This work was supported by the National Institutes of Health grant R35GM119528 to Roberta Leonardi.

All experimental procedures on mice (C57BL/6J, males, 9-11 weeks of age) were approved by the Institutional Animal Care and Use Committees (IACUC) of West Virginia University.
1. Hepatocyte isolation
<ol>
	<li>Preparation
	<ol>
		<li>In the days before the hepatocyte isolation, prepare the buffers and cell culture media listed in Table 1. Set up a water bath with the temperature set to 37 &#176;C close to where the surgery will be performed.</li>
		<li>On the day of the hepatocyte isolation, under a laminar flow hood, transfer 35 mL of Buffer 1 to a sterile 50 mL centrifuge tube and 70 mL of Buffer 2 to a 100 mL sterile beaker or bottle.</li>
		<li>Add antibiotics gentamicin (50 &#181;g/mL) and penicillin/streptomycin (1x) to both buffers.</li>
		<li>Transfer 20 mL of Buffer 2 as prepared in step 1.1.3 to a 100 mm cell culture dish and place on ice.</li>
		<li>Transfer the remaining 50 mL of Buffer 2 to a sterile 50 mL centrifuge tube. Place the 50 mL tubes containing the antibiotic-supplemented Buffers 1 and 2 in a water bath set at 37 &#176;C and let them warm up for at least 15 min before starting the perfusion. 
		NOTE: If conducting multiple hepatocytes isolations in a session, scale up the number of antibiotic-supplemented aliquots of Buffers 1 and 2 to prepare accordingly.</li>
		<li>Thaw an aliquot of collagenase solution and keep on ice. 
		NOTE: If properly stored, there is no significant loss of enzyme activity in collagenase solutions frozen and thawed up to 3 times and used within 3 weeks from preparation.</li>
		<li>Prepare the surgical instruments and peristaltic pump. Sterilize the lines of the peristaltic pump by circulating 15 mL of 70% ethanol, followed by 15 mL of sterile water.</li>
		<li>Connect a 22 G needle to the exit line (Figure 1A). The hollowed filter of a catheter works well as a connector. Fill the lines with Buffer 1 and inspect the lines, connector, and needle to ensure no air bubbles are trapped.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62904/62904fig1v2.jpg" /> 
Figure 1: Perfusion apparatus and perfused liver. (A) Peristaltic pump with outlet line connected to the needle used to cannulate and perfuse the liver. (B) Successful cannulation is indicated by immediate and homogeneous blanching of the liver. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62904/62904fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Liver perfusion and dissociation
	<ol>
		<li>Anesthetize a mouse via isoflurane inhalation with medical grade air as carrier gas, using 4% isoflurane for induction and 1.5% isoflurane to maintain anesthesia. Verify the depth of anesthesia by assessing the loss of pedal reflex.</li>
		<li>When there is no response to toe pinching, place the mouse in a supine position on a surgery board, outstretch the limbs, and secure them to the board with pins.</li>
		<li>Liberally spray the abdomen and chest of the mouse with 70% ethanol.</li>
		<li>Using forceps, pull up the skin and abdominal wall near the base of the abdomen and cut laterally, on either side of the midline and up to the diaphragm, to expose the organs.</li>
		<li>Expose the inferior vena cava (IVC) by moving the intestines to the right side and gently flipping the lobes of the liver up. Insert a small cylindrical object, such as a needle cap, under the back of the mouse to slightly tilt the IVC and facilitate its cannulation (Figure 1B).</li>
		<li>Start the pump at the lowest speed and, with Buffer 1 flowing, insert the needle into the IVC.</li>
		<li>Cut the portal vein to relieve the pressure and allow for drainage of blood and perfusion buffers, then immediately increase the flow rate to 7 mL/min. If done correctly, the liver will uniformly blanch within a few seconds (Figure 1B).</li>
		<li>For more consistent results, hold the needle in position by hand for the entire duration of the perfusion.</li>
		<li>Perfuse the liver with warm Buffer 1. To avoid introducing air bubbles, ensure that the line inserted in the tube containing Buffer 1 remains continuously submerged.</li>
		<li>While perfusion occurs, add 130 &#181;L of collagenase solution to Buffer 2 and mix by pipetting up and down or stirring with a 5 mL or 10 mL serological pipette.</li>
		<li>As the volume in the tube containing Buffer 1 decreases to about 5 mL, slowly add 5 mL of Buffer 2 to Buffer 1 by pipetting on the side of the tube. The goal is to avoid introducing air bubbles in the line while changing from Buffer 1 to Buffer 2.</li>
		<li>Wait until the volume decreases again to 5 mL and slowly add another 5 mL of Buffer 2. Repeat one more time. As Buffer 2 replaces Buffer 1 and the dissociation starts, the liver will swell.</li>
		<li>Add the remaining Buffer 2 to the tube originally containing Buffer 1. Stop the perfusion when there is about 5-10 mL of Buffer 2 left in the tube. 
		NOTE: While Buffer 2 is perfusing the liver, the portal vein can be intermittently clamped with forceps for 5 s. This step is optional, but the resultant increase in pressure throughout the liver can improve its dissociation and, thus, the final hepatocyte yield.</li>
		<li>Carefully excise the liver and transfer it to the 100 mm culture dish containing the 20 mL of ice-cold Buffer 2 set aside at step 1.1.4.</li>
		<li>Under the laminar flow hood, gently break the liver apart using surgical scissors and tweezers.</li>
		<li>Add about 20 mL of ice-cold M199 to the hepatocyte suspension and filter it through a 100 &#181;m cell strainer using the plunger of a syringe to gently promote the release of additional hepatocytes from larger liver pieces.</li>
