Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

The overall goal of this procedure is to assess fatty acid metabolism in primary mouse hepatocytes. This is accomplished by first perfusing, a mouse liver with digestion, medium, removing it, and then isolating the hepatocytes. The second step is to culture hepatocytes on collagen coated plates.

Next, the hepatocytes are treated with the experimental interventions to be tested. The final step is to incubate the cells in the presence of a radio labeled substrate. Ultimately, liquid scintillation analysis is used to show effects of various pharmacologic or genetic interventions on either fatty acid oxidation or lipogenesis in primary hepatocytes.

This method can help answer several key questions in the field of metabolism and diabetes, including how specific pharmacologic and genetic interventions affect hepatocyte biology. Visual demonstration of this method is critical for two reasons. One, specific perfusion conditions that affect hepatocyte health are challenging to obtain, and two, quantification of release carbon dioxide.

Gas is not intuitive. Demonstrating this procedure will be Tom Akey, a graduate student from my laboratory. Set up the peristaltic pump by placing one end of sterile tubing into prewarm liver perfusion, medium, and the other into a waste container.

Run the pump until the entire length of tubing is filled with medium. Following the sacrifice of a mouse of interest, spray the mouse abdomen liberally with 70%ethanol. Then use blunt end scissors to make a midline incision through the dermis, the length of the abdomen, and reflect laterally.

Make a similar incision in the peritoneum to expose the viscera. Using a blunt instrument, gently displace the intestines. To expose the abdominal vasculature, locate the abdominal inferior vena cava and place a suture underneath the blood vessel distal to the renal vein, distal to the suture.

Place a needle and catheter into the inferior vena cava and advance it beyond the suture. With the needle still in place, tie the suture around the catheter to hold it in place and then carefully remove the needle. If done correctly, blood will flow through the catheter using a pipette.

Fill the remaining area in the catheter with perfusion medium and ensure that no air is present. Then with great care, attach the tubing to the catheter. Next, carefully puncture the diaphragm with sharp tip scissors and make a lateral incision to expose the pleural cavity.

Taking care to avoid the gallbladder and pleural vasculature, place a bulldog clamp around the thoracic inferior vena cava just proximal to the hepatic vein. Then cut the portal vein and turn on the pump at three to four milliliters per minute. Perfuse the liver with liver perfusion medium for five minutes using approximately 20 milliliters of perfusion medium.

During this time, observe the immediate color change of the liver from red to a gray tan color. If portions of the liver remain red, this like indicates poor perfusion and the likelihood of successful isolation is greatly diminished. Following the five minute perfusion, stop the pump and transfer the tubing to liver digest medium.

Restart the pump and perfuse the liver for another 10 to 15 minutes. At three to four milliliters per minute until the medium is exhausted. At the end of the perfusion, stop the pump.

The liver should have a pinkish hue and appear somewhat enlarged. At this point, excise the liver by careful dissection. Transfer to a 10 centimeter tissue culture dish and remove the gallbladder.

Next, add 10 milliliters of plating medium to the liver, and begin to gently scrape the liver using a scalpel. To remove the hepatocytes, filter the suspension with a 100 micron cell strainer and transfer it to a 50 milliliter conical tube. Wash the plate with an additional 10 milliliters of plating medium and pull the liquid in the 50 milliliter conical tube.

Then pellet the cells at four degrees Celsius, centrifusion for five minutes At 350 Gs, aspirate the medium and resuspend the cell pellet in 10 milliliters of plating medium. Next, add 10 milliliters of 90%colloidal silica coated with polyvinyl perone mixed gently, and then pellet the cells as before. After centrifugation, aspirate the layer of dead cells floating on the top of the mixture.

Then wash the live pelleted cells twice with 20 milliliters of plating. Medium resus. Suspend the final cell pellet in 10 milliliters of plating medium and count the cells using a hemo cytometer place 90, 000 cells per well into collagen coated 24 Well culture dishes incubate the plates at 37 degrees Celsius and 5%carbon dioxide for two hours, 16 to 20 hours prior to beginning the assay.

Wash cells two times with warm PBS and change the cells to serum free serum starvation. Medium with 20 nanomoles of glucagon. Incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide on the morning of the assay.

Prepare pre incubation medium and then change out serum starvation. Medium for the pre incubation medium. Incubate the cells in this mixture at 37 degrees Celsius and 5%carbon dioxide for two hours during the incubation.

