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1.
J Endocrinol ; 214(2): 225-32, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22582094

RESUMO

Zinc ions are essential for the formation of insulin crystals in pancreatic ß cells, thereby contributing to packaging efficiency of stored insulin. Zinc fluxes are regulated through the SLC30A (zinc transporter, ZNT) family. Here, we investigated the effect of metabolic stress associated with the prediabetic state (zinc depletion, glucotoxicity, and lipotoxicity) on ZNT expression and human pancreatic islet function. Both zinc depletion and lipotoxicity (but not glucotoxicity) downregulated ZNT8 (SLC30A8) expression and altered the glucose-stimulated insulin secretion index (GSIS). ZNT8 overexpression in human islets protected them from the decrease in GSIS induced by tetrakis-(2-pyridylmethyl) ethylenediamine and palmitate but not from cell death. In addition, zinc supplementation decreased palmitate-induced human islet cell death without restoring GSIS. Altogether, we showed that ZNT8 expression responds to variation in zinc and lipid levels in human ß cells, with repercussions on insulin secretion. Prospects for increasing ZNT8 expression and/or activity may prove beneficial in type 2 diabetes in humans.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Ilhotas Pancreáticas/metabolismo , Adulto , Proteínas de Transporte de Cátions/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Quelantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Lipídeos/farmacologia , Lipídeos/toxicidade , Ácido Palmítico/farmacologia , Ácido Palmítico/toxicidade , Transfecção , Zinco/farmacologia , Transportador 8 de Zinco
2.
Mol Endocrinol ; 26(3): 399-413, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22301783

RESUMO

The NR4A orphan nuclear receptors Nur77, Nurr1, and Nor1 exert multiple cellular and metabolic functions. These transcriptional regulators are activated in response to extracellular stresses, including lipotoxic fatty acids (FA) and proinflammatory cytokines. The contribution of NR4As to ß-cell pathophysiology is, however, unknown. We have therefore examined the role of NR4As as downstream contributors to FA-induced ß-cell dysfunctions. Human pancreatic islets and insulinoma ß-cells were used to determine transcriptional programs elicited by NR4A, which were compared to those triggered by palmitate treatment. Functional studies evaluated the consequence of an increased NR4A expression on insulin biosynthesis and secretion and cell viability in insulinoma ß-cells. FA and cytokine treatment increased NR4A expression in pancreatic ß-cells, with Nur77 being most highly inducible in murine ß-cells. Nur77, Nurr1, or Nor1 modulated common and distinct clusters of genes involved notably in cation homeostasis and insulin gene transcription. By altering zinc homeostasis, insulin gene transcription, and secretion, Nur77 was found to be a major transcriptional mediator of part of FA-induced ß-cell dysfunctions. The repressive role of Nur77 in insulin gene regulation was tracked down to protein-protein interaction with FoxO1, a pivotal integrator of the insulin gene regulatory network. The present study identifies a member of the NR4A nuclear receptor subclass, Nur77/NR4A1, as a modulator of pancreatic ß-cell biology. Together with its previously documented role in liver and muscle, its role in ß-cells establishes Nur77 as an important integrator of glucose metabolism.


Assuntos
Glucose/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Animais , Linhagem Celular , Cromogranina A/metabolismo , Ácidos Graxos/fisiologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/genética , Secreção de Insulina , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Pâncreas/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , Estresse Fisiológico
3.
FEBS Lett ; 584(13): 2845-51, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20447400

RESUMO

Farnesoid X receptor (FXR) is highly expressed in liver and intestine where it controls bile acid (BA), lipid and glucose homeostasis. Here we show that FXR is expressed and functional, as assessed by target gene expression analysis, in human islets and beta-cell lines. FXR is predominantly cytosolic-localized in the islets of lean mice, but nuclear in obese mice. Compared to FXR+/+ mice, FXR-/- mice display a normal architecture and beta-cell mass but the expression of certain islet-specific genes is altered. Moreover, glucose-stimulated insulin secretion (GSIS) is impaired in the islets of FXR-/- mice. Finally, FXR activation protects human islets from lipotoxicity and ameliorates their secretory index.


Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ácido Palmítico/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Western Blotting , Células Cultivadas , Humanos , Técnicas In Vitro , Isoxazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Obesidade/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética
4.
BMC Biotechnol ; 10: 28, 2010 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-20353585

RESUMO

BACKGROUND: In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. RESULTS: This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability). As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. CONCLUSIONS: Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.


Assuntos
Inativação Gênica , Células Secretoras de Insulina/metabolismo , RNA Interferente Pequeno/genética , Transfecção , Animais , Apoptose , Proteínas de Transporte de Cátions/genética , Sobrevivência Celular , Células Cultivadas , Eletroporação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportador 8 de Zinco
5.
Transplantation ; 89(9): 1154-60, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20098354

RESUMO

BACKGROUND.: The original Edmonton protocol used fresh islets, but for obvious logistic advantages most transplant centers have implemented pretransplant culture in human albumin. The aim of this study was to improve current pretransplant human islet culture techniques. METHODS.: Clinical-grade purified human islets from a total of 24 donors were directly resuspended after isolation in CMRL 1066-based media at 37 degrees C, and media additions and renewal were tested. At days 1 and 5 of culture, in vitro quality controls included islet viability, insulin content and function, apoptosis, and in vivo islet potency assay in nude mice. RESULTS.: Replacing human albumin with human AB serum improved 1- and 5-day preservation of islet function and viability which was further enhanced with antioxidant Stem Ease, leading to the iCulture medium (enriched CMRL: pyruvate, zinc sulfate, insulin, transferrin, selenium, 2.5% human AB serum and Stem Ease). Major damage occurs in the first day of culture and frequent media renewal (25% vol/hr) in this period further improved viability, apoptosis, islet recovery, and function in vitro and in vivo, compared with only changing medium after overnight culture. CONCLUSIONS.: The described human islet culture technique (iCulture medium+renewal) seems to be the best choice for clinical human islet culture when short (1 day) or long (5 days) periods are used. Media choice and dilution play a major role in the function and survival of human islets in culture.


Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Animais , Apoptose , Sangue , Contagem de Células , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Divisão Celular , Sobrevivência Celular , Meios de Cultura , Glucose/farmacologia , Humanos , Insulina/análise , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Nus , Albumina Sérica/fisiologia
6.
Biochem Biophys Res Commun ; 391(1): 305-9, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19913512

RESUMO

Neurogenin 3 is necessary for endocrine cell development in the embryonic pancreas and has been shown to induce transdifferentiation duct cells from adult pancreas toward a neuro-endocrine phenotype. Here we discovered that the demethylating agent 5'-Azadeoxycytidine (AZA) induced Ngn3 expression and endocrine differentiation from the PANC-1 human ductal cell line. The expression of markers specific to mature islet cells, i.e., glucagon and somatostatin, was also observed. In addition, we demonstrated that growth factors (betacellulin and soluble factors released during pancreas embryogenesis) increased the level of maturation. Our studies revealed that the PANC-1 model system may provide a basis for elucidating the ductal/endocrine differentiation.


Assuntos
Azacitidina/análogos & derivados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Transdiferenciação Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/biossíntese , Ductos Pancreáticos/efeitos dos fármacos , Azacitidina/farmacologia , Diferenciação Celular , Linhagem Celular , Decitabina , Humanos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Fatores de Transcrição/biossíntese
7.
Biochem Biophys Res Commun ; 390(4): 1288-93, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19878649

RESUMO

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic delta-cells, with no expression in alpha- and beta-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in beta-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9(+/+) and Pcsk9(-/-) mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Serina Endopeptidases/biossíntese , Células Secretoras de Somatostatina/enzimologia , Animais , Linhagem Celular , Colesterol/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/metabolismo , Serina Endopeptidases/genética , Células Secretoras de Somatostatina/efeitos dos fármacos
8.
Diabetes Care ; 32(8): 1473-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19638525

