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Background Results from the J-ROCKET AF study revealed that rivaroxaban was non-inferior to warfarin with respect to the principal safety outcomes in patients with non-valvular atrial fibrillation. This subgroup analysis evaluated whether non-major clinically relevant bleeding (NMCRB) could be a predictive factor for major bleeding (MB). Other predictive factors for MB were also obtained in both rivaroxaban and warfarin treatment groups. Methods The temporal incidence of MB was compared between the rivaroxaban and warfarin treatment groups. Assessment was made whether MB events were often preceded by NMCRB. Univariate and multivariate analyses were carried out to identify any independent predictive factors for MB in both treatment groups. Results The incidences of MB and NMCRB were 18.04% (138/639 patients) in the rivaroxaban arm, and 16.42% in the warfarin arm (124/639 patients). NMCRB preceded MB in only four patients in each treatment group (rivaroxaban: 4/117 and warfarin: 4/98). Multivariate analysis identified predictive factors for bleeding events: anemia with warfarin treatment and concomitant use of antiplatelet agents with rivaroxaban treatment. Conclusions Results from this subgroup analysis, particularly the fact that there was no repeated or sequential pattern between NMCRB and MB occurrences in both treatment groups, suggests that NMCRB might not be a predictive factor for MB. On the contrary, anemia and concomitant use of antiplatelet therapy were likely predictive factors for bleeding with warfarin and rivaroxaban treatment, respectively.

High-dose calcium channel blocker (CCB) shows strong blood pressure (BP) lowering effect. Currently available of controlled-release (CR) nifedipine 80 mg per day clinical data are limited to monotherapy and short-term or long-term retrospective studies. We report the safety and efficacy results of a 52-week, prospective open-label study, in which Japanese patients with essential hypertension were treated with CR nifedipine [80 mg per day; 40 mg bis in die (BID; twice daily)] in combination with other antihypertensive drugs. The patients with inadequate BP control despite treatment with CR nifedipine (40 mg once daily) in combination with other antihypertensive drugs were enrolled. The primary objective of this study was to assess the long-term safety of CR nifedipine (80 mg per day). Efficacy variables included changes in the mean sitting BP, the target BP achievement rate and the BP response rate. CR nifedipine (80 mg per day) was generally well tolerated, with the most common drug-related treatment-emergent adverse event being tachycardia (6.9% of patients). Serious treatment-emergent adverse events were reported in three (4.2%) patients. By week 52, the mean reductions in sitting systolic and diastolic BP were 19.4 and 13.6 mm Hg, respectively. The target BP achievement and BP response rates after 52 weeks of treatment were 32.4 and 63.4%, respectively. Based on these findings, long-term treatment with CR nifedipine at 40 mg BID in combination with antihypertensive drugs was well tolerated and effective in Japanese patients with essential hypertension.

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.

Background: The J-ROCKET AF study found that rivaroxaban was non-inferior to warfarin with respect to the principal safety outcome in patients with atrial fibrillation (AF). The aim of this subgroup analysis was to assess the safety and efficacy of rivaroxaban and warfarin in relation to patient age. Methods and Results: A total of 39.0% were elderly (aged ≥75 years). In elderly patients, the principal safety outcome occurred at 25.05%/year with rivaroxaban vs. 16.95%/year on warfarin (hazard ratio [HR], 1.49; 95% confidence interval [CI]: 1.02–2.16), whereas the primary efficacy endpoint occurred at 2.18%/year vs. 4.25%/year (HR, 0.51; 95% CI: 0.20–1.27), respectively. There were significant interactions in the principal safety outcomes of rivaroxaban compared with warfarin between the elderly and non-elderly groups, but not in the primary efficacy endpoints (P=0.04 and 0.82 for both interactions, respectively). Furthermore, in elderly patients, in the rivaroxaban group there was a trend to increase the principal safety outcome regardless of renal function. In elderly patients with preserved renal function, however, patients on rivaroxaban had a marginally favorable trend in the primary efficacy endpoint incidence rate compared with patients on warfarin. Conclusions: There is a need to carefully consider the risks and benefits of therapy with rivaroxaban in elderly patients with non-valvular AF. (Circ J 2014; 78: 1349–1356)

