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1.
Adv Exp Med Biol ; 1212: 179-220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31025308

RESUMO

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic ß-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of ß-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional ß-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Ilhotas Pancreáticas/citologia , Organoides/citologia , Medicina Regenerativa/métodos , Células-Tronco/citologia , Animais , Diferenciação Celular , Diabetes Mellitus Tipo 1/patologia , Humanos , Transplante das Ilhotas Pancreáticas/métodos
2.
Int. j. morphol ; 37(4): 1331-1334, Dec. 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1040133

RESUMO

Obesity and its comorbidities are becoming epidemic in the Western world. Beta cell mass estimation is an important indicator to track the progression of insulin resistance/type 2 diabetes, particularly in experimental studies, where it can be performed with stereological tools in an unbiased way. In this work, we present a simple protocol that can contribute to doing the practice of estimating the mass of beta cells more frequent and reproducible. As with any quantitative study, the necessary precautions regarding sampling and randomness must be respected.


La obesidad y sus comorbilidades se están convirtiendo en una epidemia en el mundo occidental. La estimación de la masa de células beta es un indicador importante para rastrear la progresión de la resistencia a la insulina/diabetes tipo 2, particularmente en estudios experimentales, donde se puede realizar con herramientas estereológicas de manera imparcial. En este trabajo presentamos un protocolo simple que puede contribuir a que la práctica de estimar la masa de células beta sea más frecuente y reproducible. Como en cualquier estudio cuantitativo, deben respetarse las precauciones necesarias con respecto al muestreo y la aleatoriedad.


Assuntos
Humanos , Técnicas Citológicas/métodos , Ilhotas Pancreáticas/citologia , Células Secretoras de Insulina
3.
Endocrinology ; 160(11): 2759-2772, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504428

RESUMO

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.


Assuntos
Glucose/metabolismo , Iodeto Peroxidase/deficiência , Envelhecimento/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Homeostase , Hiperglicemia/genética , Iodeto Peroxidase/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Masculino , Proglucagon/metabolismo , Proinsulina/metabolismo , Receptor de Insulina/metabolismo , Receptores de Glucagon/metabolismo , Peixe-Zebra
5.
Genome Biol ; 20(1): 166, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412909

RESUMO

scRNA-seq dataset integration occurs in different contexts, such as the identification of cell type-specific differences in gene expression across conditions or species, or batch effect correction. We present scAlign, an unsupervised deep learning method for data integration that can incorporate partial, overlapping, or a complete set of cell labels, and estimate per-cell differences in gene expression across datasets. scAlign performance is state-of-the-art and robust to cross-dataset variation in cell type-specific expression and cell type composition. We demonstrate that scAlign reveals gene expression programs for rare populations of malaria parasites. Our framework is widely applicable to integration challenges in other domains.


Assuntos
Análise de Sequência de RNA , Análise de Célula Única , Software , Animais , Biomarcadores/metabolismo , Análise por Conglomerados , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Camundongos Endogâmicos C57BL , Plasmodium falciparum/citologia , Plasmodium falciparum/genética , Análise de Componente Principal , Alinhamento de Sequência
6.
Nat Commun ; 10(1): 3700, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420552

RESUMO

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.


Assuntos
Ilhotas Pancreáticas/ultraestrutura , Comunicação Parácrina , Estado Pré-Diabético/patologia , Pseudópodes/ultraestrutura , Células Secretoras de Somatostatina/ultraestrutura , Animais , Glicemia/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Microscopia Intravital , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Imagem Óptica , Optogenética , Estado Pré-Diabético/metabolismo , Pseudópodes/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
J Diabetes Res ; 2019: 2813489, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467926

