Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

Este trabalho foi apoiado por uma concessão do projeto piloto do centro CREH patrocinado pela NIEHS e pela Fundação Nacional de ciências naturais da China (n. º 31572626).

Todos os experimentos com animais foram executados em conformidade com todas as orientações pertinentes, regulamentos e agências reguladoras. O protocolo que está sendo demonstrado foi realizado sob a orientação e aprovação do cuidado institucional do Animal e Comissão de utilização (IACUC) do Texas A & M Instituto de Medicina Genômica.
1. preparação da solução
<ol><li>Diluir a solução de sal equilibrada do Hank x 10 x 1 com água destilada dobro H2O e armazenar a 4 ° C.</li>
	<li>Prepare a solução de isolamento adicionando HEPES (10 mM), MgCl2 (1 mM) e glicose (5 mM). Ajustar o pH para 7,4 e manter no gelo.</li>
	<li>Prepare a solução de colagenase da colagenase tipo 4. Nota: Especificamente, colagenase tipo 4 contém 160 U por mg de peso seco. 1 g de colagenase tipo 4 é diluído em 5 mL duplo destilada H2O com uma concentração de 200 mg/mL. 2 mL da colagenase diluído é adicionado para 30 mL de solução de isolamento frio gelo e 6 mL de solução de colagenase é transferido para um tubo de 50 mL para cada rato.</li>
	<li>Prepare a solução de lavagem, adicionando 1 mM CaCl2 para a solução de isolamento.</li>
	<li>Prepare a solução de purificação com solução de diatrizoate de sódio polysucrose frio (1,1119 g/mL, 5 mL).</li>
	<li>Prepare 500 mL de meio de cultura de ilhota pela adição de L-glutamina (20 mM), penicilina (100 U/mL), estreptomicina (100 µ g/mL) e soro fetal bovino (FBS) (10%) em meio RPMI.</li>
	<li>Preparar a solução Diniz Melo-DA-mãe, dissolvendo o pó em água destilada a 200 µM e loja a-20 ° C. Nota: O ROS coloração solução recém feito diluindo a solução para a concentração final de 5 µM em RPMI Diniz Melo-DA. Este reagente deve ser protegido da luz.</li>
</ol>2. cirúrgica preparação
<ol><li>Intraperitonealmente injete (I.P.) um rato com avertin (2,5% avertin, 0,4 mL/20 g). Depois que o mouse é completamente anestesiado e não responde mais os pés hind pitadas, eutanásia o mouse e mover para o capô biológico.</li>
	<li>Coloque o mouse euthanized com a abdominal virados para cima e pulverizar a pele com álcool 70% para a esterilização.</li>
	<li>Abrir a cavidade abdominal com um U-incisão de 1 cm em cima do abdômen superior e retrair a pele no sentido rostral sobre o peito para expor na cavidade abdominal.</li>
</ol>3. remoção e perfusão pâncreas
<ol><li>Conforme ilustrado na Figura 1, encontre a localização na ampola. Use um par de pinças curvas para fixar o duodeno perto a posição do esfíncter de Oddi. Use outro par de pinças curvas para fixar a parede do duodeno para bloquear o caminho de bile no duodeno. Nota: A ampola não foi inserida nesta etapa. Esta etapa destina-se a bloquear o caminho de bile no duodeno.</li>
	<li>Reposicione o mouse com a cabeça proximal e distal em relação ao pesquisador os pés.</li>
	<li>Encontrar o ducto biliar comum e lentamente injete 3 mL de solução de colagenase com uma seringa de 5 mL e agulha de 30g. Certifique-se que o ducto biliar e o pâncreas estão inflacionados após a injeção.</li>
	<li>Remova o pâncreas distendido, usando a pinça curvada e tesouras bem para separá-lo do descendente do cólon, intestinos, estômago e baço. Coloque o pâncreas em um tubo de 50 mL contendo 3 mL de solução de colagenase e mantê-lo no gelo.</li>
