RESUMO
BACKGROUND: Due to the shortage of multi-organ donors, human pancreatic islet transplantation has now been extended to islets originating from obese subjects. In this study, our aim is to compare the respective sensitivity of human islets from lean vs obese donors to chronic high glucose or high palmitate. METHODS: Human islets were isolated from pancreases harvested from brain-dead multi-organ donors. Islets were cultured during 72 hours in the presence of moderate (16.7 mmol/L) or high (28 mmoL/L) glucose concentrations, or glucose (5.6 mmoL/L) and palmitate (0.4 mmoL/L), before measurement of their response to glucose. RESULTS: We first observed a greater insulin response in islets from obese donors under both basal and high-glucose conditions, confirming their hyperresponsiveness to glucose. When islets from obese donors were cultured in the presence of moderate or high glucose concentrations, insulin response to glucose remained unchanged or was slightly reduced, as opposed to that observed in lean subjects. Moreover, culturing islets from obese donors with high palmitate also induced less reduction in insulin response to glucose than in lean subjects. This partial protection of obese islets is associated with less induction of inducible nitric oxide synthase in islets, together with a greater expression of the transcription factor forkhead box O1 (FOXO1). CONCLUSIONS: Our data suggest that in addition to an increased sensitivity to glucose, islets from obese subjects can be considered as more resistant to glucose and fatty acid excursions and are thus valuable candidates for transplantation.
Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Obesidade/metabolismo , Palmitatos/farmacologia , Idoso , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1ß takes part and controls ß-cell function. In the present study, using the INS-1 rat pancreatic ß-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1ß signaling and biological effects on insulin secretion, ß-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1ß leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic ß-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for ß-cell secretory function. In the same conditions, IL-1ß treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1ß inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1ß concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1ß anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1ß deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.
Assuntos
Interleucina-1beta/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Expressão Gênica , Insulina/biossíntese , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Fator de Transcrição CHOP/metabolismoRESUMO
AIMS/HYPOTHESIS: Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect. METHODS: Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays. RESULTS: nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes. CONCLUSIONS/INTERPRETATION: Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.
Assuntos
Glucose/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Western Blotting , Células Cultivadas , Transportador de Glucose Tipo 4/metabolismo , Imunoprecipitação , Masculino , Células Musculares/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It is well established that, upon changing their natural desert low caloric (succulent halophilic plants) to a regular laboratory high caloric diet, sand rats undergo various phenotypic changes depending on their genetic background and including obesity and various degrees of insulin resistance. Our aim was to investigate the acute effects of Interleukin-1ß (IL-1ß) and Interferon-γ (IFN-γ) on glucose-induced insulin secretion in normal lean sand rats maintained on their natural diet and in obese insulin resistant normoglycemic or type 2 diabetic animals after a 9-month high caloric diet. Animals were fed either a low or a high caloric diet; after 9 months, pancreatic islets were isolated and incubated in the presence of increasing cytokine concentrations. At the end of the high-energy diet, animals were all over-weight, and probably due to a different genetic background, they displayed either insulin resistance, hyperinsulinemia and normoglycemia or a marked type-2 diabetic state. Pancreatic islets from obese insulin resistant normoglycemic animals were much more sensitive and responsive to IL-1ß when compared to lean controls. The cytokine was inefficient in diabetic islets. In conclusion, the markedly increased insulinotropic effect of IL-1ß in obese diabetes-resistant sand rat could participate and be involved in pancreatic ß-cell hyperactivity that compensates for insulin resistance and thereby prevent the development of type 2 diabetes in these animals.
Assuntos
Diabetes Mellitus Tipo 2/prevenção & controle , Resistência à Insulina , Interleucina-1beta/farmacologia , Obesidade/prevenção & controle , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Colesterol/sangue , Dieta , Ingestão de Alimentos , Gerbillinae , Insulina/sangue , Interferon gama/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic ß-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic ß-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Organogênese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Transporte de Elétrons , Células HeLa , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced ß-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and ß-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1ß, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning ß-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1ß and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of ß-cell function by IL-1ß. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory process. That the latter could contribute to ß-cell hyperactivity/proliferation and possibly lead to progressive ß-cell failure in these animals, deserves further investigations.
