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HDR syndrome is an autosomal dominant disorder characterized by hypoparathyroidism (H), deafness (D), and renal dysplasia (R) caused by genetic variants of the GATA3 gene. We present the case of a 38-year-old Japanese man with HDR syndrome who exhibited hypoparathyroidism, sensorineural deafness, renal dysfunction, severe symptomatic hypocalcemia with Chvostek's and Trousseau's signs, and QT prolongation on electrocardiography. He had a family history of deafness and hypocalcemia. Genetic testing revealed a novel GATA3 gene variant at exon 2 (c.48delC), which induces a frameshift resulting in termination at codon 178, causing HDR syndrome. We summarized 45 Japanese cases of HDR syndrome with regard to the mode of onset (familial or sporadic) and the age at diagnosis. In addition, we summarized all previous cases of HDR syndrome with GATA3 gene variants. Mapping of previously reported genetic variants in HDR syndrome revealed that most missense variants were observed at exons 4 and 5 regions in the GATA3 gene. These two regions contain zinc finger domains, demonstrating their functional importance in GATA3 transcription. This review of literature provides a useful reference for diagnosing HDR syndrome and predicting the related future manifestations.
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17q12 deletion syndrome is a rare chromosomal anomaly with variable phenotypes, caused by the heterozygous deletion of chromosome 17q12. We herein report a 35-year-old Japanese patient with chromosomal 17q12 deletion syndrome identified by de novo deletion of the 1.46 Mb segment at the 17q12 band by genetic analyses. He exhibited a wide range of phenotypes, such as maturity-onset diabetes of the young (MODY) type 5, structural or functional abnormalities of the kidney, liver, and pancreas; facial dysmorphic features, electrolyte disorders; keratoconus, and acquired perforating dermatosis. This case report provides valuable resources concerning the clinical spectrum of rare 17q12 deletion syndrome.
Assuntos
Doenças do Sistema Nervoso Central , Esmalte Dentário/anormalidades , Diabetes Mellitus Tipo 2 , Doenças Renais Císticas , Masculino , Humanos , Adulto , Japão , Face , HeterozigotoRESUMO
OBJECTIVE: Promoting thermogenesis in adipose tissue has been a promising strategy against obesity and related metabolic complications. We aimed to identify compounds that promote thermogenesis in adipocytes and to elucidate their functions and roles in metabolism. METHODS: To identify compounds that directly promote thermogenesis from a structurally diverse set of 4800 compounds, we utilized a cell-based platform for high-throughput screening that induces uncoupling protein 1 (Ucp1) expression in adipocytes. RESULTS: We identified one candidate compound that activates UCP1. Additional characterization of this compound revealed that it induced cellular thermogenesis in adipocytes with negligible cytotoxicity. In a subsequent diet-induced obesity model, mice treated with this compound exhibited a slower rate of weight gain, improved insulin sensitivity, and increased energy expenditure. Mechanistic studies have revealed that this compound increases mitochondrial biogenesis by elevating maximal respiration, which is partly mediated by the protein kinase A (PKA)-p38 mitogen-activated protein kinase (MAPK) signaling pathway. A further comprehensive genetic analysis of adipocytes treated with these compounds identified two novel UCP1-dependent thermogenic genes, potassium voltage-gated channel subfamily C member 2 (Kcnc2) and predicted gene 5627 (Gm5627). CONCLUSIONS: The identified compound can serve as a potential therapeutic drug for the treatment of obesity and its related metabolic disorders. Furthermore, our newly clarified thermogenic genes play an important role in UCP1-dependent thermogenesis in adipocytes.