RESUMO

OBJECTIVE To investigate the influence of primary graft function (PGF) on graft survival and metabolic control after islet transplantation with the Edmonton protocol. RESEARCH DESIGN AND METHODS A total of 14 consecutive patients with brittle type 1 diabetes were enrolled in this phase 2 study and received median 12,479 islet equivalents per kilogram of body weight (interquartile range 11,072-15,755) in two or three sequential infusions within 67 days (44-95). PGF was estimated 1 month after the last infusion by the beta-score, a previously validated index (range 0-8) based on insulin or oral treatment requirements, plasma C-peptide, blood glucose, and A1C. Primary outcome was graft survival, defined as insulin independence with A1C < or =6.5%. RESULTS All patients gained insulin independence within 12 days (6-23) after the last infusion. PGF was optimal (beta-score > or =7) in nine patients and suboptimal (beta-score < or =6) in five. At last follow-up, 3.3 years (2.8-4.0) after islet transplantation, eight patients (57%) remained insulin independent with A1C < or =6.5%, including seven patients with optimal PGF (78%) and one with suboptimal PGF (20%) (P = 0.01, log-rank test). Graft survival was not significantly influenced by HLA mismatches or by preexisting islet autoantibodies. A1C, mean glucose, glucose variability (assessed with continuous glucose monitoring system), and glucose tolerance (using an oral glucose tolerance test) were markedly improved when compared with baseline values and were significantly lower in patients with optimal PGF than in those with suboptimal PGF. CONCLUSIONS Optimal PGF was associated with prolonged graft survival and better metabolic control after islet transplantation. This early outcome may represent a valuable end point in future clinical trials.


Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas/fisiologia , Adulto , Glicemia/metabolismo , Peptídeo C/sangue , Feminino , Seguimentos , Hemoglobinas Glicadas/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Imunossupressores/uso terapêutico , Insulina , Células Secretoras de Insulina/transplante , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Pessoa de Meia-Idade , Veia Porta/cirurgia , Tacrolimo/uso terapêutico , Resultado do Tratamento
9.
Diabetes ; 58(5): 1105-15, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19223597

RESUMO

OBJECTIVE: In type 2 diabetes, chronic hyperglycemia is detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The transcription factor cAMP-responsive element-binding protein (CREB) is crucial for beta-cell survival and function. We investigated whether prolonged exposure of beta-cells to high glucose affects the functional integrity of CREB. RESEARCH DESIGN AND METHODS: INS-1E cells and rat and human islets were used. Gene expression was analyzed by RT-PCR and Western blotting. Apoptosis was detected by cleaved caspase-3 emergence, DNA fragmentation, and electron microscopy. RESULTS: Chronic exposure of INS-1E cells and rat and human islets to high glucose resulted in decreased CREB protein expression, phosphorylation, and transcriptional activity associated with apoptosis and impaired beta-cell function. High-glucose treatment increased CREB polyubiquitination, while treatment of INS-1E cells with the proteasome inhibitor MG-132 prevented the decrease in CREB content. The emergence of apoptosis in INS-1E cells with decreased CREB protein expression knocked down by small interfering RNA suggested that loss of CREB protein content induced by high glucose contributes to beta-cell apoptosis. Loading INS-1E cells or human islets with a cell-permeable peptide mimicking the proteasomal targeting sequence of CREB blocked CREB degradation and protected INS-1E cells and human islets from apoptosis induced by high glucose. The insulin secretion in response to glucose and the insulin content were preserved in human islets exposed to high glucose and loaded with the peptide. CONCLUSIONS: These studies demonstrate that the CREB degradation by the ubiquitin-proteasome pathway contributes to beta-cell dysfunction and death upon glucotoxicity and provide new insight into the cellular mechanisms of glucotoxicity.


Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Glucose/toxicidade , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Morte Encefálica , Proteína de Ligação a CREB/efeitos dos fármacos , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modulador de Elemento de Resposta do AMP Cíclico/efeitos dos fármacos , Fragmentação do DNA , Diabetes Mellitus Experimental/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Transplantation ; 86(2): 360-3, 2008 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-18645503

RESUMO

Reliable assays are critically needed to monitor graft potency in islet transplantation (IT). We tested a quantitative in vivo islet potency assay (QIVIPA) based on human C-peptide (hCP) measurements in normoglycemic nude mice after IT under the kidney capsule. QIVIPA was initially tested by transplanting incremental doses of human islets. hCP levels in mice were correlated with the number of transplanted islet equivalents (r(2) = 0.6, P<0.01). We subsequently evaluated QIVIPA in eight islet preparations transplanted in type 1 diabetic patients. Conversely to standard criteria including islet mass, viability, purity, adenosine triphosphate content, or glucose stimulated insulin secretion, hCP in mice receiving 1% of the final islet product was correlated to primary graft function (hCP increase) after IT (r(2)=0.85, P<0.01). QIVIPA appears as a reliable test to monitor islet graft potency, applicable to validate new methods to produce primary islets or other human insulin secreting cells.


Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Animais , Peptídeo C/metabolismo , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/metabolismo , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Humanos , Insulina/metabolismo , Resistência à Insulina , Camundongos , Camundongos Nus , Radioimunoensaio , Fatores de Tempo
11.
Biochem Biophys Res Commun ; 366(4): 1096-101, 2008 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-18155663

RESUMO

We explored the in vitro effects of Rosiglitazone (RZG), a PPARgamma agonist, on human pancreatic islet dysfunctions induced by chronic free fatty acid exposure. We demonstrated that RZG beneficial effects on insulin secretion and apoptosis did not imply PDX-1 or insulin gene modulation. It rather involved, through a PPARgamma-dependent mechanism, a reduction of iNOS overexpressed in lipotoxic islets. This reduction likely led to the restoration of ATP level and insulin secretion as well as the decrease in apoptosis. More interestingly, we also demonstrated that RZG beneficial effects involved PPARgamma-independent mechanisms. RZG treatment led to a limitation of oxidative stress exemplified by an increase of GPx and SOD expression. It also increased UCP2 expression that seemed to display antioxidant action in this model. Thus, RZG did not appear to exert a direct action on insulin expression but rather an indirect action on insulin secretion and apoptosis, through PPARgamma-dependent and -independent mechanisms, via regulation of nitrogen and oxygen reactive species injury.


Assuntos
Ácidos Graxos não Esterificados/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , PPAR gama/metabolismo , Tiazolidinedionas/farmacologia , Adulto , Perfilação da Expressão Gênica , Humanos , Ilhotas Pancreáticas/metabolismo , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , Ácido Palmítico/farmacologia , Rosiglitazona
12.
Transplantation ; 83(5): 532-8, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17353769

RESUMO

BACKGROUND: The effects of sirolimus on insulin secretion are still debated. Our aim was to investigate the effects of sirolimus, both (1) in vivo in healthy minipigs; and (2) in vitro on human islets. METHODS: (1) Ten minipigs were evaluated during three successive stages: (a) basal; (b) at the end of a 4-week period of treatment with sirolimus; and (c) after a 4-week period of wash-out. We evaluated insulin secretion with the acute insulin response (AIR), and glucose tolerance with the glucose disposal rate (GDR). (2) Insulin content, stimulation index, adenosine triphosphate (ATP), and apoptosis were measured in human islets treated in vitro with sirolimus at therapeutic and supratherapeutic concentrations. RESULTS: (1) Basal and stimulated insulin levels and GDR increased during sirolimus administration and returned to baseline after a wash-out period; (2) regardless of culture duration, sirolimus dose-dependently decreased apoptosis and increased insulin content. Stimulation indexes and ATP were also significantly enhanced but only at therapeutic concentrations. CONCLUSIONS: This study suggests that sirolimus, at plasma-drug concentrations usually targeted in clinical practice, (1) increases basal and stimulated insulin levels in vivo and insulin content in vitro regardless of culture duration; (2) is able to reduce apoptosis. These findings may partly underlie the improved results of islet transplantation.


Assuntos
Glicemia/metabolismo , Teste de Tolerância a Glucose , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Sirolimo/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Glucose/fisiologia , Humanos , Insulina/sangue , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Modelos Animais , Suínos , Porco Miniatura
13.
Transplantation ; 83(3): 270-6, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17297400

