We would like to acknowledge Susan Gray and Umadevi Chalasani for their help with technical aspects of the hepatocyte isolation protocol. This work was supported by NIDDK grant 5R01DK089185 (to M.P. Cooper) and the DERC Pilot and Feasibility Program at UMMS (to M.P. Cooper).

Todos experimentação animal deve ser realizado em conformidade com os regulamentos locais e federais e com a aprovação de uma IACUC e segurança de radiação administração institucional. 1. Preparação <ol><li> Vários dias antes do ensaio, descongelar a garrafa de 500 ml de fígado Digest Médio (LDM) e recongelar ~ 35 ml de partes alíquotas em tubos cónicos de 50 ml. Armazenar a -20 ° C até serem necessárias. </li><li> Um dia antes do ensaio, pré-limpas esterilizar instrumentos de dissecação por autoclave. </li><li> No dia do ensaio, tratar a quantidade necessária de placas de cultura de 24 poços com colagénio. <ol><li> Misturar 1 parte de 3 mg / mL de colagénio de cauda de rato com 50 partes de PBS. Adicionar cerca de 500 uL a cada poço de uma placa de 24 poços e incuba-se durante 3-5 minutos à temperatura ambiente (TA). Remova a placa de colágeno e permitir ao ar seco na capa. Repetir pelo menos uma vez adicional. Nota: revestimento de colágeno pode ser realizada até uma semana em anúncioVance e as placas armazenadas a 4 ° C em filme plástico selado. </li></ol></li><li> Prepare LDM para ensaio: LDM Descongelar e aquecer a 37 ° C e ajustar o pH a 7,4 utilizando KOH 1N. Filtrar usando filtro de seringa de 0,2 um e coloque em um 42 ° C banho de água de recirculação. </li><li> Aqueça 25 ml de fígado Perfusão Médio a 42 ° C em banho de recirculação de água. </li><li> Prepare a 90% de sílica coloidal revestido com uma solução de polivinilpirrolidona: Adicione 1 ml de DPBS 10x a 9 ml de sílica coloidal revestida com polivinilpirrolidona. Ajustar o pH para 7,4 utilizando HCl 0,1 N. Filtrar com um filtro de seringa estéril de 0,2 um. Armazenar à temperatura ambiente. </li><li> Quente meio de plaqueamento de 37 ° C. </li></ol> 2. Isolamento de principal do mouse hepatócitos <ol><li> Configure bomba peristáltica, tubulação, e mesa de dissecção: Esterilizar tubulação por lavagem com etanol a 5 ml de 70% em água destilada, seguido por 10 ml de água estéril. Coloque tubulação no fígado Perfusão Médio e executar bomba para enchertodo o comprimento do tubo. </li><li> Sacrifique rato pelo método aprovado institucionalmente. </li><li> Dissecar aberto a cavidade abdominal: Pulverizar o abdômen do rato generosamente com etanol 70%. Com uma tesoura de extremidade romba, fazer uma incisão na linha média através da derme do comprimento do abdómen e reflectir lateralmente. Adicione uma incisão semelhante no peritoneu para expor as vísceras. <ol><li> Utilizando um instrumento rombo, deslocar os intestinos suavemente para expor a vasculatura abdominal Localizar a veia cava inferior abdominal (IVC) e colocar uma sutura por baixo do vaso sanguíneo, distai em relação à veia renal </li></ol></li><li> Distalmente à sutura, colocar uma agulha e cateter na veia cava inferior, avançando para além do nível da sutura. Com a agulha ainda no lugar, amarrar a sutura ao redor do cateter para segurá-la no lugar. Remova cuidadosamente a agulha. Se isso for feito correctamente, o sangue com fluxo através do cateter. </li><li> Usando uma pipeta, encher o restante da área no meio de perfusão com cateter, assegurandoque nem o ar está presente. Com muito cuidado, coloque a tubulação para o cateter. </li><li> Dissecar aberto da cavidade pulmonar: refletir suavemente os lóbulos superiores do fígado para expor o diafragma. Perfurar cuidadosamente o diafragma com uma tesoura ponta afiada, em seguida, fazer uma incisão lateral para expor a cavidade pleural, tendo o cuidado de evitar a vesícula biliar e vasculatura pleural. Coloque um grampo de bulldog em torno da veia cava inferior torácica apenas proximal à veia hepática. </li><li> Corte a veia porta e ligar a bomba a 3 - 4 ml / min. Perfundir com o fígado Fígado Perfusão médio durante 5 min, utilizando aproximadamente 20 ml de meio de perfusão. Nota: O fígado deve mudar imediatamente de vermelho para cinza / tan. Se porções do fígado permanecer vermelho, isso provavelmente