		<li>Wash the 100 mm culture dish and the cell strainer with additional M199 until the collection tube is full.</li>
		<li>Centrifuge the suspension at 50 x g for 2 min at 4 &#176;C. Carefully aspirate the supernatant and gently resuspend the hepatocyte pellet in 30 mL of cold M199 by swirling.</li>
		<li>Pellet the hepatocytes as mentioned in step 1.2.18. Repeat the wash one more time.</li>
		<li>Resuspend the hepatocytes in 10 mL of warm M199 and determine the viability and yield using the trypan blue exclusion method and a hemocytometer12.</li>
		<li>Dilute the cells in M199 warmed to 37&#160;&#186;C to a final concentration of 1.0 x 106 viable cells/mL and immediately start the assay.</li>
	</ol>
	</li>
</ol>
2. Fatty acid &#946;-oxidation assay
NOTE: The assay is conducted in triplicate, and each reaction mixture contains 750,000 cells, 1.35 mg/mL bovine serum albumin (BSA), 100 &#181;M palmitic acid, and 0.4 &#181;Ci [1-14C]palmitic acid in a final volume of 2 mL. 
CAUTION: Radioactive compounds are hazardous. Purchase, handle, store, and dispose of radioactive material in accordance with Institutional, State, and Federal regulations.
<ol>
	<li>Preparation
	<ol>
		<li>In the days before the assay, prepare the palmitic acid and BSA solutions (Table 1) and store them at -20 &#176;C.</li>
		<li>On the day of the assay, complete steps 2.1.3-2.1.9 before starting the liver perfusion.</li>
		<li>Thaw the palmitic acid and BSA solutions. Prepare the substrate mixture for multiple reactions plus a 20%-30% excess, with a typical assay setup shown in Table 2.</li>
		<li>Aliquot 13.5 &#181;L of BSA solution per reaction in a microcentrifuge tube and warm to 41 &#176;C, then add 1 &#181;L of the 200 mM palmitic acid solution (BSA: palmitic acid molar ratio = 1:5) per reaction. 
		NOTE: It is preferable to dispense solutions prepared using organic solvents, such the radioactive and non-radioactive palmitic acid solutions, with a positive displacement pipette and appropriate tips.</li>
		<li>Vortex vigorously and incubate at 41 &#176;C to facilitate the formation of the soluble palmitic acid: BSA complex. Vortex occasionally during the incubation period.</li>
		<li>The mixture will initially appear cloudy but will clarify completely after 20-30 min of incubation at 41 &#176;C. Keep it at 41 &#176;C until ready to start the reactions.</li>
		<li>Aliquot 133 &#181;L of 1 M perchloric acid in 1.5 mL microcentrifuge tubes to stop the reactions. 
		CAUTION: Perchloric acid is a strong acid and a strong oxidant. Appropriate protective gear is required for handling this compound.</li>
		<li>Aliquot 485.5 &#181;L of M199 per reaction into a tube and keep it at 37 &#176;C to dilute the radioactive BSA: palmitic acid complex prepared at steps 2.1.4-2.1.6&#160;before starting the reactions.</li>
		<li>Dispense 750 &#181;L of M199 in as many 14 mL round-bottom tubes as the samples. If desired, add inhibitors of fatty acid &#946;-oxidation, such as etomoxir, rotenone, and antimycin, including a vehicle control (Table 2).</li>
		<li>During the hepatocyte wash steps, 10-15 min before starting the reactions, transfer the tubes prepared at step 2.1.9&#160;to a shaking water bath set to 37 &#176;C and shaking at 180-200 rpm.</li>
	</ol>
	</li>
	<li>Starting, stopping, and analyzing the fatty acid &#946;-oxidation reactions
	<ol>
		<li>If the viability of the hepatocytes is acceptable (typically &#8805; 75%, Figure 2), for each reaction, transfer 0.8 &#181;L of [1-14C]palmitic acid (0.5 mCi/mL) to the microcentrifuge tube containing the clarified BSA: palmitic acid solution (steps 2.1.4-2.1.6). Vortex and return to the water bath at 41 &#176;C.</li>
		<li>To equilibrate the hepatocytes to 37 &#176;C and to pre-incubate them with inhibitors (if present), immediately after the final hepatocyte resuspension (step 1.2.21), transfer 750 &#181;L of the hepatocyte suspension with a 1 mL pipette to each of the 14 mL round-bottom tubes in the shaking water bath (steps 2.1.9-2.1.10) and vortex briefly at low speed to mix.</li>
		<li>Stagger each addition by 30 s and incubate for 15 min. To save a sample for protein determination, transfer another aliquot of hepatocytes to a 1.5 mL microcentrifuge tube and spin at 3,000 x g for 5 min. 
		NOTE: While dispensing, the hepatocyte suspension needs to be continuously swirled or gently stirred with the dispensing 1 mL pipette to prevent settling and large variability in cell number across samples.</li>
		<li>Remove the supernatant and store the pellet at -80 &#176;C until ready to measure the total amount of protein in the sample to normalize the results (Figure 3).</li>
		<li>While the hepatocytes are under pre-incubation at 37 &#176;C, add the radioactive BSA: palmitic acid complex to the warm medium in 2.1.8, and keep at 37 &#176;C until ready to start the reactions. This is the final substrate mix.</li>
		<li>To start the reactions, remove the hepatocytes from the water bath and add 500 &#181;L of substrate mix.</li>
		<li>Vortex at low speed for 5 s to completely resuspend the cells and return to the water bath. Repeat with all the samples, staggering by 30 s.</li>
		<li>Incubate for 15 min. Start a set of reactions and immediately stop (see steps 2.2.10-2.2.11) to determine the background radioactivity (Table 2).</li>
		<li>Transfer duplicate aliquots (200-250 &#181;L) of the leftover substrate mix to 6 mL scintillation vials and set aside to count. Use these counts to calculate the radioactivity corresponding to the total nmoles of palmitic acid available for oxidation in 500 &#181;L of substrate mix.</li>
