E4BP4 in macrophages induces an anti-inflammatory phenotype that ameliorates the severity of colitis.

Macrophages are versatile cells of the innate immune system that work by altering their pro- or anti-inflammatory features. Their dysregulation leads to inflammatory disorders such as inflammatory bowel disease. We show that macrophage-specific upregulation of the clock output gene and transcription factor E4BP4 reduces the severity of colitis in mice. RNA-sequencing and single-cell analyses of macrophages revealed that increased expression of E4BP4 leads to an overall increase in expression of anti-inflammatory genes including Il4ra with a concomitant reduction in pro-inflammatory gene expression. In contrast, knockout of E4BP4 in macrophages leads to increased proinflammatory gene expression and decreased expression of anti-inflammatory genes. ChIP-seq and ATAC-seq analyses further identified Il4ra as a target of E4BP4, which drives anti-inflammatory polarization in macrophages. Together, these results reveal a critical role for E4BP4 in regulating macrophage inflammatory phenotypes and resolving inflammatory bowel diseases.

Correction: A consensus statement from the Japan Diabetes Society (JDS): a proposed algorithm for pharmacotherapy in people with type 2 diabetes.

[This corrects the article DOI: 10.1007/s13340-022-00605-x.].

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.

Liver-specific dysregulation of clock-controlled output signal impairs energy metabolism in liver and muscle.

The liver is the major organ maintaining metabolic homeostasis in animals during shifts between fed and fasted states. Circadian oscillations in peripheral tissues including the liver are connected with feeding-fasting cycles. We generated transgenic mice with hepatocyte specific E4BP4, D-box negative regulator, overexpression. Liver-specific E4BP4 overexpression was also achieved by adenoviral gene transfer. Interestingly, hepatic E4BP4 overexpression induced marked insulin resistance, that was rescued by DBP, a competing D-box positive regulator, overexpression. At basal conditions hepatocyte E4BP4 transgenic mice exhibited increased gluconeogenesis with reduced AKT phosphorylation in liver. In muscle, AKT phosphorylation was impaired after insulin stimulation. Such muscle insulin resistance was associated with elevated free fatty acid flux from the liver and reduced fatty acid utilization as an energy source during the inactive phase. E4BP4, one of the clock-controlled output genes, are key metabolic regulators in liver adjusting liver and muscle metabolism and insulin sensitivity in the feeding-fasting cycles. Its tuning is critical for preventing 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.

Molecular clock as a regulator of ß-cell function.

Molecular clocks are important for the circadian regulation of ß-cell function. DBP/E4BP4 plays central roles among clock-related genes in the metabolic regulation.

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 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.

Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1ß gene expression in pancreatic islet ß-cells.

Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1ß (HIF-1ß) has emerged as a potential determinant of pancreatic ß-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1(-/-) A(y)/a mice that develop severe diabetes due to ß-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.

[Insulin secretion and insulin resistance].

Aging is a critical risk factor for impaired glucose tolerance and diabetes. In Japan, 8.9 million people are reported to have diabetes, and 37% of those are over the age of 70. In this review, we summarize the current evidence on how aging affects pancreatic beta cell function, beta cell mass, insulin secretion and insulin sensitivity. The pathogenesis of type 2 diabetes (T2DM) in aging is characterized by two major features: impaired insulin secretion and peripheral insulin resistance. Understanding the mechanism that lead to impaired glucose homeostasis and T2DM in the elderly will lead to development of novel treatments that will prevent or delay diabetes, substantially improve quality of life and ultimately increase overall life span.

Convergence of the insulin and serotonin programs in the pancreatic ß-cell.

OBJECTIVE: Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. RESEARCH DESIGN AND METHODS: We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. RESULTS: We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In ß-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. CONCLUSIONS: These studies demonstrate that a common transcriptional cascade drives the differentiation of ß-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes.

The usefulness of vibration perception threshold as a significant indicator for erectile dysfunction in patients with diabetes mellitus at a primary diabetes mellitus clinic.

INTRODUCTION: The aim of this study is to clarify the relationship of erectile dysfunction (ED) and diabetes mellitus (DM) parameters (referred to with '1'), including peripheral neuropathy (referred to with '2'). METHODS: (1) The DM parameters including age, serum levels of blood sugar, hemoglobin A1c, duration of DM and number of DM complications were obtained from 145 patients at a general DM clinic. (2) The peripheral neuropathy examinations by vibration perception threshold (VPT) and Achilles tendon reflex were performed in 97 DM patients. Erectile functions in DM patients were evaluated by the International Index of Erectile Function (IIEF 5). The DM patients' parameters were compared with the IIEF 5 scores. RESULTS: (1) The data showed IIEF 5 scores were significantly correlated with patient age, duration of DM and number of DM complications. (2) IIEF 5 scores were significantly correlated with VPT time. Furthermore, multiple regression analysis revealed that patient age and VPT time were independent risk factors for predicting ED in DM patients. CONCLUSIONS: The severity of ED in DM patients depended on age, duration of DM, number of DM complications and VPT. Significantly, the age of DM patients and the measurement of VPT are considered to be simple and useful indicators to diagnose ED in DM patients.

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.

Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation.

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.

Unregulated elevation of glutamate dehydrogenase activity induces glutamine-stimulated insulin secretion: identification and characterization of a GLUD1 gene mutation and insulin secretion studies with MIN6 cells overexpressing the mutant glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to alpha-ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8--25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic beta-cells.