indica má perfusão, ea probabilidade de isolamento bem sucedido é muito reduzido. Durante a perfusão, tenha cuidado para garantir o meio não correr para fora e que não há bolhas de entrar na tubulação. </li><li> Após a 5 min de incubação, parara bomba e tubo para transferir o fígado Digest Médio. Reiniciar a bomba e perfundir o fígado por mais 10 - 15 minutos (até que o meio é esgotada). </li><li> No final da perfusão, parar a bomba. O fígado deve ter um tom rosado e aparecer um pouco alargada. Extirpar o fígado por dissecção cuidadosa. Remover a vesícula biliar e transferir o fígado a 10 cm prato de cultura de tecidos. </li><li> Em uma cabine de segurança biológica, adicionar 10 ml de chapeamento Médio (Tabela 1) para o fígado. Raspe o fígado utilizando quer uma pinça ou um escalpelo para remover os hepatócitos. Filtra-se a suspensão através de um filtro de células de 100 mm e transferência para um tubo de 50 ml. Lave a placa com um adicional de 10 ml de Galvanização Médio e piscina no tubo de 50 ml. </li><li> Agregar as células por centrifugação durante 5 min a 350 xg, 4 ° C. </li><li> Aspirar médio e pellet ressuspender em 10 ml de Galvanização Médio e adicionar 10 ml de 90% de sílica coloidal revestido com polyvinylpyrrolidone. Misture delicadamente e centrifugar como na etapa 2.11. Após centrifugação, uma camada de células mortas vai ser flutuante na parte superior da mistura, enquanto que as células vivas irá sedimentar para o fundo. </li><li> Aspirar as células mortas e meio. Lavar 2 vezes com 20 ml de chapeamento Médio, centrifugar como em 2.11. </li><li> Ressuspender as células em 10 ml de chapeamento meio. Contar as células com um hemocitómetro e colocar 9 x 10 4 células / poço em pratos de 24 poços de cultura tratados com colagénio. Incubar em 37 ° C de cultura de tecidos incubadora durante 2 horas. Cada placa pode ser usado tanto para o ensaio de oxidação de ácido gordo ou o ensaio Lipogênese. </li><li> Opcionalmente, para alterar poços Meio de Manutenção (Tabela 1) e a cultura a 37 ° C. As células podem ser cultivadas durante 2 - 3 dias, sem afectar os resultados do ensaio. </li></ol> 3. Ácidos Graxos Oxidação Assay Aviso: O uso de radioactividade pode ser perigoso. Todas as compras, armazenamento, manipulação, e disposal de material radioativo deve ser realizado em conformidade com, estado, e regulamentos e diretrizes federais institucionais. <ol><li> 16 - 20 h antes do ensaio, células de lavar 2 vezes com PBS quente. Mudar as células ao soro inanição meio isento de soro (Tabela 1) com 20 nM de glucagon e incubar durante a noite a 37 ° C. Nota: Por causa de soro fetal bovino contém uma concentração desconhecida de hormônios metabólicos (por exemplo, insulina, glucagon), soro de morrer de fome as células para remover quaisquer efeitos de confusão que isso pode ter sobre o ensaio. As células são viáveis ​​em soro fome Médio para&gt; 24 hr. Tratamento glucagon é utilizado para estimular a oxidação do ácido gordo no interior dos hepatócitos. </li><li> Na manhã do dia do ensaio, preparar a pré-incubação Médio: Ressuspender uma quantidade apropriada de palmitato de sódio em água ultrapura para fazer uma solução 100 mM e aquecimento a 70 ° C durante 10 min. Entretanto, a quantidade necessária preparar (0,5 ml por poço de uma placa de 24 poços)de DMEM com 25 mM de HEPES, 1% de BSA Fracção V, e 20 nM de glucagon. Aquece-se a 37 ° C. <ol><li> Uma vez solubilizado, Palmitato de adicionar a uma concentração final de 250 uM para a forma. Mudar hepatócitos para a pré-incubação médio e incubar a 37 ° C durante 2 horas. Reservar alguns A pré-incubação do meio no 37 ° C, para utilização posterior. </li></ol></li><li> Durante a incubação, seco, uma quantidade adequada de 14 C-palmitato (0,5 uCi / poço) por evaporação sob atmosfera de azoto. <ol><li> Por exemplo, para medir 24 amostras numa placa de 24 poços, 120 ul de transferir 0,1 uCi / ml de C-14 Palmitato de um tubo de 1,5 ml. Lentamente, evapora-se o solvente etanol por sopragem de azoto gasoso através da solução a partir de uma distância de 3-5 cm numa hotte. O solvente deverá evaporar-se em cerca de 30 - 40 min, deixando a seco C-14 Palmitato no fundo do tubo. </li></ol></li><li> Aproximadamente 