RESUMO
Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Poli(ADP-Ribose) Polimerases/fisiologia , Animais , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Resistência a Medicamentos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/genética , EstreptozocinaRESUMO
Superantigens have been implicated in the pathogenesis of type I diabetes and other immune-mediated diseases. We therefore tested the hypothesis of an abnormal reactivity of the immune system toward bacterial superantigens during the prediabetic phase. For this purpose, splenocytes from NOD (H-2g7) mice were exposed to two well-characterized superantigens: Staphylococcal aureus enterotoxin-B (SEB) and toxic shock syndrome toxin-1 (TSST-1). Cells from BALB/c (H-2d) and C57BL/6 (H-2b) mice as well as those from NON (H-2non) and NOR (H-2g7) mice were used as controls. After 72 h of co-culture with the superantigens or the mitogen concanavalin A (Con A), proliferative response and mitochondrial activity were determined. In the culture supernatants, the cytokines gamma-interferon (IFN-gamma) and interleukin 10 (IL-10) were measured. Striking similarities between NOD cells and major histocompatiblity complex (MHC)-identical NOR cells could be observed with regard to a low proliferative and mitochondrial response to SEB, accompanied by a normal response to TSST-1 and Con A, respectively. In addition, only NOD and NOR spleen cells were low producers of the T-helper 1 (Th1) cytokine IFN-gamma in response to SEB. Conversely, abnormally high IFN-gamma levels were induced by TSST-1 in NOD and NOR spleen cells. The cytokine response to Con A was also biased toward IFN-gamma in both NOD and NOR. Since IFN-gamma and IL-10 are crucial disease-promoting or -protecting mediators in prediabetic NOD mice, superantigens may affect pathogenesis by acting on the Th1/Th2 cytokine balance. The low responder status toward SEB in NOD spleen cells may be of pathogenetic relevance in view of recent findings that the insulin B-chain also interacts with the SEB binding site on MHC class II molecules. In conclusion, we show here that immune cells from mice with a diabetes-associated MHC type respond differently to common environmental superantigens than do immune cells from control strains.
Assuntos
Toxinas Bacterianas , Diabetes Mellitus Tipo 2/imunologia , Enterotoxinas/farmacologia , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Superantígenos/farmacologia , Animais , Concanavalina A/farmacologia , Feminino , Interferon gama/biossíntese , Interleucina-10/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Baço/imunologia , Staphylococcus aureusRESUMO
In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the beta-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote beta-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.
Assuntos
Resposta ao Choque Térmico , Ilhotas Pancreáticas/metabolismo , Ratos Endogâmicos BB/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Haplótipos , Proteínas de Choque Térmico/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Complexo Principal de Histocompatibilidade , Nitroprussiato/farmacologia , Ratos , Ratos Endogâmicos F344/fisiologia , Ratos Wistar/fisiologia , Estreptozocina/farmacologia , Xantina Oxidase/farmacologiaAssuntos
Morte Celular/fisiologia , Dano ao DNA , Ilhotas Pancreáticas/fisiologia , NAD/metabolismo , Espécies Reativas de Oxigênio , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipoxantina/metabolismo , Hipoxantina/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Niacinamida/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Wistar , Xantina Oxidase/metabolismoRESUMO
When cultures of pancreatic islet cells are exposed to the nitric oxide donor sodium nitroprusside, to enzymatically generated reactive oxygen intermediates or to streptozotocin cell lysis occurs after 4-12 h. We report here that a heat shock at 43 degrees C for 90 min reduces cell lysis from nitric oxide (0.45 mM sodium nitroprusside) by 70%, from reactive oxygen intermediates (12 mU xanthine oxidase and 0.05 mM hypoxanthine) by 80% and from streptozotocin (1.5 mM) by 90%. Heat shock induced resistance was observed immediately after termination of the 90 min culture at 43 degrees C and correlated with enhanced expression of hsp70. The occurrence of DNA strand breaks, a major early consequence of nitric oxide, reactive oxygen intermediates, or streptozotocin action, was not suppressed by heat shock treatment. However, the depletion of NAD+, the major cause of radical induced islet cell death, was suppressed after heat shock (P < 0.01). We conclude that pancreatic islet cells can rapidly activate defence mechanisms against nitric oxide, reactive oxygen intermediates and streptozotocin by culture at 43 degrees C. Islet cell survival is due to the prevention of lethal NAD+ depletion during DNA repair, probably by slowing down poly(ADP-ribose)polymerase activation.
