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1.
Diabetes Obes Metab ; 12(9): 790-7, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20649631

RESUMO

AIM: The aim of this study was to evaluate the efficacy of two group-based lifestyle interventions in ameliorating the risk factors of metabolic syndrome (MS) and insulin resistance. METHODS: Ninety-eight subjects who had at least one component of MS were randomized into standard intervention (SI) (4-month intervention; n = 50) and extended intervention (EI) (10-month intervention; n = 48) groups, and 39 subjects were followed up for a control group. The effects of intervention were evaluated after 10, 22 and 34 months. RESULTS: At month 10, the standard and EI groups showed improved body mass index (BMI) (SI, -0.28; EI, -0.47; control, -0.09), high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and A1c and a decreased mean number of components of MS (SI, -0.37; EI, -0.51; control, 0.08). At month 34, the effects on BMI (SI, -0.66; EI, -0.60; control, -0.05) and HDL-cholesterol were sustained for both the intervention groups. In controls, the increases in fasting plasma glucose and the mean number of components of MS from the baseline to month 34 were greater than those in the standard and EI groups. Whole body insulin sensitivity index and hepatic insulin resistance index were also improved at month 10. CONCLUSIONS: Group-based lifestyle intervention could be an efficient way to prevent MS. Its effects were sustainable, at least in part, for 2 years. These effects may be mediated by an improvement in insulin sensitivity.


Assuntos
Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Síndrome Metabólica/prevenção & controle , Comportamento de Redução do Risco , Adulto , Idoso , Feminino , Humanos , Japão/epidemiologia , Masculino , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/etiologia , Pessoa de Meia-Idade , Fatores de Risco
2.
Diabetologia ; 46(2): 231-40, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12627322

RESUMO

AIMS/HYPOTHESIS: Resistin is a peptide secreted by adipocytes and recognized as a hormone that could link obesity to insulin resistance. This study was designed to examine the effect and mechanism(s) of insulin on resistin expression in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were stimulated with insulin and resistin mRNA expression was examined by Northern blot analysis. In some experiments, the insulin signal was blocked by several chemical inhibitors or overexpression of a dominant negative form (Deltap85) of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). RESULTS: Insulin treatment caused a reduction of resistin mRNA in time-dependent and dose-dependent manners in 3T3-L1 adipocytes. Pre-treatment with PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, or SB203580, an inhibitor of p38 mitogen-activated protein-kinase (p38 MAP-kinase) pathway, did not influence insulin-induced reduction of resistin mRNA. Inhibition of PI 3-kinase by LY294002 or Deltap85 also failed to block insulin-induced reduction of resistin mRNA. Cycloheximide, a protein synthesis inhibitor, completely blocked insulin-induced reduction of resistin mRNA. Actinomycin D, a RNA synthesis inhibitor, also blocked insulin-induced reduction of resistin mRNA, and the decreasing rate of resistin mRNA in cells treated with insulin alone was faster than that with actinomycin D. CONCLUSION/INTERPRETATION: Insulin downregulates resistin mRNA via PI 3-kinase, ERK or p38 MAP-kinase independent pathways in 3T3-L1 adipocytes. The downregulation mechanism of resistin mRNA by insulin would be an indirect event through the synthesis of novel protein(s) that could accelerate the degradation of resistin mRNA.


Assuntos
Adipócitos/metabolismo , Hormônios Ectópicos/genética , Insulina/farmacologia , Proteínas , RNA Mensageiro/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo , Insulina/administração & dosagem , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Neural , Fosfatidilinositol 3-Quinases/metabolismo , Resistina , Transdução de Sinais , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno
3.
Biochem Biophys Res Commun ; 280(2): 428-34, 2001 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-11162534

RESUMO

The liver is a major target organ of insulin and is important for glucose homeostasis. We analyzed the tissue specific regulation of the insulin receptor gene in the liver by studying the cis-acting element and trans-acting factor of the human insulin receptor gene in human hepatoma cell line, HepG2 cells. In the chloramphenicol acetyl transferase (CAT) assay with chimeric plasmids containing various deletions and insertions of the human insulin receptor promoter/CAT gene, a HepG2 cell specific cis-acting element was identified between nt -592 to -577 of the promoter. In electrophoretic mobility shift assay and UV cross-link analysis, a 35-kDa nuclear protein that bound to 5'-TCCCTCCC-3' (nt -588 to -581) sequence was identified in HepG2 cells as well as in rat hepatocytes. This nuclear protein, designated as hepatocyte-specific transcription factor of the insulin receptor gene (HTFIR), might play an important role in tissue-specific expression of the insulin receptor gene in the liver.


Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Regiões Promotoras Genéticas/genética , Receptor de Insulina/genética , Elementos de Resposta/genética , Transativadores/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Células CHO , Cricetinae , DNA/genética , DNA/metabolismo , Genes Reporter/genética , Humanos , Mutação/genética , NF-kappa B/metabolismo , Ligação Proteica , Ratos , Transfecção , Células Tumorais Cultivadas , Raios Ultravioleta
4.
Protein Eng ; 13(10): 691-5, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11112507

RESUMO

We prepared five mutant lysozymes in which glycines whose dihedral angles are located in the region of the left-handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, were mutated to an alanine residue. From analyses of their thermal stabilities using differential scanning calorimetry, most of them were more destabilized than the native lysozyme, except for the G102A mutant, which has a stability similar to that of the native lysozyme at pH 2.7. As for the destabilized mutant lysozymes, their X-ray crystallographic analyses showed that their global structures did not change but that the local structures changed slightly. By examining the dihedral angles at the mutation sites based on X-ray crystallographic results, it was found that the dihedral angles at these mutation sites tended to adopt favorable values in a Ramachandran plot and that the extent and direction of their shifts from the original value had similar tendencies. Therefore, the change in dihedral angles may be the cause of the slight local structural changes around the mutation site. On the other hand, regarding the mutation of G102A, the global structure was almost identical with that of the native structure but the local structure was drastically changed. Therefore, it was suggested that the drastic local conformational change might be effective in releasing the unfavorable interaction of the native state at the mutation site.


Assuntos
Substituição de Aminoácidos , Anti-Infecciosos/química , Clara de Ovo , Muramidase/química , Alanina/química , Animais , Anti-Infecciosos/metabolismo , Varredura Diferencial de Calorimetria , Galinhas , Cristalografia por Raios X , Feminino , Glicina/química , Modelos Moleculares , Muramidase/genética , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Estrutura Secundária de Proteína , Termodinâmica
5.
J Biochem ; 128(3): 349-54, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10965031

RESUMO

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.


Assuntos
Liases de Carbono-Enxofre/química , Pseudomonas putida/enzimologia , Fosfato de Piridoxal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Liases de Carbono-Enxofre/genética , Cristalografia por Raios X , Metionina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Difração de Raios X
6.
Science ; 289(5480): 739-45, 2000 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-10926528

RESUMO

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/química , Rodopsina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/química , Cristalografia por Raios X , Ligação de Hidrogênio , Luz , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsina/metabolismo , Bases de Schiff , Estereoisomerismo , Visão Ocular
7.
Metabolism ; 49(7): 920-30, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10910005

RESUMO

Acute exercise induces glucose uptake in skeletal muscle in vivo, but the molecular mechanism of this phenomenon remains to be identified. In this study, we evaluated the involvement of bradykinin in exercise-induced glucose uptake in humans and rats. In human studies, plasma bradykinin concentrations increased significantly during an ergometer exercise (20 minutes) in 8 healthy normoglycemic subjects and 6 well-controlled type 2 diabetic patients (mean hemoglobin A1c [HbA1c], 6.4% +/- 0.6%), but not in 6 poorly controlled type 2 diabetics (mean HbA1c, 11.6% +/- 2.6%). In rat studies, plasma bradykinin concentrations also significantly increased after 1 hour of swimming in nondiabetic and mildly diabetic (streptozotocin [STZ] 45 mg/kg intravenously [IV]) rats, but not in rats with severe diabetes (STZ 65 mg/kg IV). Glucose influx (maximum velocity [Vmax]) and GLUT-4 translocation in skeletal muscle of nondiabetic rats significantly increased after 1 hour of swimming, but these increases were abrogated by subcutaneous infusion of bradykinin B2 receptor antagonist HOE-140 (400 microg x kg(-1) x d(-1)). Insulin-stimulated tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity in response to insulin injection (20 U/kg IV) in the portal vein were significantly attenuated in exercised rats pretreated with HOE-140 compared with saline-treated exercised rats. Our results suggest that plasma bradykinin concentrations increase in response to acute exercise and this increase is affected by blood glucose status in diabetic patients. Moreover, the exercise-induced increase in bradykinin may be involved in modulating exercise-induced glucose transport through an increase of GLUT-4 translocation, as well as enhancement of the insulin signal pathway, during the postexercise period in skeletal muscle, resulting in a decrease of blood glucose.


