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Objective: Islet beta-cells loose their ability to synthesize insulin under diabetic conditions, which is at least partially due to the decreased activity of insulin transcription factors such as MafA. Although an in vitro study showed that reactive oxygen species (ROS) decrease MafA expression, the underlying mechanism still remains unclear. In this study, we examined the effects of c-Jun, which is known to be upregulated by ROS, on the expression of MafA under diabetic conditions.Research Design and Methods: To examine the protein levels of MafA and c-Jun, we performed histological analysis and Western blotting using diabetic db/db mice. In addition, to evaluate the possible effects of c-Jun on MafA expression, we performed adenoviral overexpression of c-Jun in the MIN6 beta-cell line and freshly isolated islets. RESULTS MafA expression was markedly decreased in the islets of db/db mice, while in contrast c-Jun expression was increased. Costaining of these factors in the islets of db/db mice clearly showed that MafA and insulin levels are decreased in c-Jun-positive cells. Consistent with these results, overexpression of c-Jun significantly decreased MafA expression, accompanied by suppression of insulin expression. Importantly, MafA overexpression restored the insulin promoter activity and protein levels that were suppressed by c-Jun. These results indicate that the decreased insulin biosynthesis induced by c-Jun is principally mediated by the suppression of MafA activity.Conclusions: It is likely that the augmented expression of c-Jun in diabetic islets decreases MafA expression and thereby reduces insulin biosynthesis, which is often observed in type 2 diabetes. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Islet β-cells loose their ability to synthesize insulin under diabetic conditions, which is at least partially due to the decreased activity of insulin transcription factors such as MafA. Although an in vitro study showed that reactive oxygen species (ROS) decrease MafA expression, the underlying mechanism still remains unclear. In this study, we examined the effects of c-Jun, which is known to be upregulated by ROS, on the expression of MafA under diabetic conditions. RESEARCH DESIGN AND METHODS--To examine the protein levels of MafA and c-Jun, we performed histological analysis and Western blotting using diabetic db/db mice. In addition, to evaluate the possible effects of c-Jun on MafA expression, we performed adenoviral overexpression of c-Jun in the MIN6 β-cell line and freshly isolated islets. RESULTS--MafA expression was markedly decreased in the islets of db/db mice, while in contrast c-Jun expression was increased. Costaining of these factors in the islets of db/db mice clearly showed that MafA and insulin levels are decreased in c-Jun-positive cells. Consistent with these results, overexpression of c-Jun significantly decreased MafA expression, accompanied by suppression of insulin expression. Importantly, MafA overexpression restored the insulin promoter activity and protein levels that were suppressed by c-Jun. These results indicate that the decreased insulin biosynthesis induced by c-Jun is principally mediated by the suppression of MafA activity. CONCLUSIONS--It is likely that the augmented expression of c-Jun in diabetic islets decreases MafA expression and thereby reduces insulin biosynthesis, which is often observed in type 2 diabetes. Diabetes 59:1709-1720, 2010 [ABSTRACT FROM AUTHOR]

Background: Vascular complication of type 1 diabetes is associated with multiple factors. However, biomarkers which directly reflect vascular complication have not been elucidated. We have developed a method to detect unmethylated status of pancreatic β cell specific CpG sequences of insulin gene from cell-free DNA (cfDNA) in the circulation. Transmembrane protein ROBO4 is highly expressed in vascular endothelial cells, and it has been reported that the CpG sites in the promoter region are specifically unmethylated. Aim: To develop a novel PCR method to specifically detect unmethylated CpG sites of ROBO4 gene promoter region in serum cfDNA and to evaluate this method in the supernatant of vascular endothelial cell culture and serum of patients with type 1 diabetes. Material and Methods: After bisulfite conversion of cfDNA extracted from human umbilical vein endothelial cells (HUVEC) and patient serum, unmethylated ROBO4 gene was detected by the dual Amplifications Refractory Mutation Systems PCR, which amplifies only if 4 CpG sites (-185, -144, -118 and -103bp of transcription start sites of ROBO4) are all unmetylated. These methods were applied to 83 patients with type 1 diabetes (duration 16.1±13.9 years) and clinical characteristics were compared. Results: In HUVEC culture supernatant under constant and intermittent high glucose condition, the detection of ROBO4 gene copy number increased depending on the number of dead cells and cfDNA concentration. A positive correlation (p <0.05) with the mean intima-media thickness (IMT) and MCV were observed for the ROBO4 copy numbers in patient serum cfDNA. According to the multiple regression analysis, unmethylated ROBO4 copy number (p <0.001) and age (p <0.001) were selected as the determinant factors of mean IMT. Conclusion: Novel PCR method for ROBO4 could detect vascular endothelial cell injury and atherosclerosis quantitatively in patients with type 1 diabetes. Disclosure: A. Kuroda: Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc., Sanofi. M.Y. Yamada: None. Y. Takashi: None. M. Ohishi: None. H. Mori: None. M. Ishizu: None. R. Suzuki: None. O. Yinhua: None. Y. Tominaga: None. M. Matsuhisa: Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Sanofi, Takeda Pharmaceutical Company Limited. Funding: Japan Society for the Promotion of Science [ABSTRACT FROM AUTHOR]

