RESUMEN
12-lipoxygenase (12-LOX) is one of several enzyme isoforms responsible for the metabolism of arachidonic acid and other poly-unsaturated fatty acids to both pro- and anti-inflammatory lipid mediators. Mounting evidence has shown that 12-LOX plays a critical role in the modulation of inflammation at multiple checkpoints during diabetes development. Due to this, interventions to limit pro-inflammatory 12-LOX metabolites either by isoform-specific 12-LOX inhibition, or by providing specific fatty acid substrates via dietary intervention, has the potential to significantly and positively impact health outcomes of patients living with both type 1 and type 2 diabetes. To date, the development of truly specific and efficacious inhibitors has been hampered by homology of LOX family members; however, improvements in high throughput screening have improved the inhibitor landscape. Here, we describe the function and role of human 12-LOX, and mouse 12-LOX and 12/15-LOX, in the development of diabetes and diabetes-related complications, and describe promise in the development of strategies to limit pro-inflammatory metabolites, primarily via new small molecule 12-LOX inhibitors.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Complicaciones de la Diabetes/enzimología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Humanos , Células Secretoras de Insulina/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Transducción de SeñalRESUMEN
Adipose tissue (AT) inflammation is an emerging factor contributing to cardiovascular disease. STAT4 is a transcription factor expressed in adipocytes and in immune cells and contributes to AT inflammation and insulin resistance in obesity. The objective of this study was to determine the effect of STAT4 deficiency on visceral and peri-aortic AT inflammation in a model of atherosclerosis without obesity. Stat4(-/-)Apoe(-/-) mice and Apoe(-/-) controls were kept either on chow or Western diet for 12 weeks. Visceral and peri-aortic AT were collected and analyzed for immune composition by flow cytometry and for cytokine/chemokine expression by real-time PCR. Stat4(-/-)Apoe(-/-) and Apoe(-/-) mice had similar body weight, plasma glucose, and lipids. Western diet significantly increased macrophage, CD4+, CD8+, and NK cells in peri-aortic and visceral fat in Apoe(-/-) mice. In contrast, in Stat4(-/-)Apoe(-/-) mice, a Western diet failed to increase the percentage of immune cells infiltrating the AT. Also, IL12p40, TNFa, CCL5, CXCL10, and CX3CL1 were significantly reduced in the peri-aortic fat in Stat4(-/-)Apoe(-/-) mice. Importantly, Stat4(-/-)Apoe(-/-) mice on a Western diet had significantly reduced plaque burden vs Apoe(-/-) controls. In conclusion, STAT4 deletion reduces inflammation in peri-vascular and visceral AT and this may contribute via direct or indirect effects to reduced atheroma formation.
Asunto(s)
Aterosclerosis/metabolismo , Grasa Intraabdominal/metabolismo , Macrófagos/metabolismo , Paniculitis/metabolismo , Factor de Transcripción STAT4/metabolismo , Animales , Aorta , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Polaridad Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Dieta Occidental/efectos adversos , Femenino , Grasa Intraabdominal/inmunología , Grasa Intraabdominal/patología , Macrófagos/inmunología , Macrófagos/patología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Ratones Noqueados , Ovario , Paniculitis/etiología , Paniculitis/inmunología , Paniculitis/patología , Distribución Aleatoria , Factor de Transcripción STAT4/genética , Organismos Libres de Patógenos EspecíficosRESUMEN
Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in sustained inflammation in diabetes and atherosclerosis. 12- and 15-lipoxygenase (LO), such as 12/15-LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12- and 15-LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue-specific knockout of 12/15-LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12- and 15-LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12- and 15-LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)-12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1) in islets and IL-17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.