The global ROCKET AF phase III trial evaluated rivaroxaban 20 mg once daily (o.d.) for stroke prevention in atrial fibrillation (AF). Based on rivaroxaban pharmacokinetics in Japanese subjects and lower anticoagulation preferences in Japan, particularly in elderly patients, the optimal dose regimen for Japanese AF patients was considered. The aim of this analysis was dose selection for Japanese patients from a pharmacokinetic aspect by comparison of simulated exposure in Japanese patients with those in Caucasian patients. As a result of population pharmacokinetics-pharmacodynamics analyses, a one-compartment pharmacokinetic model with first-order absorption and direct link pharmacokinetic-pharmacodynamic models optimally described the plasma concentration and pharmacodynamic models (Factor Xa activity, prothrombin time, activated partial thromboplastin time, and HepTest), which were also consistent with previous works. Steady-state simulations indicated 15 mg rivaroxaban o.d. doses in Japanese patients with AF would yield exposures comparable to the 20 mg o.d. dose in Caucasian patients with AF. In conclusion, in the context of the lower anticoagulation targets in Japanese practice, the population pharmacokinetic and pharmacodynamic modeling supports 15 mg o.d. as the principal rivaroxaban dose in J-ROCKET AF.

Background: In the Japanese Rivaroxaban Once-daily oral direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation (J-ROCKET AF) study, rivaroxaban 15mg once daily was given to patients with creatinine clearance (CrCl) ≥50ml/min (preserved renal function), and was reduced to 10mg once daily in patients with CrCl 30–49ml/min (moderate renal impairment). The aim of this subanalysis was to assess the safety and efficacy of the adjusted dose of rivaroxaban compared with warfarin in a cohort with moderate renal impairment. Methods and Results: Compared with patients with preserved renal function, those with moderate renal impairment (22.2% of all randomized patients) had higher rates of bleeding and stroke events irrespective of study treatment. Among those with moderate renal impairment, the principal safety endpoint occurred at 27.76%/year with rivaroxaban vs. 22.85%/year with warfarin (hazard ratio [HR], 1.22; 95% confidence interval [CI]: 0.78–1.91) and the rate of the primary efficacy endpoint was 2.77%/year vs. 3.34%/year (HR, 0.82; 95% CI: 0.25–2.69), respectively. There were no significant interactions between renal function and study treatment in the principal safety and the primary efficacy endpoints (P=0.628, 0.279 for both interactions, respectively). Conclusions: The safety and efficacy of rivaroxaban vs. warfarin were consistent in patients with moderate renal impairment and preserved renal function. (Circ J 2013; 77: 632–638)

SummaryPlasma proteins mediate thrombogenesis, inflammation, endocardial injury and structural remodelling in atrial fibrillation (AF). We hypothesised that anti-coagulation with rivaroxaban, a direct factor Xa inhibitor, would differentially modulate biologically-relevant plasma proteins, compared with warfarin, a multi-coagulation protein antagonist. We performed unbiased liquid chromatography/tandem mass spectroscopy and candidate multiplexed protein immunoassays among Japanese subjects with non-valvular chronic AF who were randomly assigned to treatment with 24 weeks of rivaroxaban (n=93) or warfarin (n=94). Nine metaproteins, including fibulin-1 (p=0.0033), vitronectin (p=0.0010), haemoglobin α(p=0.0012), apolipoproteins C-II (p=0.0017) and H (p=0.0023), complement C5 precursor (p=0.0026), coagulation factor XIIIA (p=0.0026) and XIIIB (p=0.0032) subunits, and 10 candidate proteins, including thrombomodulin (p=0.0004), intercellular adhesion molecule-3 (p=0.0064), interleukin-8 (p=0.0007) and matrix metalloproteinase-3 (p=0.0003), were differentially expressed among patients with and without known clinical risk factors for stroke and bleeding in AF. Compared with warfarin, rivaroxaban treatment was associated with a greater increase in thrombomodulin (Δ0.1 vs. 0.3 pg/ml, p=0.0026) and a trend towards a reduction in matrix metalloproteinase-9 (Δ2.2 vs. –4.9 pg/ml, p=0.0757) over 24 weeks. Only modest correlations were observed between protein levels and prothrombin time, factor Xa activity and prothrombinase-induced clotting time. Plasma proteomics can identify distinct functional patterns of protein expression that report on known stroke and bleeding risk phenotypes in an ethnically-homogeneous AF population. The greater upregulation of thrombomodulin among patients randomised to rivaroxaban represents a proof-of-principle that pharmacoproteomics can be employed to discern novel effects of factor Xa inhibition beyond standard pharmacodynamic measures.