Assuntos
Bradicinina/fisiologia , Diabetes Mellitus/metabolismo , Exercício Físico , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Adulto , Animais , Transporte Biológico , Glicemia/análise , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Transportador de Glucose Tipo 4 , Humanos , Insulina/sangue , Proteínas Substratos do Receptor de Insulina , Masculino , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Wistar
8.
Diabetes Res Clin Pract ; 48(3): 155-70, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10802154

RESUMO

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.


Assuntos
Bradicinina/farmacologia , Insulina/fisiologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Isoenzimas/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Receptor B2 da Bradicinina , Receptor de Insulina/metabolismo , Receptores da Bradicinina/metabolismo , Frações Subcelulares/metabolismo , Fatores de Tempo , Transfecção , Tirosina/metabolismo
9.
Diabetes Res Clin Pract ; 44(2): 83-92, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10414926

RESUMO

Intracellular mechanisms through which insulin inhibits glucagon secretion remain to be elucidated in glucagon secreting cells. In this study, we confirmed that, in In-R1-G9 cells, a pancreatic alpha cell line, insulin stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and activated phosphatidylinositol 3-kinase (PI3-kinase). We further studied, using wortmannin, an inhibitor of PI3-kinase, whether the inhibitory effect of insulin on glucagon secretion was mediated through PI3-kinase pathway in these cells. In static incubation studies, insulin significantly inhibited glucagon secretion at 2, 6 and 12 h, which was completely abolished by pretreatment with wortmannin. In perifusion studies, insulin significantly suppressed glucagon secretion after 10 min, which was also blocked by wortmannin. Insulin also reduced glucagon mRNA at 6 and 12 h but not at 2 h. Wortmannin also abolished insulin-induced reduction of glucagon mRNA. Insulin increased the amount of 85 kDa subunit of PI3-kinase in plasma membrane fraction (PM), with a reciprocal decrease of the kinase in cytosol fraction (CY). Insulin also increased PI3-kinase activity in PM, but not in CY. Our results suggest that insulin suppressed glucagon secretion by inhibiting glucagon release and gene expression. Both actions were mediated by activation of PI3-kinase. Recruitment and activation of PI3-kinase in plasma membrane might be relevant at least in part to insulin-induced inhibition of glucagon release.


Assuntos
Androstadienos/farmacologia , Glucagon/genética , Glucagon/metabolismo , Insulina/farmacologia , Ilhotas Pancreáticas/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Substratos do Receptor de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/metabolismo , Fosforilação , Fatores de Tempo , Wortmanina
10.
Endocr J ; 46(1): 43-58, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10426567

RESUMO

Pancreatic and duodenal homeobox gene-1 (PDX-1) is a transcription factor which regulates the insulin gene expression. In this study, we tried to elucidate the role of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MIN6 cells. Electrophoretic mobility shift assay (EMSA) and chloramphenicol acetyltransferase (CAT) assay demonstrated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20 mmol/l glucose more than with 2 mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosphatase treatment, suggesting the involvement of PDX-1 phosphorylation in this event. In an in vitro phosphorylation study, PDX-1 was phosphorylated by protein kinase C (PKC), but not by cAMP dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK). Furthermore, increased PDX-1 function induced by high glucose was blocked by calphostin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Gö 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MIN6 cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKC zeta antibody confirmed the presence of PKC zeta, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC zeta activity was significantly increased by glucose stimulation. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypical PKC including PKC zeta, resulting in transcriptional activation of the human insulin gene promoter.


