This protocol was developed to provide a robust method for the measurement of total mitochondrial and peroxisomal fatty acid beta-oxidation in primary mouse hepatocytes. The use of intact cells maintains organelle integrity and the regulatory mechanisms in place. Additionally, assaying freshly isolated hepatocytes minimizes changes in gene expression with respect to the liver of origin.
The surgery can be challenging. You have to be confident, diligent, and focused. Once the cannulation is successful, try and relax, but pay attention and monitor the quality of the perfusion.
To start the procedure, liberally spray the abdomen and chest of the anesthetized mouse with 70%ethanol. Then use the forceps to 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. Expose the inferior vena cava, or 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 the cannulation. After starting the pump with buffer one at the lowest speed, insert the needle into the IVC. Then cut the portal vein to relieve the pressure and allow drainage of blood and perfusion buffers.
Before increasing the flow rate to seven mL per minute. Perfuse the liver with warm buffer one and ensure that the line inserted in the tube containing buffer one remains continuously submerged to avoid introducing air bubbles. While perfusion occurs, add 130 microliters of collagenase solution to buffer two, and mix by pipetting with a serological pipette.
As the volume in the tube containing buffer one decreases to about five mL, slowly add five mL of buffer two to buffer one by pipetting on the side of the tube. Repeat the addition twice before adding the remaining buffer two to the tube. Stop the perfusion when about 5 to 10 mL of buffer two are left in the tube.
Then excise the liver and transfer it to the 100-mL culture dish containing 20 mL of ice-cold buffer two. Under the laminar flow hood, gently break the liver tissue apart using surgical scissors and tweezers. Next add about 20 mL of ice-cold M199 buffer to the hepatocytes suspension and filter it through a 100-micron cell strainer using the plunger of a syringe to gently promote the release of additional hepatocytes from the larger liver pieces.
Wash the 100-mL culture dish and the cell strainer with M199 buffer to collect the cell suspension until the collection tube is full. Then centrifuge the suspension at 50 x g for two minutes at four degrees Celsius. Aspirate the supernatant before resuspending the hepatocyte pellet in 30 mL of cold M199 by swirling.
Repeat the wash once. Thaw the palmitic acid and bovine serum albumin, or BSA, solutions before preparing the substrate mixture for multiple reactions. Aliquot 13.5 microliters of BSA solution per reaction in a microcentrifuge tube.
After warming the tube to 41 degrees Celsius, add one microliter of the 200 mM palmitic acid solution per reaction. Vortex the tube vigorously before and during incubation at 41 degrees Celsius for 20 to 30 minutes to facilitate the formation of the soluble palmitic acid-BSA complex. During this time, aliquot 133 microliters of 1 M of perchloric acid which will be used to stop the reactions in separate 1.5-mL microcentrifuge tubes.
Before starting the reactions, aliquot and incubate 485.5 microliters of M199 buffer per reaction into a tube at 37 degrees Celsius to dilute the prepared radioactive BSA-palmitic acid complex. Next dispense 750 microliters of M199 with or without inhibitors in the separate 14-mL round bottom tubes as the samples. During the hepatocyte wash steps, 10 to 15 minutes before starting the reactions, transfer the tubes to a shaking water bath set to 37 degrees Celsius at 180 to 200 rotations per minute.
If the viability of the hepatocytes is at least 75%complete the preparation of the substrate mix by transferring 0.8 microliters of carbon-14 palmitic acid per reaction to the microcentrifuge tube containing the clarified BSA-palmitic acid solution. Vortex before returning the tube to the water bath at 41 degrees Celsius. Immediately after the final hepatocyte resuspension, transfer 750 microliters of the hepatocyte suspension to each of the 14-mL round bottom tubes in the shaking water bath using a one mL pipette and vortex the tubes briefly at the low speed before transferring to an incubator, staggering each addition by 30 seconds.
Transfer a separate aliquot of hepatocytes to a 1.5-mL microcentrifuge tube to spin at 3000 x g for five minutes. Remove the supernatant before storing the pellet at minus 80 degrees Celsius to measure the total amount of protein in the sample to normalize the results. While the hepatocytes are under pre-incubation at 37 degrees Celsius, add the radioactive BSA-palmitic acid complex to the warm medium to keep at 37 degrees Celsius until ready to start the reactions.
To start the reactions, remove the hepatocytes from the water bath and add 500 microliters of the substrate mix to the hepatocytes. Vortex the cells at a low speed for five seconds before returning to the water bath to incubate for 15 minutes. Start a set of reactions and immediately stop to determine the background radioactivity.
Transfer and set aside the duplicate aliquots of 200 to 250 microliters of the leftover substrate mix in 6-mL scintillation vials to perform the counting. To stop the reactions, remove the hepatocytes from the water bath to resuspend by vortexing at a moderate speed and then transfer 400 microliters of the hepatocyte suspension to the microcentrifuge tubes containing perchloric acid. Immediately cap and vortex the tubes before repeating the sequence for all the samples, staggering by 30 seconds.
After spinning down the 1.5-mL microcentrifuge tubes at 13, 000 x g for 10 minutes, transfer 300 microliters of the supernatant to a 6-mL scintillation vial and add 4 mL of the scintillation fluid to count the radioactivity in the samples and the substrate mix aliquots in a scintillation counter. In the study, the liver perfusion yielded 30 to 40 million cells per liver, with average viability of 80%It was also observed that lowering the glucose concentration in the fasting group had no negative effect on the yield or viability of the hepatocytes. The hepatocyte suspension, isolated from fed and fasted male mice, were assayed for fatty acid beta-oxidation capacity in the presence and absence of etomoxir, a potent inhibitor of CPT1 and mitochondrial fatty acid oxidation.
The counts per minute, or CPM, associated with the background radioactivity vary with the batch of palmitic acid. However, CPM was still significantly lower than the samples incubated with the substrate mix. The hepatocytes isolated from the fasted mice show a robust increase in the rates of both mitochondrial and peroxisomal fatty acid beta-oxidation.
The data was further studied to determine the rate at which palmitic acid is oxidized in the hepatocytes isolated from the fed and fasted mice. Prevent bubbles from entering the line. Keep the needle steady during the perfusion.
Be gentle when handling the hepatocytes. Keep them in suspension when dispensing them. The hepatocytes isolated from this procedure can be used as a suspension to assay other metabolic pathways, such as fatty acid synthesis, or they can be plated for cell culture experiments.