Asunto(s)
Citocinas/fisiología , Inflamación/fisiopatología , Resistencia a la Insulina/fisiología , Islotes Pancreáticos/fisiopatología , Enfermedades Vasculares/fisiopatología , Tejido Adiposo/fisiología , Animales , Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Humanos , Mediadores de Inflamación/fisiología , RatonesRESUMEN
Type 1 diabetes (T1D) results from T helper type 1 (Th1)-mediated autoimmune destruction of insulin-producing ß cells. Novel experimental therapies for T1D target immunomodulation, ß cell survival and inflammation. We examined combination therapy with the dipeptidyl peptidase-IV inhibitor MK-626 and the histone deacetylase inhibitor vorinostat in the non-obese diabetic (NOD) mouse model of T1D. We hypothesized that combination therapy would ameliorate T1D by providing protection from ß cell inflammatory destruction while simultaneously shifting the immune response towards immune-tolerizing regulatory T cells (T(regs)). Although neither mono- nor combination therapies with MK-626 and vorinostat caused disease remission in diabetic NOD mice, the combination of MK-626 and vorinostat increased ß cell area and reduced the mean insulitis score compared to diabetic control mice. In prediabetic NOD mice, MK-626 monotherapy resulted in improved glucose tolerance, a reduction in mean insulitis score and an increase in pancreatic lymph node T(reg) percentage, and combination therapy with MK-626 and vorinostat increased pancreatic lymph node T(reg) percentage. We conclude that neither single nor combination therapies using MK-626 and vorinostat induce diabetes remission in NOD mice, but combination therapy appears to have beneficial effects on ß cell area, insulitis and T(reg) populations. Combinations of vorinostat and MK-626 may serve as beneficial adjunctive therapy in clinical trials for T1D prevention or remission.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/administración & dosificación , Inhibidores de Histona Desacetilasas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Triazoles/administración & dosificación , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos NOD , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Factor de Crecimiento Transformador beta1/metabolismo , VorinostatRESUMEN
AIMS/HYPOTHESIS: IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. METHODS: In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1ß and IFN-γ). RESULTS: The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. CONCLUSIONS/INTERPRETATION: These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Interleucina-12/biosíntesis , Islotes Pancreáticos/metabolismo , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Animales , Anticuerpos Neutralizantes/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/inmunología , Interferón gamma/metabolismo , Interleucina-12/antagonistas & inhibidores , Interleucina-12/metabolismo , Islotes Pancreáticos/inmunología , Ratones , ARN Mensajero/metabolismo , Propiedades de Superficie , Técnicas de Cultivo de Tejidos , Donantes de TejidosRESUMEN
Adipose tissue inflammation and reduced pancreatic ß-cell function are key issues in the development of cardiovascular disease and progressive metabolic dysfunction in type 2 diabetes mellitus. The aim of this study was to determine the effect of the DPP IV inhibitor sitagliptin on adipose tissue and pancreatic islet inflammation in a diet-induced obesity model. C57Bl/6J mice were placed on a high-fat (60% kcal fat) diet for 12 wk, with or without sitagliptin (4 g/kg) as a food admix. Sitagliptin significantly reduced fasting blood glucose by 21% as well as insulin by â¼25%. Sitagliptin treatment reduced body weight without changes in overall body mass index or in the epididymal and retroperitoneal fat mass. However, sitagliptin treatment led to triple the number of small adipocytes despite reducing the number of the very large adipocytes. Sitagliptin significantly reduced inflammation in the adipose tissue and pancreatic islet. Macrophage infiltration in adipose tissue evaluated by immunostaining for Mac2 was reduced by sitagliptin (P < 0.01), as was the percentage of CD11b+/F4/80+ cells in the stromal vascular fraction (P < 0.02). Sitagliptin also reduced adipocyte mRNA expression of inflammatory genes, including IL-6, TNFα, IL-12(p35), and IL-12(p40), 2.5- to fivefold as well as 12-lipoxygenase protein expression. Pancreatic islets were isolated from animals after treatments. Sitagliptin significantly reduced mRNA expression of the following inflammatory cytokines: MCP-1 (3.3-fold), IL-6 (2-fold), IL-12(p40) (2.2-fold), IL-12(p35) (5-fold, P < 0.01), and IP-10 (2-fold). Collectively, the results indicate that sitagliptin has anti-inflammatory effects in adipose tissue and in pancreatic islets that accompany the insulinotropic effect.
