Caloric restriction recovers impaired ß-cell-ß-cell gap junction coupling, calcium oscillation coordination, and insulin secretion in prediabetic mice.

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine whether caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high-fat diet (HFD), showing indications of prediabetes after 2 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose, and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

Characterization of the Goto-Kakizaki (GK) Rat Model of Type 2 Diabetes.

The Goto-Kakizaki (GK) rat is a model of type 2 diabetes mellitus, produced originally by selective inbreeding for a hyperglycemic trait. These rats are characterized as having insulin resistance and an insulin secretory defect. Pancreatic islets from these mice show abnormal morphology in the distribution of glucagon-secreting α cells and insulin-secreting ß cells, which may affect paracrine functions involved in the secretory response. This chapter describes the characterization of GK rat islets using immunofluorescence microscopy and Western blot analyses to demonstrate the effects on islet architecture and how this translates to changes in expression of predominant islet proteins.

Preparation of Islets from Rat Pancreas and Assessment of Islet Function.

The islets of Langerhans release vital hormones involved in the regulation of blood sugar and other aspects of metabolism. The islets are housed in diffuse clusters of cells within the exocrine pancreas and, therefore, purification of these cells for research or transplant purposes is a difficult undertaking. Here, a detailed protocol is presented for purification of islets from rat pancreas using limited collagenase digestion and step gradient centrifugation techniques. In addition, a method for assessing islet viability is presented using perifusion under both basal and stimulatory glucose conditions, with measurement of the hormone released using an immunoassay for insulin.

Chronic treatment with hydroalcoholic extract of Plathymenia reticulata promotes islet hyperplasia and improves glycemic control in diabetic rats / Tratamento crônico com extrato hidroalcoólico de Plathymenia reticulata promove hiperplasia de ilhotas e controle glicêmico em ratos diabéticos

ABSTRACT Objective: To investigate the anti-hyperglycemic effects of Plathymenia reticulata hydroalcoholic extract and related changes in body weight, lipid profile and the pancreas. Methods: Diabetes was induced in 75 adult male Wistar rats via oral gavage of 65mg/Kg of streptozotocin. Rats were allocated to one of 8 groups, as follows: diabetic and control rats treated with water, diabetic and control rats treated with 100mg/kg or 200mg/kg of plant extract, and diabetic and control rats treated with glyburide. Treatment consisted of oral gavage for 30 days. Blood glucose levels and body weight were measured weekly. Animals were sacrificed and lipid profile and pancreatic tissue samples analyzed. Statistical analysis consisted of ANOVA, post-hoc Tukey-Kramer, paired Student's t and χ2 tests; the level of significance was set at 5%. Results: Extract gavage at 100mg/kg led to a decrease in blood glucose levels in diabetic rats in the second, third (198.71±65.27 versus 428.00±15.25) and fourth weeks (253.29±47.37 versus 443.22±42.72), body weight loss (13.22±5.70 versus 109.60±9.95) and lower cholesterol levels (58.75±3.13 versus 80.11±4.01) in control rats. Extract gavage at 200mg/Kg led to a decrease in glucose levels on the fourth week in diabetic rats, body weight loss in the second, third and fourth weeks in control rats, and lower cholesterol levels in diabetic and control rats. Islet hyperplasia (p=0.005) and pancreatic duct dilation (p=0.047) were observed in diabetic and control rats. Conclusion: Plathymenia extract reduced blood glucose levels in diabetic rats, and body weight in control rats, and promoted pancreatic islet hyperplasia in diabetic and control rats.

RESUMO Objetivo: Avaliar o efeito anti-hiperglicêmico do extrato hidroalcoólico de Plathymenia reticulata, alterações no peso, lipídeos e efeito sobre o pâncreas. Métodos: O diabetes foi induzido pela administração de estreptozotocina 65mg/kg, em 75 ratos Wistar adultos machos, divididos em 8 grupos diferentes: ratos diabéticos e controle + água, ratos diabéticos e controle + 100mg/kg ou 200mg/kg de extrato, ratos diabéticos e controle + gliburida. O tratamento foi realizado por gavagem (oral) por 30 dias. Níveis de glicose e peso foram verificados semanalmente. Os animais foram sacrificados, e amostras de lipídeos e do pâncreas foram analisadas. A análise estatística incluiu ANOVA, post-hoc Tukey-Kramer, teste t de Student pareado e teste do χ2, com nível de significância de 5%. Resultados: O extrato 100mg/kg promoveu redução nos níveis de glicose sanguínea em ratos diabéticos na segunda, terceira (198,71±65,27 versus 428,00±15,25) e quarta semanas (253,29±47,37 versus 443,22±42,72), perda de peso (13,22±5,70 versus 109,60±9,95) e diminuição do colesterol (58,75±3,13 versus 80,11±4,01) em ratos controle. Com extrato de 200mg/kg, houve redução dos níveis de glicose na quarta semana, nos ratos diabéticos; de peso na segunda, terceira e quarta semanas, nos ratos controle; e de colesterol nos animais diabéticos e controle. Ocorreram hiperplasia de ilhotas (p=0,005) e dilatação dos ductos pancreáticos (p=0,047) em ratos diabéticos e controles. Conclusão: O extrato de Plathymenia reduziu os níveis de glicose em ratos diabéticos e de peso em ratos controle, além de ter promovido hiperplasia de ilhotas pancreáticas em diabéticos e controles.

