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.
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.
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 stores1,2.
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 animal8,9. 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 acid10,11.
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
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. 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.
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.
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 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 β-oxidation in the peroxisomes. Thus, the contribution of mitochondrial fatty acid oxidation to total fatty acid β-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 β-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 β-oxidation, consistent with the known activation of these pathways16,17,18,19.
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) ± SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student's t-test. * p < 0.05. Please click here to view a larger version of this figure.
Figure 3: Fatty acid β-oxidation capacity in hepatocytes isolated from fed and fasted male mice and assayed in suspension. Freshly isolated hepatocytes were pre-incubated with etomoxir (45 µM, +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 β-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) ± SEM. Hepatocytes isolated from fed and fasted mice were compared using an unpaired two-tailed Student's t-test. * p < 0.05; ** p < 0.01. Please click here to view a larger version of this figure.
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 <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 (±10-20 µL) 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 β-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 β-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 β-oxidation in intact hepatocytes and without the addition of exogenous cofactors, thus preserving the endogenous regulatory mechanisms of this pathway.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health grant R35GM119528 to Roberta Leonardi.
(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 |