RESUMO

While the autoimmune character of T1D (type 1 diabetes) is being challenged, it is currently recognized that inflammation plays a key role in its development. We hypothesized that glucotoxicity could contribute to ß-cell mass destruction through participation in islet inflammation. We evaluated the potential of empagliflozin (EMPA) and GABA (gamma-aminobutyric acid) to protect ß-cell mass against glucotoxicity and to increase ß-cell mass after diagnosis of T1D. Empagliflozin is a SGLT2 (sodium-dependent glucose cotransporter) inhibitor which thereby blocks glucose recapture by the kidney and promotes glucose excretion in urine. GABA is an inhibitory neurotransmitter, which stimulates α-to-ß cell transdifferentiation. In streptozotocin-treated mice, empagliflozin and/or GABA were delivered for a period of five days or three weeks. As compared to untreated T1D mice, EMPA-treated T1D mice had decreased FFA (free fatty acid) levels and improved glucose homeostasis. EMPA-treated T1D mice had higher islet density, with preserved architecture, compared to T1D mice, and EMPA-treated T1D mice also differed from T1D mice by the total absence of immune cell infiltration within islets. Islets from EMPA-treated mice were also less subjected to ER (endoplasmic reticulum) stress and inflammation, as shown by qPCR analysis. Glucose homeostasis parameters and islet area/pancreas area ratio improved, as compared to diabetic controls, when T1D mice were treated for three weeks with GABA and EMPA. T1D EMPA+GABA mice had higher glucagon levels than T1D mice, without modifications of glucagon area/islet area ratios. In conclusion, empagliflozin and GABA, used in monotherapy in streptozotocin-induced diabetic mice, have positive effects on ß-cell mass preservation or proliferation through an indirect effect on islet cell inflammation and ER stress. Further research is mandatory to evaluate whether empagliflozin and GABA may be a potential therapeutic target for the protection of ß-cell mass after new-onset T1D.


Assuntos
Compostos Benzidrílicos/farmacologia , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Glucosídeos/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Compostos Benzidrílicos/administração & dosagem , Glicemia/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Esquema de Medicação , Quimioterapia Combinada , Intervenção Médica Precoce , Teste de Tolerância a Glucose , Glucosídeos/administração & dosagem , Injeções Intraperitoneais , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Estreptozocina , Fatores de Tempo , Ácido gama-Aminobutírico/administração & dosagem
8.
Cell Tissue Bank ; 20(3): 389-401, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31270642

RESUMO

Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into insulin-producing cells (IPCs). Bio-scaffolds derived from decellularized organs can act as a carrier for seed cells and may have broad applications in regenerative medicine. This study investigated the effect of native pancreatic stroma obtained from decellularized pancreas on the proliferation, migration and differentiation of BMSCs into IPCs, and explored the potential underlying molecular mechanism. The decellularized pancreas bio-scaffold was obtained by perfusion with Triton X-100/ammonium hydroxide, followed by digestion with a mixture of pepsin and hydrochloric acid to prepare the stroma solution. Islet-like cells were differentiated from BMSCs by a three-step induction method. The differences on the cytological behavior with or without stroma were evaluated by morphological observation, insulin release assay, qRT-PCR assay and western blot analysis. Our results showed that, stroma derived from decellularized pancreas could promote the proliferation and migration of BMSCs. Furthermore, the formation of IPCs could also be promoted, which possessed similar morphology to endogenous islets. During the induced differentiation process, the presence of stroma significantly increased the expression of insulin 1, insulin 2 and Pdx-1, as well as insulin release. This was accompanied by an increase in the phosphorylation of Akt and ERK in third stage cell clusters, which was prevented by the addition of the inhibitors PD98059 and LY294002, respectively. In summary, decellularized pancreatic stroma could promote the proliferation, migration and differentiation of BMSCs into IPCs, and this involved the activation of Akt and ERK signal pathways.


Assuntos
Insulina/biossíntese , Ilhotas Pancreáticas/citologia , Células-Tronco Mesenquimais/citologia , Pâncreas , Tecidos Suporte , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Cromonas/farmacologia , Flavonoides/farmacologia , Glucose/metabolismo , Proteínas de Homeodomínio/biossíntese , Morfolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa , Transdução de Sinais , Transativadores/biossíntese
9.
Eur J Pharmacol ; 858: 172518, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31265840

RESUMO

Minocycline functions as a therapeutic drug in different diseases because of its cytoprotective properties. In the present study, we examined the potential of minocycline to decrease the islet loss in pre-transplantation culture stage. Pancreatic islets were isolated from the deceased donors and treated by 0, 2, 10, and 20 µM minocycline for 24 and 72 h. After that, the incubated islets were evaluated for viability and function. Apoptosis markers including Bax, Bcl2, and caspase-3 were determined at gene and protein level. On the other hand, TUNEL assay was used to confirm apoptosis. The functionality of the islets was investigated using glucose-induced insulin and c-peptide secretion assay. After 72 h of incubation, the viability of human islet was drastically decreased, whereas supplementation with minocycline inhibited the cells death. In this regard, the expression of Bax and active Caspase-3 was downregulated, whereas the expression of Bcl2 was upregulated. These consequences suggest that pancreatic islets undergo apoptosis in vitro and minocycline can decelerate or inhibit this process. Our findings identified minocycline as a cytoprotective molecule for preventing human islets death in pre-transplantation culture.