Influence of Flavonoids on Mechanism of Modulation of Insulin Secretion.

BACKGROUND: The development of alternatives for insulin secretion control in vivo or in vitro represents an important aspect to be investigated. In this direction, natural products have been progressively explored with this aim. In particular, flavonoids are potential candidates to act as insulin secretagogue. OBJECTIVE: To study the influence of flavonoid on overall modulation mechanisms of insulin secretion. METHODS: The research was conducted in the following databases and platforms: PubMed, Scopus, ISI Web of Knowledge, SciELO, LILACS, and ScienceDirect, and the MeSH terms used for the search were flavonoids, flavones, islets of Langerhans, and insulin-secreting cells. RESULTS: Twelve articles were included and represent the basis of discussion on mechanisms of insulin secretion of flavonoids. Papers in ISI Web of Knowledge were in number of 1, Scopus 44, PubMed 264, ScienceDirect 511, and no papers from LILACS and SciELO databases. CONCLUSION: According to the literature, the majority of flavonoid subclasses can modulate insulin secretion through several pathways, in an indication that corresponding molecule is a potential candidate for active materials to be applied in the treatment of diabetes. SUMMARY: The action of natural products on insulin secretion represents an important investigation topic due to their importance in the diabetes controlIn addition to their typical antioxidant properties, flavonoids contribute to the insulin secretionThe modulation of insulin secretion is induced by flavonoids according to different mechanisms. Abbreviations used: KATP channels: ATP-sensitive K+ channels, GLUT4: Glucose transporter 4, ERK1/2: Extracellular signal-regulated protein kinases 1 and 2, L-VDCCs: L-type voltage-dependent Ca+2 channels, GLUT1: Glucose transporter 1, AMPK: Adenosine monophosphate-activated protein kinase, PTP1B: Protein tyrosine phosphatase 1B, GLUT2: Glucose transporter 2, cAMP: Cyclic adenosine monophosphate, PKA: Protein kinase A, PTK: Protein tyrosine kinase, CaMK II: Ca2+/calmodulin-dependent protein kinase II, GSIS: Glucose-stimulated insulin secretion, Insig-1: Insulin-induced gene 1, IRS-2: Insulin receptor substrate 2, PDX-1: Pancreatic and duodenal homeobox 1, SREBP-1c: Sterol regulatory element binding protein-1c, DMC: Dihydroxy-6'-methoxy-3',5'-dimethylchalcone, GLP-1: Glucagon-like peptide-1, GLP-1R: Glucagon-like peptide 1 receptor.

Light-emitting diode modulates carbohydrate metabolism by pancreatic duct regeneration.

Pancreatic lesions can produce metabolic disorders. Light-emitting diode (LED) has been used as a safe and effective phototherapy for cell proliferation and regeneration. We investigate the effects of phototherapy using LED irradiation on the pancreas after the injection of streptozotocin (STZ) to induce experimental diabetes and evaluate that the ß cells can regenerate in the pancreas in an in vivo model and observe its implications on the control of carbohydrate metabolism. Twenty Wistar rats were randomized into three groups: non-diabetic control, diabetic control, and diabetic treated with LED. Except for the non-diabetic control group, all were induced to diabetes type I by streptozotocin injection. Treated groups were irradiated by LED: λ = 805 nm; 40 mW, 22 s; spot diameter 5 mm, spot area 0.196 cm2, 0.88 J that it was applied on pancreas projection area for 5 consecutive days and monitored for 30 days. Diabetic group treated with LED showed regeneration of islets and ducts (p = 0.001) on the pancreas. Intraperitoneal insulin tolerance test showed differences between the diabetic control and diabetic treated groups (p = 0.03). In diabetic control group, the hepatic glycogen content was 296% lower when compared with diabetic treated with LED. Furthermore, in the diabetic control group, the glycogen content of the gastrocnemius muscle was 706% smaller when compared with diabetic treated with LED. This study shows that LED was able to modify morphological and metabolic features and also altered carbohydrate metabolism on irradiated pancreas in experimental model of diabetes.

Human islet xenotransplantation in rodents: A literature review of experimental model trends

Among the innovations for the treatment of type 1 diabetes, islet transplantation is a less invasive method of treatment, although it is still in development. One of the greatest barriers to this technique is the low number of pancreas donors and the low number of pancreases that are available for transplantation. Rodent models have been chosen in most studies of islet rejection and type 1 diabetes prevention to evaluate the quality and function of isolated human islets and to identify alternative solutions to the problem of islet scarcity. The purpose of this study is to conduct a review of islet xenotransplantation experiments from humans to rodents, to organize and analyze the parameters of these experiments, to describe trends in experimental modeling and to assess the viability of this procedure. In this study, we reviewed recently published research regarding islet xenotransplantation from humans to rodents, and we summarized the findings and organized the relevant data. The included studies were recent reports that involved xenotransplantation using human islets in a rodent model. We excluded the studies that related to isotransplantation, autotransplantation and allotransplantation. A total of 34 studies that related to xenotransplantation were selected for review based on their relevance and current data. Advances in the use of different graft sites may overcome autoimmunity and rejection after transplantation, which may solve the problem of the scarcity of islet donors in patients with type 1 diabetes.