Assuntos
Ingestão de Alimentos/imunologia , Ilhotas Pancreáticas/metabolismo , Obesidade/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular/fisiologia , Ingestão de Alimentos/fisiologia , Imunofluorescência , Técnicas In Vitro , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Obesidade/imunologia , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase em Tempo Real , Receptores Tipo I de Interleucina-1/metabolismo , Receptores de Interleucina-6/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: The aim of this study was to investigate if the metabolic alterations observed after static magnetic field (SMF) exposure participates in the development of a pre-diabetic state. A comparison study using the insulin resistant animal model, the Zucker rat and the SMF-exposed Wistar rat was carried out. MATERIALS AND METHODS: Zucker rats were compared to Wistar rats either exposed to a 128 mT or 0 mT SMF (sham exposed) and analysed. This moderate-intensity SMF exposure of Wistar rats was performed for 1 h/day during 15 consecutive days. RESULTS: Wistar rats exposed to the SMF showed increased levels of carbohydrate and lipid metabolites (i.e., lactate, glycerol, cholesterol and phospholipids) compared to sham-exposed rats. Zucker rats displayed a normoglycemia associated with a high insulin level as opposed to Wistar rats which presented hyperglycemia and hypoinsulinemia after exposure to the SMF. During the glucose tolerance test, unexposed Zucker rats and Wistar rats exposed to the SMF exhibited a significantly higher hyperglycemia compared to sham-exposed Wistar rats suggesting an impairment of glucose clearance. In muscle, glycogen content was lower and phospholipids content was elevated for both unexposed Zucker rats and Wistar rats exposed to the SMF compared to Wistar rats sham control. CONCLUSIONS: This study provides evidence that the metabolic alterations following exposure to a static magnetic field of moderate intensity could trigger the development of a pre-diabetic state.
Assuntos
Campos Eletromagnéticos , Animais , Diabetes Mellitus Experimental/prevenção & controle , Glucose/metabolismo , Teste de Tolerância a Glucose , Glicogênio/química , Metabolismo dos Lipídeos/efeitos da radiação , Fígado/metabolismo , Masculino , Músculos/metabolismo , Oxigênio/química , Ratos , Ratos Wistar , Ratos Zucker , Especificidade da Espécie , TemperaturaRESUMO
BACKGROUND: We previously demonstrated that the PDS gene is involved in the genetic susceptibility to autoimmune thyroid diseases (AITD) in Tunisia. In the same population, we now investigated the presence of anti-pendrin auto-antibodies (aAbs) in AITD patients' sera. METHODS: Thirty seven Tunisian AITD patients and 19 healthy subjects from families previously linked to the PDS gene, 75 unrelated patients and 20 healthy unrelated subjects were included in our study. The detection of anti-pendrin aAbs in patients' sera was performed by ELISA using membrane protein extracts of CHO cells expressing pendrin (CHO-hPDS) and by immunofluorescence using transient COS-7 cells expressing a GFP tagged pendrin. CHO cells transfected with human TPO in the same ELISA conditions were used as positive control. RESULTS: The majority of AITD patients' sera were positive for the presence of anti-TPO aAbs. In contrast, no reactivity was detected with CHO-hPDS membrane protein extracts. Likewise, no significant immunostaining was found on transfected COS-7cells upon exposure to patients' and controls' sera. CONCLUSIONS: Our data point to the absence of anti-pendrin aAbs in Tunisian AITD patients' sera.