Assuntos
Temperatura Alta , Ilhotas Pancreáticas/efeitos dos fármacos , Óxido Nítrico/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Estreptozocina/toxicidade , Adenosina Difosfato Ribose/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Técnicas In Vitro , Ilhotas Pancreáticas/fisiologia , Masculino , NAD/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.
Assuntos
Dano ao DNA , Ilhotas Pancreáticas/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Óxido Nítrico/toxicidade , Poli(ADP-Ribose) Polimerases/genética , Espécies Reativas de Oxigênio/toxicidade , Animais , Benzamidas/farmacologia , Núcleo Celular/enzimologia , Células Cultivadas , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Cinética , Linfócitos/citologia , Linfócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , NAD/metabolismo , Nitroprussiato/farmacologia , Poli(ADP-Ribose) Polimerases/biossíntese , Xantina Oxidase/farmacologiaRESUMO
We show here that preincubation of pancreatic islet cells with alpha-tocopherol significantly improves their resistance to toxic doses of nitric oxide (NO). No protection was afforded by other antioxidants such as vitamin C or glutathione-monoethyl ester. The pathway of NO induced islet cell death involves DNA damage and excessive activation of poly(ADP-ribose)polymerase leading to irreversible depletion of intracellular NAD+. alpha-Tocopherol was found to interfere at early steps of this pathway, by preventing the occurrence of DNA strand breaks. This indicates that alpha-tocopherol directly interacts with NO or its reactive intermediates. We conclude that alpha-tocopherol is not only part of the cellular defence system against oxygen radicals but also protects eukaryotic cells from NO toxicity.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Óxido Nítrico/toxicidade , Vitamina E/farmacologia , Animais , Ácido Ascórbico/farmacologia , Morte Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glutationa/análogos & derivados , Glutationa/farmacologia , Ilhotas Pancreáticas/citologia , Cinética , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos WistarRESUMO
Morphological and biochemical evidence indicates that in several cell types, lysozyme is found in both lysosomes and the medium. Here we report that in calcitriol-treated human promonocytes U937, in which approx. two-thirds of the synthesized lysozyme is secreted, most of the intracellular lysozyme co-localizes with cathepsin D in lysosomal organelles. In the presence of NH4Cl the lysosomal targeting of procathepsin D, but not that of lysozyme, is inhibited. In the presence of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA; 'TPA'), the lysosomal packaging of lysozyme is almost completely inhibited, while that of procathepsin D is only partially so. However, the inhibition of the lysosomal targeting of procathepsin D by NH4Cl and 4 beta-PMA is additive. The targeting of lysozyme is partially inhibited in the presence of R-59022, an inhibitor of diacylglycerol kinase, whereas it is not affected by 4 alpha-phorbol 12-myristate 13-acetate, an isomer of 4 beta-PMA that does not activate protein kinase C. It is concluded that in U937 cells both carbohydrate-dependent and -independent recognition contributes to the lysosomal targeting of soluble proteins. We suggest that the carbohydrate-independent traffic of proteins to lysosomal compartments is controlled by a signalling pathway involving protein kinase C.