Streptozotocin diabetogenic action in an experimental neonatal induction model.

INTRODUCTION: The use of experimental models is essential to study the pathophysiological mechanisms of diabetes.  OBJECTIVES: To compare in adult Wistar rats the diabetogenic action of streptozotocin according to the moment and route of administration during the neonatal period by evaluating biochemical, metabolic and histological variables.  MATERIALS AND METHODS: Eight groups of neonatal female Wistar rats (n=10) were formed. We evaluated the induction with streptozotocin (100 mg/kg of body weight) on days 2 and 5 after birth, as well as the administration routes (subcutaneous or intraperitoneal). Controls were injected with sodium citrate buffer. Blood glucose level, body weight, food and water intake were monitored for 12 weeks. We also performed tolerance tests for oral glucose and glycosylated hemoglobin, and a histopathological pancreas morphometric study.  RESULTS: The mortality rate was about 100% among rats given streptozotocin on their fifth day of life. All rats receiving the drug on day 2 of life survived, and they showed a marked hyperglycemia, polyphagia, polydipsia and decreased body weight gain in addition to increased glycosylated hemoglobin rates and impaired results in the oral glucose tolerance test. Histopathological lesions of the pancreas as well as a decreased number of islets were significantly more frequent in rats receiving the drug subcutaneously on day 2, which confirms that streptozotocin administered subcutaneously produces greater damage.  CONCLUSIONS: Subcutaneous injection of streptozotocin in a dose of 100 mg/kg of body weight in the second day after birth induced moderate diabetes in adult Wistar rats more effectively.

Acción de la estreptozotocina en un modelo experimental de inducción neonatal de la diabetes / Streptozotocin diabetogenic action in an experimental neonatal induction model

Introducción. El estudio de la diabetes mediante modelos experimentales es fundamental para entender los mecanismos fisiopatológicos de la enfermedad. Objetivos. Comparar en ratas Wistar adultas la acción de inducción de la diabetes de la estreptozotocina según el momento y la vía de inoculación del fármaco durante el periodo neonatal mediante la evaluación de variables bioquímicas, metabólicas e histológicas. Materiales y métodos. Se conformaron ocho grupos con ratas Wistar hembras recién nacidas (n=10). Se evaluó la inducción con estreptozotocina (100 mg/kg de peso corporal) según el día (segundo y quinto después del nacimiento) y la vía de inoculación (subcutánea o intraperitoneal). Los controles se inyectaron con solución tampón de citrato sódico. Durante 12 semanas se evaluaron la glucemia, el peso, y la cantidad de alimento y de agua consumida. Se hicieron pruebas de tolerancia a la glucosa oral, se evaluó la hemoglobina ´glicosilada´, y se hizo el estudio morfométrico e histopatológico del páncreas. Resultados. Casi todos los animales inoculados con estreptozotocina en el quinto día murieron, en tanto que todos los inoculados en el segundo día sobrevivieron. La administración subcutánea de estreptozotocina en el segundo día produjo hiperglucemia, polifagia, polidipsia y disminución de la ganancia de peso corporal, así como alteración de los valores de hemoglobina ´glicosilada´ y en la prueba de tolerancia a la glucosa. Las lesiones histopatológicas del páncreas, así como la disminución del número de islotes, se observaron con mayor frecuencia con la estreptozotocina aplicada de forma subcutánea en el segundo día, lo cual corroboró que el efecto de este agente inoculado de forma subcutánea causa mayor daño. Conclusiones. La inyección subcutánea de una dosis de 100 mg/kg de estreptozotocina en el segundo día después del nacimiento logró mayor efectividad en la inducción de diabetes moderada en ratas Wistar adultas.

Introduction: The use of experimental models is essential to study the pathophysiological mechanisms of diabetes. Objectives: To compare in adult Wistar rats the diabetogenic action of streptozotocin according to the moment and route of administration during the neonatal period by evaluating biochemical, metabolic and histological variables. Materials and methods: Eight groups of neonatal female Wistar rats (n=10) were formed. We evaluated the induction with streptozotocin (100 mg/kg of body weight) on days 2 and 5 after birth, as well as the administration routes (subcutaneous or intraperitoneal). Controls were injected with sodium citrate buffer. Blood glucose level, body weight, food and water intake were monitored for 12 weeks. We also performed tolerance tests for oral glucose and glycosylated hemoglobin, and a histopathological pancreas morphometric study. Results: The mortality rate was about 100% among rats given streptozotocin on their fifth day of life. All rats receiving the drug on day 2 of life survived, and they showed a marked hyperglycemia, polyphagia, polydipsia and decreased body weight gain in addition to increased glycosylated hemoglobin rates and impaired results in the oral glucose tolerance test. Histopathological lesions of the pancreas as well as a decreased number of islets were significantly more frequent in rats receiving the drug subcutaneously on day 2, which confirms that streptozotocin administered subcutaneously produces greater damage. Conclusions: Subcutaneous injection of streptozotocin in a dose of 100 mg/kg of body weight in the second day after birth induced moderate diabetes in adult Wistar rats more effectively.

Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.

Comparative efficacy of Belzer or Euro-Collins solutions for pancreatic preservation during cold ischemic storage in rats

To compare the efficacy of different types of solutions (Belzer or Euro-Collins) for the preservation of rat pancreas during cold ischemia. METHODS: Thirty Wistar rats were divided into three groups according to the perfusion or storage solution: Group E (perfusion and storage in Euro-Collins solution); Group B (perfusion and storage in Belzer solution) and Group BE (Perfusion in Belzer solution and storage in Euro-Collins solution). After perfusion, the pancreas was excised and stored at 4˚C for 18 hours. Amylase was measured at 6, 12 and 18h, and histological analysis of the pancreas was performed after 18h of cold storage. RESULTS: Amylase was elevated and comparable in Groups E and BE after 12 and 18 hours of ischemia (p<0.05). In the exocrine pancreas, histological differences in the amount of necrosis (p=0.049), lymphocytic infiltrate (p<0.001) and neutrophilic infiltrate (p=0.004) were observed, with more favorable features present in Group B. In the endocrine pancreas, Group B showed less edema (p<0.001), but other parameters were similar among all groups. CONCLUSION: The Euro-Collins solution is inferior to the Belzer solution for the preservation of rat pancreas during cold ischemia.

Comparative efficacy of Belzer or Euro-Collins solutions for pancreatic preservation during cold ischemic storage in rats

To compare the efficacy of different types of solutions (Belzer or Euro-Collins) for the preservation of rat pancreas during cold ischemia. METHODS: Thirty Wistar rats were divided into three groups according to the perfusion or storage solution: Group E (perfusion and storage in Euro-Collins solution); Group B (perfusion and storage in Belzer solution) and Group BE (Perfusion in Belzer solution and storage in Euro-Collins solution). After perfusion, the pancreas was excised and stored at 4˚C for 18 hours. Amylase was measured at 6, 12 and 18h, and histological analysis of the pancreas was performed after 18h of cold storage. RESULTS: Amylase was elevated and comparable in Groups E and BE after 12 and 18 hours of ischemia (p<0.05). In the exocrine pancreas, histological differences in the amount of necrosis (p=0.049), lymphocytic infiltrate (p<0.001) and neutrophilic infiltrate (p=0.004) were observed, with more favorable features present in Group B. In the endocrine pancreas, Group B showed less edema (p<0.001), but other parameters were similar among all groups. CONCLUSION: The Euro-Collins solution is inferior to the Belzer solution for the preservation of rat pancreas during cold ischemia.(AU)

Role of AKT / mTORC1 pathway in pancreatic β - cell proliferation / Participación de la vía de señalización AKT/mTORC1 en la proliferación de las células β del páncreas

Growth factors, insulin signaling and nutrients are important regulators of β-cell mass and function. The events linking these signals to regulation of β-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein β-subunit-like protein (GβL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic β cell mass and proliferation. mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in β-cells in vivo. This information can be used to develop alternative approaches to expand β-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in β-cell function.

Factores de crecimiento y nutrientes son reguladores muy importantes de la masa y función de las células β, pero las vías de señalización que unen estas señales a estos procesos no han sido completamente elucidadas. Estudios recientes han demostrado que la proteína mTOR integra señales provenientes de factores de crecimiento y disponibilidad de nutrientes con procesos celulares como transcripción, traducción, organización del citoesqueleto y metabolismo mitocondrial. mTOR puede hacer parte de dos complejos diferentes, mTORC1 y mTORC2. En el complejo mTORC1, la proteina mTOR la cual es sensible a rapamicina y se encuentra asociada a las proteínas Raptor, G β L, deptor y PRAS40, activa reguladores claves en la síntesis de proteínas, tales como la proteína cinasa ribosomal S6 (S6K) y la proteína de unión al factor eucariótico de iniciación 4E. El presente trabajo recopila información reciente sobre la participación de la vía de señalización AKT/mTORC1 en la regulación de la proliferación y masa de las células β del páncreas. mTORC1 regula la progresión del ciclo celular en células β, mediante la modulación de los niveles de las ciclinas D2 y D3 y la actividad del complejo Cdk4/ ciclina D. Estos estudios que revelan nuevos puntos de control del ciclo celular en células β, pueden ser utilizados en el desarrollo de nuevos enfoques para expandir la masa de células β, sin el riesgo de inducir una transformación oncogénica. Los resultados relacionados en el presente trabajo aportan información muy valiosa para el desarrollo de nuevas estrategias terapéuticas para el tratamiento la diabetes tipo 2.

Role of AKT/mTORC1 pathway in pancreatic ß-cell proliferation.

Growth factors, insulin signaling and nutrients are important regulators of ß-cell mass and function. The events linking these signals to regulation of ß-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein ß-subunit-like protein (GßL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic ß cell mass and proliferation. mTORC1 is a major regulator of ß-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in ß-cells in vivo. This information can be used to develop alternative approaches to expand ß-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in ß-cell function.