Assuntos
Autoanticorpos/sangue , Proteínas de Membrana Transportadoras/imunologia , Tireoidite Autoimune/sangue , Tireoidite Autoimune/imunologia , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Família , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Transportadores de Sulfato , Tireoidite Autoimune/diagnóstico , Transfecção , TunísiaRESUMO
BACKGROUND AND AIMS: Increasing environmental pollution may participate in the growing incidence of metabolic disorders. Static magnetic fields (SMFs) are an emerging environmental health issue due to increased exposure in residential and commercial areas; however, their metabolic effects in serum and skeletal muscle are largely unknown. The aim of this study was to investigate the effect of SMF exposure on glucose and lipid metabolism in serum and skeletal muscles of rats. METHODS: Twelve 6- to 7-week-old male Wistar rats were randomly divided into two groups: rats exposed to 128 mT SMF and sham-exposed rats. This moderate-intensity exposure was performed for 1 h/day for 15 consecutive days. RESULTS: Animals exposed to 128 mT SMF displayed significant changes in both glucose (i.e., increases in plasma glucose and lactate and decrease in plasma insulin levels) and lipid (i.e., increases in plasma glycerol, cholesterol and phospholipids but not triglyceride levels) metabolism. During intraperitoneal glucose tolerance tests, SMF-exposed rats displayed significantly higher hyperglycemia compared to sham-exposed rats despite similar insulin levels in both groups. In tissues, SMF exposure induced significant alterations in enzyme activities only in glycolytic muscles and caused a significant decrease in quadriceps and liver glycogen content together with increased phospholipid levels. CONCLUSIONS: This study provides evidence that subacute SMF exposure of moderate intensity induces important alterations of glucose and lipid metabolisms, which deserve further investigations to evaluate long-term consequences.
Assuntos
Glucose/metabolismo , Metabolismo dos Lipídeos , Magnetismo , Músculo Esquelético/metabolismo , Animais , Peso Corporal , Glicogênio/metabolismo , Fígado/metabolismo , Masculino , Oxirredução , Ratos , Ratos WistarRESUMO
BACKGROUND: Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis. METHODOLOGY: We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats). PRINCIPAL FINDINGS: Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid). CONCLUSIONS/SIGNIFICANCE: We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.
Assuntos
Resistência à Insulina , Fígado/fisiologia , Óxido Nítrico/fisiologia , Ácido Oleico/administração & dosagem , Animais , Inibidores Enzimáticos/farmacologia , Comportamento Alimentar , Glucose/metabolismo , Hipotálamo/enzimologia , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , RatosRESUMO
Extracellular adenosine triphosphate is able to modulate pancreatic beta-cell function, acting on P2 purinergic ionotropic (P2X) and metabotropic (P2Y) receptors. Physiologically, ATP entrains beta-cells into a common rhythm by coordinating Ca(2+) oscillations; it plays a central role in insulin secretion pulsatility. ATP also triggers a positive feedback signal amplifying glucose-induced insulin release, which argues for a potential pharmacological application. ATP has consistently been shown to increase cytoplasmic free calcium concentration, notably in human tissue. Acting on P2X receptors, of which different molecular subtypes are expressed in beta-cells, it leads to a transient insulin release that may involve a closure of K(ATP) channels or a rapidly decaying inward current. Activation of G-protein-coupled P2Y receptors triggers different signalling pathways and amplifies insulin release in a glucose-dependent way. It has recently been shown that pancreatic beta-cells express different molecular subtypes of receptors, which may explain the complex interaction of P2Y ligands on high- and low-affinity binding sites. Despite the complexity of this purinergic pharmacology, consistent pre-clinical data suggest the potential of P2Y receptor agonists as drug candidates for type 2 diabetes.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Secreção de InsulinaRESUMO
Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35)S]thiotriphosphate) (ATP-alpha-[(35)S]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[(35)S] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[(35)S] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y(1) receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites. Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Receptores Purinérgicos P2/genética , Tionucleotídeos/metabolismoRESUMO
We previously showed that pancreatic beta-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting beta-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nomega-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.