Asunto(s)
Tejido Adiposo/patología , Antiinflamatorios , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inflamación/patología , Islotes Pancreáticos/patología , Pirazinas/farmacología , Triazoles/farmacología , Adipocitos/patología , Adipocitos/ultraestructura , Adiposidad/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Citocinas/metabolismo , Citometría de Flujo , Glucosa/metabolismo , Glucosa/farmacología , Intolerancia a la Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Insulina/metabolismo , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Infiltración Neutrófila/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fosfato de SitagliptinaRESUMEN
Lipid-induced insulin resistance is associated with intracellular accumulation of inhibitory intermediates depending on the prevalent fatty acid (FA) species. In cultured myotubes, ceramide and phosphatidic acid (PA) mediate the effects of the saturated FA palmitate and the unsaturated FA linoleate, respectively. We hypothesized that myriocin (MYR), an inhibitor of de novo ceramide synthesis, would protect against glucose intolerance in saturated fat-fed mice, while lisofylline (LSF), a functional inhibitor of PA synthesis, would protect unsaturated fat-fed mice. Mice were fed diets enriched in saturated fat, n-6 polyunsaturated fat, or chow for 6 wk. Saline, LSF (25 mg/kg x d), or MYR (0.3 mg/kg x d) were administered by mini-pumps in the final 4 wk. Glucose homeostasis was examined by glucose tolerance test. Muscle ceramide and PA were analyzed by mass spectrometry. Expression of LASS isoforms (ceramide synthases) was evaluated by immunoblotting. Both saturated and polyunsaturated fat diets increased muscle ceramide and induced glucose intolerance. MYR and LSF reduced ceramide levels in saturated and unsaturated fat-fed mice. Both inhibitors also improved glucose tolerance in unsaturated fat-fed mice, but only LSF was effective in saturated fat-fed mice. The discrepancy between ceramide and glucose tolerance suggests these improvements may not be related directly to changes in muscle ceramide and may involve other insulin-responsive tissues. Changes in the expression of LASS1 were, however, inversely correlated with alterations in glucose tolerance. The demonstration that LSF can ameliorate glucose intolerance in vivo independent of the dietary FA type indicates it may be a novel intervention for the treatment of insulin resistance.
Asunto(s)
Ceramidas/metabolismo , Grasas de la Dieta/farmacología , Músculo Esquelético/efectos de los fármacos , Ácidos Fosfatidicos/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Línea Celular , Grasas de la Dieta/administración & dosificación , Ácidos Grasos/administración & dosificación , Ácidos Grasos/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/farmacología , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/prevención & control , Inmunosupresores/farmacología , Insulina/sangre , Ácido Linoleico/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Oxidorreductasas/metabolismo , Palmitatos/farmacología , Pentoxifilina/análogos & derivados , Pentoxifilina/farmacología , Triglicéridos/metabolismoRESUMEN
CONTEXT: Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by catalyzing the oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets. OBJECTIVE: We compared the effects of 12-LO products on human islet viability and function. DESIGN: Human islets were treated with stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE and then examined for insulin secretion and islet viability. The p38-MAPK (p38) and JNK stress-activated pathways were investigated as mechanisms of 12-LO-mediated islet inhibition in rodent and human islets. RESULTS: Insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE at 1 nm reduced viability activity by 32% measured by MTT assay and increased cell death by 50% at 100 nm in human islets. These effects were partially reversed with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE increased phosphorylated p38-MAPK (pp38) protein activity in human islets. Injecting 12-LO siRNA into C57BL/6 mice reduced 12-LO and pp38-MAPK protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets. CONCLUSIONS: These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human beta-cells from inflammatory injury.