Name: determination of fatty acid oxidation and lipogenesis in mouse primary hepatocytes
Uploaded: 2015-08-27T15:30:00.000+00:00
Duration: 12 min 11 s
Description: Watch this Scientific Journal Video about Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes at JoVE.com

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).

Alle Tierversuche sollten in Übereinstimmung mit den örtlichen und nationalen Vorschriften und mit der Zustimmung eines institutionellen IACUC und Strahlungssicherheit Verwaltung durchgeführt werden. 1. Vorbereitung <ol><li> Einige Tage vor dem Test, tauen die 500 ml Flasche Liver Digest Medium (LDM) und wieder einfrieren ~ 35 ml Aliquots in 50 ml konischen Röhrchen. Lagerung bei -20 ° C, bis sie benötigt. </li><li> Einen Tag vor dem Assay, pre-sterilisiert sauberen Dissektion Tools von Autoklaven. </li><li> Am Tag des Assays, behandeln die notwendige Menge von 24-Well-Kulturplatten mit Collagen. <ol><li> 1 Teil von 3 mg / ml Rattenschwanzkollagen mit 50 Teilen PBS. In etwa 500 &amp; mgr; l in jede Vertiefung einer 24-Well-Platte und Inkubation für 3-5 Minuten bei Raumtemperatur (RT). Entfernen Sie die Kollagen und lassen Sie die Platte trocken in der Haube in die Luft. Wiederholen wenigstens ein weiteres Mal. Hinweis: Collagen-Beschichtung kann bis zu einer Woche in ad durchgeführt werdenVance und Platten bei 4 ° C in Plastikfolie abgedichtet gelagert. </li></ol></li><li> Bereiten LDM für Assay: Thaw LDM und warm bis 37 ° C und pH-Wert auf 7,4 mit 1 N KOH. Filter mit 0,2 um Spritzenfilter und in einen Umluft 42 ° C Wasserbad. </li><li> Warm 25 ml Leberperfusion Medium auf 42 ° C im Umluft Wasserbad. </li><li> Vorbereitung 90% kolloidales Siliciumdioxid, das mit Polyvinylpyrrolidon-Lösung überzogen: 1 ml 10x DPBS zu 9 ml kolloidaler Kieselsäure mit Polyvinylpyrrolidon beschichtet. Einstellen des pH auf 7,4 unter Verwendung von 0,1 N HCl. Filter mit einer sterilen 0,2 &amp; mgr; m Spritzenfilter filtriert. Lagern bei RT. </li><li> Warm Plating Medium auf 37 ° C. </li></ol> 2. Isolierung von Primär Maushepatozyten <ol><li> Einrichten peristaltische Pumpe, Schläuche und Dissektionstisch: Sterilisieren Schlauch durch Spülen mit 5 ml 70% Ethanol in destilliertem Wasser, gefolgt von 10 ml sterilem Wasser. Zeigen Schlauch in Leberperfusion Medium und führen Pumpe zu füllengesamte Rohrlänge. </li><li> Opfern Maus durch die institutionell genehmigten Methode. </li><li> Sezieren offen die Bauchhöhle: Sprühen Sie die Mausunterleib zügig mit 70% Ethanol. Verwendung blunt-end Schere, einen Mittelschnitt durch die Dermis die Länge der Bauch und seitlich reflektieren. Einen ähnlichen Schnitt in der Bauchfell, um die Eingeweide aus. <ol><li> Mit einem stumpfen Gegenstand vorsichtig zu verdrängen den Darm zu entlarven die Bauchgefäß Suchen Sie die Bauch untere Hohlvene (IVC) und legen Sie eine Naht unter dem Blutgefäß, distal der Nierenvenen </li></ol></li><li> Distal des Nahtmaterials Platzieren einer Nadel und der Katheter in die IVC, Vorschieben über das Niveau des Fadens. Mit der Nadel noch vorhanden, binden die Naht um den Katheter, um es in Position zu halten. Die Nadel vorsichtig entfernen. Wenn es richtig mit Fluss durch den