Assuntos
Apoptose/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Minociclina/farmacologia , Caspase 3/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
10.
Bull Exp Biol Med ; 167(1): 140-144, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31183647

RESUMO

We studied therapeutic activity of co-transplantation of allogeneic pancreatic islet cells and mesenchymal bone marrow progenitors on TiNi scaffolds in Wistar rats with experimental alloxan-induced diabetes mellitus. In preliminary experiments with co-culturing of cells in different proportions followed by their transplantation on tissue-engineered constructs, the optimum ratio of these cells was determined - 3:1. Regeneration was assessed by biochemical methods by the blood levels of glucose and glycosylated hemoglobin on days 15, 30, and 5. In the group with combined cell transplantation on TiNi scaffold, normalization of the studied biochemical parameters occurred earlier than after monotherapy with allogenic islet cells and was associated with an increase in animal lifespan. Normalization of the parameters of bone marrow hemopoiesis, in particular, the number of myelokaryocytes and erythroblasts was also noted.


Assuntos
Aloxano/toxicidade , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diabetes Mellitus Experimental/terapia , Ilhotas Pancreáticas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Níquel/farmacologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Titânio/farmacologia , Animais , Materiais Biocompatíveis/química , Diabetes Mellitus Experimental/induzido quimicamente , Masculino , Níquel/química , Ratos , Ratos Wistar , Titânio/química
11.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(6): 246-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189778

RESUMO

l-Glutamate is one of the most abundant amino acids in the body and is a constituent of proteins and a substrate in metabolism. It is well known that glutamate serves as a primary excitatory neurotransmitter and a critical neuromodulator in the brain. Recent studies have shown that in addition to its pivotal role in neural functions, glutamate plays many important roles in a variety of cellular functions, including those as intracellular and extracellular signals. In pancreatic islets, glutamate is now known to be required for the normal regulation of insulin secretion, such as incretin-induced insulin secretion. In this review, we primarily discuss the physiological and pathophysiological roles of glutamate as intracellular and extracellular signals in the functions of pancreatic islets.


Assuntos
Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Espaço Intracelular/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais , Animais , Humanos
12.
Acta Biomater ; 94: 351-360, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31200117

RESUMO

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced ß-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. STATEMENT OF SIGNIFICANCE: 1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices. 2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways. 3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity. 4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.


Assuntos
Elastina/química , Transplante das Ilhotas Pancreáticas/métodos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Animais , Glicemia/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Diabetes Mellitus Experimental , Regulação da Expressão Gênica , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Resultado do Tratamento
13.
Acta Histochem ; 121(5): 638-645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31146895

RESUMO

In the human pancreas, various forms of endocrine cell arrangement are found: single endocrine cells, endocrine cell clusters, and mantel, bipolar and mosaic cell (mixed) islets. Our aim was to analyse the distribution and dynamics of insulin-, glucagon- and somatostatin-containing cells within the various forms of endocrine pancreas arrangement during human prenatal development and in adults and to suggest a mechanism of change in the endocrine cell ratio in adult islets. Pancreatic autopsies derived from human foetuses from the 10th to the 40th weeks of development and from adults were examined using histological, immunohistochemical and morphometric methods. During development, the human endocrine pancreas undergoes not only de novo differentiation of endocrine cells and islet formation, but morphogenetic restructuring, which is revealed as a change of the α-, ß- and δ-cell ratio in the islets. In particular, increased proportion of glucagon- and somatostatin-containing cells and decreased proportion of ß-cells were shown in the largest mosaic islets in adults. Our results indicate that the distribution and proportion of α-, ß- and δ-cells depend on the islets size and vascularisation. Studying of the mechanism of such restructuring may contribute to the development of new approaches in the treatment of diabetes mellitus.


