Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

In our lab, we investigate how the liver responds to common as well as rare liver diseases, and how these adaptation may predispose to a pre-diabetogenic state. The liver response to changes in nutrient availability and concentrations of hormones with increased transcription of relevant genes, but importantly also with acute and non-transcriptional regulation. With the perfused liver, we can quantify all aspects of metabolism, including the release of metabolites in a minute-to-minute resolution.

Advantages of this technique compared to other in vitro models are that the liver is left in situ with an intact hepatic architecture and that the liver is viable for at least three hours, allowing each mouse liver to be its own control, vastly increasing the translational relevance. Using the mouse perfused liver system, we are able to identify and characterize novel signaling cascade of the hormone glucagon. This may be important as glucagon, as well as other hormone, may play a vital role in the dysmetabolic conditions of the liver in patients with fatty liver disease and diabetes.

To begin, place the anesthetized C57 Black 6 JRJ mouse supine on a heated operation table. Confirm the absence of reflex responses to toe pinching. Spray the mouse with 70%ethanol to prevent hair from sticking to the scissors.

Using scissors, make an abdominal incision at the base, cutting upwards on both sides to the rib cage to expose the abdominal cavity. Use a cotton swab to move the intestines to the right, exposing the portal vein. Using curved forceps, place two ligatures under the portal vein and loosely tie each ligature.

Next, insert a 0.7 millimeter catheter into the portal vein. When perforated, remove the needle from the catheter and guide the catheter through the vein until the tip of the catheter is close to the liver. Then, tighten the ligatures.

Observe the liver. Set the flow rate from the roller pump, and attach the perfusion tube. Initiate the perfusion of the liver at 37 degrees Celsius.

Once the liver turns pale, use scissors to cut the rib cage and diaphragm. Then, using fine point forceps, place ligature under the suprahepatic inferior vena cava. Next, insert a catheter into the suprahepatic inferior vena cava through the right atrium of the heart.

When perforated, remove the needle and guide the catheter through the vein closer to the liver. Then, tighten the ligature. Attach a tube for the collection of perfusion effluent and secure all tubes with waterproof tape.

Using a vessel clamp adapter, place a vessel clamp across the infrahepatic vena cava immediately above the right renal vein to prevent dead mixture. Next, increase the perfusion flow rate to 3.5 milliliters per minute. Start the timer and pressure recording.

Cover the liver with a sterile drape moistened with saline. Equilibrate for 30 minutes, and measure the volume of effluent. After 30 minutes of equilibration, initiate the experiment for the first baseline sample collection in the fraction collector.

Monitor the bubble trap regularly and refill with perfusion buffer when close to empty. Collect a buffer sample via a three-way stopcock immediately before it enters the organ and from the collecting catheter inserted in the inferior vena cava. Using a blood gas analyzer, measure the collected sample to confirm that the organ is metabolically active.

After 15 minutes of baseline perfusion, using a syringe pump, start the first stimulation by infusing a test substance through a three-way stopcock at desired flow rate. After the simulation, collect baseline samples for 20 to 30 minutes before starting the second stimulation. The production of urea in the perfused liver with steady baseline periods preceding each of two stimulation periods suggested that the response of urea to two consecutive stimulations with mixed amino acids is consistent.

A steady basal release of glucose and non-esterified fatty acids was observed before a robust increase in glucose and non-esterified fatty acids during the glucagon infusion. The mean value at the basal state and during the glucagon infusion suggests that glucagon rapidly stimulates hepatic glycogenolysis and lipolysis. However, without steady basal periods, it becomes impossible to distinguish one urea response from the next.