		<li>To stop the reactions, remove the hepatocytes from the water bath, resuspend the hepatocytes by vortexing at moderate speed, and then transfer 400 &#181;L of the hepatocyte suspension to the microcentrifuge tubes containing perchloric acid.</li>
		<li>Immediately cap the tubes. Repeat this sequence for all the samples, staggering by 30 s.</li>
		<li>Vigorously vortex the 1.5 mL microcentrifuge tubes and spin them&#160;down the 1.5 mL microcentrifuge tubes at 13,000 x g for 10 min.</li>
		<li>Transfer 300 &#181;L of the supernatant to a 6 mL scintillation vial, add 4 mL of scintillation fluid, and count the radioactivity in the samples and the substrate mix aliquots (step 2.2.9) in a scintillation counter. 
		CAUTION: After centrifuging, open the tubes under a fume hood to avoid breathing the 14C-CO2 produced by the complete oxidation of 14C-acetyl-CoA generated by fatty acid &#946;-oxidation and released by the acidic conditions.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers/Media Components</td>
			<td>Amount&#160;</td>
			<td>Final Concentration</td>
			<td>Instructions</td>
		</tr>
		<tr>
			<td colspan="4">Solution C</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&#160;1.79 g</td>
			<td>480 mM</td>
			<td rowspan="3">Add water to 50 mL.&#160; Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>MgSO4 heptahydrate</td>
			<td>&#160;1.48 g</td>
			<td>120 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>&#160;0.81 g</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td colspan="4">Krebs-Henseleit Buffer (KHB), calcium-free</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>7.0 g</td>
			<td>&#160;120 mM</td>
			<td rowspan="5">Add water to 900 mL, adjust the pH to 7.4, and bring the final volume to 1 L. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.0 g</td>
			<td>&#160;24 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES pH 7.45</td>
			<td>5 mL</td>
			<td>&#160;5 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1 or 2 g</td>
			<td>&#160;5.6 or 11 mM</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td>10 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Buffer 1</td>
		</tr>
		<tr>
			<td>KHB</td>
			<td>500 mL</td>
			<td></td>
			<td rowspan="2">Mix components and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>50 mM EGTA</td>
			<td>1.0 mL</td>
			<td>&#160;0.1 mM</td>
		</tr>
		<tr>
			<td colspan="4">Buffer 2</td>
		</tr>
		<tr>
			<td>KHB</td>
			<td>500 mL</td>
			<td></td>
			<td rowspan="2">Mix components and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>1 M CaCl2 dihydrate</td>
			<td>686 &#181;L</td>
			<td>&#160;1.4 mM</td>
		</tr>
		<tr>
			<td colspan="4">Gentamicin solution</td>
		</tr>
		<tr>
			<td>Gentamicin sulphate</td>
			<td>0.5 g</td>
			<td>50 mg/mL</td>
			<td>Add water to 10 mL and filter sterilize. Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">Collagenase solution</td>
		</tr>
		<tr>
			<td>Collagenase I and II blend</td>
			<td>10 mg</td>
			<td>7 mg/mL</td>
			<td>Dissolve the entire content of the vial in 1.43 mL of water.&#160; Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">M199</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>1 pouch</td>
			<td></td>
			<td rowspan="4">Add water to 900 mL and adjust the pH to 7.2-7.4. Bring the final volume to&#160; 1 L and filter sterilize. Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.2 g</td>
			<td>&#160;26 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES (cell culture grade)</td>
			<td>25 mL</td>
			<td>&#160;25 mM</td>
		</tr>
		<tr>
			<td>Extra glucose (only for fed mice)</td>
			<td>1 g</td>
			<td>&#160;11 mM</td>
		</tr>
		<tr>
			<td colspan="4">BSA solution</td>
		</tr>
		<tr>
			<td>Fatty acid-free BSA</td>
			<td>400 mg</td>
			<td>20% (w/v)</td>
			<td>Dissolve in 2 mL of water.&#160; Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">Non-radioactive palmitic acid solution</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>103 mg</td>
			<td>200 mM</td>
			<td>Dissolve in 2 mL of ethanol, store at -20 &#176;C</td>
		</tr>
		<tr>
			<td colspan="4">1 M Perchloric acid</td>
		</tr>
		<tr>
			<td>70% Perchloric acid</td>
			<td>3.5 mL</td>
			<td>1 M</td>
			<td>Dilute to 40 mL with water. Store at room temperature&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers, media, and other solutions required for the hepatocyte isolation and the fatty acid &#946;-oxidation assay
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Reaction number</td>
			<td colspan="2">M199 &#177; Inhibitors</td>
			<td></td>
			<td rowspan="2">Hepatocyte suspension (&#181;L)</td>
			<td></td>
			<td rowspan="2">Substrate mix (&#181;L)</td>
			<td></td>
		</tr>
		<tr>
			<td>Volume (&#181;L)</td>
			<td>Etomoxir</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1</td>
			<td rowspan="9">750</td>
			<td rowspan="3">-</td>
			<td rowspan="9">Pre-warm at 37 &#176;C</td>
			<td rowspan="9">750</td>
			<td rowspan="9">Pre-incubate at 37 &#176;C for 15 min</td>
			<td rowspan="9">500</td>
			<td rowspan="6">Incubate at 37 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
		</tr>
		<tr>
			<td>4</td>
			<td rowspan="3">+</td>
		</tr>
		<tr>
			<td>5</td>
		</tr>
		<tr>
			<td>6</td>
		</tr>
		<tr>
			<td>7</td>
			<td rowspan="3">+</td>
			<td rowspan="3">Stop immediately</td>
		</tr>
		<tr>
			<td>8</td>
		</tr>
		<tr>
			<td>9</td>
		</tr>
	</tbody>
</table>
Table 2: Example of the experimental setup for a hepatocyte suspension assayed in triplicate in the presence and absence of etomoxir.