Assuntos
Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Pâncreas/citologia , Células Secretoras de Somatostatina/citologia , Desenvolvimento Embrionário , Humanos
14.
Ann Anat ; 224: 153-160, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31108190

RESUMO

The effect of empagliflozin (EMPA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i), on the structure of endocrine pancreas in pre-diabetes (Pre-DM) is not yet elucidated. In the current study the relatively enlarged islets of Langerhans seen in the Pre-DM group was restored to control size by administration of EMPA. In addition the disbalance in the percentage of ß-cells and α-cells in islets of the Pre-DM was corrected in the Pre-DM + EMPA group with reversal of the significantly increased islet mass, ß-cell mass and neogenesis. Administrating EMPA in Pre-DM decreased level of caspase-3, increased that of Bcl-2 to control level and reduced the significantly increased inflammatory cytokines to levels approximated to those of the control group. In Pre-DM + EMPA group, EMPA had efficiently restored the significantly impaired glucose hemostasis to levels nearly similar to those of the control animals. This may indicate that the modulatory effect of EMPA on cells of the islets in Pre-DM is associated with a local pleotropic effect on inflammatory cytokines.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Experimental/patologia , Glucosídeos/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Estado Pré-Diabético/patologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Animais , Área Sob a Curva , Compostos Benzidrílicos/farmacologia , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Proliferação de Células , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Glucagon/sangue , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/efeitos dos fármacos , Teste de Tolerância a Glucose , Glucosídeos/farmacologia , Homeostase , Imuno-Histoquímica , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/patologia , Masculino , Estado Pré-Diabético/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
15.
Int J Mol Sci ; 20(10)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121855

RESUMO

BACKGROUND: Islet autophagy plays a role in glucose/lipid metabolism in type 2 diabetes mellitus. Meanwhile, fibroblast growth factor 21 (FGF21) has been found to regulate insulin sensitivity and glucose homeostasis. Whether FGF21 induces islet autophagy, remains to be elucidated. This study aimed to explore the physiological roles and signaling pathways involved in FGF21-stimulated islet autophagy under glucolipotoxic conditions. METHODS: C57/BL6J mice were fed a standard diet or high-fat diet (HFD) for 12 weeks, and islets were isolated from normal and FGF21 knockout (KO) mice. Isolated islets and INS-1E cells were exposed to normal and high-concentration glucose and palmitic acid with/without FGF21 or AMPK inhibitor compound C. Real-time PCR, Western blot and immunohistochemistry/transmission electron microscopy were performed for the expression of targeted genes/proteins. RESULTS: HFD-treated mice showed increases in fasting plasma glucose, body weight and impaired glucose tolerance; islet protein expression of FGF21 was induced after HFD treatment. Protein expression levels of FGF21 and LC3-II (autophagy marker) were induced in mouse islets treated with high concentrations of palmitic acid and glucose, while phosphorylation of AMPK was reduced, compared with controls. In addition, induction of LC3-II protein expression was reduced in islets isolated from FGF21 KO mice. Furthermore, exogenous administration of FGF21 diminished phosphorylation of AMPK and stimulated protein expression of LC3-II. Consistently, compound C significantly induced increased expression of LC3-II protein. CONCLUSIONS: Our data indicate that glucolipotoxicity-induced FGF21 activation mediates islet autophagy via AMPK inhibition, and further consolidate the evidence for the FGF21/analog being a pharmacotherapeutic target for obesity and its related T2DM.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Fatores de Crescimento de Fibroblastos/metabolismo , Ilhotas Pancreáticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Ilhotas Pancreáticas/citologia , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais
16.
Diabetes ; 68(6): 1121-1129, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31109941

RESUMO

Although it is well established that type 2 diabetes (T2D) is generally due to the progressive loss of ß-cell insulin secretion against a background of insulin resistance, the actual correlation of reduced ß-cell mass to its defective function continues to be debated. There is evidence that a compensatory increase in ß-cell mass, and the consequent insulin secretion, can effectively cope with states of insulin resistance, until hyperglycemia supervenes. Recent data strongly indicate that the mechanisms by which islets are able to compensate in response to insulin resistance in peripheral tissues is secondary to hyperplasia, as well as the activation of multiple cellular machineries with diverse functions. Importantly, islet cells exhibit plasticity in altering their endocrine commitment; for example, by switching from secretion of glucagon to secretion of insulin and back (transdifferentiation) or from an active secretory state to a nonsecretory quiescent state (dedifferentiation) and back. Lineage tracing (a method used to track each cell though its differentiation process) has demonstrated these potentials in murine models. A limitation to drawing conclusions from human islet research is that most studies are derived from human autopsy and/or organ donor samples, which lack in vivo functional and metabolic profiling. In this review, we specifically focus on evidence of islet plasticity in humans-from the normal state, progressing to insulin resistance to overt T2D-to explain the seemingly contradictory results from different cross-sectional studies in the literature. We hope the discussion on this intriguing scenario will provide a forum for the scientific community to better understand the disease and in the long term pave the way for personalized therapies.