Among various glycosylated proteins, glycosylated hemoglobin (HbA[sub 1c]) is most widely used to evaluate the long-term glycemic control and the risk for the development of complications in patients with type 1 and type 2 diabetes. Glycosylated serum albumin has been repotted to be a useful indicator of glycemic control for diabetic patients. However, clinical utility of glycosylated albumin has not been fully evaluated, especially the relation with the degree of daily glycemic profile. We, therefore, compared HbA[sub 1c] and glycosylated albumin to the daily profiles of blood glucose in patients with type 1 and type 2 diabetes. Sixty three type 1 diabetic patients (age 25.5±5.3 years, duration 15.6±6.3 years) and fifty eight type2 diabetic patients (age 59.3±11.0 years, duration 12.8±7.5 years) were enrolled in this study. These patients were not suffered from advanced micro and macro diabetic angiopathy. The daily profile of glucose levels were determined by the self-monitoring blood glucose testing before and 2-hour after meals and at bedtime. Glycosylated serum albumin levels were determined by using the enzymatic glycosylated albumin assay kit (Lucica GA). Glycosylated serum albumin (GA) was highly correlated with HbA[sub 1C] in type 1 (GA 24.4 ± 4.7%, HbA[sub 1C] 7.3 ± 1.1%, r = 0.88, P< 0.0001) and type 2 diabetic patients (GA 26.4 ± 7.4%, HbA[sub 1C] 9.1 ± 2.1%, r=0.79, P<0.0001). Interestingly, the ratio of glycosylated albumin to HbA[sub 1c] in type 1 diabetes was significantly higher than that of type 2 diabetes (3.3 ± 0.3 vs 2.9 ± 0.5). Glycosylated albumin was significantly correlated with maximum blood glucose in both type 1 (r=0.421, P<0.005) and type 2 diabetes (r=0.719. P<0.0001). The stepwise multivariate regression analysis revealed in type 1 and type 2 diabetes that maximum blood glucose is independently correlated with glycosylated albumin among the various index determined from glucose profile, such as mean glucose level and MAGE. In conclusion, glycosylated serum albumin, as well as HbA[sub 1C], is a reliable indicator of averaged glucose levels in patients with type 1 and type 2 diabetes. Glycosylated albumin may partially reflect the degree of higher excursion of glucose. [ABSTRACT FROM AUTHOR]

Islet β cells loose its ability to synthesize insulin under diabetic condition at least partially due to decreased activity of insulin transcription factors including MafA. Although in vitro study showed reactive oxygen species generated by high glucose decreased the expression of MafA, its underlying mechanism is still unclear. Our objective is to analyze the possible mechanism by which MafA is affected in diabetic β cells, Immunohistochemistry revealed that protein level of MafA was decreased in the islets of db/db mice but that expression of c-Jun, which is up-regulated by reactive oxygen species, was increased contrastively. Co-staining of those factors in islets of db/db mice clearly showed the lower expression of MafA and insulin in the c-Jun positive cells. Consistent with these results, co-transfection of c-Jun expression vector with insulin reporter plasmid into MIN6 β cell line decreased the insulin promoter activity to 25% as reported previously. Adenoviral overexpression of c-Jun in MIN6 cells also markedly and selectively decreased MafA on the analysis of both western blotting and immunocytochmistry. On the other hand, putting mutation in basic region or transactivation domain of c-Jun mined its effects on MafA and insulin. Importantly, MafA overexpression restored the insulin promoter activity and insulin protein amount suppressed by c-Jun overexpression on reporter gene analysis and immunocytochemistry, respectively. These results indicate that decreased insulin synthesis induced by c-Jun is principally mediated by suppression of MafA activity. Interestingly, c-Jun had almost no effects on the level of MafAmRNA. Furthermore, MafA promoter activity using reporter plasmids including various length of MafA enhancer and promoter region was not affected by c-Jun. Thus, we conclude that augmented expression of c-Jun in diabetic islets decreases MafA activity followed by reduced insulin synthesis, and that MafA expression is regulated by c-Jun mainly at the post-transcriptional level. [ABSTRACT FROM AUTHOR]

It is well known that activating protein-1 (AP-1) is involved in a variety of cellular function such as proliferation, differentiation, apoptosis, and ontogenesis. While AP-1 is a dimmer complex consisting of different subunits, c-Jun is known as one of its major components. Also, it has been shown that mice lacking c-Jun are embryonic lethal and that c-Jun is essential for liver and heart development, but a role of c-Jun in pancreas is not well known. Pancreas has its embryological origin in two buds developing on the dorsal and ventral side of the duodenum. The ventral bud arises immediately adjacent to the hepatic diverticulum, and the dorsal bud arises on the opposite side of the gut tube. The aim of this study was to examine a possible role c-Jun in pancreas. First, c-Jun was strongly expressed in pancreatic duct-like structures at embryonic day15.5, while lower level expression was observed in some population of adult pancreas, implying that c-Jun might play some role during pancreas development. Second, to address this point, we generated pancreas-specific c-Jun knock-out mice (Ptf1a-Cre; c-Jun flox/flox mice) by crossing Ptf1a-Cre knock-in mice with e-Jun floxed mice. It is noted that Ptf1a is a transcription factor which expression is confined to pancreatic stem/progenitor cells, which give rise to all three types of pancreatic tissue: endocrine, exocrine, and duct. The deletion of c-Jun in Ptf1a-Cre c-Jun flox/flox pancreas was reconfirmed by immunohistochemistry at embryonic day 15.5 and RT-PCR at adult stage. Contrary to our expectation, however, there was no morphological difference in pancreas between Ptf1a-Cre; c-Jun flox/flox mice and control littermates. Also, there was no difference in body weight, pancreas weight, and insulin expression between two groups. Furthermore, to examine whether deletion of c-Jun influences glucose tolerance, we performed intraperitoneal glucose tolerance test, but there was no clear difference in glucose tolerance between Ptf1a-Cre; e-Jun flox/flox and control mice. These results indicate that c-Jun is dispensable for pancreas development at least after Ptf1a promoter is activated. [ABSTRACT FROM AUTHOR]