Takuma Kondo

Perilipin Overexpression in White Adipose Tissue Induces a Brown Fat-Like Phenotype

Background Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model. Methodology and Principal Findings When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size. Conclusions These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.

PHYSICAL INTERACTION AND FUNCTIONAL ANTAGONISM BETWEEN THE RNA POLYMERASE II ELONGATION FACTOR ELL AND P53

ELL was originally identified as a gene that undergoes translocation with the trithorax-likeMLL gene in acute myeloid leukemia. Recent studies have shown that the gene product, ELL, functions as an RNA polymerase II elongation factor that increases the rate of transcription by RNA polymerase II by suppressing transient pausing. Using yeast two-hybrid screening with ELL as bait, we isolated the p53 tumor suppressor protein as a specific interactor of ELL. The interaction involves respectively the transcription elongation activation domain of ELL and the C-terminal tail of p53. Through this interaction, ELL inhibits both sequence-specific transactivation and sequence-independent transrepression by p53. Thus, ELL acts as a negative regulator of p53 in transcription. Conversely, p53 inhibits the transcription elongation activity of ELL, suggesting that p53 is capable of regulating general transcription by RNA polymerase II through controlling the ELL activity. Elevated levels of ELL in cells resulted in the inhibition of p53-dependent induction of endogenous p21 and substantially protected cells from p53-mediated apoptosis that is induced by genotoxic stress. Our observations indicate the existence of a mutually inhibitory interaction between p53 and a general transcription elongation factor ELL and raise the possibility that an aberrant interaction between p53 and ELL may play a role in the genesis of leukemias carrying MLL-ELL gene translocations.

A Comparison of the Effects of the GLP-1 Analogue Liraglutide and Insulin Glargine on Endothelial Function and Metabolic Parameters : A Randomized, Controlled Trial Sapporo Athero-Incretin Study 2 (SAIS2)

Objectives GLP-1 improves hyperglycemia, and it has been reported to have favorable effects on atherosclerosis. However, it has not been fully elucidated whether GLP-1 is able to improve endothelial function in patients with type 2 diabetes. Therefore, we investigated the efficacy of the GLP-1 analogue, liraglutide on endothelial function and glycemic metabolism compared with insulin glargine therapy. Materials and Methods In this multicenter, prospective randomized parallel-group comparison study, 31 diabetic outpatients (aged 60.3 ± 10.3 years with HbA1c levels of 8.6 ± 0.8%) with current metformin and/or sulfonylurea treatment were enrolled and randomly assigned to receive liraglutide or glargine therapy once daily for 14 weeks. Flow mediated dilation (FMD), a comprehensive panel of hemodynamic parameters (Task Force Monitor), and serum metabolic markers were assessed before and after the treatment period. Results A greater reduction (worsening) in %FMD was observed in the glargine group, although this change was not statistically different from the liraglutide group (liraglutide; 5.7 to 5.4%, glargine 6.7 to 5.7%). The augmentation index, C-peptide index, derivatives of reactive oxygen metabolites and BMI were significantly improved in the liraglutide group. Central systolic blood pressure and NT-proBNP also tended to be improved in the liraglutide-treated group, while improvements in HbA1c levels were similar between groups. Cardiac index, blood pressure and most other metabolic parameters were not different. Conclusions Regardless of glycemic improvement, early liraglutide therapy did not affect endothelial function but may provide favorable effects on beta-cell function and cardioprotection in type 2 diabetics without advanced atherosclerosis. Trial Registration UMIN Clinical Trials Registry System as trial ID UMIN000005331.

Problems in diagnosing atypical Gitelman’s syndrome presenting with normomagnesaemia

Objective  Gitelman’s syndrome, recognized as a variant of Bartter’s syndrome, is characterized by hypokalaemic metabolic alkalosis in combination with hypomagnesaemia and hypocalciuria. Overlapping biochemical features in Gitelman’s syndrome and Bartter’s syndrome has been observed. Here, we investigated the clinical, biochemical, and genetic characteristics of five, chronic, nonhypertensive and hypokalaemic Japanese patients.

Ovarian morphology and prevalence of polycystic ovary syndrome in Japanese women with type 1 diabetes mellitus

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder including polycystic ovary morphology (PCOM), ovulatory dysfunction and hyperandrogenism. PCOS is frequently associated with type 2 diabetes mellitus; however, it is unknown whether PCOM and PCOS are prevalent in Japanese patients with type 1 diabetes mellitus. The purpose of our study was to determine the frequency of PCOM and PCOS in women with type 1 diabetes mellitus.