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Resistência à Insulina , Obesidade , Proteína Desacopladora 1 , Animais , Camundongos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Obesidade/complicações , Obesidade/tratamento farmacológico , Termogênese/fisiologia , Proteína Desacopladora 1/antagonistas & inibidoresRESUMO
Gicerin/CD146 is a cell adhesion molecule which belongs to the immunoglobulin (Ig) superfamily. We have reported the existence of gicerin/CD146 in the nervous system, heart, lung and smooth muscles of blood vessels. In this study, we make a cardiac hypertrophy model rat by constricting the rat aorta (AAC, ascending aortic constriction) and examined the effect on the expression of gicerin/CD146 in the heart. We found that the expression level of gicerin/CD146 was increased by the AAC treatment. Next, stretch stimulation was applied to myocardial cell line H9c2 cells to confirm that gicerin/CD146 may participate in the cellular hypertrophy model. We also treated the cells with inhibitors of MAP pathway enzymes. In cultured myocardial cells, the expression level of gicerin/CD146 was increased by the stretch stimulation and decreased by inhibiting the MAP pathway. Based on the above findings, it is suggested that the expression of gicerin/CD146 is involved in cardiac hypertrophy, and that the MAP pathway may be involved in the expression of gicerin/CD146 RNA in the cardiomyocyte. In addition, the expression level of gicerin/CD146 RNA in neonatal rats was upregulated after birth. Therefore, it is suggested that gicerin/CD146 might participate in the increase of myocardial cell volume both in the pathway of cardiac hypertrophy and in the developmental growth of heart.
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Antígeno CD146/metabolismo , Cardiomegalia/metabolismo , Regulação da Expressão Gênica , Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Animais , Cardiomegalia/patologia , Linhagem Celular , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
AIMS/INTRODUCTION: Recent studies have suggested C-X-C motif chemokine ligand 14 (CXCL14), secreted from adipose tissue, to play an important role in the pathogenesis of metabolic syndrome. However, the clinical significance of CXCL14 in humans has not been elucidated. This study aimed to assess correlations between serum CXCL14 levels and clinical parameters in patients with type 2 diabetes mellitus. MATERIALS AND METHODS: In total, 176 individuals with type 2 diabetes mellitus were recruited. Serum CXCL14 concentrations were determined by enzyme-linked immunosorbent assay. We examined the associations of serum CXCL14 levels with laboratory values, abdominal computed tomography image information, surrogate markers used for evaluating the pathological states of diabetes, obesity and atherosclerosis. RESULTS: Serum CXCL14 levels correlated positively with body mass index, waist circumference, subcutaneous and visceral fat areas, and serum alanine transaminase, uric acid, total cholesterol, low-density lipoprotein cholesterol, triglycerides and C-peptide (CPR) levels. In contrast, CXCL14 levels correlated inversely with age, pulse wave velocity and serum adiponectin levels. Multiple linear regression analysis showed serum levels of CPR (ß = 0.227, P = 0.038) and the fatty liver index (ß = 0.205, P = 0.049) to be the only parameters showing independent statistically significant associations with serum CXCL14 levels. CONCLUSIONS: Serum CXCL14 levels were independently associated with serum CPR and fatty liver index in patients with type 2 diabetes mellitus. In these patients, a high serum CPR concentration might reflect insulin resistance rather than ß-cell function, because CXCL14 showed simple correlations with obesity-related parameters. Collectively, these data suggested that serum CXCL14 levels in type 2 diabetes patients might be useful predictors of elevated serum CPR and hepatic steatosis.
Assuntos
Peptídeo C/sangue , Quimiocinas CXC/sangue , Diabetes Mellitus Tipo 2/sangue , Fígado Gorduroso/sangue , Resistência à Insulina/genética , Adiponectina/sangue , Fatores Etários , Idoso , Alanina Transaminase/sangue , Biomarcadores/sangue , Índice de Massa Corporal , Colesterol/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Fígado Gorduroso/genética , Feminino , Humanos , Gordura Intra-Abdominal , Modelos Lineares , Lipoproteínas LDL/sangue , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/genética , Análise de Onda de Pulso , Triglicerídeos/sangue , Ácido Úrico/sangue , Circunferência da Cintura/genéticaRESUMO
OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadotropic hypogonadism (HHG) caused by mutations of the NR0B1/DAX1 gene. We aimed to search for the presence of any NR0B1/DAX1 gene mutations in a referred patient and to further characterize the phenotypes of the identified mutation. CASE PRESENTATION: Herein, we report a Japanese patient with a novel missense mutation of the NR0B1/DAX1 gene resulting in adult-onset AHC and HHG. The patient was referred with diffuse skin pigmentation at 28 years of age, presented partial adrenal insufficiency and had undiagnosed incomplete HHG. Urological examination revealed severe oligospermia and testicular microlithiasis. RESULTS: The NR0B1/DAX1 gene mutation was identified by exome sequencing as a novel missense mutation (c.884A>T, p.Leu295His). We conducted in silico modeling of this mutant NR0B1/DAX1 protein (p.Leu295His) which affected the conserved hydrophobic core of the putative ligand-binding domain (LBD). In addition, functional analysis revealed that this mutant showed a decreased ability as a transcriptional repressor to suppress target genes, such as STAR and LHB. Furthermore, this mutant showed functionally impaired repression of steroidogenesis in human adrenocortical H295R cells. CONCLUSIONS: We identified a novel missense mutation of the NR0B1/DAX1 gene in a patient suffering from late-onset AHC and HHG, who presented with oligospermia and testicular microlithiasis. This mutant NR0B1/DAX1 protein was found to have reduced repressor activity, according to in vitro studies, clinically consistent with the patient's phenotypic features.