Assuntos
Desdiferenciação Celular , Plasticidade Celular , Transdiferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Glucagon/citologia , Humanos , Resistência à Insulina , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Tamanho do Órgão
17.
Cell Biol Int ; 43(8): 846-851, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31115951

RESUMO

Lelio Orci has made seminal contributions to our understanding of pancreatic islet structure and function. He introduced quantitative criteria to structural analysis in the study of endocrine pancreas in a series of works performed in collaboration with Albert Renold, Roger Unger, and Donald Steiner. Orci has moved islet cell morphology from the primitive era of histochemistry and electron microscopy into the modern era of cell biology, applying the most advanced techniques and covering every aspect of normal and pathological structure-function relationships. In collaboration with James Rothman in New York and Randy Schekman in Berkley, Orci discovered that the transport steps from the endoplasmic reticulum to the Golgi complex, and within the Golgi, are mediated by two sets of vesicles coated with protein envelopes different from clathrin.


Assuntos
Retículo Endoplasmático , Complexo de Golgi , Ilhotas Pancreáticas , Distinções e Prêmios , Transporte Biológico , Clatrina/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , História do Século XX , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Itália , Suíça
18.
Nat Commun ; 10(1): 2078, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064983

RESUMO

Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.


Assuntos
Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Redes Reguladoras de Genes/genética , Ilhotas Pancreáticas/metabolismo , Proteínas de Ligação a RNA/genética , Adulto , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Diabetes Mellitus Tipo 2/patologia , Elementos Facilitadores Genéticos/genética , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Glucose/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Conformação Molecular , Locos de Características Quantitativas/genética , Proteínas de Ligação a RNA/metabolismo
19.
PLoS One ; 14(5): e0216136, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075114

RESUMO

BACKGROUND: Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome. METHODS: Islet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry. RESULTS: No beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I. CONCLUSIONS: The Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.


Assuntos
Ilhotas Pancreáticas/citologia , Animais , Colágeno/metabolismo , Colagenases/metabolismo , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Doadores de Tecidos
20.
PLoS One ; 14(5): e0216254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075154

RESUMO

BACKGROUND: Pancreatic islet xenotransplantation is a potential treatment for diabetes mellitus, and porcine pancreas may provide a readily available source of islets. Islets in juvenile pigs are smaller than those in young adult pigs, but the insulin content is very similar. In addition, as juvenile pigs are more easily reared in uncontaminated conditions, many researchers have conducted studies using pancreatic islets from juvenile pigs. We aimed to analyze the distributions of endocrine cell clusters by comprehensively evaluating juvenile porcine pancreatic development and to propose an appropriate age at which islets could be isolated from the juvenile porcine pancreas. METHODS: Splenic (SL) and duodenal lobe (DL) samples were collected from the pancreases of pigs aged 0-180 days (n = 3/day after birth). The chronological changes in endocrine cell clustering were analyzed in relation to morphological changes, cell characterization, numbers, islet areas, and gene expression. RESULTS: In juvenile pigs aged 0-21 days, the pancreas contained numerous endocrine cells, and compact islets appeared from 21 days of age. Well-defined small islets were seen at 28 days of age, and the clusters were denser in the SL than in the DL. At 35 days of age, the islets were morphologically similar to those observed at 180 days of age, and the greater number of islets was similar to that seen at 90 days of age. The differences in the islets' cytoarchitecture between the lobes were negligible. The expression of ß-cell-related genes was higher in the juvenile pancreas than in the adult pancreas, and the expression of neurogenin-3 decreased dramatically over time. CONCLUSIONS: These findings may have implications for attempts to refine the most appropriate age for islet isolation from porcine donors. Focusing on porcine pancreatic islets isolated at around 35 days after birth may offer benefits regarding their xenotransplantation potential.


Assuntos
Fatores Etários , Células Endócrinas/citologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/crescimento & desenvolvimento , Transplante Heterólogo/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Análise por Conglomerados , Diabetes Mellitus/terapia , Humanos , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/metabolismo , Suínos
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