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(79), e50615 (2013).</li><li>Lilly, K., Chung, C., Kerner, J., VanRenterghem, R., Bieber, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+etomoxiryl-CoA+on+different+carnitine+acyltransferases.">Effect of etomoxiryl-CoA on different carnitine acyltransferases.</a> Biochemical Pharmacology. 43 (2), 353-361 (1992).</li><li>Yu, X. X., Drackley, J. K., Odle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+mitochondrial+and+peroxisomal+beta-oxidation+of+palmitate+change+during+postnatal+development+and+food+deprivation+in+liver,+kidney+and+heart+of+pigs.">Rates of mitochondrial and peroxisomal beta-oxidation of palmitate change during postnatal development and food deprivation in liver, kidney and heart of pigs.</a> Journal of Nutrition. 127 (9), 1814-1821 (1997).</li><li>Yu, X. X., Drackley, J. K., Odle, J., Lin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+hepatic+mitochondrial+and+peroxisomal+beta-oxidation+to+increasing+palmitate+concentrations+in+piglets.">Response of hepatic mitochondrial and peroxisomal beta-oxidation to increasing palmitate concentrations in piglets.</a> Biology of the Neonate. 72 (5), 284-292 (1997).</li><li>Veerkamp, J. H., van Moerkerk, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+fatty+acid+oxidation+in+rat+and+human+tissues.+Effect+of+nutritional+state,+clofibrate+treatment+and+postnatal+development+in+the+rat.">Peroxisomal fatty acid oxidation in rat and human tissues. Effect of nutritional state, clofibrate treatment and postnatal development in the rat.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 875 (2), 301-310 (1986).</li><li>Hakvoort, T. B., 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=Interorgan+coordination+of+the+murine+adaptive+response+to+fasting.">Interorgan coordination of the murine adaptive response to fasting.</a> Journal of Biological Chemistry. 286 (18), 16332-16343 (2011).</li><li>Sokolovic, 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=The+transcriptomic+signature+of+fasting+murine+liver.">The transcriptomic signature of fasting murine liver.</a> BMC Genomics. 9, 528 (2008).</li><li>Kersten, S., 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=Peroxisome+proliferator-activated+receptor+alpha+mediates+the+adaptive+response+to+fasting.">Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.</a> Journal of Clinical Investigation. 103 (11), 1489-1498 (1999).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Ng, I. C., 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=Isolation+of+Primary+Rat+Hepatocytes+with+Multiparameter+Perfusion+Control.">Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control.</a> Journal of Visualized Experiments: JoVE. (170), e62289 (2021).</li><li>Shen, L., Hillebrand, A., Wang, D. Q., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> Journal of Visualized Experiments: JoVE. (64), e3917 (2012).</li><li>Fulgencio, J. P., Kohl, C., Girard, J., Pegorier, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metformin+on+fatty+acid+and+glucose+metabolism+in+freshly+isolated+hepatocytes+and+on+specific+gene+expression+in+cultured+hepatocytes.">Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.</a> Biochemical Pharmacology. 62 (4), 439-446 (2001).</li><li>Leonardi, R., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pank1+deletion+in+leptin-deficient+mice+reduces+hyperglycaemia+and+hyperinsulinaemia+and+modifies+global+metabolism+without+affecting+insulin+resistance.">Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.</a> Diabetologia. 57 (7), 1466-1475 (2014).</li><li>Bougarne, N., 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=PPARalpha+blocks+glucocorticoid+receptor+alpha-mediated+transactivation+but+cooperates+with+the+activated+glucocorticoid+receptor+alpha+for+transrepression+on+NF-kappaB.">PPARalpha blocks glucocorticoid receptor alpha-mediated transactivation but cooperates with the activated glucocorticoid receptor alpha for transrepression on NF-kappaB.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (18), 7397-7402 (2009).</li><li>Korelova, K., Jirouskova, M., Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+3D+collagen+sandwich+culture+of+primary+mouse+hepatocytes+to+study+the+role+of+cytoskeleton+in+bile+canalicular+formation+in+vitro.">Isolation and 3D collagen sandwich culture of primary mouse hepatocytes to study the role of cytoskeleton in bile canalicular formation in vitro.</a> Journal of Visualized Experiments: JoVE. (154), e60507 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