We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced fl-cDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.

Glucose-induced insulin secretion from beta-cells is often impaired in diabetic condition and by exposure to diabetogenic pharmacological agents. In pancreatic beta-cells, intracellular glucose metabolism regulates exocytosis of insulin granules, according to metabolism-secretion coupling in which glucose-induced mitochondrial ATP production plays an essential role. Impaired glucose-induced insulin secretion often results from impaired glucose-induced ATP elevation in beta-cells. Mitochondrial ATP production is driven by the proton-motive force including mitochondrial membrane potential (DeltaPsi(m)) generated by the electron transport chain. These electrons are derived from reducing equivalents, generated in the Krebs cycle and transferred from cytosol by the shuttles. Here, roles of the determinants of mitochondrial ATP production in impaired glucose-induced insulin secretion are discussed. Cytosolic alkalization, H(+) leak in the inner membrane by uncoupler (e.g. free fatty acid exposure), decrease in the supply of electron donors including NADH and FADH(2) to the respiratory chain, and endogenous mitochondrial ROS (e.g. Na(+)/K(+)-ATPase inhibition) all reduce hyperpolarlization of DeltaPsi(m) and ATP production, causing decresed glucose-induced insulin release. The decrease in the supply of NADH and FADH(2) to the respiratory chain derives from impairments in glucose metabolism including glycolysis (e.g. MODY2 and exposure to NO) and the shuttles (e.g. diabetic state and exposure to ketone body).

Background: In Japan, the standard of care for the treatment of pulmonary embolism (PE) and/or deep vein thrombosis (DVT) consists of intravenous unfractionated heparin (UFH) followed by warfarin, which was recently compared with rivaroxaban, an oral factor Xa inhibitor, in randomized trials. Aim: To examine the length of hospital stay in patients with PE and/or DVT receiving rivaroxaban compared to Japanese standard therapy in the Japanese (J)-EINSTEIN PE and DVT program. Methods: Open-label, randomized clinical trials that compared 3, 6, or 12 months of rivaroxaban with UFH and warfarin in patients with acute, confirmed symptomatic proximal PE and/or DVT. Decisions regarding hospital admission and/or discharge were left to the clinical judgment of attending physicians. Analyses were conducted in the intention-to-treat (ITT) population. Results: In the ITT population (N= 97), overall patient characteristics were similar in both treatment arms. The median length of stay in rivaroxaban patients was 10.0 days (interquartile range [IQR] 6.0 to 15.0 days) while it was 15.0 days (IQR 9.0 to 22.0) for patients on standard therapy (p = 0.016). All of the four DVT patients who were not hospitalized for the index event were in the rivaroxaban arm. Conclusions: Our results suggest that treatment with rivaroxaban may significantly reduce the length of hospital stay in patients hospitalized for PE and/or DVT compared with the current standard of care in Japan, thereby reducing the burden on patients and the healthcare system. The limitations of our study include small sample size and the generalizability of the findings to the real-world setting. Further research is warranted to identify PE and/or DVT patients in Japanese clinical practice who may potentially be managed as outpatients.

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca2+concentration, because even in the absence of ATP at low Ca2+, the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an α-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca2+, suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.