Inhibition of Small Maf Function in Pancreatic β-Cells Improves Glucose Tolerance Through the Enhancement of Insulin Gene Transcription and Insulin Secretion

The large-Maf transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) has been found to be crucial for insulin transcription and synthesis and for pancreatic β-cell function and maturation. However, insights about the effects of small Maf factors on β-cells are limited. Our goal was to elucidate the function of small-Maf factors on β-cells using an animal model of endogenous small-Maf dysfunction. Transgenic (Tg) mice with β-cell-specific expression of dominant-negative MafK (DN-MafK) experiments, which can suppress the function of all endogenous small-Mafs, were fed a high-fat diet, and their in vivo phenotypes were evaluated. Phenotypic analysis, glucose tolerance tests, morphologic examination of β-cells, and islet experiments were performed. DN-MafK-expressed MIN6 cells were also used for in vitro analysis. The results showed that DN-MafK expression inhibited endogenous small-Maf binding to insulin promoter while increasing MafA binding. DN-MafK Tg mice under high-fat diet conditions showed improved glucose metabolism compared with control mice via incremental insulin secretion, without causing changes in insulin sensitivity or MafA expression. Moreover, up-regulation of insulin and glucokinase gene expression was observed both in vivo and in vitro under DN-MafK expression. We concluded that endogenous small-Maf factors negatively regulates β-cell function by competing for MafA binding, and thus, the inhibition of small-Maf activity can improve β-cell function.

Epalrestat Improves Diabetic Wound Healing via Increased Expression of Nerve Growth Factor

AIMS/INTRODUCTION Aldose reductase inhibitors (ARIs) are a useful therapy for diabetic neuropathy. Nerve damage is associated with delayed wound healing of skin ulcers in diabetic patients. Therefore, we hypothesized that ARI supplementation would improve diabetic wound healing. MATERIALS AND METHODS Control and streptozotocin-induced diabetic mice were fed either control diet or diet containing the ARI Epalrestat (40 mg/kg). After 12 weeks, we created skin wounds on the backs of the mice. Wound healing was determined by measuring the reduction in wound area. RESULTS The wound gap of the diabetic group was significantly larger 9 days after creating the wounds when compared to the other groups (p<0.01). Interestingly, wound healing in the diabetic mice fed Epalrestat was comparable to the non-diabetic mice. To clarify the mechanism(s) behind this improved wound healing, mRNA expression of growth factors reported to be involved in wound healing were examined. Among the growth factors investigated, only the expression of nerve growth factor (NGF) was -significantly decreased (54.0%) in the healing lesions of diabetic mice. Similarly, NGF protein expression was decreased in diabetic mice and recovered in Epalrestat treated diabetic mice. Inhibition of NGF via 2 separate inhibitors (K252a and BSO) reduced the ability of Epalrestat to improve wound healing in diabetic mice. CONCLUSIONS These findings suggest that Epalrestat is a potential therapy for improving diabetic wound healing and the mechanism involves upregulation of NGF.

Expression and Regulation of Neuromedin B in Pituitary Corticotrophs of Male Melanocortin 2 Receptor-Deficient Mice

The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R(-/-) mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R(-/-) mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R(-/-) mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R(-/-) than in the wild-type (WT) mice. We detected NMB expression in the MC2R(-/-) pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R(-/-) mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Effects of 50 mg vildagliptin twice daily vs. 50 mg sitagliptin once daily on blood glucose fluctuations evaluated by long-term self-monitoring of blood glucose

To date, several clinical trials have compared differences in glucose fluctuation observed with dipeptidyl peptidase-4 inhibitor treatment in patients with type 2 diabetes mellitus. However, most patients were assessed for limited periods or during hospitalization. The aim of the present study was to evaluate the effects of switching from sitagliptin to vildagliptin, or vice versa, on 12-week glucose fluctuations using self-monitoring of blood glucose in the standard care setting. We conducted a multicenter, prospective, open-label controlled trial in Japanese patients with type 2 diabetes. Thirty-two patients were treated with vildagliptin (50 mg) twice daily or sitagliptin (50 mg) once daily and were allocated to one of two groups: vildagliptin treatment for 12 weeks before switching to sitagliptin for 12 weeks, or vice versa. Daily profiles of blood glucose were assessed several times during each treatment period, and the mean amplitude of glycemic excursions and M-value were calculated. Metabolic biomarkers such as hemoglobin A1c (HbA1c), glycated albumin, and 1,5-anhydroglucitol were also assessed. With vildagliptin treatment, mean amplitude of glycemic excursions was significantly improved compared with sitagliptin treatment (57.9 ± 22.2 vs. 68.9 ± 33.0 mg/dL; p=0.0045). M-value (p=0.019) and mean blood glucose (p=0.0021) were also lower with vildagliptin, as were HbA1c, glycated albumin, and 1,5-anhydroglucitol. There were no significant differences in other metabolic parameters evaluated. Reduction of daily blood glucose profile fluctuations by vildagliptin was superior to that of sitagliptin in Japanese patients with type 2 diabetes.