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Pentoxifilina/análogos & derivados , Pentoxifilina/farmacología , Fosforilación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIMS/HYPOTHESIS: Beta cell inflammation and cytokine-induced toxicity are central to autoimmune diabetes development. Lipid mediators generated upon lipoxygenase (LO) activation can participate in inflammatory pathways. 12LO-deficient mice are resistant to streptozotocin-induced diabetes. This study sought to characterise the cellular processes involving 12LO-activation lipid inflammatory mediator production in cytokine-treated pancreatic beta cells. METHODS: Islets and beta cell lines were treated with a combination of IL-1beta, IFN-gamma and TNF-alpha, or the 12LO product 12(S)-hydroxyeicosatetraenoic acid (HETE). Insulin secretion was measured using an enzyme immunoassay, and cell viability was evaluated using an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. 12LO activity was evaluated and 12LO protein levels were determined using immunoblotting with a selective leucocyte type 12LO antibody. Cellular localisation of 12LO was evaluated using immunocytochemistry. RESULTS: Basal expression of leucocyte type 12LO protein was found in human and mouse islets and in several rodent beta cell lines. In mouse beta-TC3 cells, and in human islets, cytokines induced release of 12-HETE within 30 min. Cytokine addition also induced a rapid translocation of 12LO protein from the cytosol to the nucleus of beta-TC3 cells as shown by subcellular fractionation and immunostaining. Cytokine-induced cell death and inhibition of insulin secretion were partially reversed by baicalein, a 12LO inhibitor. 12(S)-HETE inhibited beta-TC3 cell insulin release in a time- and concentration-dependent manner. Incubating beta-TC3 cells with 100 nmol/l of 12(S)-HETE resulted in a 57% reduction in basal insulin release (6 h), and a 17% increase in cell death (18 h) as compared with untreated cells. 12(S)-HETE activated the stress-activated protein kinase c-Jun N-terminal kinase and p38 within 15 min, as judged by increased kinase protein phosphorylation. CONCLUSIONS/INTERPRETATION: The data suggest that inflammatory cytokines rapidly activate 12LO and show for the first time that cytokines induce 12LO translocation. The effects of 12-HETE on insulin secretion, cytotoxicity and kinase activation were similar to the effects seen with cytokines. The results provide mechanistic information of cytokine-induced toxic effects on pancreatic beta cells and support the hypothesis that blocking 12LO activation could provide a new therapeutic way to protect pancreatic beta cells from autoimmune injury.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Citocinas/toxicidad , Islotes Pancreáticos/enzimología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Línea Celular , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Transporte de Proteínas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Pancreatic islet transplantation can replace insulin-secreting beta cells in patients with diabetes mellitus. However, current methodology for isolating islets from a pancreas only retrieves a portion of the total islets. Within these limited number of islets, nearly 50% of beta cells lose biological function before transplantation. Protecting and improving beta-cell viability and function was the goal of this study. Previously we observed that an anti-inflammatory compound, lisofylline (LSF), protects beta cells from cytotoxicity during diabetes development. In this study, we demonstrated that human islets treated in vitro with LSF retained beta-cell glucose responsiveness and insulin secretion in the presence of multiple proinflammatory cytokines. In addition, LSF treatment in vitro enhanced basal insulin production in beta cells, suggesting that LSF can directly improve beta-cell function. LSF reduced beta-cell apoptosis induced by proinflammatory cytokines by 50%. Importantly, 30% fewer LSF-treated islets were sufficient to achieve insulin independence in a murine islet transplantation model. These results demonstrate the ability of LSF-like compounds to protect and enhance beta-cell function, suggesting the potential of using LSF or its analogs in islet transplantation.