We examined the effects of the novel hypoglycaemic agent JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] on insulin secretion using rat pancreatic islets, and analysed the mechanism of its effect. JTT-608 augmented 8.3 mM glucose-induced insulin secretion dose-dependently, and there was a stimulatory effect of 100 μM JTT-608 at both moderate and high concentrations (8.3, 11.1 and 16.7 mM) of glucose, but not at low concentrations (3.3 and 5.5 mM). In perifusion experiments, both phases of insulin release were enhanced, and the effect was eliminated 10 min after withdrawal of the agent. In the presence of 200 μM diazoxide and a depolarizing concentration (30 mM) of K+, there was an augmentation of insulin secretion by 100 μM JTT-608, not only under high levels of glucose but also under low levels, and the effects were abolished by 10 μM nitrendipine. JTT-608 augmented insulin secretion from electrically permeabilized islets in the presence of stimulatory concentrations (0.3 and 1.0 μM) of Ca2+, and the intracellular Ca2+ concentration ([Ca2+]i) response under 16.7 mM glucose, 200 μM diazoxide, and 30 mM K+ was also increased. The cyclic AMP content in the islets was increased by 100 μM JTT-608, and an additive effect to 1 μM forskolin was observed, but not to 50 μM 3-isobutyl-1-methylxanthine (IBMX). JTT-608 inhibited phosphodiesterase (PDE) activity dose-dependently. We conclude that JTT-608 augments insulin secretion by enhancing Ca2+ efficacy and by increasing Ca2+ influx. This appears to be a result of the increased intracellular cyclic AMP concentration due to PDE inhibition. Keywords: JTT-608, pancreatic islets, insulin secretion, Ca2+ sensitivity, Ca2+ influx, cyclic AMP, phosphodiesterase inhibitor Introduction An impaired β-cell insulin response to glucose makes an important contribution to the metabolic derangement in non-insulin-dependent diabetes mellitus (NIDDM) (Ward et al., 1984), so an improvement of insulin secretion is effective in the treatment for patients with NIDDM who have sufficient functional β-cell reserve. To evaluate therapeutic strategies, it is essential to understand the mechanism of insulin secretion induced by glucose and of its enhancement by physiological factors. Glucose metabolism raises the ATP/ADP ratio in the β-cell and closes the ATP-sensitive K+ channels (KATP channels), leading to membrane depolarization and subsequent activation of the voltage-dependent Ca2+ channels (VDCCs). Elevation of the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ influx through the VDCCs triggers exocytosis of insulin granules (Ashcroft & Rorsman, 1989). Circulating peptides such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secreted from the intestine, as well as glucagon from pancreatic α-cells, are well known to be important physiological potentiators of insulin secretion. The insulinotropic effect of these peptides is due to the increase of the cyclic AMP concentration in the β-cells and subsequent activation of protein kinase A, which increases Ca2+ influx (Grapengiesser et al., 1991; Lu et al., 1993; Yada et al., 1993) through the activated VDCCs (Britsch et al., 1995; Ding & Gromada, 1997; Gromada et al., 1998) and enhances Ca2+ efficacy in the exocytotic system (Ding & Gromada, 1997; Gromada et al., 1998). There are several ways to stimulate insulin secretion: potentiation of glucose metabolism, inhibition of KATP channel activity, increase of Ca2+ influx by VDCC activation, and enhancement of Ca2+ efficacy in insulin exocytosis. However, the only method clinically used presently in NIDDM treatment is blocking the KATP channels by sulphonylureas and other agents. A novel oral insulinotropic agent which acts on a site distinct from that of the sulphonylureas, therefore, might be a helpful supplemental clinical modality. We have recently identified such a novel hypoglycemic agent, JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] (Shinkai et al., 1998). An in vivo study shows that the agent increases plasma insulin and lowers plasma glucose in both normal and NIDDM rat models. Interestingly, these effects were not observed in the basal state, but only at a stimulatory concentration of glucose, in contrast to the effects of the sulphonylureas, which also affect the basal glucose level (Ohta et al., 1999a, 1999b). JTT-608, therefore, acts at a site distinct from the sulphonylureas to stimulate insulin release, by a mechanism which remains to be clarified. In the present study, we have analysed the mechanism of the augmentation of insulin release by JTT-608. Insulin secretory capacity was examined in both static incubation and perifusion experiments. The mechanism distal to Ca2+ influx was investigated using the [Ca2+]i response in fluorescence measurements and insulin release from electrically permeabilized islets in which [Ca2+]i can be manipulated.

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene ( GIPR ) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic β cells. GIPR −/− mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR +/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR −/− mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo , and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

The effect of 1α,25-dihydroxylumisterol3 (1α,25(OH)2lumisterol3) on insulin release from rat pancreatic β-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1α,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mm glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1β,25-dihydroxyvitamin D3 (1β,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1α,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx throu...