RESUMO

BACKGROUND: The influence of the preservation solution used for in situ perfusion of the donor and pancreas storage on islet isolation has received little attention. METHODS: In this prospective controlled trial, we compared the outcome of human islet isolation from pancreata perfused with University of Wisconsin (UW) solution or Celsior, an alternative colloid-free extracellular solution. RESULTS: At the 1-year interim analysis, the viability and insulin secretion of islets isolated from donors perfused with UW (n=19) or Celsior (n=5) were identical. However, total islet recovery (IEQ) and isolation yield (IEQ/g) were 1.8-fold and 2.1-fold inferior in the Celsior group (P<0.05 vs. UW). Overall, 13 (68%) of islet preparations were effectively transplanted from the UW group vs. none from the Celsior group (P=0.01). The clinical study was discontinued and the causes of these differences were further explored in the pig (n=14). In contrast to UW, Celsior induced cell swelling and pancreas edema after only four hours of cold storage. These abnormalities were delayed when the donor was perfused with Solution de Conservation d'Organes et de Tissus (SCOT), an extracellular solution containing polyethylene glycol. CONCLUSIONS: Our results suggest that colloid-free preservation solutions might be suboptimal for pancreas perfusion and cold storage prior to islet isolation and transplantation. Because pancreata are now frequently recovered for islet transplantation, preliminary experimental and clinical data about islet isolation should be obtained prior to the routine implementation of new preservation solutions for abdominal perfusion during multiorgan recovery.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/efeitos dos fármacos , Doadores Vivos , Soluções para Preservação de Órgãos/química , Soluções para Preservação de Órgãos/farmacologia , Adenosina/farmacologia , Adolescente , Adulto , Alopurinol/farmacologia , Animais , Estudos de Casos e Controles , Coloides/análise , Dissacarídeos/farmacologia , Eletrólitos/farmacologia , Glutamatos/farmacologia , Glutationa/farmacologia , Histidina/farmacologia , Humanos , Insulina/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Manitol/farmacologia , Pessoa de Meia-Idade , Estudos Prospectivos , Rafinose/farmacologia , Resultado do Tratamento
14.
Endocrinology ; 148(4): 1605-14, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17204559

RESUMO

Prolonged exposure of beta-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E beta-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca2+]i), which was KATP-channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca2+]i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To confirm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis.


Assuntos
Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Glucose/toxicidade , Insulina/metabolismo , Animais , Compartimento Celular , Células Cultivadas , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ratos , Distribuição Tecidual , Proteínas de Transporte Vesicular/metabolismo
15.
J Cell Sci ; 119(Pt 20): 4199-206, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16984975

RESUMO

Insulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Glucagon/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Zinco/metabolismo , Zinco/farmacologia , Transportador 8 de Zinco
16.
Diabetes ; 55(6): 1605-13, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16731822

RESUMO

Peroxisome proliferator-activated receptor (PPAR) alpha is a transcription factor controlling lipid and glucose homeostasis. PPARalpha-deficient (-/-) mice are protected from high-fat diet-induced insulin resistance. However, the impact of PPARalpha in the pathophysiological setting of obesity-related insulin resistance is unknown. Therefore, PPARalpha(-/-) mice in an obese (ob/ob) background were generated. PPARalpha deficiency did not influence the growth curves of the obese mice but surprisingly resulted in a severe, age-dependent hyperglycemia. PPARalpha deficiency did not aggravate peripheral insulin resistance. By contrast, PPARalpha(-/-) ob/ob mice developed pancreatic beta-cell dysfunction characterized by reduced mean islet area and decreased insulin secretion in response to glucose in vitro and in vivo. In primary human pancreatic islets, PPARalpha agonist treatment prevented fatty acid-induced impairment of glucose-stimulated insulin secretion, apoptosis, and triglyceride accumulation. These results indicate that PPARalpha improves the adaptative response of the pancreatic beta-cell to pathological conditions. PPARalpha could thus represent a promising target in the prevention of type 2 diabetes.


Assuntos
Resistência à Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , PPAR alfa/fisiologia , Pâncreas/metabolismo , Adulto , Fatores Etários , Animais , Apoptose/efeitos dos fármacos , Peso Corporal , Relação Dose-Resposta a Droga , Expressão Gênica , Glucose/farmacologia , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Técnicas In Vitro , Insulina/metabolismo , Resistência à Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Pessoa de Meia-Idade , PPAR alfa/genética , Palmitatos/farmacologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Triglicerídeos/metabolismo
17.
Biochem Biophys Res Commun ; 333(4): 1153-9, 2005 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-15979049