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Pentoxifilina/análogos & derivados , Animales , Antiinflamatorios no Esteroideos/farmacología , Células Cultivadas , Citocinas/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Interferón gamma/farmacología , Interleucina-1/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos , Ratones , Ratones Endogámicos NOD , Pentoxifilina/farmacología , Proteínas Recombinantes/farmacología , Trasplante Heterólogo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Autoimmune-mediated cytotoxicity may cause pancreatic islet transplant failure, leading to recurrent diabetes. Protection of islet grafts depends on immunosuppressive control, which may also prevent autoimmune recurrence of diabetes. In this study, we compared the survival of syngeneic islet transplants using different strains of donor mice. We observed extended functional survival in the islet grafts from donors lacking the genetic background and potential of autoimmunity. Without immunosuppression, the islet grafts of NOR and immune-deficient NOD. Scid donors functioned up to 3 weeks in syngeneic islet transplants compared to 3-day survivals with the grafts from NOD donors. T-cell proliferation and activation markers, CD44 and CD69, were upregulated in NOD donors, suggesting that T-cell activation had occurred prior to pancreas procurement. Systemic delivery of a recombinant adenoassociated viral vector (AAV) encoding the viral (vIL-10) IL-10 gene (AAV vIL-10) in NOD recipients protected syngeneic islets from autoimmune destruction. Alternatively, pretreatment of NOD donor mice with AAV vIL-10 prolonged islet graft survival in untreated NOD recipients. Both studies indicate the effectiveness of vIL-10 gene therapy in autoimmune regulation. These results suggest that a donor factor may exist in autoimmune-prone donors. Therefore, autoimmune recurrence of diabetes may result from donor immune cells transferred during islet transplantation. The AAV vIL-10 gene therapy suppressed previously activated donor T cells and protected the grafted islets from autoimmune-mediated destruction.
Asunto(s)
Supervivencia de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Trasplante Isogénico/inmunología , Animales , Diabetes Mellitus Tipo 1/cirugía , Ratones , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones SCID , Especificidad de la EspecieRESUMEN
Pancreatic islet transplantation can replace functional insulin-secreting beta cells for patients with type 1 diabetes. More than 300 patients who have received islet transplantation have returned to a euglycemic condition without using insulin. Therefore, islet transplantation has gained public attention and interest. Unfortunately, shortages in organ donations, suboptional antirejection regimens, and difficulties in islet isolation limit clinical utilization of this therapy. Recently, successful islet transplantation has been reported using a centralized islet isolation facility. The advantage of this experience is that it avoids the high costs in building an isolation facility and maintaining an experienced technical team. However, a private airplane carrier was required for transporting islets back to the transplantation site in a remote hospital. The cost of this specialized transportation was still too high to be considered as a routine procedure. In this study, we report our experience using commercial carriers to deliver isolated human islets from an established isolation facility to a remote medical center.
Asunto(s)
Islotes Pancreáticos/citología , Recolección de Tejidos y Órganos/métodos , Animales , Aviación , Supervivencia Celular , Diabetes Mellitus Experimental/cirugía , Diabetes Mellitus Tipo 1/cirugía , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos NOD , Ratas , Donantes de Tejidos , TransportesAsunto(s)
Supervivencia de Injerto/fisiología , Trasplante de Islotes Pancreáticos/fisiología , Trasplante Heterólogo/fisiología , Animales , Femenino , Rechazo de Injerto/prevención & control , Trasplante de Islotes Pancreáticos/instrumentación , Trasplante de Islotes Pancreáticos/métodos , Masculino , Ratones , Ratones Endogámicos NOD , Modelos Animales , Ratas , Ratas Wistar , Trasplante Heterólogo/instrumentación , Trasplante Heterólogo/métodosRESUMEN
