Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

Summary

Hepatic insulin clearance is critical for regulating glucose homeostasis. This article describes a user-friendly hepatic perfusion procedure for directly evaluating the hepatic insulin clearance rate in situ in mice.

Abstract

Hepatic insulin clearance is essential for maintaining glucose homeostasis and is closely linked to metabolic disorders such as obesity, insulin resistance, and diabetes. Accurate measurement of insulin clearance is vital for understanding the underlying mechanisms of these conditions. This protocol presents a straightforward and user-friendly hepatic perfusion procedure in mice, specifically designed to directly evaluate the hepatic insulin clearance rate. The method involves precise cannulation of the portal vein and suprahepatic inferior vena cava to create an in situ perfusion system that mimics physiological conditions. The protocol guides researchers through every stage of the procedure, from surgical preparation and setting up the perfusion system to sample collection and analysis. Detailed instructions are provided, along with representative results and important tips for optimizing the procedure. A video tutorial accompanies the written protocol, offering visually in-depth instructions and illustrations, making it an accessible and comprehensive reference for scientists exploring the molecular mechanisms behind hepatic insulin metabolism and clearance.

Introduction

The discovery of insulin has become one of the milestones of the last century. Much is known about the regulation of insulin synthesis, secretion, and its physiological functions in metabolic tissues. However, there has been less focus on insulin degradation and its regulatory mechanisms. Insulin metabolism can be understood as the interplay between beta cell function, insulin resistance (IR) or sensitivity, and insulin clearance. Alongside insulin secretion, hepatic insulin clearance plays a crucial role in maintaining the homeostatic level of insulin necessary for reaching peripheral target tissues and facilitating proper insulin action¹. Mul....

Protocol

This protocol was approved by the Nanjing Medical University Animal Care and Use Committee (IACUC-2105018) and followed the guidelines of the Institutional Animal Care and Use Committee. All C57BL/6N mice were maintained on a 12-h light/dark cycle with free access to food and water. Six-week-old mice were randomly divided into a Chow diet (CD) group and a High-fat diet (HFD) group. The HFD group was fed a 60% high-fat diet and continued on this diet until 10 weeks of age. The average body weight was 28.55 g ± 1.2 g .......

Discussion

Critical steps in the protocol
The above-described surgical procedures should be performed with gentle care to avoid creating any lesions in the liver. Moreover, the fragile structure of the liver vein vessel wall renders it vulnerable to puncture and subsequent bleeding if not handled with care during cannulation. Softer silicone tubes are utilized in this protocol to minimize damage to blood vessels. It is recommended that catheterization be performed by an experienced surgeon who should practice.......

Materials

Name	Company	Catalog Number	Comments
60% high-fat diet	Research Diets, USA	D12492
Alanine aminotransferase Assay Kit	Nanjing Jiancheng Bioengineering Institute	C009-2-1
Anhydrous Glucose	Sangon Biotech	50-99-7	500 G
Aspartate aminotransferase Assay Kit	Nanjing Jiancheng Bioengineering Institute	C010-2-1
Bovine Serum Albumin	GeminiBio	700-107P	Fatty Acid-Free
Contour TS Blood Glucose Meter	Bayer	PH220800019
Contour TS Blood Glucose Test Strips	Bayer	DP38M3F05A
Heparin Sodium	Changzhou Qian hong Bio-pharma	H32022088	12500 U/2mL
Human insulin	Novo Nordisk	S20191007	300 U/3mL
Human insulin immunoassay kit	Ezassay Biotechnology	HM200
KRBH buffer (Sugar, BSA free)	coolaber	SL65501	500 mL
Membrane oxygenator	Xi'an Xijing Medical Appliance	5
Microscopic scissors	Shanghai Jinzhong	YBC020
Micro-serrefine clamp	Ningbo Medical Needle	180709
Microsurgery forceps	Shanghai Jinzhong	WA3010, WA3020
Needle type filter	N-buliv	LG05-133-2
Povidone-iodine Solution	Shanghai likang Disinfectant Hi-Tech	20231016J
pump 11 Elite	Harvard Apparatus	PC5 70-4500
Retractor	Globalebio (Beijing) Technology	GEKK-10mm	10 mm
Silicone Tubing	scientific commodities	#BB518-12	0.31 mm × 0.64 mm
Silicone Tubing	Fisher Scientific	#11-189-15A	ID 0.5 mm
Sodium Chloride Injection	Baxter	S2402023	4.5 g/500 mL
Surgical silk suture	Yangzhou Huanyu Medical Equipment	6-0
Temperature modulation	Xi'an Xijing Medical Appliance	6
Thermostatic water bath	Jiaxing Junsi Electronics	HIH-1	220 V 50 HZ
Three-way Joint	YISAI	AQTCY1.6	ID 0.4 mm
Xylazine Hydrochloride Injection	ShengXin	20240106	200 mg/2mL
Zoletil 50	Virbac	WK001	250 mg/5mL