RESUMO

In vivo lineage tracing experiments in mice have recently cast doubt on the potential islet neogenesis from ductal precursors in adult mammals. We examined, in human obesity, a model for pancreatic endocrine tissue plasticity, the gene and protein expression of PBX-1-a transcription factor expressed in regenerating rat ductules and potentially implicated in the pancreatic development, alone or in association with PDX-1. When comparing gene expression, by quantitative real-time RT-PCR, in pancreatic exocrine tissue from obese non-diabetic subjects with increased islet mass, we found that Pbx-1 and Pdx-1 were up-regulated (5.9+/-1.2 and 2.4+/-0.6 versus non-obese). Immunohistochemistry confirmed PBX-1 over-expression and its cytoplasmic sequestration in ductal cells of obese subjects, associated with pronounced islet neogenesis (cytokeratin 19/chromogranin A double labeling). cDNA microarray analysis also showed up-regulation of other genes implicated in islet regeneration, including betacellulin, laminin, TGFa, NeuroD1, Pax6, substantiating the role of the islet neogenesis pathway in human obesity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Regeneração , Transativadores/metabolismo , Adaptação Fisiológica , Cadáver , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/patologia , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Fator de Transcrição 1 de Leucemia de Células Pré-B , Distribuição Tecidual
18.
Gene Expr ; 12(2): 83-98, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15892450

RESUMO

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.


Assuntos
Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Células-Tronco/metabolismo , Adulto , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Imunoensaio , Ilhotas Pancreáticas/citologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Biochem Biophys Res Commun ; 320(1): 27-33, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15207697

RESUMO

Generating human insulin-secreting cells for cell therapy of diabetes represents a highly competitive world challenge. Human ductal cells can give rise to islets in vivo and in vitro. The goal of this study was to devise a rapid sorting method to highly purify human ductal cells from pancreatic tissue using a pan-ductal membrane antibody carbohydrate antigen 19-9 (CA19-9). Human pancreatic sections confirmed antibody specificity. The human exocrine fraction (30% ductal cells) was sorted with magnetic bead technology or by FACS. Immunocytochemistry post-sorting determined ductal cell content. The manual magnetic bead technique resulted in 74%+/-2 (n = 4) CA19 positive cells. Whereas the automated AutoMACS technique (n = 5) yielded 92.6%+/-0.5 CA19-9 positive cells with only a minor beta cell contamination (0.2%+/-0.03); cell yield post-sorting was 12.9%+/-2.5 (1.69+/-0.41 x 10(6) cells) with 51.7%+6.5 (n = 5) viability post-sorting. The FACS (n = 6) resulted in 97.1%+/-0.82 CA19-9 positive cells, a cell yield of 25.5%+/-5.6 (5.03+/-1.0 x 10(6)), with 72.1%+/-6.1 viability post-sorting.


Assuntos
Antígeno CA-19-9/imunologia , Separação Imunomagnética/métodos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Pâncreas/citologia , Pâncreas/fisiologia , Células Cultivadas , Humanos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/fisiologia
20.
Cell Transplant ; 12(7): 799-807, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14653626

RESUMO

The need for transplantable beta cells with a stable phenotype has given rise to several strategies including the expansion of existing pancreatic islets and/or growth of new ones. In vitro studies of beta cell proliferation on extracellular matrices plus growth factors have highlighted a possible cell expansion technique; however, the technique was accompanied with loss of insulin secretion. Herein we showed that human islet cell proliferation was marked by a decreased expression of specific differentiation markers, particularly insulin, insulin promoting factor-1 (IPF-1), and glucokinase. After a 6-day expansion period, we tried to reexpress the beta cell differentiation markers with compounds known for their differentiation and/or insulin-secreting properties. Sodium butyrate was a potent factor of IPF-1, insulin, and glucokinase gene reexpression; it also clearly induced secretion of gastrin, a known neogenic factor. Other compounds, namely TGF-beta, calcitriol, GLP-1, and activin A, efficiently enhanced the glucose sensor machinery, particularly Glut-1 and glucokinase, thus triggering glucose responsiveness. Our results indicate that specific beta cell gene expression may be induced after expansion and dedifferentiation. This rekindles interest in human beta cell expansion. The possible stabilization of specialized genes needed by beta cells to fulfill their role as nutrient sensors and metabolic regulators may also be of interest to ensure graft maintenance and efficiency.


Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Diferenciação Celular , Divisão Celular , Células Cultivadas , Gastrinas/análise , Regulação da Expressão Gênica , Glucose/fisiologia , Humanos , Imunoensaio , Insulina/análise , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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