AIMS/HYPOTHESIS: Pro-inflammatory cytokines are increased during the active stages of Type I (insulin-dependent) diabetes mellitus. The aim of this study was to investigate the applicability of using a new anti-inflammatory compound, Lisofylline, to prevent diabetes in non-obese diabetic (NOD) mice. Lisofylline has previously been shown to block Th1 cell differentiation and to reduce IL-1 beta-induced dysfunction in rat islets. METHODS: Lisofylline was added to isolated NOD islets in vitro, with or without IL-1 beta. Insulin secretion and DNA damage of the islets was assessed. Lisofylline was administered to female non-obese diabetic mice starting at 4, 7 and 17 weeks of age for 3 weeks. Cytokines and blood glucose concentrations were monitored. Histology and immunohistochemistry were carried out in pancreatic sections. Splenocytes isolated from donor mice were intravenously injected into immunodeficient NOD (NOD.scid) mice. RESULTS: In vitro, Lisofylline preserved beta-cell insulin secretion and inhibited DNA damage of islets in the presence of IL-1 beta. In vivo, Lisofylline suppressed IFN-gamma production, reduced the onset of insulitis and diabetes, and inhibited diabetes after transfer of splenocytes from Lisofylline-treated donors to NOD.scid recipients. However, cotransfer of splenocytes from both Lisofylline-treated and diabetic NOD donors did not suppress diabetes in recipient mice. CONCLUSION/INTERPRETATION: Lisofylline prevents the onset of autoimmune diabetes in NOD mice by a mechanism that does not seem to enhance the function of regulatory T cells, but could be associated with suppression of proinflammatory cytokines and reduction of cellular infiltration in islets. This study suggests that Lisofylline could have therapeutic benefits in preventing the onset of Type I diabetes.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Diabetes Mellitus Tipo 1/prevención & control , Pentoxifilina/análogos & derivados , Pentoxifilina/uso terapéutico , Animales , Glucemia/análisis , Citocinas/sangre , Daño del ADN , Diabetes Mellitus Tipo 1/epidemiología , Femenino , Incidencia , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Transfusión de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Bazo/inmunologíaRESUMEN
AIMS/HYPOTHESIS: We previously showed that vascular smooth muscle cells and endothelial cells cultured under high glucose conditions produced more 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the 12-lipoxygenase (12-LO) product of arachidonate metabolism, relative to normal glucose. Because the lipoxygenase (LO) pathway has been associated with oxidant stress and the pathogenesis of atherosclerosis, we now examined 12-LO activation in vivo in a pig model of diabetes-induced accelerated atherosclerosis which displays human characteristics. METHODS: The animal model was developed in pigs who were fed a normal or high fat diet and given streptozotocin injections to produce normolipemic-normoglycaemic (NLNG), normolipemic-hyperglycaemic (NLHG), hyperlipemic-normoglycaemic (HLNG) and hyperlipemic-hyperglycaemic (HLHG) pigs. Tissue samples were obtained from key arterial beds to examine 12-LO expression at 20 weeks after the pigs began their diet. RESULTS: All HG pigs maintained threefold higher serum glucose concentrations. The HL groups developed atherosclerosis but diabetic HLHG pigs showed markedly accelerated atherosclerosis (twofold) relative to non-diabetic HLNG pigs. Immunostaining showed progressive increases in 12-LO in arteries in the order NLNG, NLHG, HLNG and HLHG. Leukocyte-type 12-LO protein (immuno-blotting) as well as mRNA expression (by competitive PCR) in abdominal and coronary arteries were significantly greater in HLHG pigs than in all the other three groups. Furthermore, increased oxidant stress was observed in monocytes from NLHG and HLNG pigs, and greatly potentiated in HLHG pigs. CONCLUSIONS/INTERPRETATION: These results are consistent with the hypothesis that 12-LO activation plays a key role in accelerated atherosclerosis due to diabetes and hyperlipemia.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Arteriosclerosis/fisiopatología , Diabetes Mellitus Experimental/fisiopatología , Estrés Oxidativo/fisiología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/orina , Animales , Aorta Abdominal/patología , Araquidonato 12-Lipooxigenasa/genética , Arteriosclerosis/patología , Glucemia/metabolismo , Colesterol/sangre , Vasos Coronarios/patología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/orina , Dieta Aterogénica , Progresión de la Enfermedad , Hiperlipidemias/fisiopatología , Masculino , Reacción en Cadena de la Polimerasa , Porcinos , Triglicéridos/sangreRESUMEN