Effects of voglibose (an alpha-glucosidase inhibitor) and pioglitazone (an insulin sensitizer) on glycemic control and on the function of pancreatic islets were evaluated using Goto-Kakizaki (GK) rats with non-insulin-dependent diabetes mellitus (NIDDM). Five week administration (8-13 weeks of age in GK rats) of voglibose alone (added to the chow at a concentration of 10 ppm), pioglitazone alone (10 mg/kg daily p.o.), or both of the agents together significantly improved fasting plasma glucose levels and those at 120 min in oral glucose tolerance tests. Insulin secretory capacity in response to glucose of the isolated islets, assessed by batch incubation, was significantly improved in the voglibose and in the voglibose plus pioglitazone groups. Eight-week administration (5-13 weeks of age) of voglibose and voglibose plus pioglitazone successfully lowered the fasting levels of plasma glucose and triglyceride. The glucose-responsiveness in insulin release from the islets was also significantly recovered by the therapy. The treatment increased the insulin content of the islets to almost twice that in untreated controls. Thus, treatment by these drugs can not only effectively ameliorate the metabolic derangement of NIDDM in GK rats, but it can also restore the deteriorated islet function, possibly through protection from glucose toxicity.

The alterations of the ATP concentration in response to fuel secretagogues such as glucose, glyceraldehyde, and ketoisocaproate (KIC) were investigated in a single islet. The intraislet ATP concentration was transiently elevated and then decreased to a level slightly higher than basal. To assess the ATP content under conditions of reduced ATP consumption, the Na-K pump blocker ouabain was used. The elevation of ATP concentration was found unexpectedly to be suppressed under ouabain in the islets, even when incubated with any of the secretagogues. High glucose did not elevate the intracellular creatine phosphate during incubation with ouabain. Since the suppression rate for the intraislet ATP elevation was considerably smaller with KIC than with glyceraldehyde and glucose, we conclude that ouabain inhibits ATP production, at least in part, in the glycolytic pathway through a feedback mechanism.

To investigate the possible involvement of some intracellular metabolic signaling other than the ATP derived from glucose metabolism under protein kinase A (PKA) activation, we measured the insulin secretory capacity stimulated by glucose and other fuel secretagogues using diazoxide-treated pancreatic islets. Under these conditions, we found a signal from a site proximal to glyceraldehyde-3-phosphate (GA-3-P) in the glycolysis to be necessary for glucose-induced insulin secretion. By using several different glycolytic enzyme inhibitors, we found that this proximal signal is derived from glucose-6-phosphate (G-6-P), and that metabolic signaling distal to GA-3-P also is necessary. Mycophenolic acid completely inhibited the augmented glucose-induced insulin secretion, which guanosine could reverse, indicating that the proximal signaling is coupling with endogenous GTP production. In this novel system of metabolic signaling, endogenous GTP derived from G-6-P in the glycolysis elicits the augmentation of glucose-induced insulin secretion under PKA activation in diazoxide-treated pancreatic islets.

As we had an opportunity to take blood samples from a totally thyroidectomized patient who had attempted suicide by taking 2,000 microg of Levothyroxine (L-T4), the serum levels of thyroid hormones were sequentially measured to investigate the metabolism of circulating thyroid hormones in an athyreotic human. The serum concentrations of most thyroid hormones reached a peak on the second day, but the serum T3 level showed a peak one day later. The maximum concentrations of T4 (315 microg/l), FT4 (48.8 ng/l) and rT3 (0.80 microg/l) were very high, while the peak T3 level (1.92 microg/l) did not exceed the upper limit of the normal range. The serum T4 and rT3 levels returned to their normal range 13-17 days after the suicide attempt. The TSH level was suppressed rapidly and reached its nadir (0.044 mU/l) on the 6th day. During this period, the T1/2 and MCR of serum T4 were 10.4 days and 0.64 l/day, respectively, which values were almost equivalent to those observed during 15 days after discontinuation of the maintenance L-T4 therapy. In summary, the oral intake of a large amount of L-T4 at one time does not induce a proportional increase in the T3 level in an athyreotic person. The MCR of serum T4 is decreased and the T1/2 of serum T4 is prolonged, probably due to the lack of intrathyroidal deiodination. These findings support the conclusion that the D1 activity in the thyroid is one of the major determinants in the metabolic clearance of serum T4.