15 minutos antes do final da incubação, ressuspender o 14C-Palmitato em NaOH 0,1 N (12,5 mL / uCi). Incubar a 70 ° C durante 10 min. Adicione três volumes de quente Pré-Incubação Médio e misture pipetando cima e para baixo. </li><li> Pico de cada poço com 25 uL de 14 C diluída-palmitato. Misturar agitando suavemente a placa e incubar a 37 ° C durante 90 min. Esta é a placa de ensaio. </li><li> Durante a incubação, preparar a Placa de filtro de papel (Figura 1). <ol><li> Remova a tampa de uma estéril placa de 24 poços. Coloque um 2 cm x 2 cm pedaço de papel de filtro na parte inferior de cada um dos poços. Sobrepor a placa com um pedaço de 4 &quot;x 7&quot; parafilme. </li><li> Usando um grande objecto rectangular, tal como uma caixa de ponta de micropipeta, esfregue o parafilme nos poços para perfurar o parafilme as aberturas de poços e criar uma vedação ao longo do resto da placa. Remova os círculos perfuradas de parafilm agora cobre as cavidades. A placa deve agora ser firmemente coberto com filme plástico em todas as áreas, exceto o bem openings. </li></ol></li><li> 10 minutos antes do final da incubação, adicionar 200 uL de NaOH 3 N a cada poço da placa de papel de filtro, certificando-se o papel de filtro absorve o líquido todo. <ol><li> Opcionalmente antes da congelação, a transferência do médio para uma nova placa de 24 poços. Após lavagem uma vez com PBS, lisar as células remanescentes em HCl 0,1 N e calcular o teor de proteína por ensaio BCA. </li></ol></li><li> No final da incubação, snap-congelar a placa de ensaio em azoto líquido. Tenha cuidado para garantir que cada poço está completamente congelado antes de prosseguir. </li><li> Adicionar 100 ul de ácido perclórico a 70% a cada poço da placa de ensaio. Cubra imediatamente com a Placa de filtro de papel. Coloque as placas num agitador orbital e rock na velocidade orbital de 80 rpm à temperatura ambiente durante 2 horas. </li><li> Após a incubação, processar as amostras: <ol><li> Para medir a fracção de CO 2, transferir os quadrados de papel de filtro para 4 ml de fluido de cintilação líquida num cintilaçãofrasco e medida 14 do sinal C. </li><li> Para medir o material solúvel em ácido, transferir 400 ul de meio para um tubo de microcentrífuga de 1,5 ml. Centrifugar a velocidade máxima para 10 min. Adiciona-se 100 ul do sobrenadante resultante a 500 mL de 2: 1 de clorofórmio-metanol (v / v), e agitar com vortex brevemente. <ol><li> Adicionar 250 mL de água à mistura, e novamente vórtice. Centrifugar as amostras durante 10 min a 3.000 x g. Transferir 200 ul da fase superior a 4 ml de fluido de cintilação líquida num frasco de cintilação de 14 e medir o sinal C. </li></ol></li></ol></li></ol> 4. Lipogênese Assay <ol><li> A noite antes de iniciar o ensaio, lavar as células 2 vezes com PBS quente. Alterar os hepatócitos ao soro fome Médio com 100 nM de insulina. Incubar durante a noite a 37 ° C. Nota: Por causa de soro fetal bovino contém uma concentração desconhecida de hormônios metabólicos (por exemplo, insulina, glucagon), Soro de morrer de fome as células para remover quaisquer efeitos de confusão que isso pode ter sobre o ensaio. As células são viáveis ​​em soro fome Médio para&gt; 24 hr. A insulina é usada neste ensaio para estimular a lipogénese. </li><li> Faça Lipogênese Médio: Soro Fome médio com 100 nM de insulina, 10 ï¿½ de etilo frio e 0,5 uCi de 3 H-Acetato por poço. Mudar para células Lipogênese Médio e incubar a 37 ° C durante 2 horas. Inclui quaisquer compostos de interesse a ser testado. </li><li> Após o período de incubação, lavar as células 2 vezes com PBS. Lisar as células por raspagem em 120 ul de 0,1 N de HCl. Reserva de 10 uL para o ensaio de proteína (por avaliar ensaio BCA), e transferência de 100 uL para tubos de microcentrífuga de 1,5 ml. </li><li> Extrair lípidos pela adição de 500 ul de 2: 1 de clorofórmio-metanol (v / v). Vortex brevemente e incubar à TA durante 5 min. Adicionar 250 uL de água, vortex e incubar à temperatura ambiente durante um adicional de 5 min. Centrifugar as amostras 10 min a 3000 xg, RT. Cuidadosamentetransferência de fase inferior a 4 ml de fluido de cintilação líquida num frasco de cintilação e medir a actividade de 3 h. </li></ol>

