Mutation of the Pax6 gene causes impaired glucose-stimulated insulin secretion.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Y Fujitani
Publications and source records attributed to Y Fujitani.
Ectopically expressed PDX-1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis.
To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into beta-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken beta-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.
Animals↗
IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation.
BACKGROUND AND OBJECTIVES: Lung remodelling is a recognized feature of chronic asthma. In the present study, we have used IL-5-deficient mice to evaluate the role of this cytokine and eosinophilic inflammation in the initial stages of the structural changes occurring in the lung after antigen challenge. METHODS: Ovalbumin-sensitized wild type and IL-5-deficient mice were daily challenged for 5 consecutive days and killed 3 or 7 days after the last challenge to study the inflammatory and remodelling events, respectively. RESULTS: Wild type mice challenged with ovalbumin exhibited an accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid, associated with a production of BAL cellular fibronectin. Histological analysis also revealed an antigen-specific increase in epithelial and alveolar cell proliferation together with an increase in mucus producing epithelial cells. Eosinophilic infiltration and the associated lung remodelling were totally abrogated in IL-5-deficient mice. In wild type mice, treated intranasally with 1 microg of murine IL-5 for 5 consecutive days, no BAL eosinophilia and structural changes of the lungs could be observed. CONCLUSION: Our results demonstrate that eosinophil accumulation, but not IL-5 alone, plays a central role in the initial stages of the lung remodelling process and suggests that therapies directed at inhibiting eosinophilic inflammation may be beneficial in treating chronic asthma.
Animals↗
A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese.
Pax4 is one of the transcription factors that play an important role in the differentiation of islet beta-cells. We scanned the Pax4 gene in 200 unrelated Japanese type 2 diabetic patients and found a missense mutation (R121W) in 6 heterozygous patients and 1 homozygous patient (mutant allele frequency 2.0%). The mutation was not found in 161 nondiabetic subjects. The R121W mutation was located in the paired domain and was thought to affect its transcription activity through lack of DNA binding. Six of seven patients had family history of diabetes or impaired glucose tolerance, and four of seven had transient insulin therapy at the onset. One of them, a homozygous carrier, had relatively early onset diabetes and slowly fell into an insulin-dependent state without an autoimmune-mediated process. This is the first report of a Pax4 gene mutation that exhibits loss of function and seems to be associated with type 2 diabetes. This work provides significant implications for the Pax4 gene as one of the predisposing genes for type 2 diabetes in the Japanese.
Adult↗
Effect of a novel bifunctional endothelin receptor antagonist, IRL 3630A, on guinea pig respiratory mechanics.
This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[N-(3, 5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)- alanyl]-( L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET(A) and ET(B) receptors, giving apparent K(i) values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca(2+) increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET(A) receptors and human Girardi heart (hGH) cells expressing endothelin ET(B) receptors. In guinea pig isolated tracheas having both endothelin ET(A) and ET(B) receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2)=7.1), which was partially or scarcely suppressed by the endothelin ET(A) receptor antagonist cyclo[-(D)-Trp-(D)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ET(B) receptor antagonist N-(3, 5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-phenylalanyl-(L)-t ryptop han (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 microg/kg inhibited endothelin-1 (1.3 microg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1, 3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4, 5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET(A) receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 microg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.
Airway Resistance↗
Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression.
Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-gamma). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.
Administration, Intranasal↗
Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells.
Extracellular stimuli trigger adipocyte differentiation by inducing the complex cascades of transcription. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma (PPARgamma) play crucial roles in this process. Although ectopic expression of these factors in NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in adipogenic conversion, little is known as to hormonal factors that regulate adipogenesis in these cells. In this report we demonstrate that PRL, a lactogenic hormone, enhances C/EBPbeta and PPARbeta mRNA expression and augments adipogenic conversion of NIH-3T3 cells. Moreover, we show that ectopic expression of the PRL receptor in NIH-3T3 cells results in efficient adipocyte conversion when stimulated with PRL and a PPARgamma ligand, as evidenced by expression of the adipocyte differentiation-specific genes as well as the presence of fat-laden cells. We further demonstrate that signal transducer and activator of transcription 5 (Stat5), a PRL signal transducer, activates aP2 promoter in a PRL-dependent manner. These results suggest that PRL acts as an adipogenesis-enhancing hormone in NIH-3T3 cells.