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Heparan sulfate proteoglycans (HSPGs) comprise a core protein to which extracellular glycosaminoglycan chains are attached. Syndecan-4, one of the major HS-containing core proteins, is distributed on the cell surface, where they interact with various protein ligands and regulate a wide range of biological activities. Here, we propose that the core protein of HSPGs is involved in the insulin secretory response. To investigate the participation of HSPGs in the insulin-secretion mechanism, MIN6 cells, a mouse pancreatic ß-cell line, were subcloned. The subcloned MIN6 cells were selected based on their insulin secretory response, the expression of HS and core proteins. The results from these screening experiments indicated that only syndecan-4-expressing subclones are able to secrete insulin in response to glucose. Silencing of syndecan-4 reduced glucose-induced insulin secretion, whereas the overexpression of syndecan-4 increased the insulin secretory response. These data indicate that the HSPG syndecan-4 plays important role(s) in the insulin secretory response.
Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Sindecana-4/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sindecana-4/genéticaRESUMO
A possible cure for diabetes is explored by using non-pancreatic cells such as fetal hepatocytes. The expression of insulin and transcription factors for insulin is investigated in mouse fetal liver. We detected mRNAs for insulin I (Ins1) and insulin II (Ins2) and proinsulin- and mature insulin-positive cells in mouse fetal liver by reverse transcription plus the polymerase chain reaction and immunohistochemistry. Glucagon, somatostatin and pancreatic polypeptide were not expressed throughout development. Mouse Ins2 and Ins1 promoters were transiently activated in mouse fetal hepatocytes of embryonic days 13.5 and 16.5, respectively. Pancreatic and duodenal homeobox 1 (Pdx1) mRNA was not expressed during development of the liver. In contrast, mRNAs and proteins of neurogenic differentiation (NeuroD)/ß cell E-box transactivator 2 (Beta2) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MafA) were almost simultaneously expressed with insulin genes in the liver. Ins2 and Ins1 promoters were activated in hepatoma cells by the transfection of the expression vector for NeuroD/Beta2 alone and by the combination of NeuroD/Beta2 and MafA, respectively. These results indicate that the expression of NeuroD/Beta2 and MafA is linked temporally with the transcription of Ins2 and Ins1 genes in mouse fetal liver and suggest the potential usage of fetal hepatocytes to make insulin-producing ß cells by introducing transcription factors.
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Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Insulina/genética , Fígado/embriologia , Fígado/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Glucagon/metabolismo , Hepatócitos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/metabolismo , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/genéticaRESUMO
Reg (regenerating gene) product, Reg protein, is induced in pancreatic ß-cells and acts as autocrine/paracrine growth factor for regeneration via the cell surface Reg receptor. However, high concentrations of Reg I protein induced ß-cell apoptosis. In the present study, we found that hepatocyte growth factor (HGF) attenuated the ß-cell apoptosis induced by the high concentrations of Reg I protein and that the combined stimulation of interleukin-6 (IL-6) and dexamethasone (Dx) induced the accumulation of HGF mRNA as well as Reg I mRNA in ß-cells. The accumulation of the HGF mRNA was caused by the activation of the HGF promoter. Deletion analysis revealed that the region of -96 to -92 of the HGF gene was responsible for the promoter activation by IL-6+Dx. The promoters contain a consensus transcription factor binding sequence for signal transducer and activator of transcription (STAT). Site-directed mutations of STAT-binding motif in the region markedly attenuated the HGF promoter activity. Chromatin immunoprecipitation assay showed that STAT3 is located at the active HGF promoter in response to IL-6+Dx stimulation. These results strongly suggest that the combined stimulation of IL-6 and glucocorticoids induces the activation of both Reg and HGF genes and that the anti-apoptotic effects of HGF against the Reg I-induced apoptosis may help ß-cell regeneration by Reg I protein.