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	<li>Clark, J. M., Brancati, F. L., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+and+etiology+of+elevated+aminotransferase+levels+in+the+United+States.">The prevalence and etiology of elevated aminotransferase levels in the United States.</a> The American journal of gastroenterology. 98, 960-967 (2003).</li><li>Lazo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+nonalcoholic+Fatty+liver+disease+in+the+United+States:+the+third+national+health+and+nutrition+examination+survey,+1988-1994.">Prevalence of nonalcoholic Fatty liver disease in the United States: the third national health and nutrition examination survey, 1988-1994.</a> American journal of epidemiology. 178, 38-45 (2013).</li><li>Angulo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonalcoholic+fatty+liver+disease.">Nonalcoholic fatty liver disease.</a> The New England journal of medicine. 346, 1221-1231 (2002).</li><li>Adams, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+nonalcoholic+fatty+liver+disease:+a+population-based+cohort+study.">The natural history of nonalcoholic fatty liver disease: a population-based cohort study.</a> Gastroenterology. 129, 113-121 (2005).</li><li>Feldstein, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+apoptosis+and+fas+expression+are+prominent+features+of+human+nonalcoholic+steatohepatitis.">Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis.</a> Gastroenterology. 125, 437-443 (2003).</li><li>Day, C. P., James, O. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steatohepatitis:+a+tale+of+two+'hits'.">Steatohepatitis: a tale of two 'hits'.</a> Gastroenterology. 114, 842-845 (1998).</li><li>Donnelly, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+fatty+acids+stored+in+liver+and+secreted+via+lipoproteins+in+patients+with+nonalcoholic+fatty+liver+disease.">Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.</a> The Journal of clinical investigation. 115, 1343-1351 (2005).</li><li>Lambert, J. E., Ramos-Roman, M. A., Browning, J. D., Parks, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+de+novo+lipogenesis+is+a+distinct+characteristic+of+individuals+with+nonalcoholic+fatty+liver+disease.">Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.</a> Gastroenterology. 146, 726-735 (2014).</li><li>Parks, E. J., Hellerstein, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+patient-oriented+research.+Recent+advances+in+liver+triacylglycerol+and+fatty+acid+metabolism+using+stable+isotope+labeling+techniques.">Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques.</a> Journal of lipid research. 47, 1651-1660 (2006).</li><li>Befroy, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+assessment+of+hepatic+mitochondrial+oxidative+and+anaplerotic+fluxes+in+humans+using+dynamic+13C+magnetic+resonance+spectroscopy.">Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy.</a> Nature medicine. 20, 98-102 (2014).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malaria journal. 6, 169 (2007).</li></ol>

The authors indicate they have no conflicts of interest.

O tempo de sacrifício para perfusão deve ser inferior a 3 minutos para perfusão ideal e digestão com colagenase do fígado. Uma vez que a perfusão com Perfusão Médio é iniciada, o fígado deve imediatamente alterar a aparência do vermelho ao pálido. Após aproximadamente 10 minutos de incubação com LDM, o fígado aparece inchado e rosa. No caso em que a perfusão é insuficiente, o fígado podem não exibir estas mudanças, e este será normalmente resultam num rendimento mais baixa de hepatócitos. <p c...

Non-alcoholic fatty liver disease is one of the leading causes of liver disease in Westernized cultures1,2. Lipid accumulation within the liver is associated with cell death, fibrosis, and liver failure via yet unknown mechanisms3-6. In fatty liver disease, hepatocyte-mediated &#946;-fatty acid oxidation and de novo lipogenesis are important determinants of net lipid accumulation7,8. This article will, therefore, focus on hepatocyte isolation, followed by quantification of &#946;-fatty acid oxidation and de novo lipogenesis.
Numerous methodologies have been developed to interrogate hepatocyte lipid metabolism. Though it is possible to measure metabolism of fat in vivo using stable isotopes9,10, these methods are costly, and require large numbers of animals. Additionally, the ability to investigate the effect of exogenous chemicals is limited due to the nature of in vivo experimentation. In contrast, the isolation of primary hepatocytes from mouse liver provides an affordable avenue to pursue11. Furthermore, studying hepatocytes in culture allows investigators to study the effects of varying chemicals on lipid processing while circumventing the difficulties of in vivo experimentation. Finally, isolated hepatocytes avoid any confounding from varying genetics since they are derived from the liver of a single animal.
Here we isolate and culture of hepatocytes, and we measure &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled palmitate. The protocol detailed below is straight forward, effective, and reproducible.

<table><tbody><tr><td>Liver Perfusion Medium</td><td>Life Technologies</td><td>17701038</td><td></td></tr><tr><td>Liver Digest Medium</td><td>Life Technologies</td><td>17703034</td><td>Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>10X DPBS</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140079&amp;nbsp;</td><td></td></tr><tr><td>Collagen</td><td>Life Technologies</td><td>A1048301&amp;nbsp;</td><td></td></tr><tr><td>Colloidal silica coated with polyvinylpyrrolidone</td><td>GE Life Sciences</td><td>17-0891-01</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Cellgro</td><td>30-001-CI</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I0516-5ML</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D2915-100MG</td><td></td></tr><tr><td>Albumin (BSA), Fraction V</td><td>MP Biomedicals</td><td>103703</td><td></td></tr><tr><td>24-Well Culture Dish</td><td>Corning Falcon</td><td>353047&amp;nbsp;</td><td></td></tr><tr><td>Tygon S3 Tubing&amp;nbsp;</td><td>Cole Parmer</td><td>06460-34</td><td></td></tr><tr><td>Male Leur Lock to 200 Barb Connectors</td><td>Cole Parmer</td><td>45518-00</td><td></td></tr><tr><td>24 G x 3/4&quot; Catheter</td><td>SurFlo</td><td>SROX2419CA</td><td></td></tr><tr><td>Perma-Hand Silk Suture</td><td>Ethicon</td><td>683G</td><td></td></tr><tr><td>Cell Strainer</td><td>Corning Falcon</td><td>08-771-2</td><td></td></tr><tr><td>IsoTemp 3013HD Recirculating Water Bath</td><td>Fisher</td><td>13-874-3</td><td></td></tr><tr><td>MasterFlex C/L Peristaltic Pump</td><td>MasterFlex</td><td>HV-77122-24</td><td></td></tr><tr><td>Microclamp</td><td>Roboz</td><td>RS-7438</td><td>Pre-sterilize in autoclave</td></tr><tr><td>5&amp;rdquo; Straight, Blunt-Blunt Operating Scissors</td><td>Roboz</td><td>RS-6810</td><td>Pre-sterilize in autoclave</td></tr><tr><td>24 mm Blade Straight, Sharp-point Microdissecting Scissors</td><td>Roboz</td><td>RS-5912</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm Tip Microdissecting Forceps</td><td>Roboz</td><td>RS-5130</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm Tip Full Curve Microdissecting Forceps</td><td>Roboz</td><td>RS-5137</td><td>Pre-sterilize in autoclave</td></tr><tr><td>60 ml Syringe</td><td>Becton Dickinson</td><td>309653</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Corning Falcon</td><td>352070</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td></td><td></td><td></td></tr><tr><td>Serological pipets</td><td></td><td></td><td></td></tr><tr><td>1,000, 200, 20 &amp;mu;l pipet and tips</td><td></td><td></td><td></td></tr></tbody></table>