Assuntos
Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Homeodomínio/genética , Proteína Quinase C/metabolismo , Transativadores/genética , Animais , Carbazóis/farmacologia , Linhagem Celular , DNA/metabolismo , Humanos , Indóis/farmacologia , Insulina/genética , Ilhotas Pancreáticas , Camundongos , Naftalenos/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Transfecção
11.
Diabetologia ; 41(12): 1492-501, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9867217

RESUMO

We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.


Assuntos
Adenoma/metabolismo , Glucoquinase/genética , Insulina/genética , Insulina/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Neoplasias Hipofisárias/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Hormônio Liberador da Corticotropina/farmacologia , Diazóxido/farmacologia , Expressão Gênica , Glucose/farmacologia , Transportador de Glucose Tipo 2 , Glibureto/farmacologia , Humanos , Hipoglicemiantes , Secreção de Insulina , Nifedipino/farmacologia , Cloreto de Potássio/farmacologia , Transfecção , Células Tumorais Cultivadas
12.
Biosci Biotechnol Biochem ; 62(8): 1539-45, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9757561

RESUMO

We purified and characterized a thermophilic beta-galactosidase from Thermus sp. A4 isolated from the Atagawa hot spring (Shizuoka, Japan). The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was extremely thermostable and retained its full activity after incubation at 70 degrees C for 20 h. The Km observed were 5.9 mM for ortho-nitrophenyl beta-D-galactopyranoside and 19 mM for lactose. We cloned and analyzed the complete sequence of the gene encoding this enzyme. It was found to consist of 645 amino acid residues. We propose that this enzyme and seven other unclassified beta-galactosidases are new members of family 42 of the glycosyl hydrolases.


Assuntos
Thermus/enzimologia , beta-Galactosidase/química , Sequência de Aminoácidos , Sequência de Bases , Metabolismo dos Carboidratos , Cromatografia em Gel , Clonagem Molecular , DNA Bacteriano/química , Laticínios , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Lactose/metabolismo , Dados de Sequência Molecular , Peso Molecular , Nitrofenilgalactosídeos/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Thermus/química , beta-Galactosidase/genética , beta-Galactosidase/isolamento & purificação
13.
Diabetes ; 47(4): 621-31, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9568696

RESUMO

It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.


Assuntos
DNA Mitocondrial/biossíntese , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Leucina/farmacologia , Compostos de Sulfonilureia/farmacologia , Trifosfato de Adenosina/biossíntese , Cálcio/metabolismo , Respiração Celular , Etídio/farmacologia , Glucose/metabolismo , Histocitoquímica , Humanos , Insulina/biossíntese , Insulina/genética , Secreção de Insulina , Líquido Intracelular/metabolismo , Ilhotas Pancreáticas/citologia , Mitocôndrias/enzimologia , Oxirredução , Fosforilação , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas
15.
J Biochem ; 122(3): 512-7, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9348077

RESUMO

His 15 of hen lysozyme is located at the protein surface and is partly buried by the neighboring residues. The side chain of His 15 forms hydrogen bonds with surrounding residues and these hydrogen bonds are somewhat buried. A series of mutant lysozymes at the position 15 (Gly, Ala, Val, and Phe) was prepared, and their stabilities were analyzed by GdnHCl denaturation and X-ray crystallography. The mutants were less stable than the wild type at pH 5.5 and 35 degrees C. In H15G and H15A, X-ray crystallography revealed two fixed water molecules at the mutated region, which formed similar hydrogen bonds to those in the wild type. On the other hand, it was suggested that the hydrogen bonds were disrupted and that several unfavorable van der Waals' contacts occurred in H15V and H15F. Therefore, we concluded that His 15 stabilized the lysozyme structure by forming hydrogen bonds and the best packing with the neighboring residues. Moreover, we found that the method of protein stabilization by increasing the hydrophobicity of an amino acid residue was not always effectively applicable, especially when the residue had formed a hydrogen bond.


