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1.
Am J Transplant ; 21(4): 1493-1502, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32986297

RESUMO

Many variables impact islet isolation, including pancreas ischemia time. The ischemia time upper limit that should be respected to avoid a negative impact on the isolation outcome is not well defined. We have performed a retrospective analysis of all islet isolations in our center between 2008 and 2018. Total ischemia time, cold ischemia time, and organ removal time were analyzed. Isolation success was defined as an islet yield ≥200 000 IEQ. Of the 452 pancreases included, 288 (64%) were successfully isolated. Probability of isolation success showed a significant decrease after 8 hours of total ischemia time, 7 hours of cold ischemia time, and 80 minutes of organ removal time. Although we observed an impact of ischemia time on islet yield, a probability of isolation success of 50% was still present even when total ischemia time exceeds 12 hours. Posttransplantation clinical outcomes were assessed in 32 recipients and no significant difference was found regardless of ischemia time. These data indicate that although shorter ischemia times are associated with better islet isolation outcomes, total ischemia time >12 hours can provide excellent results in appropriately selected donors.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Soluções para Preservação de Órgãos , Humanos , Isquemia , Pâncreas , Estudos Retrospectivos
2.
Hum Mol Genet ; 26(18): 3453-3465, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28911206

RESUMO

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.


Assuntos
Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Difosfato de Adenosina/metabolismo , Aminoácidos/genética , Animais , Glicemia/metabolismo , Hiperinsulinismo Congênito/genética , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperinsulinismo/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Polimorfismo de Nucleotídeo Único/genética
3.
EMBO J ; 28(18): 2786-93, 2009 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-19680222

RESUMO

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B-Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B-Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B-Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B-Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B-Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B-Cdc2 but also by the regulation of PP2A by GW.


Assuntos
Regulação Enzimológica da Expressão Gênica , Mitose , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/metabolismo , Animais , Núcleo Celular/metabolismo , Humanos , Masculino , Microcistinas/metabolismo , Modelos Biológicos , Ácido Okadáico/metabolismo , Oócitos/metabolismo , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Espermatozoides/metabolismo , Proteínas de Xenopus/química
4.
Proc Natl Acad Sci U S A ; 107(28): 12564-9, 2010 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-20538976

RESUMO

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.


Assuntos
Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclina B/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ciclo Celular/genética , Ciclina B1 , Humanos , Mitose/efeitos dos fármacos , Ácido Okadáico/metabolismo , Ácido Okadáico/farmacologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
5.
Biomedicines ; 11(3)2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-36979966

RESUMO

This study evaluates the influence of a gelatin sponge on adipose-derived stromal cells (ASC). Transcriptomic data revealed that, compared to ASC in a monolayer, a cross-linked porcine gelatin sponge strongly influences the transcriptome of ASC. Wound healing genes were massively regulated, notably with the inflammatory and angiogenic factors. Proteomics on conditioned media showed that gelatin also acted as a concentrator and reservoir of the regenerative ASC secretome. This secretome promoted fibroblast survival and epithelialization, and significantly increased the migration and tubular assembly of endothelial cells within fibronectin. ASC in gelatin on a chick chorioallantoic membrane were more connected to vessels than an empty sponge, confirming an increased angiogenesis in vivo. No tumor formation was observed in immunodeficient nude mice to which an ASC gelatin sponge was transplanted subcutaneously. Finally, ASC in a gelatin sponge prepared from outbred rats accelerated closure and re-vascularization of ischemic wounds in the footpads of rats. In conclusion, we provide here preclinical evidence that a cross-linked porcine gelatin sponge is an optimal carrier to concentrate and increase the regenerative activity of ASC, notably angiogenic. This formulation of ASC represents an optimal, convenient and clinically compliant option for the delivery of ASC on ischemic wounds.

6.
J Cell Sci ; 123(Pt 13): 2281-91, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20554897

RESUMO

Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.


Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Fator Promotor de Maturação/metabolismo , Metáfase/fisiologia , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Xenopus laevis , Fosfatases cdc25/metabolismo
7.
Mol Cell Endocrinol ; 510: 110815, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32315719

RESUMO

Syndecans (SDC) are important multifunctional components of the extracellular matrix mainly described in endothelial cells. We studied the expression and regulation of SDC in cultured MIN6B1 cells and pancreatic islets. qRT-PCR revealed that syndecan-4 (SDC4) was the predominant isoform expressed in MIN6B1 cells and islets compared to other forms of SDC. Immunofluorescence in mouse and human pancreas sections revealed that SDC4 is mainly expressed in ß-cells compared to other pancreatic cells. Exposure of MIN6B1 and human islets to IL-1ß dose-dependently induced a rapid and transient expression of SDC4 while SRC and STAT3 inhibitors decreased this effect. Exposure of human islets to Il-1ß caused an increase of SDC4 shedding, however treatment with STAT3 and SRC inhibitors inhibited this effect. These results indicate that SDC4 is upregulated by IL-1ß through the SRC-STAT3 pathway and this pathway is also involved in SDC4 shedding in islets.


Assuntos
Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , Sindecana-4/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sindecana-4/genética , Regulação para Cima/genética , Quinases da Família src/metabolismo
8.
Sci Rep ; 10(1): 7011, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332867

RESUMO

Hypoxia, IL-1ß production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1ß production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1ß released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1ß in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1ß released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.


Assuntos
Inflamassomos/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/metabolismo , Morte Celular/genética , Morte Celular/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/cirurgia , Interleucina-1beta/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos SCID , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
9.
Sci Rep ; 9(1): 19350, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852918

RESUMO

TLR4 is a transmembrane receptor of the innate immune system that recognizes LPS from gram-negative bacteria. Its stimulation induces pro-inflammatory responses and modulates adaptive immunity. Our aim is to determine the role of TLR4 in the activation and proliferation of T lymphocytes in the onset of autoimmune diabetes, using the non-obese diabetic (NOD) mouse model. Antigen-specific activation and proliferation of diabetogenic T cells were assessed in vitro by Carboxyfluorescein succinimidyl ester (CFSE) dilution, in presence of vehicle or CLI-095, a cyclohexene derivative that inhibits TLR4 signaling. NOD mice were treated with vehicle or CLI-095 and sacrificed either before or after the onset of autoimmune diabetes. T lymphocyte activation and proliferation were evaluated in treated and control mice. Insulitis was analyzed by histology and diabetes incidence was determined in treated and control mice. Our results demonstrate that TLR4 blockade decreases CD4+ T lymphocyte activation and auto-antigen-specific proliferation both in vitro and in vivo, decreases the infiltrative insulitis and finally prevents the onset of spontaneous diabetes. Taken together, our data demonstrate that TLR4 signaling contributes to the development and maintenance of autoimmune diabetes. The immunomodulatory effect of CLI-095 could be part of a preventive strategy targeting patients at risk for type 1 diabetes.


Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Receptor 4 Toll-Like/antagonistas & inibidores , Transferência Adotiva , Animais , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Epitopos/metabolismo , Feminino , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T/metabolismo , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/metabolismo
10.
J Endocrinol ; 229(2): 123-32, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26931137

RESUMO

The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.


Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Células Secretoras de Polipeptídeo Pancreático/citologia , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Adolescente , Adulto , Idoso , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Polipeptídeo Pancreático/metabolismo , Somatostatina/metabolismo , Distribuição Tecidual , Adulto Jovem
11.
PLoS One ; 10(2): e0117130, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25647668

RESUMO

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic ß-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in ß-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in ß-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet ß-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in ß-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.


Assuntos
Glutamato Descarboxilase/metabolismo , Células Secretoras de Insulina/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Glutamato Descarboxilase/análise , Complexo de Golgi/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/metabolismo
12.
Science ; 330(6011): 1673-7, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-21164014

RESUMO

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.


Assuntos
Mitose , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Interfase , Dados de Sequência Molecular , Oócitos , Peptídeos/química , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Xenopus laevis
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