Assessing Insulin Clearance in Mice via In Situ Liver Perfusion

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.

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.

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....

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 .......

This protocol outlines the procedure for liver infusion to calculate hepatic insulin clearance directly. This model is reliable and reproducible. An example of the results obtained from an experiment is shown in Figure 3. After a 10-min equilibration period, KRBH buffer supplemented with 4.0 ng/mL human insulin was perfused through the portal vein for 30 min. Perfusion fluid was collected from the catheter in the suprahepatic inferior vena cava at 2-min intervals, and the concentration of hu.......

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.......

This work was supported by the National Natural Science Foundation of China (82200948, 82270921, 82170882).