Assuntos
Apoptose , Dexametasona/farmacologia , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Células Secretoras de Insulina/patologia , Interleucina-6/farmacologia , Litostatina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Citoproteção/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Litostatina/farmacologia , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to function as a second messenger for intracellular Ca(2+) mobilization in various vertebrate and invertebrate tissues. In this study, we isolated two Xenopus laevis cDNAs (frog cd38 and cd157 cDNAs) homologous to the one encoding the human cADPR-metabolizing enzyme CD38. Frog CD38 and CD157 are 298-amino acid proteins with 35.9 and 27.2 % identity to human CD38 and CD157, respectively. Transfection of expression vectors for frog CD38 and CD157 into COS-7 cells revealed that frog CD38 had NAD(+) glycohydrolase, ADP-ribosyl cyclase (ARC), and cADPR hydrolase activities, and that frog CD157 had no enzymatic activity under physiological conditions. In addition, when recombinant CD38 and frog brain homogenate were electrophoresed on an SDS-polyacrylamide gel, ARC of the brain homogenate migrated to the same position in the gel as that of frog CD38, suggesting that frog CD38 is the major enzyme responsible for cADPR metabolism in amphibian cells. The frog cd38 gene consists of eight exons and is ubiquitously expressed in various tissues. These findings provide evidence for the existence of the CD38-cADPR signaling system in frog cells and suggest that the CD38-cADPR signaling system is conserved during vertebrate evolution.
Assuntos
ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase/genética , Antígenos CD/genética , ADP-Ribose Cíclica/biossíntese , Proteínas de Xenopus/genética , Xenopus laevis/genética , ADP-Ribosil Ciclase/biossíntese , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase 1/biossíntese , ADP-Ribosil Ciclase 1/química , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos CD/química , Sequência de Bases , Encéfalo/enzimologia , Células COS , Chlorocebus aethiops , Clonagem Molecular , Sequência Conservada , ADP-Ribose Cíclica/metabolismo , Evolução Molecular , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Hidrólise , Nucleotídeos de Inosina/química , Cinética , Dados de Sequência Molecular , NAD/análogos & derivados , NAD/química , Especificidade de Órgãos , Filogenia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/químicaRESUMO
UNLABELLED: Aims/Introduction: Heparan sulfate (HS) mediates a variety of molecular recognition events that are essential for differentiation, morphogenesis and homeostasis through various HS forms that result from differential sulfate modification. Recently, we found that HS is localized exclusively around ßß-cells in islets of adult mice and is required for insulin secretion. The aim of this study was to examine the contribution of HS sulfate groups to insulin secretion. MATERIALS AND METHODS: Glucose-induced insulin secretion (GIIS) was examined in mouse pancreatic islets, the mouse pancreatic ß-cell line MIN6 cells and its derivative MIN6T3 cells after removal of sulfate groups by sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation. Quantitative reverse transcription polymerase chain reaction was used for analyzing messenger ribonucleic acid (mRNA) expression of HS modification enzymes. Expression of HS 3-O-sulfotransferase isoform-1 (Hs3st1) was silenced and GIIS was examined. RESULTS: Impaired insulin secretion by islets, MIN6 cells and MIN6T3 cells was observed after treatment with sodium chlorate. Sodium chlorate-treatment upregulated the mRNA expression of sulfotransferases expressed in MIN6T3 cells. Expression of the Hs3st1 was strongly upregulated by sodium chlorate-treatment, and its silencing by RNA interference reduced GIIS in MIN6T3 cells. CONCLUSIONS: Our data suggest that the 3-O-sulfate group of HS that is modified by Hs3st1 plays a significant role(s) in the insulin secretory pathway, selectively through an interaction with factor(s) upstream of membrane depolarization in ß-cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00205.x, 2012).
