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

Summary

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

Abstract

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Introduction

The liver is an essential organ in metabolism. It plays a key role in the control of whole-body energy balance by regulating glucose, lipid, and amino acid metabolism. The increase in liver diseases worldwide is emerging as a major global health burden, and more knowledge is needed about the pathophysiology and its consequences for liver functions.

Various in vitro models have been developed for research on the liver to complement in vivo studies. Isolated and cultured primary hepatocytes from rodents and humans are widely used. Non-parenchymal cells can be separated from hepatocytes using differential and gradient centrif....

Protocol

All animal experiments were conducted with permission from the Danish Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark (permit 2018-15-0201-01397), and the local ethics committee in accordance with the EU directive 2010/63/EU, the National Institutes of Health (publication No. 85-3) and following the guidelines of Danish legislation governing animal experimentation (1987). This is a terminal procedure, and the cause of death is exsanguination and perforation of the diaphragm under deep anesthesia.

1. Experimental animals

1. Obtain mice of desired strain, age, and gender. This study used ....

Results

A steady baseline is required to determine whether a stimulus or substrate leads to the release of the molecule of interest. Figure 3A shows an example of a successful experiment. Production of urea in the perfused liver is measured in 2 min intervals and shown as mean ± SEM. The baseline periods preceding each of the two stimulation periods are steady. The mean urea production during the two stimulation periods and the respective preceding baselines are.......

Discussion

The isolated perfused mouse liver is a strong research tool for studies of the dynamics and molecular mechanisms of hepatic metabolism. The possibility of minute-to-minute sample collection provides a detailed evaluation of the direct effect of a test compound on the liver. Compared to in vivo studies, the perfused liver allows us to study liver metabolism in an isolated manner avoiding extra-hepatic factors carried by the blood and with complete control over the experimental conditions. The advantages of liver .......