Assuntos
Dineínas/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Linhagem Celular , Dineínas do Citoplasma , Dineínas/fisiologia , Glucose/farmacologia , Homeostase , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos WistarRESUMO
PURPOSE: A series of C2-substituted ATP analogues was previously shown to have potent insulin-secreting properties, yet with poor tissue-selectivity for the pancreatic beta-cell. The present study was designed to evaluate the binding profile on beta-cell membranes and the effects on insulin release and pancreatic vascular resistance of a second generation of P2Y(1) receptor agonists, based on C2-substitution of the adenosine 5'-O-(1-boranotriphosphate) scaffold. MATERIALS AND METHODS: Functional experiments were performed in the rat isolated pancreas model; binding studies with ATP-alpha-[(35)S] were performed in membrane homogenates from the rat insulinoma INS-1 cell line. The diastereoisomers of the compounds are designated by A and B. RESULTS: Under 8.3 mmol l(-1) glucose, 2-methylthio-ATP-alpha-B, A isomer, induced a biphasic and concentration dependent insulin response; its maximal efficacy reaches ninefold the baseline secretion and its EC(50) is 28.1 nmol l(-1). No significant effect of this isomer was observed on vascular resistance, whereas the B isomer, which was a less potent insulin secretagogue, consistently induced a transient vasoconstriction. Interestingly, the insulin response induced by 2-methylthio-ATP-alpha-B, A isomer, was clearly glucose-dependent. This drug competes with ATP-alpha-[(35)S] binding in a complex two sites interaction model, with a K(0.5) value of 17.7 nmol l(-1). 2-Chloro-ATP-alpha-B had a similar insulin-secreting profile as 2-methylthio-ATP-alpha-B, with a lower tissue-selectivity. The non-substituted ATP-alpha-B analog, A isomer, was less potent than the C2-substituted derivatives (A isomers) and had a vasorelaxant effect. CONCLUSIONS: We conclude that 2-methylthio-ATP-alpha-B, A isomer, is a potent and tissue-selective P2Y receptor agonist with high efficacy. Its insulin-releasing action is glucose-dependent, which gives interest to this compound as a drug candidate for treating type 2 diabetes.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Boranos/farmacologia , Insulina/metabolismo , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Secreção de Insulina , Masculino , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y1 , Tionucleotídeos/metabolismo , Resistência Vascular/efeitos dos fármacosRESUMO
By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Antígenos CD4/imunologia , Dineínas/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Dineínas/genética , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Linfócitos T/efeitos dos fármacosRESUMO
The aim of this study was to determine whether epiregulin, a novel member of EGF-related growth factor family, was able to affect proliferation and secretory function of rat insulinoma INS-1E and RINm5F cell lines. A 24 h treatment with epiregulin resulted in a stimulation of INS-1E and RINm5F cells proliferation; this effect was completely blocked in the presence of an anti-epiregulin antibody which did not affect basal DNA synthesis in the absence of added ligand. In acute experiments, epiregulin was able to potentiate insulin release in the presence of glucose or arginine, in the two cell lines. Finally, in the two cell lines expressing ErbB receptors, we demonstrated that only EGFR/ErbB1 was activated by epiregulin. Thus, epiregulin appears as a new growth and insulinotropic factor in pancreatic beta cell lines.
Assuntos
Proliferação de Células , Fator de Crescimento Epidérmico/fisiologia , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , DNA/biossíntese , Fator de Crescimento Epidérmico/imunologia , Epirregulina , Receptores ErbB/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , RatosRESUMO
Blood glucose concentration is controlled by a number of hormone and neurotransmitter signals, either increasing or reducing glucose levels in the case of hypoglycemia or hyperglycemia, respectively. The pancreatic beta-cell responds to an increase in circulating glucose levels by a cascade of metabolic and electrophysiological events leading to the secretion of insulin. Type 2 diabetes is a metabolic disorder characterized by chronic hyperglycemia; the progressive pancreatic beta-cell dysfunction, with altered insulin production and secretion, is a major pathophysiological determinant of the disease together with the resistance of insulin-sensitive tissues to the action of the hormone. Hence, drugs which stimulate or enhance insulin secretion will reduce plasma glucose concentrations; this lowering of hyperglycemia will, in turn, reduce the occurrence of long-term complications. K(ATP) channels play a critical role in insulin secretion and can be considered as transducers of glucose-induced metabolic changes into biophysical events leading to the exocytosis of insulin granules. All currently marketed insulin secretagogues, sulfonylureas and glinides, target the beta-cell K(ATP) channels and reduce their opening probability. They induce insulin release regardless of the plasma glucose concentration, thus favoring the occurrence of hypoglycemia in the fasting state. Despite the intensive use of current drugs, many patients suffering from type 2 diabetes still exhibit poor glycemic control, others fail to respond to the treatment, and some develop serious complications. Therefore, there is a real need for innovative compounds, either enhancing insulin secretion from the pancreas or improving insulin action on the hormone-sensitive tissues. Here, we overview the existing and novel approaches targeting the beta-cell to enhance the release of insulin, with special emphasis on new ways of amplifying insulin secretion in a glucose-dependent manner.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Humanos , Hipoglicemiantes/uso terapêutico , Secreção de Insulina , Ilhotas Pancreáticas/metabolismoRESUMO
Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
Assuntos
Aminopeptidases/metabolismo , Glucagon/biossíntese , Glucagon/metabolismo , Ilhotas Pancreáticas/enzimologia , Metaloendopeptidases/metabolismo , Fragmentos de Peptídeos/biossíntese , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Animais , Cálcio/farmacologia , Células Cultivadas , Cobalto/farmacologia , Endopeptidases/genética , Endopeptidases/metabolismo , Hipoglicemia/metabolismo , Metaloendopeptidases/genética , Camundongos , Fenantrolinas/farmacologia , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Zinco/farmacologiaRESUMO
Cellulose membrane supported peptide arrays, prepared according to the Spot method, allow the rapid identification and characterization of protein-protein interaction sites. Here, the method was used to screen reactive peptides from different proteins that bind to a single molecule, the PIN protein. PIN possesses two binding grooves, that have been shown to interact with several targets, including neuronal NO synthase, dynein intermediate chain, myosin V, the proapoptotic protein Bim, the scaffolding proteins DAP1alpha and gephyrin, and the transcription factor NRF-1. Arrays of peptides representing sequences of these targets were probed for reactivity with GST-tagged PIN, enabling the precise identification of binding motifs. Binding motifs were then minimized to seven or eight amino acid long peptides: YSKETQT for dynein IC, CDKSTQT for Bim, KDTGIQVD for nNOS, QSVGVQV for DAP1alpha and EDKNTMTD for myosin V. Alascan and substitution analysis provided proof that the Gln residue is critical for the interaction and cannot be easily replaced. Positions -1 and +1, just flanking the pivotal Gln, are also important; they consist of hydrophobic residues (Thr, Val) that could only be replaced by hydrophobic or aromatic amino acids. Position -4 is also critical for binding, with its Asp or Ser being replaceable to some extent. Alignment of sequences of proteins known to bind PIN shows that the most frequent amino acids in the motif are DKGTQT, consistent with the Spot results. We postulate that the degenerate character of binding to PIN is based on the propensity of several sequences to adopt a beta-strand conformation that allows the Gln residue to position itself in the PIN channel and on the conformational breathing of the PIN binding groove.
Assuntos
Dineínas/química , Análise Serial de Proteínas , Proteínas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Celulose , Dineínas do Citoplasma , Dineínas/síntese química , Indicadores e Reagentes , Dados de Sequência Molecular , Miosina Tipo V/química , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase Tipo I , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo , RatosRESUMO
We previously showed that pancreatic beta-cells express a neuronal isoform of nitric oxide synthase (nNOS) that controls insulin secretion by exerting two enzymatic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now bring evidence that two inhibitors of nNOS, N-omega-nitro-l-arginine methyl ester (l-NAME) and 7-nitroindazole (7-NI), increase glucose-induced insulin secretion but affect beta-cell function differently. In the presence of l-NAME, insulin response is monophasic, whereas 7-NI preserves the normal biphasic secretory pattern. In addition, the alterations of beta-cell functional response induced by the inhibitors also differ by their sensitivity to a substitutive treatment with sodium nitroprusside, a chemical NO donor. These differences are probably related to the nature of the two inhibitors. Indeed, using low-temperature SDS-PAGE and real-time analysis of nNOS dimerization by surface plasmon resonance, we could show that 7-NI, which competes with arginine and tetrahydrobiopterin (BH(4)), an essential cofactor for nNOS dimer formation, inhibits dimerization of the enzyme, whereas the substrate-based inhibitor l-NAME stabilizes the homodimeric state of nNOS. The latter effect could be reproduced by the two endogenous inhibitors of NOS, N-omega-methyl-l-arginine and asymmetric dimethylarginine, and resulted interestingly in a reduced ability of the protein inhibitor of nNOS (PIN) to dissociate nNOS dimers. We conclude that intracellular factors able to induce abnormalities in the nNOS monomer/dimer equilibrium could lead to pancreatic beta-cell dysfunction.