The majority of the patients enrolled in the rivaroxaban vs. warfarin in Japanese patients with atrial fibrillation (J-ROCKET AF) trial had hypertension. In this subgroup analysis, we investigated differences in the safety and efficacy of rivaroxaban and warfarin in subjects with and without hypertension. The baseline blood pressure (BP) measurements of patients with hypertension in the rivaroxaban and warfarin groups were 130/77 mm Hg and 131/77 mm Hg, respectively, whereas those of patients without hypertension were 123/74 mm Hg and 124/73 mm Hg, respectively. The incidence rates of the principal safety outcomes in the rivaroxaban and warfarin groups were 18.39% per year and 16.81% per year, respectively, among patients with baseline hypertension (hazard ratio (HR): 1.10; 95% confidence interval (CI): 0.84–1.45) and 16.71% per year and 15.00% per year, respectively, among patients without hypertension at baseline (HR: 1.14; 95% CI: 0.66–1.97), indicating no significant interaction (P=0.933). The incidence rates of the primary efficacy endpoints in the rivaroxaban group and the warfarin group were 0.54% per year and 2.24% per year, respectively, in patients without baseline hypertension (HR: 0.25; 95% CI: 0.03–2.25), and 1.45% per year and 2.71% per year, respectively, in patients with baseline hypertension (HR: 0.54; 95% CI: 0.25–1.16), indicating no significant interaction (P=0.509). In conclusion, the safety and efficacy profile of rivaroxaban was similar to that of warfarin, independent of baseline hypertensive status.

Background Results from a trial of rivaroxaban versus warfarin in 1280 Japanese patients with atrial fibrillation (J-ROCKET AF) revealed that rivaroxaban was noninferior to warfarin with respect to the principal safety outcome. In this subanalysis, we investigated the safety and efficacy of rivaroxaban and warfarin in relation to patients' CHADS 2 scores. Results The mean CHADS 2 score was 3.25, and the most frequent scores were 3 and 4. No statistically significant interactions were observed between principal safety outcome event rates and CHADS 2 scores with respect to treatment groups ( P value for interaction = .700). Irrespective of stratification into moderate- and high-risk groups based on CHADS 2 scores of 2 and 3 or more, respectively, no differences in principal safety outcome event rates were observed between rivaroxaban- and warfarin-treated patients (moderate-risk group: hazard ratio [HR], 1.06; 95% confidence interval [CI], .58-1.95; high-risk group: HR, 1.11; 95% CI, .86-1.45; P value for interaction = .488). The primary efficacy end point rate in the rivaroxaban-treated group was numerically lower than in the warfarin-treated group, regardless of risk group stratification (moderate-risk group: HR, .46; 95% CI, .09-2.37; high-risk group: HR, .49; 95% CI, .22-1.11; P value for interaction = .935). Conclusion This subanalysis indicated that the safety and efficacy of rivaroxaban compared with warfarin were similar, regardless of CHADS 2 score.

This study was designed to confirm the appropriateness of the dose setting for a Japanese phase III study of rivaroxaban in patients with non-valvular atrial fibrillation (NVAF), which had been based on model simulation employing phase II study data. The previously developed mixed-effects pharmacokinetic/pharmacodynamic (PK-PD) model, which consisted of an oral one-compartment model parameterized in terms of clearance, volume and a first-order absorption rate, was rebuilt and optimized using the data for 597 subjects from the Japanese phase III study, J-ROCKET AF. A mixed-effects modeling technique in NONMEM was used to quantify both unexplained inter-individual variability and inter-occasion variability, which are random effect parameters. The final PK and PK-PD models were evaluated to identify influential covariates. The empirical Bayes estimates of AUC and C(max) from the final PK model were consistent with the simulated results from the Japanese phase II study. There was no clear relationship between individual estimated exposures and safety-related events, and the estimated exposure levels were consistent with the global phase III data. Therefore, it was concluded that the dose selected for the phase III study with Japanese NVAF patients by means of model simulation employing phase II study data had been appropriate from the PK-PD perspective.

