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).

All animal experimentation should be carried out in accordance with local and federal regulations and with the approval of an institutional IACUC and radiation safety administration.
1. Preparation
<ol>
	<li>Several days prior to the assay, thaw the 500 ml&#160;bottle of Liver Digest Medium (LDM) and refreeze ~35 ml&#160;aliquots in 50 ml&#160;conical tubes. Store at -20 &#176;C until needed.</li>
	<li>One day prior to the assay, pre-sterilize clean dissection tools by autoclave.</li>
	<li>On the day of the assay, treat the necessary amount of 24-well culture plates with collagen.
	<ol>
		<li>Mix 1 part of 3 mg/ml&#160;rat tail collagen with 50 parts of PBS. Add approximately 500 &#181;l&#160;to each well of a 24-well plate and incubate for 3-5 min at room temperature (RT). Remove the collagen and allow plate to air dry in hood. Repeat at least one additional time. 
		Note: Collagen coating can be performed up to one week in advance and plates stored at 4 &#176;C sealed in plastic wrap.</li>
	</ol>
	</li>
	<li>Prepare LDM for assay: Thaw LDM and warm to 37 &#176;C and adjust pH to 7.4 using 1 N KOH. Filter using 0.2 &#181;m syringe filter and place in a recirculating 42 &#176;C water bath.</li>
	<li>Warm 25 ml&#160;of Liver Perfusion Medium to 42 &#176;C in recirculating water bath.</li>
	<li>Prepare 90% colloidal silica coated with polyvinylpyrrolidone solution: Add 1 ml&#160;of 10x DPBS to 9 ml&#160;of colloidal silica coated with polyvinylpyrrolidone. Adjust pH to 7.4 using 0.1 N HCl. Filter with a sterile 0.2 &#181;m syringe filter. Store at RT.</li>
	<li>Warm Plating Medium to 37 &#176;C.</li>
</ol>
2. Isolation of Primary Mouse Hepatocytes
<ol>
	<li>Set up peristaltic pump, tubing, and dissection table: Sterilize tubing by flushing with 5 ml&#160;70% ethanol in distilled water, followed by 10 ml&#160;of sterile water. Place tubing in Liver Perfusion Medium and run pump to fill entire length of tubing.</li>
	<li>Sacrifice mouse by the institutionally approved method.</li>
	<li>Dissect open the abdominal cavity: Spray the mouse abdomen liberally with 70% ethanol. Using blunt-end scissors, make a midline incision through the dermis the length of the abdomen and reflect laterally. Make a similar incision in the peritoneum to expose the viscera.
	<ol>
		<li>Using a blunt instrument, gently displace the intestines to expose the abdominal vasculature Locate the abdominal inferior vena cava (IVC) and place a suture underneath the blood vessel, distal to the renal vein</li>
	</ol>
	</li>
	<li>Distal to the suture, place a needle and catheter into the IVC, advancing it beyond the level of the suture. With the needle still in place, tie the suture around the catheter to hold it in place. Carefully remove the needle. If done correctly, blood with flow through the catheter.</li>
	<li>Using a pipet, fill the remaining area in the catheter with perfusion medium, ensuring that no air is present. With great care, attach the tubing to the catheter.</li>
	<li>Dissect open the lung cavity: gently reflect the superior liver lobes to expose the diaphragm. Carefully puncture the diaphragm with sharp tip scissors, then make a lateral incision to expose the pleural cavity, taking care to avoid the gall bladder and pleural vasculature. Place a bulldog clamp around the thoracic inferior vena cava just proximal to the hepatic vein.</li>
	<li>Cut the portal vein and turn on the pump at 3 - 4 ml/min. Perfuse the liver with Liver Perfusion Medium for 5 min using approximately 20 ml&#160;of perfusion medium. 
	Note: The liver should immediately change from red to grey/tan. If portions of the liver remain red, this likely indicates poor perfusion, and the likelihood of successful isolation is greatly diminished. During the perfusion, be careful to ensure the medium does not run out and that no bubbles enter the tubing.</li>
	<li>Following the 5 min incubation, stop the pump and transfer the tubing to Liver Digest Medium. Restart the pump and perfuse the liver for another 10 - 15 min (until the medium is exhausted).</li>
	<li>At the end of the perfusion, stop the pump. The liver should have a pinkish hue and appear somewhat enlarged. Excise the liver by careful dissection. Remove the gall bladder and transfer the liver to 10 cm tissue culture dish.</li>
	<li>In a biosafety cabinet, add 10 ml&#160;of Plating Medium ( Table 1) to the liver. Gently scrape the liver using either forceps or a scalpel to remove the hepatocytes. Filter the suspension using a 100 &#181;m cell strainer and transfer to a 50 ml&#160;conical tube. Wash the plate with an additional 10 ml&#160;of Plating Medium and pool in the 50 ml&#160;conical tube.</li>
	<li>Pellet the cells by centrifuging for 5 min at 350 x g, 4 &#176;C.</li>
	<li>Aspirate medium and resuspend pellet in 10 ml&#160;of Plating Medium and add 10 ml&#160;of 90% colloidal silica coated with polyvinylpyrrolidone. Mix gently and centrifuge as in step 2.11. After centrifugation, a layer of dead cells will be floating on the top of the mixture, while the live cells will pellet to the bottom.</li>
	<li>Aspirate dead cells and medium. Wash 2 times with 20 ml&#160;of Plating Medium, centrifuge as in 2.11.</li>
	<li>Resuspend cells in 10 ml&#160;of Plating Medium. Count cells with a hemocytometer and place 9 x 104 cells/well in collagen-treated 24-well culture dishes. Incubate in a 37 &#176;C tissue culture incubator for 2 hr. Each plate can be used for either the Fatty Acid Oxidation assay OR the Lipogenesis assay.</li>
	<li>Optionally, change wells to Maintenance Medium (Table 1) and culture at 37 &#176;C. Cells can be cultured for 2 - 3 days without affecting assay results.</li>
</ol>
3. Fatty Acid Oxidation Assay
Warning: Use of radioactivity can be hazardous. All purchasing, storage, handling, and disposal of radioactive material should be carried out in accordance with institutional, state, and federal regulations and guidelines.