Factores de crecimiento y nutrientes son reguladores muy importantes de la masa y función de las células ß, pero las vías de señalización que unen estas señales a estos procesos no han sido completamente elucidadas. Estudios recientes han demostrado que la proteína mTOR integra señales provenientes de factores de crecimiento y disponibilidad de nutrientes con procesos celulares como transcripción, traducción, organización del citoesqueleto y metabolismo mitocondrial. mTOR puede hacer parte de dos complejos diferentes, mTORC1 y mTORC2. En el complejo mTORC1, la proteina mTOR la cual es sensible a rapamicina y se encuentra asociada a las proteínas Raptor, G ß L, deptor y PRAS40, activa reguladores claves en la síntesis de proteínas, tales como la proteína cinasa ribosomal S6 (S6K) y la proteína de unión al factor eucariótico de iniciación 4E. El presente trabajo recopila información reciente sobre la participación de la vía de señalización AKT/mTORC1 en la regulación de la proliferación y masa de las células ß del páncreas. mTORC1 regula la progresión del ciclo celular en células ß, mediante la modulación de los niveles de las ciclinas D2 y D3 y la actividad del complejo Cdk4/ ciclina D. Estos estudios que revelan nuevos puntos de control del ciclo celular en células ß, pueden ser utilizados en el desarrollo de nuevos enfoques para expandir la masa de células ß, sin el riesgo de inducir una transformación oncogénica. Los resultados relacionados en el presente trabajo aportan información muy valiosa para el desarrollo de nuevas estrategias terapéuticas para el tratamiento la diabetes tipo 2.

Islet transplantation in rodents. Do encapsulated islets really work? / Transplante de ilhotas de Langerhans em modelos experimentais em roedores. Ilhotas encapsuladas realmente funcionam?

CONTEXT: Diabetes mellitus type I affects around 240 million people in the world and only in the USA 7.8 percent of the population. It has been estimated that the costs of its complications account for 5 percent to 10 percent of the total healthcare spending around the world. According to World Health Organization, 300 million people are expected to develop diabetes mellitus by the year 2025. The pancreatic islet transplantation is expected to be less invasive than a pancreas transplant, which is currently the most commonly used approach. OBJECTIVES: To compare the encapsulated and free islet transplantation in rodents looking at sites of islet implantation, number of injected islets, viability and immunosuppression. METHODS: A literature search was conducted using MEDLINE/PUBMED and SCIELO with terms about islet transplantation in the rodent from 2000 to 2010. We found 2,636 articles but only 56 articles from 2000 to 2010 were selected. RESULTS: In these 56 articles used, 34 percent were encapsulated and 66 percent were nonencapsulated islets. Analyzing both types of islets transplantation, the majority of the encapsulated islets were implanted into the peritoneal cavity and the nonencapsulated islets into the liver, through the portal vein. In addition, the great advantage of the peritoneal cavity as the site of islet transplantation is its blood supply. Both vascular endothelial cells and vascular endothelial growth factor were used to stimulate angiogenesis of the islet grafts, increasing the vascularization rapidly after implantation. It also has been proven that there is influence of the capsules, since the larger the capsule more chances there are of central necrosis. In some articles, the use of immunosuppression demonstrated to increase the life expectancy of the graft. CONCLUSION: While significant progress has been made in the islets transplantation field, many obstacles remain to be overcome. Microencapsulation provides a means to transplant islets without immunosuppressive agents and may enable the performance of xenotransplantation. The use of alternative donor sources, fewer islets per capsule and the appropriate deployment location, such as the peritoneal cavity, may give a future perspective to the application of immunoprotective capsules and viability in clinical practice. A variety of strategies, such as genetic engineering, co-encapsulation, improvement in oxygen supply or the establishment of hypoxia resistance will also improve the islet transplantation performance. It remains to be determined which combination of strategies with encapsulation can fulfill the promise of establishing a simple and safe transplantation as a cure for diabetes.

CONTEXTO: Diabetes mellitus tipo I afeta cerca de 240 milhões de pessoas no mundo e 7,8 por cento só nos EUA. Foi estimado que o custo de suas complicações fosse de 5 por cento-10 por cento dos custos mundiais em saúde. De acordo com a OMS (Organização Mundial de Saúde), espera-se que cerca de 300 milhões de pessoas desenvolvam o diabetes mellitus até o ano de 2025. É esperado que o transplante de ilhotas pancreáticas seja menos invasivo que o transplante pancreático, opção atual de maior uso. OBJETIVOS: Comparar as ilhotas encapsuladas e as ilhotas livres em roedores nos seguintes aspectos: local de implantação das ilhotas, número de ilhotas, viabilidade e imunossupressão. MÉTODOS: A pesquisa bibliográfica foi conduzida com o uso de citações do MEDLINE/PUBMED e SCIELO que apresentassem termos sobre transplante de ilhotas em roedores no período de 2000 a 2010. Foram achados 2.636 artigos, mas somente 56 desse período foram selecionados. RESULTADOS: Nos 56 artigos utilizados, 34 por cento eram encapsulados e 66 por cento eram não-encapsulados. Analisando ambos os tipos de transplante de ilhotas, a maioria delas encapsuladas, foi implantada na cavidade peritonial e as não-encapsuladas, através da veia porta, no fígado. A grande vantagem da cavidade peritonial como local de transplante era a oferta sanguínea. As células endoteliais e o fator de crescimento endotelial foram usados para estimular a angiogênese nas ilhotas, aumentando a vascularização rapidamente após a implantação. Foi também provada a influência das cápsulas, dado que quanto maior a cápsula maior era a chance de necrose central. Em alguns artigos, o uso de imunossupressão demonstrou aumento da expectativa de vida do enxerto. CONCLUSÃO: Enquanto algum progresso significativo não tenha sido obtido no campo de transplante de ilhotas, restam ainda muitos obstáculos a serem vencidos. A microencapsulação viabiliza o transplante de ilhotas sem o uso de imunossupressores, o que pode permitir o xenotransplante. O uso de fontes doadoras alternativas, menor quantidade de ilhotas por cápsula e local de implantação adequado, como a cavidade peritonial, podem dar melhor perspectiva na aplicação de cápsulas imunoprotegidas, aumentando viabilidade na prática clínica. Uma série de estratégias, como engenharia genética, coencapsulamento, melhora da oferta de oxigênio ou o estabelecimento de resistência à hipóxia também podem aprimorar os resultados do transplante de ilhotas. Deve-se determinar ainda qual a combinação de estratégias com relação ao uso de ilhotas encapsuladas que possam cumprir com as promessas de um transplante simples e seguro para a cura do diabetes.

