Effects of pemafibrate on left ventricular diastolic function in patients with type 2 diabetes mellitus: a pilot study.

Aims/introduction: Diabetic cardiomyopathy (DCM) is characterized predominantly by diastolic dysfunction. The multiple mechanisms underlying DCM include altered energy substrate utilization. Recent studies indicate that PPARα plays an important role in the pathogenesis of lipotoxic cardiomyopathy. Pemafibrate is known to be a selective PPARα modulator (SPPARMα). We thus investigated the effects of pemafibrate on cardiac diastolic function in patients with type 2 diabetes. Materials and methods: Seventeen patients with type 2 diabetes (T2D) and hypertriglyceridemia were screened and treated with pemafibrate at a dose of 0.2 mg/day for 8-16 weeks. Fourteen patients were eligible for analysis. Echocardiography was used for assessment of diastolic function. Early diastolic filling velocity (E), late atrial filling velocity (A) and the E/A ratio were included in this study. Peak early diastolic annular velocities (e') were also assessed using color tissue Doppler images. The primary endpoints were changes in the ratio of E to A (E/A), e', and the ratio of E to e' (E/e') from baseline. Results: Pemafibrate significantly increased average e' (7.24 ± 0.58 vs 7.94 ± 0.67, p = 0.019) and a significant reduction in E/e' (9.01 ± 0.94 vs 8.20 ± 0.91, p = 0.041). The increase in e' was significantly related to increases in fasting blood glucose (r = 0.607, p = 0.021) and non-esterified fatty acid (r = 0.592, p = 0.026). Conclusion: Pemafibrate improved diastolic function in patients with T2D and hypertriglyceridemia, suggesting that PPARα activation by pemafibrate prevents the development of DCM at an early stage.

Metabolic state switches between morning and evening in association with circadian clock in people without diabetes.

AIMS/INTRODUCTION: Understanding morning-evening variation in metabolic state is critical for managing metabolic disorders. We aimed to characterize this variation from the viewpoints of insulin secretion and insulin sensitivity, including their relevance to the circadian rhythm. MATERIALS AND METHODS: A total of 14 and 10 people without diabetes were enrolled, and underwent a 75-g oral glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp study, respectively. Participants completed the OGTT or hyperinsulinemic-euglycemic clamp at 08.00 hours and 20.00 hours in random order. Before each study, hair follicles were collected. In mice, phosphorylation levels of protein kinase B were examined in the liver and muscle by western blotting. RESULTS: Glucose tolerance was better at 08 .00 hours, which was explained by the higher 1-h insulin secretion on OGTT and increased skeletal muscle insulin sensitivity on hyperinsulinemic-euglycemic clamp. Hepatic insulin sensitivity, estimated by the hepatic insulin resistance index on OGTT, was better at 20.00 hours. The 1-h insulin secretion and hepatic insulin resistance index correlated significantly with Per2 messenger ribonucleic acid expression. The change (evening value - morning value) in the glucose infusion rate correlated significantly with the change in non-esterified fatty acid, but not with clock gene expressions. The change in non-esterified fatty acid correlated significantly with E4bp4 messenger ribonucleic acid expression and the change in cortisol. In mice, phosphorylation of protein kinase B was decreased in the liver and increased in muscle in the beginning of the active period as, expected from the human study. CONCLUSIONS: Glucose metabolism in each tissue differed between the morning and evening, partly reflecting lipid metabolism, clock genes and cortisol levels. Deeper knowledge of these associations might be useful for ameliorating metabolic disorders.

Adrenomedullin has a cytoprotective role against endoplasmic reticulum stress for pancreatic ß-cells in autocrine and paracrine manners.