Dry 0.5 micro curies per well of C 14 palmitate by evaporating the ethanol solvent under nitrogen approximately 15 minutes before the end of the incubation resuspend the C 14 palmitate at 12.5 microliters per micro curie in 0.1. Normal sodium hydroxide incubate the mixture at 70 degrees Celsius for 10 minutes. Then add three volumes of warm pre incubation, medium, and mix by pipetting up and down.

Spike each well with 25 microliters of diluted C 14 palmitate mix by gently rocking the plate and incubate at 37 degrees Celsius for 90 minutes. This is the assay plate During the incubation, remove the cover from a sterile 24 well plate and place one two centimeter by two centimeter piece of filter paper in the bottom of each of the wells. Then overlay the plate with a piece of param measuring four inches by seven inches using a large rectangular object such as a micro pipetter tip box.

Rub the paraform on the wells to perforate the paraform at the well openings and create a seal over the remainder of the plate. Then remove the perforated circles of parfum. Now covering the wells 10 minutes before the end of the incubation, add 200 microliters of three normal sodium hydroxide to each well of the filter paper plate, making sure the filter paper absorbs all of the liquid.

At the end of the incubation snap freeze the assay plate in liquid nitrogen. Next, add 100 microliters of 70%per caloric acid to each well of the assay plate. Immediately cover with a filter paper plate and place the plates on an orbital shaker.

Rock the plates at an orbital speed of 80 RPM at room temperature for two hours. Then measure the carbon dioxide fraction by transferring the filter paper squares into four milliliters of liquid scintillation fluid in scintillation vials, and measure the C 14 signal on a scintillation counter to measure the acid soluble material. Transfer 400 microliters of medium to a 1.5 milliliter micro fuge tube centrifuge at maximum speed for 10 minutes, and then add 100 microliters of the resulting supernatant to 500 microliters of a two to one mixture of chloroform and methanol.

Vortex the mixture briefly and then add 250 microliters of water to the mixture and vortex. Again, centrifuge samples for 10 minutes at the thousand Gs.Then transfer 200 microliters of the upper phase to four milliliters of liquid scintillation fluid in a scintillation vial, and measure the C 14 signal following serum starving of cells. Change the cells into lipogenesis medium.

Add any compounds of interest to be tested, and then incubate the cells at 37 degrees Celsius and 5%carbon dioxide for two hours after the incubation period. Wash the cells two times with PBS before lysing the cells by adding 120 microliters of 0.1 normal hydrochloric acid. To each, well scrape the wells to release the lysate and then pipette 100 microliters of the lysate into a 1.5 milliliter micro fuge tube.

Next, add 500 microliters of a two to one mixture of chloroform and methanol to each tube. Vortex briefly, and then incubate at room temperature for five minutes. After incubation at 250 microliters of water vortex, and then incubate the sample for an additional five minutes at room temperature.

Then centrifuge the samples for 10 minutes at 3000 Gs.Carefully transfer the lower phase of the separated liquid into four milliliters of liquid scintillation fluid in a scintillation vial, and measure the tritium activity using a scintillation counter. This procedure typically results in the isolation of 10 to 30 million hepatocytes per mouse. After overnight incubation, healthy cells appear hexagonal, many of which will be nucleated.

Unhealthy cultures often contain granulations or blebs, which are indicative of cell death. Results of a fatty acid oxidation assay are shown here where the addition of F-C-C-P-A cellular respiration promoter and oligo mycin, a respiration inhibitor were compared with a vehicle control to measure complete oxidation. The ratio of carbon dioxide to acid soluble material is calculated.

Substances that promote cellular respiration such as FCCP will shift this ratio towards carbon dioxide indicating more oxidation via the TC.A cycle inhibitors of the respiratory chain will diminish oxidation on the whole when measured. For lipo activity compounds, which enhance fatty acid oxidation such as FCCP or diminish a TP synthesis, such as oligo mycin, will reduce lipo activity in cultured hepatocytes when compared with vehicle controls. While attempting this procedure, it's important to ensure profusion occurs completely and in a timely manner following animal sacrifice.

Additionally, during the fatty acid oxidation assay, complete freezing of the medium is critical for reproducible measurements of carbon dioxide gas. After watching this video, you should have a good understanding of how to isolate primary hepatocytes as well as use these cells to analyze fatty acid oxidation and lipogenesis in vitro.