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

Podsumowanie

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.

Streszczenie

Fatty acid β-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 β-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 β-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 β-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid β-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 β-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Wprowadzenie

Fatty acid β-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 stores^1,2.

Oxidation of fatty acid through the β-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 β-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 α-oxidation in the peroxisomes before entering the β-oxidation pathway. Similarly, some fatty acids are first converted to dicarboxylic fatty acids by oxidation of the terminal methyl group (ω-oxidation) in the endoplasmic reticulum before being preferentially oxidized in the peroxisomes by β-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 β-oxidation pathway. β-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 β-oxidation machinery. In the fasted state, the acetyl-CoA produced through β-oxidation in hepatic mitochondria is primarily channeled to ketogenesis. Peroxisomes serve as the primary site for the β-oxidation of very long-chain, branched-chain, and dicarboxylic fatty acids. Peroxisomes do not require the carnitine shuttle to import fatty acid substrates, instead 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 β-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 animal^8,9. The following protocol describes a method to isolate primary hepatocytes and assay their capacity for fatty acid β-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 β-oxidation of [1-¹⁴C]palmitic acid^10,11.

Protokół

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

1. Preparation
   1. 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 °C close to where the surgery will be performed.
   2. 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.
   3. Add antibiotics gentamicin (50 µg/mL) and penicillin/streptomycin (1x) to both buffers.
   4. Transfer 20 mL of Buffer 2 as prepared in step 1.1.3 to a 100 mm cell culture dish and place on ice.
   5. 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 °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.
   6. 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.
   7. 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.
   8. 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.

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. Please click here to view a larger version of this figure.

2. Liver perfusion and dissociation
   1. 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.
   2. 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.
   3. Liberally spray the abdomen and chest of the mouse with 70% ethanol.
   4. 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.
   5. 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).
   6. Start the pump at the lowest speed and, with Buffer 1 flowing, insert the needle into the IVC.
   7. 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).
   8. For more consistent results, hold the needle in position by hand for the entire duration of the perfusion.
   9. 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.
   10. While perfusion occurs, add 130 µL of collagenase solution to Buffer 2 and mix by pipetting up and down or stirring with a 5 mL or 10 mL serological pipette.
   11. 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.
   12. 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.
   13. 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.
   14. 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.
   15. Under the laminar flow hood, gently break the liver apart using surgical scissors and tweezers.
   16. Add about 20 mL of ice-cold M199 to the hepatocyte suspension and filter it through a 100 µm cell strainer using the plunger of a syringe to gently promote the release of additional hepatocytes from larger liver pieces.
   17. Wash the 100 mm culture dish and the cell strainer with additional M199 until the collection tube is full.
   18. Centrifuge the suspension at 50 x g for 2 min at 4 °C. Carefully aspirate the supernatant and gently resuspend the hepatocyte pellet in 30 mL of cold M199 by swirling.
   19. Pellet the hepatocytes as mentioned in step 1.2.18. Repeat the wash one more time.
   20. Resuspend the hepatocytes in 10 mL of warm M199 and determine the viability and yield using the trypan blue exclusion method and a hemocytometer¹².
   21. Dilute the cells in M199 warmed to 37 ºC to a final concentration of 1.0 x 10⁶ viable cells/mL and immediately start the assay.

2. Fatty acid β-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 µM palmitic acid, and 0.4 µCi [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.