During the liver perfusion, it is critical to avoid the introduction of air bubbles, as they block the microcapillaries in the liver, preventing or restricting the buffer circulation and overall decreasing the hepatocyte yield and viability20,21. Precautions, such as closely inspecting the buffer-filled inlet line before cannulation of the IVC and avoiding lifting the inlet line off the tube containing Buffer 1 to switch to Buffer 2, as described herein, can successfully decrease the number of failed perfusions (viability &#60;70%). The use of bubble traps in the perfusion system can also significantly reduce this risk20,21.
The collagenase activity is another critical parameter for the isolation of hepatocytes, historically requiring testing and optimization of each new batch acquired12,20. The use of a highly purified and defined blend of collagenases dramatically reduces the batch-to-batch variability, eliminating the need to test each new batch. Furthermore, when these blends are used, small adjustments in the volume used (&#177;10-20 &#181;L) are usually sufficient to restore high yields or viability of the hepatocyte preparations.
Hepatocytes are delicate cells. All resuspension and dispensing steps should be done gently by swirling or pipetting slowly to reduce shear damage and lysis. The use of wide-bore tips can also further minimize hepatocyte damage. Vortexing at steps 2.2.2 and 2.2.7 of the assay should be done at the lowest setting possible that still ensures good mixing of the components.
Compared to traditional protocols20,21,22, one of the major changes introduced in the hepatocyte isolation procedure described here is the replacement of the intravenous catheter insertion and ligation with the insertion of a hypodermic needle held in position by the operator. This modification provides two main advantages. First, it decreases the risk of introducing air bubbles when connecting the end of the intravenous catheter to the line, as the buffer is already flowing when the hypodermic needle is inserted in the IVC. Second, it decreases the risk of perforating the IVC during manipulations of the catheter, such as the retraction of the needle or its securing with sutures. One of the drawbacks of this modification is that it is usually necessary to hold the needle in position by hand for the duration of the perfusion to ensure the consistent success of the procedure. This can be tiring for the person performing the surgery and could limit the number of consecutive perfusions that can be done in a session. To limit the movements of the person holding the needle, which could cause inadvertent perforation of the IVC, it is advisable to work in pairs, with one person conducting the surgery and another person changing the perfusion buffers without interruptions in the perfusion. Multiple back-to-back perfusions would require a third person to start the fatty acid &#946;-oxidation assay within 1-2 min of each completed hepatocyte isolation.
Similar to other hepatocyte isolation methods, the procedure described here yields hepatocytes that can be used in suspension or in culture to assess a variety of other liver processes, including additional metabolic pathways and changes in gene expression due to various treatments23,24,25. To culture the hepatocytes, steps 1.2.20-1.2.21 can be easily modified by resuspending the cells in the appropriate medium, followed by plating in cell culture dishes and incubation20,22,26. Furthermore, while not required for the &#946;-oxidation assay, if needed by other applications, the percentage of viable hepatocytes can be increased by removing the dead cells through a Percoll layer22,26.
In conclusion, this protocol describes a robust assay to measure the rate of fatty acid &#946;-oxidation in intact hepatocytes and without the addition of exogenous cofactors, thus preserving the endogenous regulatory mechanisms of this pathway.