Assuntos
Muramidase/química , Mutação , Animais , Fenômenos Químicos , Físico-Química , Galinhas , Cristalografia por Raios X , Estabilidade Enzimática , Guanidina , Muramidase/genética , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
Biosci Biotechnol Biochem ; 61(10): 1710-7, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9362117

RESUMO

To obtain genes with sequence similarity to aminopeptidase T (AP-T) of Thermus aquaticus YT-1, we cloned the genes encoding aminopeptidase Th (AP-Th) from Thermus thermophilus HB8 and aminopeptidase II (APII) from Bacillus stearothermophilus NCIB8924. The AP-Th gene encoded a polypeptide of 408 amino acid residues and the deduced molecular weight of this subunit was 45,015. The APII gene encoded a polypeptide of 413 amino acid residues with a deduced molecular weight of 46,207. The extent of amino acid sequence similarity between AP-Th and AP-T was 86%, and that between APII and AP-T was 43%. The substrate specificities of these expressed enzymes were similar, and each efficiently hydrolyzed leucyl- or phenyl-peptide substrates. Since the deduced amino acid sequence of these enzymes show no similarity to other known aminopeptidases, they appear to comprise an independent family of peptidases, designated the AP-T family. However, a conserved region within the enzymes of the AP-T family shows similarity to the active site signature of the leucyl aminopeptidase family, suggesting that these enzymes may belong to the leucyl aminopeptidase superfamily.


Assuntos
Aminopeptidases/genética , Proteínas de Bactérias , Geobacillus stearothermophilus/enzimologia , Leucil Aminopeptidase/genética , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Meios de Cultura , Escherichia coli/genética , Fluorometria , Regulação Enzimológica da Expressão Gênica , Geobacillus stearothermophilus/genética , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Thermus thermophilus/genética
17.
J Biochem ; 122(1): 25-31, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9276667

RESUMO

Hen lysozyme, with three alpha-helices (A, B, and C), is a c-type lysozyme. In these lysozymes, Ser24 and Asp88 located at the N-cap position in the B- and C-helix, respectively, are mostly conserved, but residue 4 at the N-cap position in A-helix is variable. To investigate the effect of mutation at position 4 on the stability of hen lysozyme, we prepared five mutant lysozymes and examined their stabilities and structures. Gly4Pro lysozyme (G4P), in which Gly4 was replaced by Pro, was less stable by 8.8 kJ/mol than the wild-type lysozyme, possibly because the side chain at position 7 is shifted away from the A-helix. The other mutant lysozymes were of almost equal stability to the wild-type lysozyme, although the hydrogen bonds of the amide groups at positions N1-N3 in the A-helix were absent or altered. These results indicated that various mutations at the N-cap position in the A-helix would be allowed as long as the negative charge of Glu7 at the N-terminus stabilized the A-helix.


Assuntos
Muramidase/química , Muramidase/genética , Mutação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Estabilidade Enzimática , Feminino , Glicina , Guanidina , Guanidinas/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Muramidase/metabolismo , Conformação Proteica , Desnaturação Proteica
18.
Diabetes ; 46(6): 929-36, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9166661

RESUMO

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and M209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and M209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in [3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and M209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and M209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and M209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.


Assuntos
Regulação da Expressão Gênica/genética , Insulina/farmacologia , Mutação/genética , Fosfoproteínas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , DNA/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Proteínas Substratos do Receptor de Insulina , Camundongos , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismo , Fosforilação , Testes de Precipitina , Ratos , Timidina/metabolismo
19.
J Biochem ; 121(6): 1076-81, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9354379

RESUMO

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.


Assuntos
Muramidase/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Galinhas , Cristalografia por Raios X , Eletroquímica , Estabilidade Enzimática , Feminino , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Desnaturação Proteica , Homologia de Sequência de Aminoácidos , Termodinâmica
20.
Diabetes ; 46(3): 354-62, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9032089

RESUMO

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/biossíntese , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Células CHO , Cloranfenicol O-Acetiltransferase/biossíntese , Cricetinae , Humanos , Proteínas Substratos do Receptor de Insulina , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas
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