1. Preparation
   1. In the days before the assay, prepare the palmitic acid and BSA solutions (Table 1) and store them at -20 °C.
   2. On the day of the assay, complete steps 2.1.3-2.1.9 before starting the liver perfusion.
   3. 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.
   4. Aliquot 13.5 µL of BSA solution per reaction in a microcentrifuge tube and warm to 41 °C, then add 1 µL 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.
   5. Vortex vigorously and incubate at 41 °C to facilitate the formation of the soluble palmitic acid: BSA complex. Vortex occasionally during the incubation period.
   6. The mixture will initially appear cloudy but will clarify completely after 20-30 min of incubation at 41 °C. Keep it at 41 °C until ready to start the reactions.
   7. Aliquot 133 µL 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.
   8. Aliquot 485.5 µL of M199 per reaction into a tube and keep it at 37 °C to dilute the radioactive BSA: palmitic acid complex prepared at steps 2.1.4-2.1.6 before starting the reactions.
   9. Dispense 750 µL of M199 in as many 14 mL round-bottom tubes as the samples. If desired, add inhibitors of fatty acid β-oxidation, such as etomoxir, rotenone, and antimycin, including a vehicle control (Table 2).
   10. During the hepatocyte wash steps, 10-15 min before starting the reactions, transfer the tubes prepared at step 2.1.9 to a shaking water bath set to 37 °C and shaking at 180-200 rpm.
2. Starting, stopping, and analyzing the fatty acid β-oxidation reactions
   1. If the viability of the hepatocytes is acceptable (typically ≥ 75%, Figure 2), for each reaction, transfer 0.8 µL 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 °C.
   2. To equilibrate the hepatocytes to 37 °C and to pre-incubate them with inhibitors (if present), immediately after the final hepatocyte resuspension (step 1.2.21), transfer 750 µL 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.
   3. 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.
   4. Remove the supernatant and store the pellet at -80 °C until ready to measure the total amount of protein in the sample to normalize the results (Figure 3).
   5. While the hepatocytes are under pre-incubation at 37 °C, add the radioactive BSA: palmitic acid complex to the warm medium in 2.1.8, and keep at 37 °C until ready to start the reactions. This is the final substrate mix.
   6. To start the reactions, remove the hepatocytes from the water bath and add 500 µL of substrate mix.
   7. 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.
   8. 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).
   9. Transfer duplicate aliquots (200-250 µL) 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 µL of substrate mix.
   10. To stop the reactions, remove the hepatocytes from the water bath, resuspend the hepatocytes by vortexing at moderate speed, and then transfer 400 µL of the hepatocyte suspension to the microcentrifuge tubes containing perchloric acid.
   11. Immediately cap the tubes. Repeat this sequence for all the samples, staggering by 30 s.
   12. Vigorously vortex the 1.5 mL microcentrifuge tubes and spin them down the 1.5 mL microcentrifuge tubes at 13,000 x g for 10 min.
   13. Transfer 300 µL 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 β-oxidation and released by the acidic conditions.

Buffers/Media Components	Amount	Final Concentration	Instructions
Solution C
KCl	1.79 g	480 mM	Add water to 50 mL.  Store at 4 °C
MgSO4 heptahydrate	1.48 g	120 mM
KH2PO4	0.81 g	119 mM
Krebs-Henseleit Buffer (KHB), calcium-free
NaCl	7.0 g	120 mM	Add water to 900 mL, adjust the pH to 7.4, and bring the final volume to 1 L. Store at 4 °C
NaHCO3	2.0 g	24 mM
1 M HEPES pH 7.45	5 mL	5 mM
Glucose	1 or 2 g	5.6 or 11 mM
Solution C	10 mL
Buffer 1
KHB	500 mL		Mix components and filter sterilize. Store at 4 °C
50 mM EGTA	1.0 mL	0.1 mM
Buffer 2
KHB	500 mL		Mix components and filter sterilize. Store at 4 °C
1 M CaCl2 dihydrate	686 µL	1.4 mM
Gentamicin solution
Gentamicin sulphate	0.5 g	50 mg/mL	Add water to 10 mL and filter sterilize. Aliquot and store at -20 °C
Collagenase solution
Collagenase I and II blend	10 mg	7 mg/mL	Dissolve the entire content of the vial in 1.43 mL of water.  Aliquot and store at -20 °C
M199
M199	1 pouch		Add water to 900 mL and adjust the pH to 7.2-7.4. Bring the final volume to  1 L and filter sterilize. Store at 4 °C
NaHCO3	2.2 g	26 mM
1 M HEPES (cell culture grade)	25 mL	25 mM
Extra glucose (only for fed mice)	1 g	11 mM
BSA solution
Fatty acid-free BSA	400 mg	20% (w/v)	Dissolve in 2 mL of water.  Aliquot and store at -20 °C
Non-radioactive palmitic acid solution
Palmitic acid	103 mg	200 mM	Dissolve in 2 mL of ethanol, store at -20 °C
1 M Perchloric acid
70% Perchloric acid	3.5 mL	1 M	Dilute to 40 mL with water. Store at room temperature