AIMS/INTRODUCTION: Pancreatic ß-cells are sensitive to endoplasmic reticulum (ER) stress, which has a major role in the context of ß-cell death. Adrenomedullin (ADM) has been shown to exert a cytoprotective effect under various pathophysiological conditions. Several studies have suggested that thiazolidinediones have protective effects on ß-cells. During the course to elucidate the molecular mechanisms by which pioglitazone prevents ß-cell death, ADM emerged as a candidate. Here, we studied the regulation of ADM expression, including the effects of pioglitazone, and its role in pancreatic islets. MATERIALS AND METHODS: We analyzed ADM expression in islet cell lines treated with pioglitazone. The effects of ER stress on ADM and ADM receptor expressions were investigated by analyzing thapsigargin-treated MIN6 cells and islets isolated from Wfs1-/- and db/db mice. To study the anti-apoptotic effect of ADM, ER stress-exposed MIN6 cells were treated with ADM peptides or transfected with ADM expression plasmid. RESULTS: Pioglitazone increased the production and secretion of ADM in islets through peroxisome-proliferator activated receptor-γ-dependent mechanisms. Thapsigargin treatment increased expressions of both ADM and ADM receptor, composed of Ramp2, Ramp3 and Crlr, in MIN6 cells. ADM and ADM receptor expressions were also increased in isolated islets from Wfs1-/- and db/db mice. ADM peptides and ADM overexpression protected MIN6 cells from thapsigargin-induced apoptosis. CONCLUSIONS: ER stress stimulates ADM production and secretion in islets. ADM signaling might protect ß-cells from ER stress-induced apoptosis, and might be one of the self-protective mechanisms. ß-Cell protection by pioglitazone is partly through induction of ADM. ADM-based therapy could be a novel strategy for treating diabetes.

Clock Gene Dysregulation Induced by Chronic ER Stress Disrupts ß-cell Function.

In Wfs1-/-Ay/a islets, in association with endoplasmic reticulum (ER) stress, D-site-binding protein (Dbp) expression decreased and Nuclear Factor IL-3 (Nfil3)/E4 Promoter-binding protein 4 (E4bp4) expression increased, leading to reduced DBP transcriptional activity. Similar alterations were observed with chemically-induced ER stress. Transgenic mice expressing E4BP4 under the control of the mouse insulin I gene promoter (MIP), in which E4BP4 in ß-cells is expected to compete with DBP for D-box, displayed remarkable glucose intolerance with severely impaired insulin secretion. Basal ATP/ADP ratios in MIP-E4BP4 islets were elevated without the circadian oscillations observed in wild-type islets. Neither elevation of the ATP/ADP ratio nor an intracellular Ca2+ response was observed after glucose stimulation. RNA expressions of genes involved in insulin secretion gradually increase in wild-type islets early in the feeding period. In MIP-E4BP4 islets, however, these increases were not observed. Thus, molecular clock output DBP transcriptional activity, susceptible to ER stress, plays pivotal roles in ß-cell priming for insulin release by regulating ß-cell metabolism and gene expressions. Because ER stress is also involved in the ß-cell failure in more common Type-2 diabetes, understanding the currently identified ER stress-associated mechanisms warrants novel therapeutic and preventive strategies for both rare form and common diabetes.

[Wolfram syndrome: clinical features, molecular genetics of WFS1 gene].

Wolfram syndrome(WFS: OMIM 222300) is a rare recessive neuro-endocrine degenerative disorder, known as DIDMOAD(Diabetes Insipidus, early-onset Diabetes Mellitus, Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene(WFS1). The WFS1 protein is an endoplasmic reticulum(ER) embedded protein, which functions in ER calcium homeostasis and unfolded protein responses. Dysregulation of these cellular processes results in the development of ER stress, leading to apoptosis. In addition, abundantly present WFS1 protein in insulin secretory granules plays a role in the intra-granular acidification. However, the phenotypic pleiomorphism and molecular complexity of this disease limit the understanding of WFS. Here we review clinical features, molecular mechanisms and mutations of WFS1 gene that relate to this syndrome.

Wolfram syndrome in the Japanese population; molecular analysis of WFS1 gene and characterization of clinical features.