Katheter durchgeführt, Blut. </li><li> Unter Verwendung einer Pipette, füllen Sie die restliche Fläche in dem Katheter mit Perfusionsmedium, sicherzustellen,daß keine Luft vorhanden ist. Mit großer Sorgfalt, befestigen Sie den Schlauch mit dem Katheter. </li><li> Sezieren offen die Lunge Hohlraum: sanft spiegeln die überlegene Leberlappen, um die Membran freizulegen. Sorgfältig durchstechen die Membran mit scharfen Spitze Schere, dann machen Sie eine laterale Inzision, um die Brusthöhle freizulegen, kümmert sich um die Gallenblase und Pleura Gefäßsystem zu vermeiden. Legen Sie eine Bulldogge Schelle um den Brust untere Hohlvene gerade proximal der Lebervene. </li><li> Schneiden Sie die Pfortader und schalten Sie die Pumpe mit einem 3 - 4 ml / min. Perfusion der Leber Leberperfusion Medium für 5 min unter Verwendung von etwa 20 ml Perfusionsmedium. Hinweis: Die Leber sollten sofort von Rot zu ändern bis grau / tan. Wenn Teile der Leber rot bleiben, dürfte dies deutet auf eine schlechte Durchblutung, und die Wahrscheinlichkeit einer erfolgreichen Isolierung stark vermindert. Während der Perfusion, seien Sie vorsichtig, um das Medium nicht auslaufen zu gewährleisten, und dass keine Luftblasen geben Sie die Schläuche. </li><li> Nach 5 min Inkubation stoppendie Pumpe und übertragen Sie den Schlauch an Leber Digest Medium. Starten Sie die Pumpe und Perfusion der Leber für eine weitere 10 bis 15 min (bis das Medium erschöpft ist). </li><li> Am Ende der Durchblutung, die Pumpe stoppen. Die Leber ist ein rosa Farbton haben und scheinen etwas vergrößert. Auszuschneiden die Leber durch sorgfältige Dissektion. Entfernen Sie die Gallenblase und übertragen die Leber zu 10 cm Gewebekulturschale. </li><li> In einer Biosicherheitswerkbank, 10 ml Plating Medium (Tabelle 1) in die Leber. Kratzen Sie vorsichtig die Leber unter Verwendung entweder einer Pinzette oder einem Skalpell, um die Leberzellen zu entfernen. Filtern der Suspension unter Verwendung eines 100 &amp; mgr; m Zellsieb und in ein konisches 50 ml-Röhrchen. Waschen Sie die Platte mit weiteren 10 ml Plattierungsmedium und Pool in der 50 ml konischen Röhrchen. </li><li> Pelletieren Sie die Zellen durch Zentrifugieren für 5 min bei 350 × g, 4 ° C. </li><li> Saugen Sie mittel- und Pellet in 10 ml Plattierungsmedium und 10 ml 90% kolloidaler Kieselsäure mit po beschichtetlyvinylpyrrolidone. Vorsichtig mischen und Zentrifuge wie in Schritt 2.11. Nach der Zentrifugation wird eine Schicht aus toten Zellen werden auf der Oberseite der Mischung schwimmt, während die lebenden Zellen auf den Grund zu pelletieren. </li><li> Saugen Sie abgestorbenen Zellen und Medium. Mit 20 ml Plattierungsmedium, Zentrifuge 2.11 waschen 2 mal. </li><li> Die Zellen in 10 ml Medium Überzug. Zählen von Zellen mit einem Hämozytometer und legen 9 x 10 4 Zellen / Vertiefung in Kollagen behandelten 24-Well-Kulturschalen. Inkubieren in einem 37 ° C Brutschrank für 2 Std. Jede Platte kann entweder für die Fettsäure-Oxidation Assay oder der Lipogenese-Assay verwendet werden. </li><li> Optional Brunnen verändern Wartung Medium (Tabelle 1) und die Kultur bei 37 ° C. 3 Tage, ohne die Testergebnisse - Zellen für 2 kultiviert werden. </li></ol> 