3T3 Cells↗
Identification of oxidative stress-regulated genes in rat aortic smooth muscle cells by suppression subtractive hybridization.
A suppression subtractive hybridization technique was used to identify reactive oxygen species (ROS)-regulated genes in rat vascular smooth muscle cells. Three genes out of 89 clones, identified as fibronectin, p105 coactivator and ECA39, showed increased expression after treatment with H(2)O(2). The mRNA expressions of these three genes were induced in a time- and dose-dependent manner, independent of protein kinase C activation. Immunohistochemical staining showed that the p105 coactivator expression was markedly induced in the neointima of balloon-injured rat carotid arteries. These results suggest that ROS may play an important role in the development of atherosclerosis by regulating the gene expressions we identified in this study.
Animals↗
Oxidative stress induces p21 expression in pancreatic islet cells: possible implication in beta-cell dysfunction.
AIMS/HYPOTHESIS: Prolonged poor glycaemic control in patients with Type II (non-insulin-dependent) diabetes mellitus often causes pancreatic beta-cell dysfunction accompanied by decreases in insulin biosynthesis and beta-cell proliferation. This is well known as a clinical concept called glucose toxicity. Whereas oxidative stress is provoked under diabetic conditions, we examined the possible implication of cyclin-dependent kinase (Cdk) inhibitor p21 (WAF1/CIP1/Sdi1) in beta-cell dysfunction mediated by oxidative stress. METHODS: Oxidative stress was induced in isolated rat pancreatic islet cells by treatment with H(2)O(2) and mRNA expression of p21 and insulin was examined by northern blot analyses. Also, the expression of p21 and insulin mRNA was examined in Zucker diabetic fatty rat. In islet cells p21 was overexpressed using adenovirus and its effect on insulin gene transcription was examined. RESULTS: When oxidative stress was charged on isolated rat pancreatic islet cells, p21 mRNA expression was induced whereas insulin mRNA was decreased. Also, when diabetes developed in Zucker diabetic fatty rats, p21 expression was induced and the insulin mRNA expression was reduced. As support for the implication of p21 in impairment of beta-cell function, the p21 overexpression in the islet cells suppressed the insulin gene transcription. CONCLUSIONS/INTERPRETATION: The expression of cyclin-dependent kinase inhibitor p21, which can be induced by oxidative stress, increases in pancreatic islet cells upon development of diabetes. By suppressing cell proliferation and insulin biosynthesis, the p21 induction is likely to be implicated in the beta-cell glucose toxicity.
Animals↗
Induction of glycation suppresses glucokinase gene expression in HIT-T15 cells.
AIMS/HYPOTHESIS: Chronic hyperglycaemia in patients with Type II (non-insulin-dependent) diabetes mellitus often leads to a decline in glucose-responsive insulin secretion from pancreatic beta cells, a phenomenon called glucose toxicity. Upon hyperglycaemia, glycation reaction occurs in the beta cells and induces oxidative stress. To understand the molecular basis of the beta-cell glucose toxicity, we investigated the possible effects of glycation on the expression and enzymatic activity of glucokinase, which plays a crucial part in glucose-responsive insulin secretion. METHODS: Glycation and reactive oxygen species were induced in HIT-T15 cells by treatment with D-ribose and effects on glucokinase gene transcription, glucokinase protein amount, glucose phosphorylation activity, and DNA-binding activities of putative glucokinase gene transcription factors were evaluated. RESULTS: When glycation was induced in HIT-T15 cells, the activity of the human glucokinase gene beta-cell-type promoter was suppressed substantially (83% reduction at 60 mmol/l D-ribose). Also, similar reductions in mRNA and protein amounts of glucokinase and in the Vmax of its enzymatic activity were observed. In agreement with the reduction in the promoter activity, the two major transcription factors of the glucokinase gene, the Pal-binding factor and PDX-1, reduced their binding to their target sequences in the glucokinase gene promoter in glycation-induced HIT cells. Because these effects of D-ribose were counteracted by aminoguanidine or N-acetylcysteine, reactive oxygen species, generated by the glycation reaction, appears to be involved in the phenomena. CONCLUSION/INTERPRETATION: The induction of the glycation reaction, which is known to occur in pancreatic beta cells in chronic hyperglycaemia, suppresses the glucokinase gene transcription and its enzymatic activity. Thus, hyperglycaemia-dependent inhibition of glucokinase activity could in part explain beta-cell glucose toxicity.
