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 show.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-way stopcock	BD	394601
Altromin breeding diet	Altromin Spezialfutter	1319
Calcium chloride dihydrate (CaCl2·2H2O)	Sigma	C8106
Catheters (0.7 mm)	BD	381812
Filter paper (pore size 2.0 µm)	Millipore	AP2029325
Glucose kit	QuantiChromTM	DIGL-100	Low concentration protocol
Ketamine	MSD Animal Health	511485	90 mg/kg
Ligature (black sterile silk)	Agnthos	14739
Magnesium sulfate (MgSO4)	Sigma	230391
Non-esterified fatty acids kit	Fujifilm Wako Chemicals	NEFA-HR(2)
Operating table, heated on tripod stand, type 873	Harvard Bioscience, Inc.	733776
Potassium chloride (KCl)	Sigma	P9541
Potassium dihydrogen phosphate(KH2PO4)	Merck	1.04877
Roller Pump, with four channels	Harvard Bioscience, Inc.	730100
Sleek tape	Mediq danmark	4001910
Sodium bicarbonate (NaHCO3)	Sigma	S5761
Sodium chloride (NaCl)	Sigma	S1679
Thermostatic Circulator	Harvard Bioscience, Inc.	730125	Bath Volume 3 L, 230 V/50 Hz
Tubing	Tygon	E3603	Inner diameter 1.59 mm, outer diameter 3.18 mm
Universal perfusion system	Harvard Bioscience, Inc.	732316	Basic unit uniper UP-100, type 834
Vamin	Fresenius Kabi	B05ABA01	Mixed amino acids
Vessel clamp adaptor	Deutsche Biomedical	DBC1002
Vessel clamps	Deutsche Biomedical	DBC1005
Windkessel	Harvard Bioscience, Inc.	732068
Xylazine	Rompun Vet	530701	10 mg/kg