Alotransplante de ilhotas pancreáticas no baço com imunossupressão com ciclosporina. Modelo experimental em cães / Pancreatic islet allograft in spleen with immunossuppression with cyclosporine. Experimental model in dogs

PURPOSE: To study the functional behavior of the allograft with immunosuppression of pancreatic islets in the spleen. METHODS: Five groups of 10 Mongrel dogs were used: Group A (control) underwent biochemical tests; Group B underwent total pancreatectomy; Group C underwent total pancreatectomy and pancreatic islet autotransplant in the spleen; Group D underwent pancreatic islet allograft in the spleen without immunosuppressive therapy; Group E underwent pancreatic islet allograft in the spleen and immunosuppression with cyclosporine. All of the animals with grafts received pancreatic islets prepared by the mechanical-enzymatic method - stationary collagenase digestion and purification with dextran discontinuous density gradient, implanted in the spleen. RESULTS: The animals with autotransplant and those with allografts with immunosuppression that became normoglycemic showed altered results of intravenous tolerance glucose (p < 0.001) and peripheral and splenic vein plasmatic insulin levels were significantly lower (p < 0.001) in animals that had allografts with immunosuppression than in those with just autotransplants. CONCLUSIONS: In the animals with immunosupression with cyclosporine subjected to allograft of pancreatic islets prepared with the mechanical-enzymatic preparation method (stationary collagenase digestion and purification with dextran discontinuous density gradient), the production of insulin is decreased and the response to intravenous glucose is altered.(AU)

OBJETIVO: Avaliar o comportamento funcional do alotransplante com imunossupressão de ilhotas pancreáticas no baço. MÉTODOS: Foram utilizados cinco grupos de 10 cães mestiços: grupo A (controle) submetido aos exames bioquímicos; grupo B, submetido à pancreatectomia total; grupo C (autotransplante) submetido à pancreatectomia total e autotransplantação de ilhotas pancreáticas no baço; grupo D, submetido à alotransplantação de ilhotas pancreáticas no baço sem terapia imunossupressiva; grupo E, submetido à alotransplantação de ilhotas no baço e imunossupressão com ciclosporina. Todos os animais transplantados receberam ilhotas pancreáticas isoladas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran e foram implantadas no baço. RESULTADOS: Animais autotransplantados e alotransplantados com imunossupressão que se tornaram normoglicêmicos apresentaram testes de tolerância à glicose intravenosa alterados (p<0,001) e o nível de insulina plasmática periférica e na veia esplênica foram significantemente menores (p<0,001) nos animais alotransplantados com imunossupressão em relação aos autotransplantados. CONCLUSÃO: Nos animais submetidos ao alotransplante de ilhotas pancreáticas com imunossupressão com ciclosporina e preparadas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran, a produção de insulina está diminuída e a resposta à sobrecarga de glicose intravenosa alterada.(AU)

Pancreatic islet allograft in spleen with immunossuppression with cyclosporine. Experimental model in dogs / Alotransplante de ilhotas pancreáticas no baço com imunossupressão com ciclosporina. Modelo experimental em cães

PURPOSE: To study the functional behavior of the allograft with immunosuppression of pancreatic islets in the spleen. METHODS: Five groups of 10 Mongrel dogs were used: Group A (control) underwent biochemical tests; Group B underwent total pancreatectomy; Group C underwent total pancreatectomy and pancreatic islet autotransplant in the spleen; Group D underwent pancreatic islet allograft in the spleen without immunosuppressive therapy; Group E underwent pancreatic islet allograft in the spleen and immunosuppression with cyclosporine. All of the animals with grafts received pancreatic islets prepared by the mechanical-enzymatic method - stationary collagenase digestion and purification with dextran discontinuous density gradient, implanted in the spleen. RESULTS: The animals with autotransplant and those with allografts with immunosuppression that became normoglycemic showed altered results of intravenous tolerance glucose (p < 0.001) and peripheral and splenic vein plasmatic insulin levels were significantly lower (p < 0.001) in animals that had allografts with immunosuppression than in those with just autotransplants. CONCLUSIONS: In the animals with immunosupression with cyclosporine subjected to allograft of pancreatic islets prepared with the mechanical-enzymatic preparation method (stationary collagenase digestion and purification with dextran discontinuous density gradient), the production of insulin is decreased and the response to intravenous glucose is altered.