Patients with diabetes are at higher risk for atherosclerotic disease than nondiabetic individuals with other comparable risk factors. Studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. In this study, diabetes was superimposed on a well-characterized swine model of atherosclerosis by injection of the beta-cell cytotoxin streptozotocin (STZ), resulting in a >80% reduction in beta-cells and an increase in plasma glucose to diabetic levels. Animals were maintained without exogenous insulin for up to 48 weeks. Plasma glucose and cholesterol levels and lesion extent and severity were quantified in swine with diabetes and hyperlipemia alone and in combination compared with controls. Diabetes had no effect on plasma cholesterol levels, but diabetic/hyperlipemic (D-HL) swine developed hypertriglyceridemia and showed a doubling in aortic sudanophilia over nondiabetic/hyperlipemic (N-HL) swine as early as 12 weeks (47.25 +/- 4.5 vs. 24.0 +/- 4.6%). At 20 weeks, coronary artery stenosis was significantly greater in D-HL than in N-HL animals (86 +/- 10 vs. 46 +/- 8%). Coronary lesions predominantly arose in the first 2-3 cm of the vessels and displayed humanoid morphology. Aortic lesions in D-HL swine had double the cholesterol content of those in N-HL swine, and incorporation of oleate into cholesteryl ester was significantly greater in grossly normal aortic areas of D-HL swine compared with N-HL and was attributed to similar elevated incorporation in monocytes. This large study demonstrates that a model of diabetes with humanoid characteristics, including hypertriglyceridemia and severe, accelerated atherosclerosis can be reproducibly induced and maintained in swine. This model should potentially be of great value in elucidating mechanisms underlying the accelerated atherosclerosis seen in human diabetic individuals.
Asunto(s)
Arteriosclerosis/fisiopatología , Diabetes Mellitus Experimental/complicaciones , Acetatos/metabolismo , Animales , Glucemia/análisis , Colesterol/sangre , Ésteres del Colesterol/biosíntesis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Islotes Pancreáticos/efectos de los fármacos , Lípidos/sangre , Monocitos/metabolismo , Ácido Oléico/metabolismo , Factores de Riesgo , Estreptozocina/farmacología , Porcinos , Triglicéridos/sangreRESUMEN
The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Glucosa/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , ARN Catalítico/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Adenoviridae/genética , Animales , Aorta , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucocitos/enzimología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/antagonistas & inhibidores , Especificidad por Sustrato/genética , PorcinosRESUMEN
BACKGROUND: Arachidonic acid-derived 12-lipoxygenase (12-LO) products have potent growth and chemotactic properties. The present studies examined whether 12-LO and fibronectin are induced in cultured rat mesangial cells (MCs) exposed to high glucose and whether they are expressed in experimental diabetic nephropathy. METHODS: To determine the effect of high glucose on MC 12-LO mRNA and protein expression, rat MCs were incubated with RPMI medium containing 100 (NG) or 450 mg/dL glucose (HG). For animal studies, rats were injected with diluent (control) or streptozotocin. The latter were left untreated (DM) or treated with insulin (DM + I). At sacrifice after four months, GAPDH, 12-LO, and fibronectin mRNA were measured by competitive reverse transcription-polymerase chain reaction (RT-PCR) in microdissected glomeruli (G). Renal sections were semiquantitatively scored (0 to 4+) for diabetic changes and for 12-LO and fibronectin by immunohistochemistry. RESULTS: 12-LO mRNA expression in MC exposed to HG (12.71 +/- 1.17 attm/microL) and DM G (1.78 +/- 0.65 x 10-3 attm/glomerulus) was significantly higher than those of MCs in NG media (6.71 +/- 0.78 attm/microL) and control G (0.34 +/- 0.12 x 10-3 attm/glomerulus, P < 0.005), respectively. Western blot revealed a 1.7- and a 2.8-fold increase in MC and G 12-LO protein expression, respectively (P < 0.05). The immunohistochemistry score for G 12-LO and diabetic nephropathy score was significantly greater in DM and DM + I than controls. MC and G GAPDH mRNA remained unchanged. CONCLUSIONS: In MCs exposed to HG and in diabetic rat glomeruli, increments in 12-LO mRNA and protein are associated with changes modeling diabetic nephropathy. These findings suggest a role for the 12-LO pathway in the pathogenesis of diabetic nephropathy.