Background The overall analysis of the rivaroxaban versus warfarin in Japanese patients with atrial fibrillation (J-ROCKET AF) trial revealed that rivaroxaban was not inferior to warfarin with respect to the primary safety outcome. In addition, there was a strong trend for a reduction in the rate of stroke/systemic embolism with rivaroxaban compared with warfarin. Methods In this subanalysis of the J-ROCKET AF trial, we investigated the consistency of safety and efficacy profile of rivaroxaban versus warfarin among the subgroups of patients with previous stroke, transient ischemic attack, or non–central nervous system systemic embolism (secondary prevention group) and those without (primary prevention group). Results Patients in the secondary prevention group were 63.6% of the overall population of J-ROCKET AF. In the secondary prevention group, the rate of the principal safety outcome (% per year) was 17.02 in rivaroxaban-treated patients and 18.26 in warfarin-treated patients (hazard ratio [HR] 0.95; 95% confidence interval [CI] 0.70-1.29), while the rate of the primary efficacy endpoint was 1.66 in rivaroxaban-treated patients and 3.25 in warfarin-treated patients (HR 0.51; 95% CI 0.23-1.14). There were no significant interactions in the principal safety and the primary efficacy endpoints of rivaroxaban compared to warfarin between the primary and secondary prevention groups ( P = .090 and .776 for both interactions, respectively). Conclusions The safety and efficacy profile of rivaroxaban compared with warfarin was consistent among patients in the primary prevention group and those in the secondary prevention group.

Background: The global ROCKET AF study evaluated once-daily rivaroxaban vs. warfarin for stroke and systemic embolism prevention in patients with atrial fibrillation (AF). A separate trial, J-ROCKET AF, compared the safety of a Japan-specific rivaroxaban dose with warfarin administered according to Japanese guidelines in Japanese patients with AF. Methods and Results: J-ROCKET AF was a prospective, randomized, double-blind, phase III trial. Patients (n=1,280) with non-valvular AF at increased risk for stroke were randomized to receive 15mg once-daily rivaroxaban or warfarin dose-adjusted according to Japanese guidelines. The primary objective was to determine non-inferiority of rivaroxaban against warfarin for the principal safety outcome of major and non-major clinically relevant bleeding, in the on-treatment safety population. The primary efficacy endpoint was the composite of stroke and systemic embolism. Non-inferiority of rivaroxaban to warfarin was confirmed; the rate of the principal safety outcome was 18.04% per year in rivaroxaban-treated patients and 16.42% per year in warfarin-treated patients (hazard ratio [HR] 1.11; 95% confidence interval 0.87–1.42; P

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.

The scarcity of available islets is an obstacle for clinically successful islet transplantation. One solution might be to increase the efficacy of the limited islets. Isolated islets are exposed to a variety of cellular stressors, and disruption of the cell-matrix connections damages islets. We examined the effect of fibronectin, a major component of the extracellular matrix, on islet viability, mass and function, and also examined whether fibronectin-treated islets improved the results of islet transplantation. Islets cultured with fibronectin for 48 hours maintained higher cell viability (0.146 +/- 0.010 vs. 0.173 +/- 0.007 by MTT assay), and also had a greater insulin and DNA content (86.8 +/- 3.6 vs. 72.8 +/- 3.2 ng/islet and 35.2 +/- 1.4 vs. 30.0 +/- 1.5 ng/islet, respectively) than islets cultured without fibronectin (control). Absolute values of insulin secretion were higher in fibronectin-treated islets than in controls; however, the ratio of stimulated insulin secretion to basal secretion was not significantly different (206.9 +/- 23.3 vs. 191.7 +/- 20.2% when the insulin response to 16.7 mmol/l glucose was compared to that of 3.3 mmol/l glucose); the higher insulin secretion was thus mainly due to larger islet cell mass. The rats transplanted with fibronectin-treated islets had lower plasma glucose and higher plasma insulin levels within 2 weeks after transplantation, and had more favorable glucose tolerance 9 weeks after transplantation. These results indicate that cultivation with fibronectin might preserve islet cell viability, mass and insulin secretory function, which could improve glucose tolerance following islet transplantation.