Fatty acid &#946;-oxidation is an essential process in lipid metabolism, providing a catabolic pathway to balance fatty acid synthesis and intake from the diet. This process generates energy for multiple organs, including the cardiac muscle, kidney cortex, and fasted liver, and utilizes fatty acids obtained from the diet, adipose tissue lipolysis, and internal triglyceride stores1,2.
Oxidation of fatty acid through the &#946;-oxidation pathway results in the sequential shortening of the fatty acyl chain by two carbons at a time, released as acetyl-CoA, and this process occurs both in the mitochondria and the peroxisomes. While most fatty acids undergo only &#946;-oxidation, some are oxidized at different carbons before entering this pathway. For example, 3-methyl-substituted fatty acids, such as phytanic acid, undergo removal of one carbon by &#945;-oxidation in the peroxisomes before entering the &#946;-oxidation pathway. Similarly, some fatty acids are first converted to dicarboxylic fatty acids by oxidation of the terminal methyl group (&#969;-oxidation) in the endoplasmic reticulum before being preferentially oxidized in the peroxisomes by &#946;-oxidation3.
Regardless of the specific organelle, a fatty acid must first be converted to a coenzyme A (CoA) thioester, or acyl-CoA, to be oxidized through the &#946;-oxidation pathway. &#946;-Oxidation of long-chain acyl-CoAs in the mitochondrial matrix requires the carnitine shuttle for their translocation, where carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of acyl-CoAs to acylcarnitines and is the rate-limiting enzyme in this process4. Once translocated to the mitochondrial matrix, the acyl-CoAs are re-formed and serve as substrates for the mitochondrial &#946;-oxidation machinery. In the fasted state, the acetyl-CoA produced through &#946;-oxidation in hepatic mitochondria is primarily channeled to ketogenesis. Peroxisomes serve as the primary site for the &#946;-oxidation of very long-chain, branched-chain, and dicarboxylic fatty acids. Peroxisomes do not require the carnitine shuttle to import fatty acid substrates, instead&#160;importing the correspondent acyl-CoAs through the activity of the ATP-binding cassette (ABC) transporters ABCD1-35. Within the peroxisomes, acyl-CoAs are then oxidized by a dedicated set of enzymes, distinct from the mitochondrial fatty acid &#946;-oxidation machinery. Both mitochondria and peroxisomes also require a supply of NAD+ and free CoA to oxidize fatty acyl chains. CoA levels in the liver have been shown to increase in response to fasting, supporting the increased rate of fatty acid oxidation which occurs in this state6. Furthermore, increased CoA degradation in the peroxisomes results in a selective decrease in peroxisomal fatty acid oxidation7. Therefore, the process of fatty acid oxidation within the cell is regulated by the expression levels and activities of enzymes involved in the activation, transport, and oxidation of fatty acids, as well as the concentrations of cofactors and other metabolites throughout multiple subcellular compartments.
Procedures using tissue homogenates to measure fatty acid oxidation destroy the cellular architecture regulating and supporting this process, leading to a collection of data that does not accurately reflect the in vivo metabolism. While techniques using plated primary hepatocytes preserve this system, culturing isolated cells for extended periods of time results in a loss of the in vivo gene expression profile that was present in the cells when they were still living within the animal8,9. The following protocol describes a method to isolate primary hepatocytes and assay their capacity for fatty acid &#946;-oxidation immediately after isolation and in suspension, using [1-14C]palmitic acid. The assay is based on the measurement of the radioactivity associated with the acid-soluble metabolites (ASM) or products, like acetyl-CoA, produced by the &#946;-oxidation of [1-14C]palmitic acid10,11.