Assuntos
Cloreto de Amônio/farmacologia , Catepsina D/metabolismo , Lisossomos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Glucuronidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Microscopia Eletrônica , Monócitos/efeitos dos fármacos , Testes de Precipitina , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Despite extensive studies on streptozotocin, alloxan and nitric oxide toxicity in pancreatic islets the mechanism of oxygen radical induced islet cell death has not been determined. The present study shows at the level of single cells that following exposure to oxygen radicals generated from xanthine oxidase DNA strand breaks occur in cell nuclei within 5-60 min and precede cell death by several hours. Similar kinetics were seen when treating islet cells with the alkylating agent streptozotocin. Immunofluorescence studies demonstrated the endogenous formation of ADP-ribose polymers in nearly all islet cell nuclei within minutes of treatment with xanthine oxidase, indicating activation of the enzyme poly(ADP-ribose) polymerase (PARP). Concomitantly, cellular NAD+ depletion was noted. Nicotinamide largely prevented NAD+ depletion and in parallel resulted in islet cell survival. These findings identify islet cell nuclear DNA as a primary target of oxygen radical toxicity and suggest related pathways of oxygen radical, nitric oxide and streptozotocin toxicity.
Assuntos
Dano ao DNA , Ilhotas Pancreáticas/efeitos dos fármacos , Niacinamida/farmacologia , Espécies Reativas de Oxigênio/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Imunofluorescência , Hipoxantina , Hipoxantinas/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Cinética , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Wistar , Xantina Oxidase/metabolismo , Xantina Oxidase/farmacologiaRESUMO
Previous studies have shown that DNA strand breaks are an early consequence of nitric oxide toxicity in pancreatic islet cells. We show here that exposure of islet cells to chemical NO donors causes the formation of ADP-ribose polymers in cell nuclei, with concomitant depletion of intracellular NAD+. Islet cell lysis was largely prevented by the ADP-ribosylation inhibitors nicotinamide, 3-aminobenzamide, and 4-amino-1,8-naphthalimide, the latter being a potent new-generation compound with high selectivity for poly(ADP-ribosyl)-ation. These findings indicate a key role of poly(ADP-ribose) polymerase activation in NO toxicity in islet cells.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , NAD/metabolismo , Óxido Nítrico/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , Animais , Benzamidas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Ativação Enzimática , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Cinética , Naftalimidas , Niacinamida/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Quinolonas/farmacologia , Ratos , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina , Fatores de TempoRESUMO
We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.
Assuntos
Amino Açúcares/biossíntese , Cloreto de Amônio/farmacologia , Antígenos CD , Glicoproteínas de Membrana/biossíntese , N-Acetilglucosaminiltransferases , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Calcitriol/farmacologia , Diferenciação Celular/fisiologia , Glucosiltransferases/metabolismo , Humanos , Proteínas de Membrana Lisossomal , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Células Tumorais CultivadasRESUMO
The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.
Assuntos
Cloreto de Amônio/farmacologia , Catepsina D/metabolismo , Hexosefosfatos/metabolismo , Lisossomos/enzimologia , Manosefosfatos/metabolismo , Calcitriol/farmacologia , Linhagem Celular , Fibroblastos/enzimologia , Humanos , Hidrólise , Monócitos/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Precursores de Proteínas/metabolismoRESUMO
Brefeldin A (BFA) has been shown to inhibit transiently the subcellular transport of cathepsin D (Oda & Nishimura (1989) Biochem. Biophys. Res. Commun. 163, 220-225). We studied the effect of this antibiotic on processing of the phosphorylated oligosaccharides in cathepsin D in human promonocytes U937. In the presence of the drug the phosphorylation of cathepsin D precursor continued at a diminished rate. The phosphorylated oligosaccharides in cathepsin D comprised mono- and bis-phosphorylated forms. The relative amounts of the two species were not changed in the presence of BFA. The uncovering of the phosphate groups and the proteolytic processing of the phosphorylated precursor were abolished. In an in vitro assay the uncovering enzyme, N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase was not inhibited by BFA. We suggest that this drug interrupts the traffic between the compartments containing N-acetylglucosaminyl phosphotransferase and N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase.