Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both alpha- and beta-cells, and MafA and MafB showed selective expression in the beta- and alpha-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of beta-cells proceeds through a MafB+ MafA- Ins+ intermediate cell to MafB- MafA+ Ins+ cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1(high)) in MafB+ Ins+ cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both beta- and alpha-cells while MafA may regulate replication/survival and function of beta-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth.

Recent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferentially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.

No abstract

Our data show that M1 macrophage content of atherosclerotic plaques is associated with clinical incidence of ischemic stroke and increased inflammation or fibrinolysis. We also show the benefits of using ultrasound to evaluate vulnerability in the plaques.

BackgroundPerilipin 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 FindingsWhen 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.ConclusionsThese 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.

ELL was originally identified as a gene that undergoes translocation with the trithorax-like MLL 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.The ELL gene (also known as MEN) was molecularly identified as a gene that was fused to the Drosophila trithorax-like MLL gene (also called ALL-1 or Hrx) in acute myeloid leukemia carrying the t(11;19)(q23;p13.1) chromosomal translocation (1, 2). The gene product, ELL, was recently demonstrated to function as an RNA polymerase II elongation factor that increases the overall rate of transcription elongation by RNA polymerase II via suppression of transient pausing by the polymerase at many sites along DNA (3).The connection between transcription elongation factors and oncogenesis was first provided by the finding that the product of VHL tumor suppressor gene, VHL, associates with the complex of the B and C regulatory subunits of another transcription elongation factor, elongin (4, 5). Binding of VHL to the elongin BC complex inhibits its ability to activate elongin A subunit in transcription elongation. The observation that many of the naturally occurring VHL mutants exhibit substantially reduced binding to the elongin BC complexes led to the suggestion that tumor suppression by VHL may involve negative regulation of elongin as a transcription elongation factor.ELL is the second transcription elongation factor found associated with human malignancy. The chimeric protein generated by MLL-ELL gene translocation contains an N-terminal AT-hook DNA binding domain and the methyltransferase-like domain of MLL that is fused to almost the entire ELL sequences (1, 2). Expression of the MLL portion of the fusion protein appears to be insufficient for inducing the leukemic phenotype ...

ObjectivesGLP-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 MethodsIn 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.ResultsA 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.ConclusionsRegardless 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 RegistrationUMIN Clinical Trials Registry System as trial ID UMIN000005331.

Mammalian MafA/RIPE3b1 is an important glucose-responsive transcription factor that regulates function, maturation, and survival of beta-cells. Increased expression of MafA results in improved glucose-stimulated insulin secretion and beta-cell function. Because MafA is a highly phosphorylated protein, we examined whether regulating activity of protein kinases can increase MafA expression by enhancing its stability. We demonstrate that MafA protein stability in MIN6 cells and isolated mouse islets is regulated by both p38 MAPK and glycogen synthase kinase 3. Inhibiting p38 MAPK enhanced MafA stability in cells grown under both low and high concentrations of glucose. We also show that the N-terminal domain of MafA plays a major role in p38 MAPK-mediated degradation; simultaneous mutation of both threonines 57 and 134 into alanines in MafA was sufficient to prevent this degradation. Under oxidative stress, a condition detrimental to beta-cell function, a decrease in MafA stability was associated with a concomitant increase in active p38 MAPK. Interestingly, inhibiting p38 MAPK but not glycogen synthase kinase 3 prevented oxidative stress-dependent degradation of MafA. These results suggest that the p38 MAPK pathway may represent a common mechanism for regulating MafA levels under oxidative stress and basal and stimulatory glucose concentrations. Therefore, preventing p38 MAPK-mediated degradation of MafA represents a novel approach to improve beta-cell function.