<ol>
	<li>16 - 20 hr prior to assay, wash cells 2 times with warm PBS. Change the cells to serum-free Serum Starvation Medium (Table 1) with 20 nM glucagon and incubate overnight at 37 &#176;C. 
	Note: Because fetal bovine serum contains an unknown concentration of metabolic hormones (e.g. insulin, glucagon), serum starve the cells to remove any confounding effects this may have on the assay. Cells are viable in Serum Starvation Medium for &#62;24 hr. Glucagon treatment is used to stimulate fatty acid oxidation within the hepatocytes.</li>
	<li>On the morning of the assay, prepare Pre-Incubation Medium: Resuspend an appropriate amount of Sodium Palmitate in ultrapure water to make a 100 mM solution and heat to 70 &#176;C for 10 min. In the meantime, prepare the requisite amount (0.5 ml&#160;per well of a 24-well plate) of DMEM with 25 mM HEPES, 1% BSA Fraction V, and 20 nM glucagon. Heat to 37 &#176;C.
	<ol>
		<li>Once solubilized, add Palmitate to a final concentration of 250 &#181;M to the medium. Change hepatocytes to Pre-Incubation Medium and incubate at 37 &#176;C for 2 hr. Reserve some Pre-Incubation Medium at 37 &#176;C for later use.</li>
	</ol>
	</li>
	<li>During the incubation, dry an appropriate amount of 14C-Palmitate (0.5 &#181;Ci/well) by evaporation under nitrogen gas.
	<ol>
		<li>For example, to measure 24 samples in a 24-well plate, transfer 120 &#181;l&#160;of 0.1 &#181;Ci/ml14 C-Palmitate to a 1.5 ml&#160;tube. Slowly evaporate the ethanol solvent by blowing nitrogen gas over the solution from a distance of 3-5 cm in a fume hood. The solvent should evaporate in approximately 30 - 40 min, leaving dry 14C-Palmitate in the bottom of the tube.</li>
	</ol>
	</li>
	<li>Approximately 15 min&#160;before the end of the incubation, resuspend the 14C-Palmitate in 0.1 N NaOH (12.5 &#181;l/&#181;Ci). Incubate at 70 &#176;C for 10 min. Add three volumes of warm Pre-Incubation Medium and mix by pipetting up and down.</li>
	<li>Spike each well with 25 &#181;l&#160;of diluted 14C-Palmitate. Mix by gently rocking the plate and incubate at 37 &#176;C for 90 min. This is the Assay Plate.</li>
	<li>During the incubation, prepare the Filter Paper Plate (Figure 1).
	<ol>
		<li>Remove the cover from a sterile 24-well plate. Place one 2 cm x 2 cm piece of filter paper in the bottom of each of the wells. Overlay the plate with a piece of 4&#8221; x 7&#8221; parafilm.</li>
		<li>Using a large rectangular object, such as a micropipettor tip box, rub the parafilm on the wells to perforate the parafilm at the well openings and create a seal over the remainder of the plate. Remove the perforated circles of parafilm now covering the wells. The plate should now be tightly covered with parafilm in all areas except the well openings.</li>
	</ol>
	</li>
	<li>10 min before the end of the incubation, add 200 &#181;l&#160;of 3 N NaOH to each well of the Filter Paper Plate, making sure the filter paper absorbs all of the liquid.
	<ol>
		<li>Optionally prior to freezing, transfer the medium to a fresh 24-well plate. After washing once with PBS, lyse the remaining cells in 0.1 N HCl and calculate protein content by BCA assay.</li>
	</ol>
	</li>
	<li>At the end of the incubation, snap-freeze the Assay Plate in liquid nitrogen. Be careful to ensure each well is completely frozen before proceeding.</li>
	<li>Add 100 &#181;l&#160;of 70% Perchloric Acid to each well of the Assay Plate. Immediately cover with the Filter Paper Plate. Place the plates on an orbital shaker and rock at orbital speed of 80 rpm at RT for 2 hr.</li>
	<li>Following the incubation, process the samples:
	<ol>
		<li>To measure the CO2 fraction, transfer the filter paper squares to 4 ml&#160;liquid scintillation fluid in a scintillation vial and measure 14C signal.</li>
		<li>To measure the acid soluble material, transfer 400 &#181;l&#160;of medium to a 1.5 ml&#160;microfuge tube. Centrifuge at maximum speed for 10 min. Add 100 &#181;l&#160;of the resulting supernatant to 500 &#181;l&#160;of 2:1 Chloroform-Methanol (v/v), and vortex briefly.
		<ol>
			<li>Add 250 &#181;l&#160;of water to the mixture, and vortex again. Centrifuge samples for 10 min at 3,000 x g. Transfer 200 &#181;l&#160;of the upper phase to 4 ml&#160;liquid scintillation fluid in a scintillation vial and measure 14C signal.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Lipogenesis Assay
<ol>
	<li>The evening prior to beginning the assay, wash the cells 2 times with warm PBS. Change the hepatocytes to Serum Starvation Medium with 100 nM insulin. Incubate overnight at 37 &#176;C. 
	Note: Because fetal bovine serum contains an unknown concentration of metabolic hormones (e.g. insulin, glucagon), serum starve the cells to remove any confounding effects this may have on the assay. Cells are viable in Serum Starvation Medium for &#62;24 hr. Insulin is used in this assay to stimulate lipogenesis.</li>
	<li>Make Lipogenesis Medium: Serum Starvation Medium with 100 nM insulin, 10 &#181;M cold acetate and 0.5 &#181;Ci 3H-Acetate per well. Change cells to Lipogenesis Medium and incubate at 37 &#176;C for 2 hr. Include any compounds of interest to be tested.</li>
	<li>After the incubation period, wash cells 2 times with PBS. Lyse cells by scraping in 120 &#181;l&#160;of 0.1 N HCl. Reserve 10 &#181;l&#160;for protein assay (assess by BCA assay), and transfer 100 &#181;l&#160;to 1.5 ml&#160;microfuge tube.</li>
	<li>Extract lipids by addition of 500 &#181;l&#160;of 2:1 chloroform-methanol (v/v). Vortex briefly and incubate at RT for 5 min. Add 250 &#181;l&#160;of water, vortex and incubate at RT for an additional 5 min. Centrifuge samples 10 min at 3,000 x g, RT. Carefully transfer lower phase to 4 ml&#160;liquid scintillation fluid in a scintillation vial and measure 3H activity.</li>
</ol>