RESUMO
ATRA and a synthetic RAR agonist tamibarotene (Am80) induce granulocytic differentiation of human acute leukemia HL-60 cells and have been used in antineoplastic therapy. ATRA induces CD38 antigen during HL-60 cell differentiation, which interacts with CD31 antigen on the vascular EC surface and may induce disadvantages in the therapy. We here examined the mechanisms of the ATRA-mediated CD38 induction and compared the difference between ATRA- and tamibarotene-mediated induction. Tamibarotene-induced HL-60 cell adhesion to ECs was 38% lower than ATRA, and NB4 cell adhesion to ECs by tamibarotene was equivalent to ATRA, which induced CD38 gene transcription biphasically in HL-60 cells, the early-phase induction via DR-RARE containing intron 1, and the delayed-phase induction via RARE lacking the 5'-flanking region. In contrast to ATRA, tamibarotene induced only the early-phase induction, resulting in its lower CD38 induction than ATRA. A PKCδ inhibitor, rottlerin, and siRNA-mediated PKCδ knockdown suppressed the ATRA-induced CD38 promoter activity of the 5'-flanking region, whereas a RAR antagonist, LE540, or RAR knockdown did not affect it. Cycloheximide and rottlerin suppressed the delayed-phase induction of CD38 expression by ATRA but did not affect the early-phase induction. Moreover, ATRA, but not tamibarotene, induced PKCδ expression without affecting its mRNA stability. The diminished effect of tamibarotene on CD38-mediated HL-60 cell adhesion to ECs compared with ATRA is likely a result of the lack of its delayed-phase induction of CD38 expression, which may be advantageous in antineoplastic therapy.
Assuntos
ADP-Ribosil Ciclase 1/genética , Benzoatos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia/patologia , Tetra-Hidronaftalenos/farmacologia , Tretinoína/farmacologia , Adesão Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células HL-60 , Humanos , Proteína Quinase C-delta/efeitos dos fármacos , Proteína Quinase C-delta/genética , Retinoides/farmacologiaRESUMO
Cyclic ADP-ribose (cADPR), a potent Ca(2+) mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca(2+) concentrations in pancreatic islets, resulting from Ca(2+) mobilization from RyRs as well as Ca(2+) influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [(3)H]ryanodine binding. Intracellular Ca(2+) release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.
Assuntos
Processamento Alternativo , Ilhotas Pancreáticas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , ADP-Ribose Cíclica/metabolismo , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Coelhos , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Distribuição TecidualRESUMO
Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet beta-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in beta-cells. These mice exhibited abnormal islet morphology with reduced beta-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.
Assuntos
Heparitina Sulfato/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Pâncreas/crescimento & desenvolvimento , Animais , Glucose/farmacologia , Heparitina Sulfato/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Pâncreas/citologia , Pâncreas/metabolismoRESUMO
REG (Regenerating gene) Ialpha protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPARgamma-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG Ialpha protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG Ialpha gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPARgamma, in PPARgamma-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPARgamma-antagonist GW9662. Although TZDs did not inhibit the REG Ialpha gene promoter activity in PPARgamma-non-expressing cells, PPARgamma overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG Ialpha gene transcription through a PPARgamma-dependent pathway. The TZD-induced REG Ialpha mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG Ialpha and PPARgamma.
Assuntos
Neoplasias Gastrointestinais/genética , Litostatina/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Litostatina/genética , PPAR gama/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genéticaRESUMO
Cyclic ADP-ribose (cADPR), accumulated in pancreatic beta-cells in response to elevated ATP levels after glucose stimulation, mobilizes Ca(2+) from the endoplasmic reticulum through the ryanodine receptor (RyR) and thereby induces insulin secretion. We have recently demonstrated in an in vitro study that cADPR activates RyR through binding to FK506-binding protein 12.6 (FKBP12.6), an accessory protein of RyR. Here we generated FKBP12.6-deficient (FKBP12.6(-/-)) mice by homologous recombination. FKBP12.6(-/-) mice showed glucose intolerance coupled to insufficient insulin secretion upon a glucose challenge. Insulin secretion in response to glucose was markedly impaired in FKBP12.6(-/-) islets, while sulfonylurea- or KCl-induced insulin secretion was unaffected. No difference was found in the glucose oxidation rate between FKBP12.6(-/-) and wild-type islets. These results indicate that FKBP12.6 plays a role in glucose-induced insulin secretion downstream of ATP production, independently of ATP-sensitive K(+) channels, in pancreatic beta-cells.