BACKGROUND: Wolfram syndrome (WFS) is a recessive neurologic and endocrinologic degenerative disorder, and is also known as DIDMOAD (Diabetes Insipidus, early-onset Diabetes Mellitus, progressive Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene (WFS1). However, the phenotypic pleiomorphism, rarity and molecular complexity of this disease complicate our efforts to understand WFS. To address this limitation, we aimed to describe complications and to elucidate the contributions of WFS1 mutations to clinical manifestations in Japanese patients with WFS. METHODOLOGY: The minimal ascertainment criterion for diagnosing WFS was having both early onset diabetes mellitus and bilateral optic atrophy. Genetic analysis for WFS1 was performed by direct sequencing. PRINCIPAL FINDINGS: Sixty-seven patients were identified nationally for a prevalence of one per 710,000, with 33 patients (49%) having all 4 components of DIDMOAD. In 40 subjects who agreed to participate in this investigation from 30 unrelated families, the earliest manifestation was DM at a median age of 8.7 years, followed by OA at a median age of 15.8 years. However, either OA or DI was the first diagnosed feature in 6 subjects. In 10, features other than DM predated OA. Twenty-seven patients (67.5%) had a broad spectrum of recessive mutations in WFS1. Two patients had mutations in only one allele. Eleven patients (27.5%) had intact WFS1 alleles. Ages at onset of both DM and OA in patients with recessive WFS1 mutations were indistinguishable from those in patients without WFS1 mutations. In the patients with predicted complete loss-of-function mutations, ages at the onsets of both DM and OA were significantly earlier than those in patients with predicted partial-loss-of function mutations. CONCLUSION/SIGNIFICANCE: This study emphasizes the clinical and genetic heterogeneity in patients with WFS. Genotype-phenotype correlations may exist in patients with WFS1 mutations, as demonstrated by the disease onset.

X-box binding protein 1 is essential for insulin regulation of pancreatic α-cell function.

Patients with type 2 diabetes (T2D) often exhibit hyperglucagonemia despite hyperglycemia, implicating defective α-cell function. Although endoplasmic reticulum (ER) stress has been suggested to underlie ß-cell dysfunction in T2D, its role in α-cell biology remains unclear. X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), and its deficiency in ß-cells has been reported to impair insulin secretion, leading to glucose intolerance. To evaluate the role of XBP1 in α-cells, we created complementary in vivo (α-cell-specific XBP1 knockout [αXBPKO] mice) and in vitro (stable XBP1 knockdown α-cell line [αXBPKD]) models. The αXBPKO mice exhibited glucose intolerance, mild insulin resistance, and an inability to suppress glucagon secretion after glucose stimulation. αXBPKD cells exhibited activation of inositol-requiring enzyme 1, an upstream activator of XBP1, leading to phosphorylation of Jun NH2-terminal kinase. Interestingly, insulin treatment of αXBPKD cells reduced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) (pY(896)) and phosphorylation of Akt while enhancing serine phosphorylation (pS(307)) of IRS1. Consequently, the αXBPKD cells exhibited blunted suppression of glucagon secretion after insulin treatment in the presence of high glucose. Together, these data indicate that XBP1 deficiency in pancreatic α-cells induces altered insulin signaling and dysfunctional glucagon secretion.

Fe and P solubilization under limiting conditions by bacteria isolated from Carex kobomugi roots at the Hasaki coast.

Our objective was simply to report a sedge species, Carex kobomugi Ohwi that has beneficial bacterial associations under low Fe and P conditions of the Hasaki coast, Japan. C. kobomugi is the dominant species in our study area and grows closest to the sea. C. kobomugi showed higher Fe and P content, while these nutrients were less available under alkaline root-zone soil. Within the roots, mycorrhizal fungal colonization was absent, and endophytic fungal colonization was low. On the contrary, endophytic bacteria (e.g. Bacillus sp., Streptomyces luteogriseus, and Pseudomonas fluorescens) were isolated, which exhibited both siderophore production and inorganic phosphate solubilization under Fe or P limited conditions. Our results suggest that colonization of root tissue by these bacteria contribute to the Fe and P uptakes by C. kobomugi by increasing availability in the soil.

Altered insulin receptor signalling and ß-cell cycle dynamics in type 2 diabetes mellitus.

Insulin resistance, reduced ß-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced ß-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in ß-cells--which also exhibits reduced ß-cell mass, the ß-cell-specific insulin receptor knockout (ßIRKO). Freshly isolated islets and ß-cell lines derived from ßIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in ßIRKO ß-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in ß-cell growth. These data provide evidence that human ß- and α-cells can enter the cell-cycle, but proliferation of ß-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in ß-cell-cycle progression are attractive therapeutic targets to enhance ß-cell regeneration in the treatment of T2DM.

Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic ß-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic ß-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type ß-cells, there was a 32% reduction in the intensity in WFS1-deficient ß-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null ß-cells relative to that in wild-type ß-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of ß-cell dysfunction in patients with Wolfram syndrome.

Progressive multifocal leukoencephalopathy in a patient with multiple myeloma.