<ol>
	<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.

The time from sacrifice to perfusion should be less than 3 min for ideal perfusion and collagenase digestion of the liver. Once perfusion with Perfusion Medium is initiated, the liver should immediately change appearance to from red to pale. After approximately 10 min of incubation with LDM, the liver will appear swollen and pink. In the event that perfusion is insufficient, the liver may not exhibit these changes, and this will typically result in a lower hepatocyte yield.
Following the washi...

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.

Hepatocyte isolations typically result in 1 - 3 x 107 total cells. After overnight incubation, the cells will appear hexagonal, many of which will be binucleated (Figure 2). Healthy cells should be devoid of granulations or blebs, which are indicative of cell death.
In general, Fatty Acid Oxidation assay is run in three to four replicates per test compound. Counts for the CO2 samples are approximately one-fifth of those derived from the acid soluble mater...

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 lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

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 Views.  University of Massachusetts Medical School. De novo lipogenesis and β-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of β-fatty acid oxidation and lipogenesis.

Video: 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.

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

Biochemistry

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.

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

Medicine

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.

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;

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.

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.

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.

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

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.

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 ...

Semi-Automated Isolation of Murine White Adipose Tissue

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.

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

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.

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

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

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Chapters in this video

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Assessing Whole-Body Lipid-Handling Capacity in Mice

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

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

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

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

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