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Nefropatías Diabéticas/enzimología , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/enzimología , Glucosa/farmacología , Animales , Araquidonato 12-Lipooxigenasa/genética , Células Cultivadas , Diabetes Mellitus Experimental , Nefropatías Diabéticas/patología , Fibronectinas/genética , Fibronectinas/metabolismo , Mesangio Glomerular/citología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Insulina/farmacología , Riñón/enzimología , Riñón/patología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: 12-Lipoxygenase (12-LO) products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells. We have also recently demonstrated increased 12-LO mRNA and protein expression in the neointima of balloon-injured rat carotid arteries. In this study, we evaluated whether 12-LO activation plays a role in neointimal thickening in this rat model by using a specific ribozyme (Rz) directed to rat 12-LO. METHODS AND RESULTS: We designed a chimeric DNA-RNA hammerhead Rz to cleave rat leukocyte-type 12-LO mRNA. This Rz dose-dependently cleaved a 166-nucleotide target 12-LO mRNA substrate in vitro and reduced 12-LO mRNA and protein expression in rat vascular smooth muscle cells. A control mutant Rz (MRz) with a point mutation in the catalytic site was inactive. To test the in vivo efficacy of the 12-LO Rz, the left common carotid arteries of rats were injured with a balloon catheter. The distal half of the injured arteries was treated with Rz or MRz mixed with lipofectin. The proximal half received only lipofectin. Twelve days after injury, intima-to-media ratios were significantly lower in the Rz-treated sections than in untreated sections from the same rat (0.742+/-0.16 versus 1.749+/-0.12, P:<0.001). In contrast, the MRz had no significant effect. CONCLUSIONS: These results indicate the important role of the leukocyte-type 12-LO pathway in restenosis in response to injury.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Enfermedades de las Arterias Carótidas/prevención & control , Leucocitos/enzimología , ARN Catalítico/uso terapéutico , Túnica Íntima/patología , Análisis de Varianza , Angioplastia Coronaria con Balón/efectos adversos , Animales , Araquidonato 12-Lipooxigenasa/genética , Enfermedades de las Arterias Carótidas/etiología , Movimiento Celular/efectos de los fármacos , Medios de Contraste/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Fluoresceína/metabolismo , Hiperplasia/etiología , Hiperplasia/prevención & control , Inhibidores de la Lipooxigenasa , Masculino , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
The 12-lipoxygenase (LO) enzyme has been implicated in playing a role in pancreatic beta cell inflammatory damage and atherosclerosis. 12-LO reacts with fatty acids to form hydroperoxides which may alter cellular growth. In this study we investigated the direct effect of mouse leukocyte type 12-LO cDNA overexpression on apoptosis in Chinese hamster ovary fibroblast cells that also stably overexpress the angiotensin II type 1a receptor. CHO-AT1a cells expressing background levels of 12-LO exhibited clear increases in growth in response to angiotensin II. In contrast, the new 12-LO transfected cells (CHO-AT1a/ML12-LO cells) displayed reduced basal and angiotensin Il-induced growth compared to CHO-AT1a cells. Furthermore, serum-deprivation resulted in a significantly greater number of non-viable cells in clones having the greatest magnitude of 12-LO overexpression. These results suggested that reduction of the proliferation rate of CHO-AT1a/ML12-LO cells was due to an increasing rate of cell death. To determine whether the increase in cell death was due to apoptosis, we evaluated nuclear DNA fragmentation, cell morphologic changes, and activation of caspase-3. Cells overexpressing 12-LO cDNA displayed all these changes characteristic of apoptosis. In addition the 12-LO product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), directly induced apoptosis in CHO-AT1a cells. These results demonstrate for the first time that 12-LO activation can lead to apoptosis in fibroblasts, suggesting a role of 12-LO in leading to inflammatory mediated cellular damage.