3. Fettsäure-Oxidationsassay Warnung: Verwenden von Radioaktivität kann gefährlich sein. Alle Einkauf, Lagerung, Handhabung, und diEntsorgungsbetrieb von radioaktivem Material sollte in Übereinstimmung mit den institutionellen, staatlichen und bundesstaatlichen Vorschriften und Richtlinien ausgeführt werden. <ol><li> 16 bis 20 h vor dem Assay, waschen Zellen 2 mal mit warmem PBS. Ändern die Zellen an serumfreies Serumentzugs-Medium (Tabelle 1) mit 20 nM Glucagon und Inkubation über Nacht bei 37 ° C. Anmerkung: Da fötales Rinderserum enthält eine unbekannte Konzentration von Stoffwechsel Hormone (zB Insulin, Glukagon), Serum hungern die Zellen keine Verwechslung sich daraus für den Assay haben kann. Zellen lebensfähig in Serumhungermedium für&gt; 24 h. Glucagon Behandlung wird verwendet, um die Oxidation von Fettsäuren in den Leberzellen zu stimulieren. </li><li> Am Morgen des Tests vorzubereiten Vorinkubationsmedium: Resuspendieren eine entsprechende Menge an Natriumpalmitat in ultrareinem Wasser, um eine 100 mM Lösung und Wärme bis 70 ° C für 10 min zu machen. In der Zwischenzeit bereiten die erforderliche Menge (0,5 ml pro Vertiefung einer 24-Well-Platte)DMEM mit 25 mM HEPES, 1% BSA Fraktion V, und 20 nM Glucagon. Wärme auf 37 ° C. <ol><li> Einmal solubilisiert hinzuzufügen Palmitate auf eine Endkonzentration von 250 &amp; mgr; M zu dem Medium. Ändern Hepatozyten zu Vorinkubationsmedium und bei 37 ° C für 2 Stunden. Behalten einige Vorinkubationsmedium bei 37 ° C für die spätere Verwendung. </li></ol></li><li> Während der Inkubation Trocknen eine geeignete Menge an 14 C-Palmitat (0,5 uCi / Vertiefung) durch Verdampfung unter Stickstoff. <ol><li> Zum Beispiel, um 24 Proben in einem 24-Well-Platte zu messen, Transfer 120 ul 0,1 &amp; mgr; Ci / ml 14 C-Palmitate in ein 1,5-ml-Tube. Langsam verdampft das Ethanol-Lösungsmittel durch Einblasen von Stickstoffgas über die Lösung in einem Abstand von 3-5 cm in einem Abzug. Das Lösungsmittel sollte in etwa 30 verdampfen - 40 min, wobei Trocken 14 C-Palmitat im Boden des Röhrchens. </li></ol></li><li> Ca. 15 min vor dem Ende der Inkubation resuspendieren 14C-Palmitate in 0,1 N NaOH (12,5 ul / &amp; mgr; Ci). Inkubieren bei 70 ° C für 10 min. In drei Bänden der warmen Vorinkubationsmedium und mischen durch Auf-und Abpipettieren. </li><li> Spike jede Vertiefung mit 25 ul verdünnt 14 C-Palmitate. Mischen Sie durch vorsichtiges Schwenken der Platte und bei 37 ° C für 90 min. Dies ist die Testplatte. </li><li> Während der Inkubation, bereiten Sie die Filter Pappteller (Abbildung 1). <ol><li> Entfernen Sie die Abdeckung aus einer sterilen 24-Well-Platte. Platzieren einer 2 cm x 2 cm großes Stück Filterpapier in den Boden einer jeden der Vertiefungen. Überlagern die Platte mit einem Stück von 4 &quot;x 7&quot; Parafilm. </li><li> Mit einem großen, rechteckigen Gegenstand, beispielsweise eine Mikropipettenspitze Box, reiben Sie die Parafilm auf den Brunnen, um den Parafilm an den gut Öffnungen perforiert und eine Abdichtung zu schaffen über den Rest der Platte. Entfernen Sie die Lochkreise Parafilm jetzt Abdecken der Brunnen. Die Platte sollte jetzt fest mit Parafilm in allen Bereichen abgedeckt werden außer dem gut openings. </li></ol></li><li> 10 min vor dem Ende der Inkubation werden 200 ul 3 N NaOH zu jeder Vertiefung der Filterpapierplatte, so dass das Filterpapier absorbiert die gesamte Flüssigkeit. <ol><li> Gegebenenfalls vor dem Einfrieren, Transfer des Mediums auf eine frische 24-Well-Platte. Nach einmaligem Waschen mit PBS, lysieren die restlichen Zellen in 0,1 N HCl und berechnen Proteingehalt durch BCA-Assay. </li></ol></li><li> Am Ende der Inkubation Schnappgefrierassayplatte in flüssigem Stickstoff. Seien Sie vorsichtig, um jede Vertiefung zu gewährleisten ist komplett eingefroren, bevor Sie fortfahren. </li><li> 100 l 70% iger Perchlorsäure in jede Vertiefung der Testplatte. Unmittelbar decken mit dem Filter Pappteller. Legen Sie die Platten auf einem Orbitalschüttler und Felsen am Umlaufgeschwindigkeit von 80 rpm bei RT für 2 h. </li><li> Nach der Inkubation der Proben zu verarbeiten: <ol><li> Um den CO 2 -Anteil zu messen, übertragen Sie die Filterpapier Quadrate, um 4 ml flüssige Szintillationsflüssigkeit in einem SzintillationszählerFläschchen und Maßnahme 14 C-Signal. </li><li> Um den säurelöslichen Materials zu messen, zu übertragen 400 ul Medium in ein 1,5 ml Mikrozentrifugenröhrchen. Zentrifuge bei maximaler Geschwindigkeit für 10 min. 100 l des resultierenden Überstandes auf 500 ul von 2: 1 Chloroform-Methanol (v / v), und kurz vortexen. <ol><li> Fügen Sie 250 ul Wasser zu der Mischung und Wirbel erneut. Zentrifuge Proben für 10 min bei 3.000 x g. Transfer 200 ul der oberen Phase auf 4 ml flüssige Scintillationsflüssigkeit in einem Szintillationsfläschchen und messen 14 C-Signal. </li></ol></li></ol></li></ol> 4. Lipogenese-Assay <ol><li> Der Abend vor dem Test, waschen Sie die Zellen 2 mal mit warmem PBS. Ändern Sie die Hepatozyten, um Serumhungermedium mit 100 nM Insulin. Inkubieren über Nacht bei 37 ° C. Anmerkung: Da fötales Rinderserum enthält eine unbekannte Konzentration von Stoffwechsel Hormone (zB Insulin, Glucagon), Zu verhungern Serum die Zellen keine verwirrende Auswirkungen dies auf den Assay haben können, zu entfernen. Zellen lebensfähig in Serumhungermedium für&gt; 24 h. Insulin wird in diesem Test verwendet werden, um die Lipogenese zu stimulieren. </li><li> Machen Lipogenese Medium: Serumhungermedium mit 100 nM Insulin, 10 &amp; mgr; M kaltem Acetat und 0,5 &amp; mgr; Ci 3 H-Acetat pro Vertiefung. Wechselzellen Lipogenese Medium und bei 37 ° C für 2 Std. Fügen Sie alle Verbindungen von Interesse getestet werden. </li><li> Nach der Inkubationszeit, waschen Zellen 2 mal mit PBS. Lysieren Zellen durch Abschaben in 120 ul 0,1 N HCl. Reserve 10 &amp; mgr; l für die Protein-Assay (Beurteilung durch BCA-Test), und Transfer 100 ul bis 1,5 ml Mikrozentrifugenröhrchen. </li><li> Extrakt Lipide durch Zugabe von 500 ul von 2: 1 Chloroform-Methanol (v / v). Vortex kurz und Inkubation bei Raumtemperatur für 5 min. Fügen Sie 250 ul Wasser, Wirbel und Inkubation bei Raumtemperatur für weitere 5 min. Centrifuge Proben 10 min bei 3.000 xg, RT. Sorgsamübertragen untere Phase bis 4 ml flüssige Szintillationsflüssigkeit in einem Szintillationsfläschchen und messen 3 H-Aktivität. </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.

Die Zeit vom Opfer Perfusion sollte weniger als 3 min für ideale Perfusion und Kollagenaseverdau der Leber. Sobald Perfusion mit Perfusionsmedium eingeleitet wird, sollte die Leber sofort Aussehen von rot bis hell ändern. Nach ca. 10 min Inkubation mit LDM, wird die Leber geschwollen und rosa angezeigt. Im Falle, dass die Perfusion unzureichend ist, kann die Leber nicht zeigen diese Änderungen, und dies wird in der Regel in einem unteren Hepatozyten Ausbeute führen. Nach den Waschschritt...

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.

Hepatozyten-Isolierungen führen in der Regel in 1-3 x 10 7 Zellen insgesamt. Nach Inkubation über Nacht werden die Zellen hexagonal, von denen viele binukleären werden (Figur 2) angezeigt werden. Gesunde Zellen sollten frei von Granulationen oder Blasen, was auf Zelltod sein. Im allgemeinen wird die Oxidation von Fettsäuren Assays in drei bis vier Wiederholungen pro Testverbindung ausgeführt. Zählungen für die CO 2 Proben sind etwa ein Fünftel d...

Watch this Scientific Journal Video about Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes at JoVE.com

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

De novo Lipogenese und β-Oxidation von Fettsäuren bilden Schlüsselstoffwechselwege in Hepatozyten, Wege, die in mehreren Stoffwechselstörungen, einschließlich Fettleber gestört werden. Hier zeigen wir Isolierung von Maus primären Hepatozyten und zu beschreiben, die Quantifizierung der β-Oxidation von Fettsäuren und Lipogenese.

Bestimmung der Fettsäureoxidation und Lipogenese in Maus primären Hepatozyten

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 Aufrufe.  University of Massachusetts Medical School.  De novo Lipogenese und β-Oxidation von Fettsäuren bilden Schlüsselstoffwechselwege in Hepatozyten, Wege, die in mehreren Stoffwechselstörungen, einschließlich Fettleber gestört werden. Hier zeigen wir Isolierung von Maus primären Hepatozyten und zu beschreiben, die Quantifizierung der β-Oxidation von Fettsäuren und Lipogenese. 

Serie: 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.

Messung der Fettsäure-β-Oxidation in einer Suspension frisch isolierter Maus-Hepatozyten

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

Forschung

Biochemie

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.

Optimierte Analyse von<em&gt; In Vivo</em&gt; und<em&gt; In Vitro</em&gt; Hepatosteatose

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

Medizin

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.

Isolierung der primäre Maustaste Hepatozyten für entstehende Protein Synthese Analyse von nicht-radioaktiven L-Azidohomoalanine Kennzeichnung Methode

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;

Induzieren und Charakterisieren der vesikulären Steatose in differenzierten HepaRG-Zellen

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.

Beurteilung der Ganzkörper-Lipid-Handling-Kapazität bei Mäusen

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.

Ein Modell der experimentellen Steatose in vitro: Hepatozytenzellkultur in lipidüberlastetem Medium

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.

Ein verbessertes zeit- und arbeitseffizientes Protokoll für die primäre Hepatozytenisolierung der Maus

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

Biologie

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.

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

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.

Messung der Lipolyserate in ex vivo murinem Fettgewebe und primären Präadipozyten, die in vitro differenziert wurden

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 ...

Halbautomatische Isolierung von weißem Fettgewebe der Maus

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Autor im Rampenlicht: Halbautomatische Isolierung der stromalen vaskulären Fraktion aus weißem Fettgewebe der Maus mit einem Gewebedissoziator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Halbautomatische Isolierung der stromalen vaskulären Fraktion aus murinem weißem Fettgewebe mit Hilfe eines Gewebedissoziators

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...