</ol>4. pâncreas digestão
<ol><li>Coloque cada tubo de 50 mL em um banho-37 ° C. Incubar durante 15-20 minutos (tempo varia com a tensão e a idade dos ratos).</li>
	<li>Vibre o tubo usando um vórtice.</li>
	<li>4-5 pâncreas digerido em um coador grande coletor estéril da piscina e use uma colher para fazer com que o pâncreas desmoronar completamente. Use uma seringa de 10 mL e agulha 23G contendo solução de lavagem para pipetar com força as células digeridas fora da tela. Recolha a solução de lavagem em um tubo de 50 mL.</li>
	<li>Centrifugar os tubos a 97 x g a 4 ° C por 1 min e decante o sobrenadante.</li>
	<li>Adicionar 25 mL de solução de lavagem para tubos e ressuspender o tecido vortexing suave. Spin para baixo os tecidos a 97 x g a 4 ° C por 1 min e decante o sobrenadante. Repita por 3 vezes. Tenha cuidado para não deslocar o tecido granulado.</li>
</ol>5. purificação de Ilhéus
<ol><li>Adicionar 10 mL de solução de purificação para a pelota de tecido lavado e ressuspender delicadamente o conteúdo.</li>
	<li>Delicadamente sobreposição de 5 mL de solução de isolamento sobre a solução de purificação. Tenha cuidado para manter uma interface líquida acentuada entre as duas soluções.</li>
	<li>Centrifugar o tubo a 560 x g a 4 ° C por 15 min com aceleração lenta e sem freio. Introduza a pipeta a interface e recolher a camada de ilhotas para novos tubos de 50 mL.</li>
	<li>Lave os ilhéus, conforme descrito na etapa 3.5.</li>
</ol>6. chapeamento e tratamento de células das ilhotas
<ol><li>Resuspenda as ilhotas em 10 mL de solução de cultura de Ilhéus, delicadamente misture bem e coloque 100 µ l/poço com uma pipeta multicanal para uma placa de 96 poços. Coloque aproximadamente 5 ilhotas por 96 bem.</li>
	<li>Coloque a placa na incubadora de cultura de tecido para 12h.</li>
	<li>Centrifugar (112 x g) a placa de 96 poços e remover o meio de cultura.</li>
	<li>Dilua os químicos ambientais em meio de cultura de Ilhéus. Adicione 100 µ l de cada um dos diluentes a química em cavidades separadas da placa. Resuspenda as ilhotas em cada poço e incubar durante 12 horas a 37 ° C (a concentração varia para os diferentes produtos químicos ambientais).</li>
</ol>7. ROS manchando e medição
<ol><li>Após o tratamento de Ilhéus, lave com 1X PBS (200 µ l de cada poço-96). Nota: A concentração das substâncias químicas é como segue: AFB1: 3 µM, atrazina: 100 µM, BaP: 10 µM, BPA: 100 µM, β-estrogênio, PCB126: 10 µM, nonilfenol e BPA: 100 µM.</li>
	<li>Use o N-acetil cisteína (NAC) como um antioxidante para saciar ROS. Adicionar 5 mM do NAC para o diluente químico induzida por ROS e incubar durante 12 horas a 37 ° C.</li>
	<li>Centrifugue a placa de 96 a 112 x g e com solução de 5 µM de sonda fluorescente (H2-Diniz Melo-DA dissolvido na mídia RPMI), incubar a 37 ° C por 60 min.</li>
	<li>Lavar as células da ilhota tratada 3 vezes com PBS e medir a fluorescência com um leitor de microplacas em 488 nm. Nota: O acúmulo de DCF nas células pode ser medido por um aumento na fluorescência 530 nm quando a amostra está animada em 488 nm.</li>
	<li>Executar a secreção (GSIS) de insulina glicose-estimulada ensaio9 na indução ROS xenobiótico tratados ilhotas e Ilhéus não tratadas para medir os efeitos de xenobióticos sobre a função fisiológica das ilhotas pancreáticas.</li>
</ol>

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Os autores declaram que eles têm não tem interesses financeiro concorrente.

Acumular evidências sugere que a exposição a substâncias químicas ambientais desempenha um papel importante no desenvolvimento do T2DM. Induzida por xenobióticos ROS tem sido reconhecida como um fator etiológico potencial, contribuindo para o desenvolvimento do T2DM. Os seres humanos são expostos a uma ampla gama de produtos químicos xenobióticos e há uma grande necessidade de técnicas de pesquisa de romance para efetivamente identificar os tóxicos do pâncreas e para investigar o mecanismo de toxicidade esp...

Aumento na prevalência de T2DM tornaram-se uma crise de saúde global nos últimos anos posando uma séria ameaça à saúde pública1. Muitos fatores foram encontrados para ser causalmente ligada ao desenvolvimento do T2DM, entre os quais, recorrentes achados sugerem que um ponto convergente comuns a esses fatores é a indução de estresse oxidativo, que leva à geração de excessiva ROS2 , 3.
Um amplo espectro de produtos químicos ambientais incluindo PCBs, dioxinas e BaP foi encontrado para induzir o estresse oxidativo, que pode prejudicar a função das células β do pâncreas e levar à resistência à insulina e T2DM4. Embora o nível fisiológico de ROS desempenha um papel importante nas funções celulares, exposição a ROS que excede a capacidade do sistema antioxidante resulta em danos de tecidos/células e leva a doenças5. Células β do pâncreas expressam um baixo nível de enzima antioxidante e assim são um alvo sensível para o dano mediada por estresse oxidativo6,7. A exposição crônica a níveis elevados de ROS foi mostrada para causar disfunção de células pancreáticas induzida por estresse5 bem como resistência à insulina no fígado e tecido adiposo8.
O objetivo geral deste projeto é desenvolver um ensaio baseada em célula de substâncias químicas de tela para seus potenciais de diabetogenic com base na sua indução de ROS em células pancreáticas. O pâncreas carece de desintoxicação metabólica e é um alvo sensível para dano induzido por xenobiótico6,7. Portanto, medindo diretamente o ROS gerado nas células do pâncreas, este ensaio deve fornecer uma aproximação direta da lesão induzida por produto químico no pâncreas. Para desenvolver este método, nós isolado de ilhotas pancreáticas do mouse, culta o ilhéu isolado sob condição de cultura celular com produtos químicos e utilizou a geração de ROS induzida por substância química como a leitura. Este procedimento é simples e eficaz na identificação de produtos químicos ROS-induzindo o ilhéu isolado; Isso pode ser desenvolvido para a investigação dos mecanismos de toxicidade que são específicos para o pâncreas em vitro.

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposição a certas substâncias químicas ambientais em humanos e animais foi encontrada para causar dano celular das células β do pâncreas que conduzirá ao desenvolvimento de diabetes mellitus tipo 2 (T2DM). Embora os mecanismos para o danos nas células β induzida em química foram clara e susceptível de ser complexo, uma constatação recorrente é que estes produtos químicos induzem o estresse oxidativo levando a geração de espécies de excessiva reativas de oxigênio (ROS) que induzem danos a célula β. Para identificar potenciais produtos químicos ambientais diabetogenic, isolamos as células de ilhotas pancreáticas de camundongos C57BL/6 e de células da ilhota cultivadas em placas de cultura de células 96 poços; em seguida, as células das ilhotas foram tratadas com produtos químicos e a geração de ROS foi detectada por 2', tintura fluorescente 7'-diclorofluoresceína (Diniz Melo-DA). Usando esse método, nós encontrou que o bisfenol A (BPA), Benzo [a] pireno (BaP) e os bifenilos policlorados (PCBs), poderia induzir altos níveis de ROS, sugerindo que eles podem potencialmente induzir danos nas células das ilhotas. Este método deve ser útil para a seleção diabetogenic xenobióticos. Além disso, as células das ilhotas culta também podem ser adaptadas para análise em vitro de toxicidade induzida por substância química nas células do pâncreas.

Uma micrografia de ilhéu isolado do saudável é mostrada na Figura 2, em que ilhéus tem uma forma redonda ou oval com relativamente uniforme tamanho (apesar de uniformidade de tamanho pode variar de cepa para cepa). Em seguida, investigamos as funções de ilhotas pancreáticas em um ensaio em vitro isolando o ilhéu e estimulando a secreção de insulina em ilhotas a cultura. A Figura 3 mostra a nossa análise típic...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Aqui nós apresentamos um protocolo para isolar células de ilhotas pancreáticas do mouse para as induções ROS de triagem pelos xenobióticos para identificar os potenciais químicos xenobióticos de diabetogenic.

Um rastreio de baseada em célula de murino ilhotas pancreáticas para produtos químicos ambientais Diabetogenic

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

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Research

JoVE Journal

Immunology and Infection

7.2K visualizações.  Hunan Agriculture University. Aqui nós apresentamos um protocolo para isolar células de ilhotas pancreáticas do mouse para as induções ROS de triagem pelos xenobióticos para identificar os potenciais químicos xenobióticos de diabetogenic.

Vídeo: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Isolamento Islet murino de pâncreas

Biologia

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análises de célula única Transcriptomic de células endócrinas no pâncreas de rato

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Biologia do Desenvolvimento

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

Análise do consumo de oxigênio em ilhotas pancreáticas de primatas não humanos

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Medicina

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A-alta conteúdo<em&gt; In Vitro</em&gt; Replicação plataforma de descoberta de células-β das ilhotas pancreáticas

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Um modelo em Ovo para testar compostos insulino-mimética

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Neurociência

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Análise quantitativa da mitofagia em células β pancreáticas murinas

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Autor em destaque: Avaliação do fluxo de mitofagia em células β pancreáticas usando abordagens complementares eficazes e robustas 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Abordagens Complementares para Interrogar o Fluxo de Mitofagia em Células β Pancreáticas

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Um rastreio de baseada em célula de murino ilhotas pancreáticas para produtos químicos ambientais Diabetogenic

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