Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Summary

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Abstract

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

Protocol

A: Surgical Catheterization of Arterial and Venous Circulation

Part 1: Arterial and Venous Catheter Preparation

1. Cut 15 cm of PE-50 (internal diameter of .58mm (.023") x an outer diameter of .965mm (.038"). Cut a 1mm section of silastic tubing (0.76mm (0.030") internal diameter x 1.65mm (0.065") outer diameter) for use as restraining bead. The restraining bead prevents the rat from pulling out the catheter once it is in place.
2. Insert the tips of micro dissecting forceps into the lumen of the restraining bead and gently hold the tips of the forceps apart to stretch the opening wider. Using another pair of micro dissecti....

Discussion

Initially developed for investigation of insulin sensitivity in humans, the clamp procedure has now been adapted to other species including laboratory rats and mice. Investigating animal models of insulin resistance provides a significant aid in understanding the pathophysiology of insulin sensitivity and associated pathologies as well as identifying therapeutic interventions that have clinical value^1,2. Several methods to evaluate insulin sensitivity in animals have been employed³. Such techniques .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Intramedic Polyethylene Tubing (PE-50)	Fisher Scientific	14-170-12B	Internal diameter of .58mm (.023") x Outer diameter of .965mm (.038")
Dow Corning Silastic Laboratory Tubing	Fisher Scientific	11-189-15C	Internal diameter of .76mm (0.030") x Outer diameter of 1.65mm (0.065")
Tygon S-50-HL Medical Tubing	Harvard Apparatus	PY2 72-1251	Internal diameter of 3.2mm (0.125") x Outer diameter of 4.7mm (0.1875")
Loctite Super Glue	Grand Toy	32237	Gel Control
Sterile Surgical Blade	VWR international	BD371610
Curved Micro Dissecting Forceps	George Tiemann & Co.	160-20	x 2
Straight Micro Dissecting Forceps	George Tiemann & Co.	160-15	x 2
Curved Hemostat	George Tiemann & Co.	105-1135	x 2
Straight Hemostat	George Tiemann & Co.	105-1130	x 2
Hemostat Tip Guards	Robbins Instruments, Inc.	15.09-2-004
Straight Surgery Scissors	George Tiemann & Co.	105-402
VENOJECT Multi-Sample Luer Adapter	Terumo Medical Corp.	810127A	21 guage, 1 in.
Sterile Catheter Introducer	BD Biosciences	406999
14-gauge Blunt Needle	BD Biosciences	511310	14 guage, 2 in.
Sterile Surgical Suture	Johnson & Johnson	1679H	Silk, size 3-0
Non-Sterile Surgical Suture	Angiotech Pharmaceuticals, Inc.	SP116	Silk, size 4-0
Cotton Swabs	VWR international	10806-005
4ply Gauze Pads	VWR international	CA43845-062
Small Animal Cordless Clippers	Harvard Apparatus	729063
Isoflurane	Halocarbon Products Corp.	IPN-45
Anesthetic Cart	Benson Medical Instruments
70 % Ethanol	Fisher Scientific	HC-1000
Betadine Antiseptic Solution	Western Drug Distribution Center, Ltd.	105267
Model 11 Plus Syringe Pump	Harvard Apparatus	702208
Stainless Steel Tubing Couplers	Harvard Apparatus	72-4434	23 gauge, 0.3 in.
Stainless Steel Tubing Plugs	Harvard Apparatus	72-4436	23 gauge, 0.5 in.
Stainless Steel Blunt Needles	Instech Laboratories, Inc	LS22	22 gauge
60 Degree Y-Connectors	Small Parts, Inc.	STCY-22-05	22 gauge
CritSpin Micro-hematocrit Centrifuge	Iris Sample Processing	CS12
Mini Centrifuge	Fisher Scientific	05-090-100
Micro Centrifuge Tubes	VWR international	53550-778
50ml polypropylene centrifuge tubes	VWR international	89004-364
1ml Plastic Slip Tip Syringes	BD Biosciences	309602
3ml Plastic Luerlok Tip Syringes	BD Biosciences	309585
Heparin Anticoagulant Injection	Western Drug Distribution Center, Ltd.	102824	Manufacturer: LEO Pharma Inc.Conc. 1000 IU
EDTA Solution	Promega Corp.	V4231	0.5 M, pH 8.0
Saline	Western Drug Distribution Center, Ltd.	ABB7983154	Manufacturer: Hospira0.9% Sodium Chloride
50% Dextrose	Vétoquinol	8DEX012D
Humulin-R	Eli Lilly	HI-210	100U/ml
1ml Insulin Syringes	BD Biosciences	309311
Fisherbrand* Hemato-Seal Sealant	Fisher Scientific	02-678
Fisherbrand* Microhematocrit Capillary Tubes	Fisher Scientific	22-362-574
One Touch Ultra Test Strips	LifeScan, Inc.	AW 085-314H
One Touch Ultra Blood Glucose Meter	LifeScan, Inc.	AW 085-314B
Sodium Pentobarbitol	Ceva Sante Animale	1715 138	Conc. 54.7mg/ml
Red Laboratory Labeling Tape	VWR international	89097-932
Blue Laboratory Labeling Tape	VWR international	89097-936
Weigh Scale	Fisher Scientific	01-913-88
Vortex	VWR international	58815-234
Timer	VWR international	62344-641