OBJETIVO: Avaliar o comportamento funcional do alotransplante com imunossupressão de ilhotas pancreáticas no baço. MÉTODOS: Foram utilizados cinco grupos de 10 cães mestiços: grupo A (controle) submetido aos exames bioquímicos; grupo B, submetido à pancreatectomia total; grupo C (autotransplante) submetido à pancreatectomia total e autotransplantação de ilhotas pancreáticas no baço; grupo D, submetido à alotransplantação de ilhotas pancreáticas no baço sem terapia imunossupressiva; grupo E, submetido à alotransplantação de ilhotas no baço e imunossupressão com ciclosporina. Todos os animais transplantados receberam ilhotas pancreáticas isoladas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran e foram implantadas no baço. RESULTADOS: Animais autotransplantados e alotransplantados com imunossupressão que se tornaram normoglicêmicos apresentaram testes de tolerância à glicose intravenosa alterados (p<0,001) e o nível de insulina plasmática periférica e na veia esplênica foram significantemente menores (p<0,001) nos animais alotransplantados com imunossupressão em relação aos autotransplantados. CONCLUSÃO: Nos animais submetidos ao alotransplante de ilhotas pancreáticas com imunossupressão com ciclosporina e preparadas pelo método mecânico-enzimático, digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran, a produção de insulina está diminuída e a resposta à sobrecarga de glicose intravenosa alterada.

Atualização no transplante de pâncreas / Pancreas transplantation: an overview

Pancreas transplantation is the only treatment able to reestablish normal glucose and glycated hemoglobin levels in insulin-dependent diabetic patients without the use of exogenous insulin. The evolution of pancreas transplantation in treatment of diabetes was determined by advances in the s of surgical technique, organ preservation and immunosuppressants. The main complication leading to graft loss is technical failure followed by acute or chronic rejection. Technical failure means graft loss within the first three months following transplantation due to vascular thrombosis (50%), pancreatitis (20%), infection (18%), fistula (6.5%) and bleeding (2.4%). Immunological complications still affect 30% of patients, and rejection is the cause of graft loss in 10% of cases. Chronic rejection is the most common late complication. Cardiovascular diseases are the most common causes of late mortality in pancreas transplantation, so it remains the most effective treatment for type 1 diabetes patients. There is a significant improvement in quality of life and in patient's survival rates. The development of islet transplantation could eliminate or minimize surgical complications and immunosuppression.

O transplante de pâncreas é o único tratamento capaz de restabelecer os níveis de glicose e hemoglobina glicada em pacientes diabéticos dependentes de insulina, sem o uso de insulina exógena. A evolução do transplante de pâncreas no tratamento de diabetes foi marcada por avanços nos campos da técnica cirúrgica, preservação de órgãos e imunossupressão. A principal complicação da perda do enxerto é a falha técnica, seguida de rejeição aguda ou crônica. Por falha técnica entende-se perda do enxerto dentro dos primeiros três meses seguintes ao transplante devido a: trombose vascular (50%), pancreatite (20%), infecção (18%), fístula (6,5%) e hemorragia (2,4%). Complicações imunológicas ainda afetam 30% dos pacientes, e a rejeição causa perda do enxerto em 10% dos casos. A rejeição crônica é a complicação tardia mais comum. Doenças cardiovasculares são a causa mais frequente de mortalidade tardia no transplante de pâncreas que continua sendo o tratamento mais eficaz para pacientes com diabetes do tipo 1. Há uma importante melhora na qualidade de vida e na sobrevida dos pacientes. O desenvolvimento de ilhotas transplantadas pode eliminar ou minimizar complicações cirúrgicas e a imunossupressão.

Isolamento de ilhotas pancreáticas e a alternativa de uso da frutose-1, 6-bisfosfato: [revisão] / Pancreatic islet isolation and the alternative use of fructose-1, 6-bisphosphate: [review]

Objetivos: revisar dados da literatura sobre isolamento de ilhotas pancreáticas para transplante e sobre as ações da frutose-1,6-bisfosfato. Fonte de dados: revisão de artigos publicados, a partir da pesquisa em bancos de dados nacionais e internacionais (SciELO, Lilacs, PubMed). Síntese dos dados: o transplante de ilhotas surge como uma alternativa para o tratamento do diabetes mellitus tipo 1. Entretanto, durante o processo de isolamento, há grande perda celular, principalmente na periferia da ilhota pancreática. As espécies reativas de oxigênio contribuem significativamente nesse processo, afetando a viabilidade das células para transplante. Vários esforços estão sendo feitos na tentativa de minimizar os danos causados pela liberação e produção destes compostos químicos. Conclusões: frente às importantes ações da frutose-1,6-bisfosfato descritas na literatura, seu emprego durante o processo de isolamento das ilhotas pancreáticas parece ser uma alternativa bastante atraente. O efeito da frutose-1,6-bisfosfato na redução da formação e liberação de radicais livres, assim como a sua ação citoprotetora, poderiam viabilizar um maior número de células, otimizando o processo de isolamento, além de auxiliar na enxertia, por diminuir a liberação de citocinas pró-inflamatórias.