<table><tbody><tr><td>(R)-(+)-Etomoxir sodium salt</td><td>Tocris Bioscience</td><td>4539/10</td><td></td></tr><tr><td>[1-14C]-Palmitic acid, 50&amp;ndash;60 mCi/mmol, 0.5 mCi/mL</td><td>American Radiolabeled Chemicals</td><td>ARC 0172A</td><td></td></tr><tr><td>1 M HEPES, sterile</td><td>Corning</td><td>25060CI</td><td></td></tr><tr><td>10 &amp;micro;L disposable capillaries/pistons for positive displacement pipette</td><td>Mettler Toledo</td><td>17008604</td><td></td></tr><tr><td>1000 &amp;micro;L, 200 &amp;micro;L, and 10 &amp;micro;L pipettes and tips</td><td></td><td></td><td></td></tr><tr><td>5 mL, 10 mL, and 25 mL serological pipettes</td><td></td><td></td><td></td></tr><tr><td>50 mL sterile centrifuge tubes</td><td>CellTreat</td><td>229421</td><td></td></tr><tr><td>70% Perchloric acid</td><td>Fisher Scientific</td><td>A2296-1LB</td><td></td></tr><tr><td>BSA, fatty acid-free</td><td>Fisher Scientific</td><td>BP9704100</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; dihydrate</td><td>MilliporeSigma</td><td>223506</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>MilliporeSigma</td><td>G7021</td><td></td></tr><tr><td>EGTA</td><td>Gold Biotechnology</td><td>E-217</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco</td><td>111000200CSPP</td><td></td></tr><tr><td>Filter System, 0.22 &amp;mu;m PES Filter, 500 mL, Sterile</td><td>CellTreat</td><td>229707</td><td></td></tr><tr><td>Gentamicin sulphate</td><td>Gold Biotechnology</td><td>G-400-25</td><td></td></tr><tr><td>HDPE, 6.5 mL scintillation vials</td><td>Fisher Scientific</td><td>03-342-3</td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Hypodermic needles 22 G, 1.5 in</td><td>BD Biosciences</td><td>305156</td><td></td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>502017</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366-1</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>MilliporeSigma</td><td>P5655</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>MilliporeSigma</td><td>5401119001</td><td>Defined blend of purified collagenase I and II with a medium concentration of thermolysin</td></tr><tr><td>M199 medium</td><td>MilliporeSigma</td><td>M5017</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; heptahydrate</td><td>MilliporeSigma</td><td>M1880</td><td></td></tr><tr><td>Microcentrifuge</td><td>Fisher Scientific</td><td></td><td>accuSpin Micro 17</td></tr><tr><td>Microdissecting Scissors</td><td>Roboz Surgical Instrument Co</td><td>RS-5980</td><td></td></tr><tr><td>NaCl</td><td>Chem-Impex International</td><td>30070</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Acros Organics</td><td>424270010</td><td></td></tr><tr><td>Palmitic acid</td><td>MilliporeSigma</td><td>P0500</td><td></td></tr><tr><td>Penicillin/streptomycin (100x)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Cytiva Life Sciences</td><td>SH30256.01</td><td></td></tr><tr><td>Positive displacement pipette MR-10, 10 &amp;micro;L</td><td>Mettler Toledo</td><td>17008575</td><td></td></tr><tr><td>Refrigerated centrifuge with inserts for 50 mL conical tubes</td><td>Eppendorf</td><td></td><td>5810 R</td></tr><tr><td>Round-bottom, 14 mL, polypropylene culture test tubes</td><td>Fisher Scientific</td><td>14-956-9A</td><td></td></tr><tr><td>Scintillation counter</td><td>Perkin Elmer</td><td></td><td>TriCarb 4810 TR</td></tr><tr><td>ScintiVerse BD cocktail</td><td>Fisher Scientific</td><td>SX18-4</td><td></td></tr><tr><td>Shaking water bath, 30 L capacity</td><td>New Brunswick Scientific</td><td></td><td>&amp;nbsp;Model G76</td></tr><tr><td>Sterile cell strainers, 100 &amp;micro;m</td><td>Fisher Scientific</td><td>22363549</td><td></td></tr><tr><td>Thumb Dressing Forceps</td><td>Roboz Surgical Instrument Co</td><td>RS-8120</td><td></td></tr><tr><td>Trypan Blue</td><td>Corning</td><td>25900CI</td><td></td></tr><tr><td>Variable-flow peristaltic pump</td><td>Fisher Scientific</td><td>138762</td><td></td></tr><tr><td>Water baths, 2&amp;ndash;2.5 L capacity</td><td></td><td></td><td></td></tr></tbody></table>