Acetylcysteine↗
Modulation of ET-1-induced contraction of human bronchi by airway epithelium-dependent nitric oxide release via ET(A) receptor activation.
1. The purpose of this work was to investigate whether endothelin-1 (ET-1) was able to induce the release of an inhibitory factor from the airway epithelium in isolated human bronchi and to identify this mediator as well as the endothelin receptor involved in this phenomenon. 2. In intact bronchi, ET-1 induced a concentration-dependent contraction (-logEC50 = 7.92+/-0.09, n = 18) which was potentiated by epithelium removal (-logEC50 = 8.65+/-0.11, n = 17). BQ-123 , an ET(A) receptor antagonist, induced a significant leftward shift of the ET-1 concentration-response curve (CRC). This leftward shift was abolished after epithelium removal. 3. L-NAME (3 x 10(-3) M), an inhibitor of nitric oxide (NO) synthase, induced a significant leftward shift of the ET-1 CRC, and abolished the potentiation by BQ-123 (10(-8) M) of ET-1-induced contraction. 4. In intact preparations, the ET(B) receptor antagonist BQ-788 induced only at 10(-5) M a slight rightward shift of the ET-1 CRC. In contrast, in epithelium-denuded bronchi or in intact preparations in the presence of L-NAME, BQ-788 displayed a non-competitive antagonism toward ET-1-induced contraction. 5. IRL 1620, a selective ET(B) receptor agonist, induced a contraction of the isolated bronchus (-logEC50=7.94+/-0.11, n= 19). This effect was not modified by epithelium removal or by BQ-123. BQ-788 exerted a competitive antagonism against IRL 1620 which was similar in the presence or absence of epithelium. 6. These results show that ET-1 exerts two opposite effects on the human airway smooth muscle. One is contractile via ETB-receptor activation, the other is inhibitory and responsible of NO release which counteracts via ETA-receptor activation the contraction.
Antihypertensive Agents↗
Effect of DNA sequence divergence on homologous recombination as analyzed by a random-walk model.
A point connecting a pair of homologous regions of DNA duplexes moves along the homology in a reaction intermediate of the homologous recombination. Formulating this movement as a random walk, we were previously successful at explaining the dependence of the recombination frequency on the homology length. Recently, the dependence of the recombination frequency on the DNA sequence divergence in the homologous region was investigated experimentally; if the methyl-directed mismatch repair (MMR) system is active, the logarithm of the recombination frequency decreases very rapidly with an increase of the divergence in a low-divergence regime. Beyond this regime, the logarithm decreases slowly and linearly with the divergence. This "very rapid drop-off" is not observed when the MMR system is defective. In this article, we show that our random-walk model can explain these data in a straightforward way. When a connecting point encounters a diverged base pair, it is assumed to be destroyed with a probability that depends on the level of MMR activity.
DNA↗
Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas.
Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.
Adenovirus E1A Proteins↗
Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity.
Oxidative stress is produced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. The aim of this study was to examine the involvement of oxidative stress in the progression of pancreatic beta-cell dysfunction in type 2 diabetes and to evaluate the potential usefulness of antioxidants in the treatment of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice, in whom antioxidant treatment (N-acetyl-L-cysteine [NAC], vitamins C plus E, or both) was started at 6 weeks of age; its effects were evaluated at 10 and 16 weeks of age. According to an intraperitoneal glucose tolerance test, the treatment with NAC retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Vitamins C and E were not effective when used alone but slightly effective when used in combination with NAC. No effect on insulin secretion was observed when the same set of antioxidants was given to nondiabetic control mice. Histologic analyses of the pancreases revealed that the beta-cell mass was significantly larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a possible cause, the antioxidant treatment suppressed apoptosis in beta-cells without changing the rate of beta-cell proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis induced by oxidative stress causes reduction of beta-cell mass. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. Furthermore, expression of pancreatic and duodenal homeobox factor-1 (PDX-1), a beta-cell-specific transcription factor, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. In conclusion, our observations indicate that antioxidant treatment can exert beneficial effects in diabetes, with preservation of in vivo beta-cell function. This finding suggests a potential usefulness of antioxidants for treating diabetes and provides further support for the implication of oxidative stress in beta-cell dysfunction in diabetes.
Acetylcysteine↗
[Sleep-promoting substances].
Explore the source record for details and available documents.
Adenosine↗
Mismatch-stimulated destruction of intermediates as an explanation for map expansion in genetic recombination.
Explore the source record for details and available documents.
Animals↗
Suppression of transcription factor PDX-1/IPF1/STF-1/IDX-1 causes no decrease in insulin mRNA in MIN6 cells.
The insulin gene transcription factor PDX-1/IPF1/STF-1/ IDX-1 plays a key role in directing beta cell-specific gene expressions. Recently, impairment of PDX-1 expression or activity has been observed in beta cell-derived HIT cells cultured under high glucose concentrations, and this has been suggested as a possible cause of the decrease in insulin gene transcription. To investigate the pathophysiological significance of PDX-1 as a determinant of the rate of insulin gene transcription, we suppressed its expression in beta cell-derived MIN6 cells using an antisense oligodeoxynucleotide (ODN) and searched for possible changes in the beta cell-specific gene expression. Treatment of MIN6 cells with an 18-mer phosphorothioate ODN complementary to a sequence starting at the translation initiation codon of PDX-1 caused a potent, concentration-dependent reduction in PDX-1 expression; addition of 2 microM antisense ODN could reduce PDX-1 expression to 14+/-4% of the control. There was also a decrease in its DNA binding to the insulin gene A element. Despite such suppression of PDX-1, Northern blot analysis revealed no decrease in the amount of insulin mRNA in the MIN6 cells. Similarly, no changes were detected in the transcription of the glucokinase or islet amyloid polypeptide gene, for which PDX-1 was shown to function as a transcription factor. Thus, our findings dispute the physiological significance of PDX-1 in determining the rate of insulin gene transcription. This means that other components constituting the transcription-controlling machinery need to be evaluated in order to understand the molecular basis of impaired insulin biosynthesis such as that observed due to glucose toxicity.
Binding Sites↗
Tec tyrosine kinase links the cytokine receptors to PI-3 kinase probably through JAK.
Tec/Btk tyrosine kinases are members of a subgroup of Src tyrosine kinase family. They are reported to be activated in response to cytokines, such as IL-3 and IL-6. Janus kinases (JAKs) are known to associate with certain cytokine receptors, e.g. gp130, the signal transducing subunit of IL-6 receptor, and the common beta chain of IL-3 receptor, which can be activated upon receptor dimerization in response to cytokines. Here we show the association between Jak1/Jak2 and Tec or Jak1 and Btk. Furthermore, Jak1 but not Jak2 induces tyrosine phosphorylation of Btk, but not Tec. These observations suggest that upon cytokine stimulation JAKs activate Tec/Btk or induce their dimerization resulting in endogenous tyrosine phosphorylation. Furthermore using a yeast two-hybrid system we have identified the target molecules for Tec, the p85 and p55PIK subunits of PI-3 kinase, and Vav. Tec associated with Vav through its SH2 domain independently of its kinase activity. In contrast the p85 and p55PIK subunits of PI-3 kinase associated with the SH2-kinase domain of Tec, dependent on Tec kinase activity. Consistent with these, IL-6 or IL-3 induced the association between Tec and the p85 subunit of PI-3 kinase in mammalian cells. These findings suggest that Tec tyrosine kinase links cytokine receptors to PI-3 kinase probably through JAKs.
Antigens, CD↗