determination of fatty acid oxidation and lipogenesis in mouse primary hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

Isolamentos de hepatócitos normalmente resultam em 1 - 3 x 10 7 células totais. Após incubação durante a noite, as células aparece hexagonal, muitos dos quais serão binucleadas (Figura 2). As células saudáveis ​​deve ser desprovido de granulações ou bolhas, que são indicativas de morte celular. Em geral, o ensaio de oxidação de ácido gordo é executado em três a quatro réplicas por composto de teste. Conta para o CO 2 amostras são ...

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Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

De novo a lipogênese e oxidação do ácido β-gordo constituem vias metabólicas importantes em hepatócitos, caminhos que são perturbados em vários distúrbios metabólicos, incluindo a doença de fígado gordo. Aqui demonstramos isolamento de hepatócitos primários de rato e descrever quantificação da oxidação de ácidos graxos e β-lipogênese.

Determinação da oxidação de ácidos gordos e Lipogênese em Rato Os hepatócitos primária

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

determination-fatty-acid-oxidation-lipogenesis-mouse-primary

Research

JoVE Journal

Biology

20.2K visualizações.  University of Massachusetts Medical School.  De novo a lipogênese e oxidação do ácido β-gordo constituem vias metabólicas importantes em hepatócitos, caminhos que são perturbados em vários distúrbios metabólicos, incluindo a doença de fígado gordo. Aqui demonstramos isolamento de hepatócitos primários de rato e descrever quantificação da oxidação de ácidos graxos e β-lipogênese. 

Vídeo: Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Medição do ácido graxo β-oxidação em uma suspensão de hepatócitos de rato recém-isolados

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Bioquímica

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análise otimizada de<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; A esteatose hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Medicina

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Isolamento dos hepatócitos primários de rato para análise de síntese de proteína nascente pelo método de rotulagem não-radioativo L-azidohomoalanine

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Induzir e caracterizar a esteatose vesicular em células HepaRG diferenciadas

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Avaliando a capacidade de manuseio de lipídios em camundongos

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

Um modelo de esteatose experimental in vitro: cultura celular hepatocitada em meio lipídidamente condicionado

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Um protocolo aprimorado de tempo e trabalho-eficiente para o isolamento primário do hepatocito do rato

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Biologia

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

<meta charset="utf8"></head><body>Avaliação da oxidação de ácidos graxos em células ósseas cultivadas

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

Avaliação da oxidação de ácidos graxos em células ósseas cultivadas

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Mensuração da Taxa de Lipólise em Tecido Adiposo Murino Ex Vivo e Pré-adipócitos Primários Diferenciados In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

Determinação da oxidação de ácidos gordos e Lipogênese em Rato Os hepatócitos primária

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