs exhibit the highest homology to those of members of the Gli and Zic subfamilies of Krüppel-like proteins. Confocal microscopic analysis demonstrated that Glis2 localizes to the nucleus. Analysis of the genomic structure of the Glis2 gene showed that it is composed of 6 exons separated by 5 introns spanning a genomic region of more than 7.5 kb. Fluorescence in situ hybridization mapped the mouse Glis2 gene to chromosome 16A3-B1. Northern blot analysis showed that the Glis2 gene encodes a 3.8-kb transcript that is most abundant in adult mouse kidney. By in situ hybridization, expression was localized to somites and neural tube, and during metanephric development predominantly to the ureteric bud, precursor of the collecting duct, and inductor of nephronic tubule formation. One-hybrid analysis using Glis2 deletion mutants identified a novel activation function (AF) at the N terminus. The activation of transcription through this AF domain was totally suppressed by two repressor functions just downstream from the AF. One of the repressor functions is contained within the first zinc finger motif. The level of transcriptional activation and repression varied with the cell line tested, which might be due to differences in cell type-specific expression of co-activators and co-repressors. Our results suggest that Glis2 behaves as a bifunctional transcriptional regulator. Both the activation and repressor functions may play an important role in the regulation of gene expression during embryonic development.

Gross hematuria rapidly deteriorated renal function in a patient with polycystic kidney disease and Klippel-Trenaunay-Weber syndrome.

A case of polycystic kidney disease (PKD) associated with Klippel-Trenaunay-Weber syndrome is described. A 58-year-old man with chronic renal failure experienced urinary retention following gross hematuria. Intermittent drainage was necessary for significant urination for five days. Thereafter his urinary retention was relieved, but renal failure progressively developed and hemodialysis was started. Right hydronephrisis and hydroureter disappeared one month later. In spite of relief of obstruction, of which the cause was likely blood clots, renal function was not restored. Obstructive nephropathy was most likely explicable for notable deterioration in renal function. Our case might have susceptibilities to PKD development in terms of angiogenesis.