Assuntos
Intolerância à Glucose/genética , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Expressão Gênica/genética , Glucose/administração & dosagem , Teste de Tolerância a Glucose , Insulina/análise , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Canais KATP/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Oxirredução , Cloreto de Potássio/farmacologia , RNA Mensageiro/metabolismo , Compostos de Sulfonilureia/farmacologia , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Ciclina D1/metabolismo , Células Secretoras de Insulina/fisiologia , Litostatina/metabolismo , Regeneração , Fator 2 Ativador da Transcrição/genética , Animais , Ciclina D1/genética , Genes Reporter , Células Secretoras de Insulina/citologia , Lectinas Tipo C/metabolismo , Litostatina/genética , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Cyclic ADP-ribose (cADPR) induces the release of Ca2+ from microsomes of pancreatic islets for insulin secretion. It has been demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP 12.6) on rat islet ryanodine receptor and that the binding of cADPR to FKBP12.6 frees the ryanodine receptor from FKBP12.6, causing it to release Ca2+ [Noguchi, N., Takasawa, S., Nata, K., Tohgo, A., Kato, I., Ikehata, F., Yonekura, H., Okamoto, H., 1997. Cyclic ADP-ribose binds to FK506-binding protein to release Ca2+ from islet microsomes. J. Biol. Chem. 272, 3133-3136.]. In this study, we cloned, characterized the structural organization of the human FKBP12.6, which is highly homologous to human FKBP12, and analyzed the promoters for FKBP12.6 and FKBP12. Human FKBP12.6 gene spanned about 16 kb in length and consisted of four exons and three introns. The positions of exon-intron junction of the FKBP12.6 gene were perfectly matched with those of FKBP12 gene except that FKBP12 has an additional exon, exon V, to code exclusively for 3'-UTR. Fluorescence in situ hybridization revealed that the FKBP12.6 gene was located on chromosome 2 p21-23, which is different from the locus (chromosome 20 p13) of the FKBP12 gene. Reporter gene analyses revealed that the regions of -58 approximately -24 of FKBP12.6 and -106 approximately -79 of FKBP12 are important for promoter activities. The promoters contain a consensus transcription factor binding sequence for Sp family in FKBP12.6 and Ets-1 in FKBP12. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoters. The DNA/protein complex on FKBP12.6 promoter was competed out by Sp1 consensus probe and the complex was supershifted by anti-Sp3 antibodies. On the other hand, the DNA/protein complex on FKBP12 promoter was competed out by Ets-1 consensus probe but not by its mutant probe, indicating that Sp3 and Ets-1 play an essential role in transcription of FKBP12.6 and FKBP12, respectively.
Assuntos
Cromossomos Humanos Par 2/genética , Éxons/genética , Íntrons/genética , Regiões Promotoras Genéticas/genética , Proteína 1A de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/genética , Regiões 3' não Traduzidas , Sequência de Bases , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hibridização in Situ Fluorescente , Rim/metabolismo , Luciferases/metabolismo , Dados de Sequência Molecular , Proteína Proto-Oncogênica c-ets-1/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismoRESUMO
Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.
Assuntos
Cromossomos Humanos Par 2/genética , Perfilação da Expressão Gênica , Proteínas/genética , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA/química , DNA/genética , DNA/isolamento & purificação , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Família Multigênica/genética , Pâncreas/metabolismo , Proteínas Associadas a Pancreatite , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sítio de Iniciação de TranscriçãoRESUMO
Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced beta-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [(3)H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control Reg(+/+) mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in beta-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [(3)H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic beta-cell mass. These results indicate that Reg plays an important role in beta-cell growth/regeneration.