Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The overall goal of this intravenous glucose tolerance test, or IVGTT, is to characterize the progression of metabolic disease in macaques, from a healthy to a dismetabolic, hyperglycemic state. This method can be used to establish cohorts of subjects stratified by their metabolic status for dietary or pharmacological investigation of the therapeutic efficacy of treatments for insulin resistance in diabetes. The main advantage of this technique is that it is easy to perform relative to other more time consuming and costly techniques.

14 to 18 hours before beginning the procedure, remove the food from the macaque's cage to avoid any post-prandial variegation in the glycemic values. The next morning, weigh the sedated animal. Then place the primate in a laterally recumbent position on a heated procedure table.

Every 15 to 20 minutes, use a pulse oximeter to measure the heart rate and peripheral capillary oxygen saturation. Use a stop watch to measure the respiratory rate, and a thermometer to measure the temperature rectally. Confirm that the mucous membrane around the gum and lips is moist and pink at every clinic parameter check as well.

After confirming the baseline health of the animal, use clippers to trim the hair from the areas where the catheters will be inserted, followed by the sterilization of both regions with alternating scrubs of chlorhexidine and 70%alcohol. When the sites are ready, insert the first catheter and attach the lure to a heparin-saline flush with a three-way stopcock. This is the sampling blood draw site.

Tape the stopcock to secure it to the leg. The placement of the catheter is critical for patency and precise serial blood draws. Therefore, it is important to consider a medial and more proximal region of the saphenous vein to easily secure and manipulate the heparinized flush in-lock system.

Then, place the second catheter, and attach a port for the dextrose infusion, followed by the administration of a one millilitre flush of heparinized saline to keep the cannula patent until the dextrose infusion. Tape the port to the arm to secure it. When both of the catheters are in place, obtain a baseline sample, and use a handheld glucometer to measure the fasting blood glucose level.

Secure a serum sample for the standard chemistry analysis, as well as a whole blood sample for a complete blood cell count to assess the overall health of the animal. Invert the whole blood sample. After each blood draw, mix the samples in EDTA tubes with the protease inhibitors.

After obtaining the baseline samples, infuse the 50%dextrose dose over 30 seconds via the dextrose infusion port. Then, flush with five milliliters of heparinized saline, so that no dextrose is left in the port. At the end of the infusion, change gloves, as residual dextrose from the infusion may contaminate the subsequent blood samples.

The first post-infusion sample time point is at time three minutes from the end of the dextrose infusion, followed by samples at T five, seven, 10, 15, and 20 minutes, with the last sample obtained at T 30 minutes. Centrifuge the samples for glucose and insulin analysis within 10 minutes of collection, then aliquot the plasma into cryovials for freezing and storage at negative 80 degrees Celsius, until downstream analysis of the samples. At the T three sample draw, use the handheld glucometer to check the blood glucose level for confirmation of the dextrose infusion.

When the last sample has been collected, remove the cannulas, applying pressure to the catheterized sites for hemostasis, and monitor the animal until it is fully recovered. Here, typical glucose and insulin curves from mature, healthy and diabetic cynomolgus macaques over the course of a 30 minute IVGTT are shown. The fasted baseline glucose values of healthy macaques can be as low as 50 to 60 milligrams per deciliter, with an initial plasma glucose spike observed at the T three minute time point.

That is dramatically lower than the typical plasma glucose spike exhibited by diabetic animals. Over the course of the procedure, the glucose levels of healthy animals often return to their baseline values, while dismetabolic and diabetic animal levels do not. The accompanying insulin curve for a healthy animal exhibits two peaks, corresponding to an initial rapid decline in blood glucose mediated in larger than normal part by the peripheral effects of insulin on glucose transport and uptake.

The magnitude of these peripheral effects, even during the first phase of the insulin response, does not equal the contribution of the liver to glucose lowering, which, during the smaller but more sustained second insulin peak, has the greatest impact on glycemic levels via the suppression of endogenous glucose production. Once an animal has become overly diabetic, insulin production in response to the dextrose bolus drops dramatically. Glycemic levels will remain elevated over the course of the procedure.

As with glucose, clearance occurs is now mediated almost entirely by non-insulin dependent mechanisms. Following this procedure, other methods like graded glucose infusion, or the hyperinsulinemic euglycemic clamp can be performed to answer additional questions about insulin sensitivity. Don't forget that working with needles exposed to macaque blood can be extremely hazardous, due to the potential for exposure to herpes B virus.

Precautions, such as wearing the proper personal protective gear, and taking extreme care when handling sharps should always be observed while performing this procedure.