We examined the effects of reduced Na(+)/K(+)-ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na(+)/K(+)-ATPase, augmented 16.7 mmol/l glucose-induced insulin release in the early period but suppressed it after a delay of 20-30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na(+)/K(+)-ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca(2+)-depleted or Na(+)-depleted conditions. That mitochondrial membrane hyperpolarization and O(2) consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, alpha-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic β-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca 2+ and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca 2+ for 30 min was increased by about 1.9-fold more than in the presence of Ca 2+ . The increment was extracellular Ca 2+ -dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni 2+ did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca 2+ . However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca 2+ , the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca 2+ . On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca 2+ . In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca 2+ is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.

Aims To explore the mechanisms underlying the impaired erythrocyte deformability (RBC-df) in diabetic patients, the relationship between erythrocyte intracellular free calcium-ion concentration ([Ca2+]i) and RBC-df, and the effects of Ca2+-channel blocker on [Ca2+]i and RBC-df were evaluated. Methods Forty-eight patients with NIDDM and 24 control subjects were enrolled in this study. [Ca2+]i was determined using fura-2, and RBC-df by filtration method expressed as Deformability Index (DI). Erythrocytes were treated with nisoldipine to evaluate the effects of a Ca2+-channel blocker. Results [Ca2+]i was significantly higher (82.6 (78.0–87.2) vs 76.6 (74.3–81.2) nmol lRBC−1, P

In two patients with lymphocytic adenohypophysitis, images of the pituitary gland were serially observed by MRI. In both cases, the pituitary gland had swollen during the late stage of the first pregnancy. In case 1, MRI findings were representative of lymphocytic adenohypophysitis. After delivery, plasma levels of PRL, ACTH and cortisol decreased markedly. The height of the pituitary gland gradually decreased from 22 mm (14 days after delivery) to 13 mm (73 days) and became rapidly smaller (4.9 mm, 115 days) following administration of massive doses of hydrocortisone for the treatment of acute adrenal insufficiency induced by painless thyroiditis. Six years later, the height was 2.5 mm. Low plasma levels of PRL and cortisol persisted. Diabetes insipidus did not develop. In case 2, MRI revealed a pituitary mass accompanied by a cystic change. Lymphocytic adenohypophysitis was confirmed by histological examination. Because pituitary function tests indicated that ACTH, PRL, GH and TSH were of low levels, hydrocortisone and L-thyroxine were orally administered. No diabetes insipidus was demonstrated. MRI disclosed that the height of the pituitary gland was 23 mm (17 days after delivery) but decreased to 17 and 5.5 mm after 44 and 128 days, respectively. Four years later immediately after the second delivery, it was 1 mm, and the patient was diagnosed as having empty sella. Long-term observation of lymphocytic adenohypophysitis demonstrated that the pituitary gland was markedly atrophied, leading to empty sella. It is believed that some of the classic cases of Sheehan's syndrome associated with empty sella may include lymphocytic adenohypophysitis.

We had a patient with asymptomatic hyper-immunoreactive glucagonemia and with no evidence of pancreatic tumor detected by radiological examinations. The glucagon level was not decreased by the administration of glucose or somatosatin analogue (SMS 201-995). Gel filtration studies revealed that most glucagon immunoreactivity was eluted at the position of 150,000 daltons [big plasma glucagon (BPG)]. Binding studies with 125I-glucagon showed that glucagon autoantibody was negative. Acid treatment of plasma and reduction of immunoglobulin G (IgG) did not result in a shift of BPG to normal glucagon (3485 daltons). Glucagon immunoreactivity determined with anti-glucagon antiserum OAL 123 (C-terminal specific antiserum used in the present radioimmunoassay kit) did not dilute out in parallel to normal glucagon (3485 daltons), and the plasma glucagon level was normal with Unger's 30K (anther C-terminal specific antiserum) and OAL 196 (N-terminal specific antiserum). The patient's IgG dose-dependently reduced the binding of 125I-glucagon to anti-glucagon antiserum OAL-123. Glucagon degrading activity (GDA) was negative in the patient's plasma. These results suggest that the patient's IgG cross-reacted with the present anti-glucagon antiserum OAL 123, and caused a spuriously high plasma immunoreactive glucagon level.