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.

The liver perfusion described here typically yields 30-40 million cells/liver with average viability of 80%, as estimated by trypan blue exclusion (Figure 2). The typical concentration of glucose in the Krebs-Henseleit buffer (KHB), which is used to prepare the perfusion Buffers 1 and 2, is 11 mM. When measuring fatty acid &#946;-oxidation in hepatocytes isolated from fasted mice, the concentration of glucose in the KHB can be lowered to better represent the fasted state. As shown in Figure 2, lowering the glucose concentration to 5.6 mM has no negative effect on the yield or viability of the hepatocytes.
Table 2 shows a typical experimental setup for a hepatocyte suspension assayed in triplicate in the presence and absence of etomoxir, a potent inhibitor of CPT1 and thus, mitochondrial fatty acid oxidation10,13. In the presence of this or other inhibitors of mitochondrial fatty acid oxidation, any residual 14C-labeled products generated by [1-14C]palmitic acid oxidation can be ascribed to the first cycle of &#946;-oxidation in the peroxisomes. Thus, the contribution of mitochondrial fatty acid oxidation to total fatty acid &#946;-oxidation can be calculated as the difference between total (-etomoxir) and peroxisomal (+ etomoxir) fatty acid oxidation7,14,15 (Figure 3).
For hepatocytes, more than 95% of the radioactivity associated with the products of the &#946;-oxidation of [1-14C]palmitic acid is found in the ASM, and the rest is released as 14C-CO210. The counts per minute (CPM) associated with the background radioactivity vary with the batch of [1-14C]palmitic acid. However, they are still significantly lower than the CPM obtained in samples allowed to incubate with the substrate mix for 15 min (Figure 3A). As expected, hepatocytes isolated from fasted mice show a robust increase in the rates of both mitochondrial and peroxisomal fatty acid &#946;-oxidation, consistent with the known activation of these pathways16,17,18,19.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62904/62904fig02.jpg" /> 
Figure 2: Viability and yield of hepatocytes isolated using the procedure described herein. Hepatocytes were isolated from male mice fed ad libitum or fasted overnight for 16-18 h, with free access to water. (A) Hepatocyte viability and (B) yield per liver. Data are reported as the mean (bars) of measurements on individual hepatocyte preparations (circles) &#177; SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student&#39;s t-test. * p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62904/62904fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62904/62904fig03.jpg" /> 
Figure 3: Fatty acid &#946;-oxidation capacity in hepatocytes isolated from fed and fasted male mice and assayed in suspension. Freshly isolated hepatocytes were pre-incubated with etomoxir (45 &#181;M, +Eto) or DMSO (vehicle, -Eto) before the addition of the substrate mix. (A) Total CPM introduced in each assay and recovered in the ASM fraction of reactions set up to estimate the background radioactivity, total (-Eto), peroxisomal (+Eto), and mitochondrial fatty acid &#946;-oxidation. These data are shown before any correction (for background, cell number, or protein levels) or any other calculations were applied. (B) Data in (A) corrected for the background, the total volume of the assay, normalized to 1 million viable cells and expressed as the rate at which palmitic acid is oxidized in hepatocytes isolated from fed and fasted mice. (C) Total protein corresponding to the estimated 750,000 hepatocytes/assay used. (D) Data in (A) corrected as in (B) but normalized to mg of protein. Data are reported as the mean (bars) of measurements on individual hepatocyte preparations (circles) &#177; SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student&#39;s t-test. * p &#60; 0.05; ** p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62904/62904fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measurement of Fatty Acid Î²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes at JoVE.com

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

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 ...

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3.8K Views.  West Virginia University. Fatty acid β-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 β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

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

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Developmental Biology

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

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Biology

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 ...

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.

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 ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport&#160;chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases.
The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

This protocol describes an easy-to-use method to examine substrate oxidation by tracking 14CO2&#160;production in vitro.

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport&#160;chain (ETC), which generate adenosine triphosphate ...

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 ...

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Here, we present an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo lipogenesis in brown adipose tissue in vivo.

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic ...

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

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Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Assessing Energy Substrate Oxidation In Vitro with ¹⁴CO₂ Trapping

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

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide