Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

The overall goal of the following experiment is to assess whole body and tissue specific insulin action in conscious unstressed mice without handling them. This is achieved by surgically implanting a catheter into the jugular vein for infusions and another in the carotid artery to obtain blood samples. After a five to seven day recovery period, mice that have been fasted for five hours are infused with insulin to achieve physiological hyperinsulinemia.

Glucose levels are then measured from the arterial catheter, and U glycemia is maintained by adjusting the rate of glucose infusion. This experiment allows the measurement of differences in whole body insulin sensitivity based on the direct relationship between the rate of glucose infusion necessary to maintain U glycemia and insulin action. The main advantage of this technique over existing methods for sampling for blood like cutting the tail, is that the arterial catheter that is surgically implanted gives us vascular access to the mouse so that we can obtain blood samples without having to handle the mouse.

So this allows us to perform these experiments with unstressed and unrestrained mice. The arterial catheter is made by inserting a beveled piece of PE 10 tubing into a six centimeter piece of astic tubing, such that the PE 10 extends 0.9 centimeters from the sel. The venous catheter is a collared six centimeter piece of SEL tubing.

The masa is constructed by inserting three centimeter pieces of PE 20 into each of two 1.3 centimeter 25 gauge stainless steel connectors, Ben, to an approximate 120 degree angle. These are inserted into a dollop of medical silicone adhesive as shown here To begin catheter implantation, place an anesthetized mouse onto a clean work surface using sterile technique. Make an incision five millimeters to the right of the midline.

Isolate the right jugular vein. Carefully ligate the cephalic, end with silk suture and loosely tie another piece of suture at the codal end. Next, cut just below the cephalic ligature with spring scissors and insert the catheter up to the restraining bead.

Tie a suture behind the bead and confirm that the catheter samples by connecting the free end of the catheter to a sampling syringe. To place the arterial catheter, make a small incision, five millimeters to the left of the sternum and cephalic to the first incision. Isolate the left carotid artery, then ligate the cephalic.

End with silk suture and loosely knot. Another piece of suture on the causal end of the exposed vessel. Next clamp the codal end of the vessel.

And cut just below the ligated end with spring scissors. Carefully insert the catheter up to the clamp. Carefully release the clamp and advance the catheter to the Astic P junction.

Tie ligatures securely to the catheter and confirm that the catheter samples by connecting the free end of the catheter to a sampling syringe. Next, flip the animal over and make a small incision between the shoulder blades tunnel, a 14 gauge needle under the skin from the incisions on the front to this incision on the back. Thread the catheters through the needle to exteriorize them at the back of the mouse.

Connect the arterial and venous catheters to the appropriate connectors on the mouse antenna for sampling access or masa. Next, insert the masa into the incision between the shoulder blades. Close both incisions with nylon suture.

Confirm the patency of both catheters. Place the mouse in a warm, clean cage for recovery and allow the mouse to recover for at least five days prior to the insulin clamp. The most difficult step in performing a hyperinsulinemic u glycemic clamp in mice is trying to maintain U glycemia.

Mice have a very rapid metabolism and their glucose levels can change appreciably during the length of the experiment. What this takes is some practice to develop a feel for how a mouses glucose levels will change throughout an experiment, and then how to adjust the glucose infusion rate to keep U glycemia. The setup for a standard clamp is shown here.

Three infuse eights, glucose insulin and washed erythrocytes are attached to a four-way connector connected to a swivel and suspended above the mouse. The infusion port of the swivel is connected to the venous catheter. Prior to connecting the mouse, fill all infuse eight lines with saline.

Next, fill the sampling line with heparinized saline. Leave a syringe with heparinized saline connected to the top of the swivel the clearing syringe. Begin fasting mice five to six hours before the start of the clamp.

Study three hours into the fast weigh the mouse and connect the venous and arterial catheters to the infusion and sampling lines respectively. 15 minutes before the start of the clamp, insert a blank glucose drip into a handheld glucometer. Next, draw approximately 50 microliters of blood into the clearing syringe to remove any saline from the line and detach the clearing syringe.

Measure the blood glucose level by touching the glucose strip to the drop of blood forming at the end of the tubing. Next, insert a syringe with a blunt needle into the sampling line and draw 50 microliters of blood. Dispense the blood into an EDTA coated tube and centrifuge the tube to collect the plasma for measurements of basal hormone and metabolites.

At this point, reinsert the clearing syringe and draw up the plunger. To remove air bubbles, reinfuse the 50 microliters of blood that was originally drawn. Write down the blood glucose value from the glucometer.

10 minutes later, use the glucometer and clearing syringe to repeat the blood glucose measurement and then draw another 50 microliters of blood to obtain another plasma sample. After the second baseline measurements are taken, fill the infusion lines for glucose, insulin and saline washed erythrocytes up to the four-way connector. Connect the four-way connector to the tubing attached to the swivel infusion port.

Begin by infusing the saline washed erythrocytes. Set the rate of infusion to replace the total volume of blood being sampled over the duration of the study. Once the erythrocyte infuse eight reaches the mouse, begin the insulin and glucose infusions.

This is time equals zero minutes. Insulin is infused at a constant predetermined rate. The initial glucose infusion rate is estimated based on the baseline blood glucose levels and previous experience.

Over the next two hours, take blood glucose readings with the glucometer and adjust the glucose infusion rate to maintain target u glycemia At 10 minute intervals, record the new glucose readings and glucose infusion rates at 100 minutes and 120 minutes, collect additional blood for the measurement of plasma insulin and any other hormones or metabolites. Shown here is the time course of glucose levels throughout the clamp procedure demonstrating how well U glycemia was maintained in both chow fed and high fat fed mice throughout the clamp. A time course of the glucose infusion rate shows that the rate necessary to maintain u glycemia is significantly lower in the high fat fed group indicating impairment in whole body insulin action.

Both fasting and clamp insulin levels are also higher in the high fat fed group, further supporting the presence of an insulin resistant phenotype. In these mice, tritiated glucose is used to estimate the rate of endogenous glucose appearance. Endo a an index of hepatic glucose production, whereas insulin completely suppresses endo A in control mice.

The suppression is impaired in mice fed a high fat diet. Similarly, TRID glucose is used to estimate the rate of whole body glucose disappearance. Rd the ability of insulin to stimulate RD is compromised in mice fed a high fat diet.

Two deoxy glucose containing carbon 14 is used to assess the glucose metabolic index RG a measure of tissue specific glucose uptake. As seen in this example, insulin stimulated glucose uptake into skeletal muscle is impaired in mice fed a high fat diet. Generally, individuals new to this method will struggle with the microsurgery needed to implant the arterial and venous catheters.

The surgery requires very specialized skills and it needs constant practice over several months to become very adept at the surgery for the hyperinsulinemic U glycemic clamp, the difficulty again is to try to maintain U glycemia in an animal model with a very high metabolism.