Progressive multifocal leukoencephalopathy (PML) is a neurological disease that affects immunodeficient patients. We describe here a case of 64-year-old man with IgD type multiple myeloma (MM) who developed progressive neurological symptoms. T(2)-weighted magnetic resonance imaging of the brain showed a hyperintense non-enhancing lesion in the left frontal lobe, and analysis of the cerebrospinal fluid by polymerase chain reaction revealed the presence of John Cunningham virus (JCV) DNA. Histopathological analysis of the autopsy brain specimen with in situ hybridization assay revealed the presence of JCV DNA in the nuclei of oligodendroglia. PML in a patient with MM is rare. However, this case report suggests that PML should be suspected and relevant diagnostic examinations should be performed when MM patients present with neurological symptoms.

Systemic AL amyloidosis with disseminated intravascular coagulation associated with hyperfibrinolysis.

Many coagulation abnormalities are known to coexist in patients with AL amyloidosis; however, disseminated intravascular coagulation (DIC) is rarely observed. We describe the case of a 61-year-old woman who presented with systemic purpura, macroscopic hematuria, and hepatosplenomegaly as the initial manifestations of systemic AL amyloidosis. A coagulation study revealed severe DIC associated with fibrinolysis. The patient was treated for DIC with gabexate mesilate (GM); however, her bleeding symptoms and thrombocytopenia continued to worsen. The treatment was changed from GM to nafamostat mesilate (NM); DIC improved gradually, and the platelet count normalized in 1 week. After the tapering and cessation of NM therapy, deterioration of DIC did not occur. She underwent autologous peripheral blood stem cell transplantation twice following high-dose melphalan therapy, and received maintenance therapy with thalidomide. Hepatosplenomegaly progression appears to have been halted, and DIC has not recurred. This is the first reported case of AL amyloidosis showing severe DIC with excessive fibrinolysis. The clinical observation that NM was considerably more effective than GM in our patient suggests that NM may be more suitable for the treatment of DIC with a hyperfibrinolytic condition in AL amyloidosis patients.

The antifungal compound totarol of Thujopsis dolabrata var. hondai seeds selects for fungi on seedling root surfaces.

Hinoki-asunaro (Thujopsis dolabrata Sieb. et Zucc. var. hondai Makino) is a tree endemic in Japan whose seeds produce several terpenoids. We hypothesized that antifungal compounds in seeds might select for fungi on the root surfaces of T. dolabrata var. hondai seedlings. We examined seed and soil fungi, their sensitivity to methanol extracts of the seeds, the fungi on root surfaces of seedlings grown in Kanuma pumice (a model mineral soil) and nursery soil, and the frequency at which each fungus was detected on the seedling root surface. We calculated correlation coefficients between fungal detection frequency on root surfaces and fungal sensitivity to seed extracts. We also isolated from the seeds the antifungal compound totarol that selected for fungi on root surfaces. Species of Alternaria, Cladosporium, Pestalotiopsis, and Phomopsis were the most frequently isolated fungi from seeds. Mortierella and Mucor were the dominant fungi isolated from Kanuma pumice, whereas Umbelopsis and Trichoderma were the main fungi isolated from nursery soil. Alternaria, Cladosporium, Mortierella, Pestalotiopsis, and Phomopsis were the dominant fungi isolated from root surfaces of seedlings grown in Kanuma pumice, and Alternaria, Cladosporium, Pestalotiopsis, Phomopsis, and Trichoderma were the main root-surface fungi isolated from seedlings grown in nursery soil. The fungal detection frequencies on root surfaces in both soils were significantly and negatively correlated with fungal sensitivity to the seed extract. A similar correlation was found between the fungal detection frequency on root surfaces and fungal sensitivity to totarol. We conclude that totarol is one factor that selects for fungi on root surfaces of T. dolabrata var. hondai in the early growth stage.

Endoplasmic reticulum stress induces Wfs1 gene expression in pancreatic beta-cells via transcriptional activation.

OBJECTIVE: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic beta-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses. DESIGN AND METHODS: Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic beta-cell-derived MIN6 cells, and Akita mouse-derived Ins2 (96Y/Y) insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted. RESULT: Islet beta-cells were the major site of Wfs1 expression. This expression was also found in delta-cells, but not in alpha-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 (96Y/Y) insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 (96Y/Y) cells than Ins2 (wild/wild) cells. CONCLUSION: Wfs1 expression, which is localized to beta- and delta-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.