Aims: To review the literature data about pancreatic islet isolation and fructose-1,6-bisphosfate. Source of data: Review of specific articles on the issue published in national and internacional databases (SciELO,Lilacs, PubMed). Summary of findings: Islets transplantation is an alternative for the treatment of type 1 diabetes mellitus. However, during the process of isolation, cell loss, mainly on the periphery of the pancreatic islet, ensues. Reactive oxygen species seem to contribute significantly in this process, affecting the viability of these cells. Various efforts are being made in an attempt to minimize the damage caused by the release and production of reactive oxygen species. Conclusions: Considering the important actions of fructose-1,6-bisphosphate which are described in the literature, its use in pancreatic islet isolation may represent an attractive alternative. The effect of fructose-1,6-bisphosphate in reducing the production and release of free radicals, as well as its role in cellular protection, could enable a greater number of viable cells, optimizing the isolation process, and also protecting the graft process, by reducing the release of proinflammatory cytokines.

Imersão do pâncreas por 24 horas em solução de colagenase a 4ºC no isolamento de ilhotas de Langerhans / Immersion of pancreas for 24 hours in collagenase solution at 4ºC in Langerhans islets isolation

Objetivos: testar se a imersão do pâncreas em solução de 1 ou 2 mg/ml de colagenase em 4ºC por 24 horas, no isolamento experimental de ilhotas de Langerhans, aumenta o rendimento de ilhotas por grama de tecido pancreático. Métodos: estudo experimental com camundongos, realizado no Laboratório de Nefrologia do Instituto de Pesquisas Biológicas do Hospital São Lucas da PUCRS, Porto Alegre, RS. Após sacrifício dos animais sob anestesia, os pâncreas foram retirados e triturados, sendo divididos em quatro grupos, conforme a técnica utilizada para isolar as ilhotas. A) colagenase 1mg/mL, a 4ºC por 24 horas e posterior aquecimento a 39ºC por 15 minutos. B) colagenase 2mg/mL com as mesmas etapas anteriores. C) colagenase 1mg/mL com aquecimento da solução no mesmo dia da retirada, a 39ºC por 15 minutos. D) colagenase 2mg/mL com aquecimento da solução no mesmo dia da retirada, a 39ºC por 15 minutos. Verificamos a viabilidade das ilhotas através do teste do azul tripano. Resultados: as medianas da quantidade de ilhotas isoladas nos grupos A, B, C e D foram 9.142, 8.285, 2.813 e 3.199 respectivamente. O teste de Kruskal- Wallis demonstrou diferença significativa, com valor de H = 17,44 com a = 0,01 e, na comparação dos grupos entre si, demonstrou que não há diferença entre as soluções de colagenase com concentrações de 1 e 2 mg/dL. Os grupos com a imersão do tecido pancreático em solução de colagenase por 24 horas obtiveram três vezes mais ilhotas, quando comparados aos submetidos à digestão imediata, conforme teste de Dunn com a<0,05. O teste do azul tripano demonstrou uma vitalidade maior que 95% em todos os grupos. Conclusões: sugere-se que a imersão em colagenase por tempo mais prolongado melhora o processo de digestão do tecido pancreático, aumentando o rendimento de ilhotas isoladas por grama de tecido pancreático. A diferença de concentração entre as soluções de colagenase não afetou o resultado.

Aims: To test whether the immersion of the pancreas in solutions of 1 or 2 mg/mL of collagenase in 4°C for 24 hours, for the isolation of Langerhans islets, rises the yield of islets/ grams of pancreatic tissue. Methods: Experimental study with mouses, performed in the Laboratory of Nephrology of the Instituto de Pesquisas Biológicas do Hospital São Lucas da PUCRS, Porto Alegre, RS. After the animals have been sacrified under anesthesia, the pancreas were removed and divided in four groups, according the technique used for isolating the islets. A) collagenase 1mg/mL, in 4ºC for 24 hours and heating for 39ºC for 15 minutes. B) collagenase 2mg/mL with the same previous described steps. C) collagenase 1mg/ mL and heating of the solution in the same day, in 39ºC for 15 minutes. D) collagenase 2mg/mL and heating of the solution in the same day, in 39ºC for 15 minutes. We verified the viability of the islet through the trypan blue test. Results: The median numbers of isolated islets in the groups A, B, C and D were 9142, 8285, 2813 e 3199, respectively. Kruskal-Wallis test showed significant difference, with the value of H = 17,44 with a = 0,01, and in the comparison between the groups, there was o difference in the solutions of collagenase with concentrations of 1 and 2 mg/dL. Groups with immersion of pancreatic tissue in collagenase solution for 24 hours had three times more islets when compared to groups submitted to immediate digestion, according to Dunn test with a <0,05. The trypan blue test showed viability higher than 95% in all groups. Conclusions: We suggest that the immersion in solution of collagenase for longer time improves the process of digestion of pancreatic tissue, rising the yield of islets/ grams of pancreatic tissue. Different concentration between the collagenase solutions did not affect the final result.