Table 1: Buffers, media, and other solutions required for the hepatocyte isolation and the fatty acid β-oxidation assay

Reaction number	M199 ± Inhibitors			Hepatocyte suspension (µL)		Substrate mix (µL)
	Volume (µL)	Etomoxir
1	750	-	Pre-warm at 37 °C	750	Pre-incubate at 37 °C for 15 min	500	Incubate at 37 °C for 15 min
2
3
4		+
5
6
7		+					Stop immediately
8
9

Table 2: Example of the experimental setup for a hepatocyte suspension assayed in triplicate in the presence and absence of etomoxir.

Wyniki

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 β-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

Dyskusje

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 viability^20,21. 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 succ...

Materiały

Name	Company	Catalog Number	Comments
(R)-(+)-Etomoxir sodium salt	Tocris Bioscience	4539/10
[1-14C]-Palmitic acid, 50–60 mCi/mmol, 0.5 mCi/mL	American Radiolabeled Chemicals	ARC 0172A
1 M HEPES, sterile	Corning	25060CI
10 µL disposable capillaries/pistons for positive displacement pipette	Mettler Toledo	17008604
1000 µL, 200 µL, and 10 µL pipettes and tips
5 mL, 10 mL, and 25 mL serological pipettes
50 mL sterile centrifuge tubes	CellTreat	229421
70% Perchloric acid	Fisher Scientific	A2296-1LB
BSA, fatty acid-free	Fisher Scientific	BP9704100
CaCl2 dihydrate	MilliporeSigma	223506
D-(+)-Glucose	MilliporeSigma	G7021
EGTA	Gold Biotechnology	E-217
Ethanol	Pharmco	111000200CSPP
Filter System, 0.22 μm PES Filter, 500 mL, Sterile	CellTreat	229707
Gentamicin sulphate	Gold Biotechnology	G-400-25
HDPE, 6.5 mL scintillation vials	Fisher Scientific	03-342-3
Hemocytometer
Hypodermic needles 22 G, 1.5 in	BD Biosciences	305156
Isoflurane	VetOne	502017
KCl	Fisher Scientific	BP366-1
KH2PO4	MilliporeSigma	P5655
Liberase TM Research Grade	MilliporeSigma	5401119001	Defined blend of purified collagenase I and II with a medium concentration of thermolysin
M199 medium	MilliporeSigma	M5017
MgSO4 heptahydrate	MilliporeSigma	M1880
Microcentrifuge	Fisher Scientific		accuSpin Micro 17
Microdissecting Scissors	Roboz Surgical Instrument Co	RS-5980
NaCl	Chem-Impex International	30070
NaHCO3	Acros Organics	424270010
Palmitic acid	MilliporeSigma	P0500
Penicillin/streptomycin (100x)	Gibco	15140122
Phosphate buffered saline (PBS)	Cytiva Life Sciences	SH30256.01
Positive displacement pipette MR-10, 10 µL	Mettler Toledo	17008575
Refrigerated centrifuge with inserts for 50 mL conical tubes	Eppendorf		5810 R
Round-bottom, 14 mL, polypropylene culture test tubes	Fisher Scientific	14-956-9A
Scintillation counter	Perkin Elmer		TriCarb 4810 TR
ScintiVerse BD cocktail	Fisher Scientific	SX18-4
Shaking water bath, 30 L capacity	New Brunswick Scientific		Model G76
Sterile cell strainers, 100 µm	Fisher Scientific	22363549
Thumb Dressing Forceps	Roboz Surgical Instrument Co	RS-8120
Trypan Blue	Corning	25900CI
Variable-flow peristaltic pump	Fisher Scientific	138762
Water baths, 2–2.5 L capacity