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

Summary

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

Abstract

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state⁵. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state ^{8, 10, 11}.

Introduction

The IVGTT is a convenient functional assay that is routinely used to determine the β-cell function in humans at different metabolic states ^{5, 7}. In animal models of T2D, it is well recognized as a tool to characterize animals that show metabolic disease progression from a healthy to a dysmetabolic hyperglycemic state ^{8, 9}. The closest animal model of T2D is demonstrated in nonhuman primates (NHPs), of which rhesus and cynomolgus macaques are notable examples. These animals naturally develop T2D with the same risk factors of age and obesity contributing to its incidence as in humans ¹⁰. Furthermore, there is a similar disease progr....

Protocol

All animal procedures were approved by the David H. Murdock Research Institute IACUC located on the North Carolina Research Campus (NCRC), under protocol 14-017, Characterization of a nonhuman primate model of diabetes and prediabetes/insulin resistance and efficacy of therapeutics to improve insulin sensitivity and metabolic function.

1. Animal Selection and Study Preparation

1. Select diet and weight stable animals based on monthly food intake and body weight records.
   NOTE: Animals that have exhibited a recent decline in appetite should not be characterized until their food intake has stabilized.
   1. For mature ani....

Results

The results shown in Figure 1 are demonstrative of typical glucose and insulin curves from mature, healthy and diabetic cynomolgus macaques over the course of a 30 min IVGTT. Data from healthy and advanced diabetic monkeys are shown in order to contrast the obvious differences between animals from both extreme ends of the range of metabolic characterization. This IVGTT protocol has been used successfully by the authors in rhesus macaques with similar results.

Discussion

The IVGTT assesses the capacity of glucose-stimulated insulin release by a single dextrose infusion based on body weight ^{5, 12, 13}. From the assay, the fasting blood glucose and insulin level is attained, and it allows an assessment of the animal's capacity to release insulin and return the elevated glucose level towards baseline. This provides the user with information to characterize the animal as a normal glucose and insulin level healthy control, a hyperinsulinemic dysmetabolic animal with normoglycemi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Allegra X-15R Centrifuge			plasma: 4 °C at 3,000 rpm for 10 min
Sorvall ST16R Centrifuge			serum: 22 °C at 3,000 rpm for 10 min
Thermo Scientific -86 °C Freezer, Forma 88000 Series		Model: 88500A
Dextrose 50% (D50)	Webster	07-8008986	I.V. glucose infusate
3 ml Luer Lock Syringe	Midwest Veterinary Suppy		serial blood draws
5 ml Luer Lock Syringe	Midwest Veterinary Suppy		heparinized saline flush
10 ml Luer Lock Syringe	Midwest Veterinary Suppy		delivery of I.V. D50
Gauze sponges 2 x 2	Midwest Veterinary Suppy	366.23000.4	Used Dry, w/ 70% Alcohol, and 2% Chlorohex Solution
4 ml serum separator tubes	Midwest Veterinary Supply	366.45000.4	blood collection tube for superchem panel
K2EDTA, 2 ml	VWR	95057-239	blood collection tubes
Aprotinin, 100 mg	Sigma	A1153-100MG	blood collection tube protease additive
22 G x 1" Catheters	Midwest Veterinary Suppy	193.75250.2	I.V. catheter
Injection Plug W/ Cap	Midwest Veterinary Suppy	001.11500.2	%50 dextrose infusion port
Porus Tape, 1/2" x 10 yd	Midwest Veterinary Suppy	001.85000.2	maintain adherance of catheters and hep. Locks
Chlorhexidine Solution 2%	Midwest Veterinary Suppy	193.08855.3	prep catheter site
70% Ethanol	VWR	71001-654	prep catheter site
tourniquet	Webster	07-8003432
3-way stopcock	Midwest Veterinary Supply	366.28510.4	hep. lock
37" extension set	Webster	07-8454200	hep. lock
Exel 50-60cc LL Syringes	Midwest Veterinary Suppy	001.12250.2	Heparinized saline flush
250 ml bag 0.9% saline	Webster	07-8365593	flush
1,000 U Heparin, 10 ml	Webster	07-883-4916
Ketamine (Ketaset) 100 mg/ml	Fort Dodge	(AV ordered)
Precision Xtra glucose test strips 50/bx	Abbott (American Diabetes Wholesale)	9381599728K7	test baseline/T3 blood glucose levels
Masimo Rad 57	DRE	6052057V	pulse-oximeter
Pavia rectal thermometer	Patterson	07-8391335
Precision Xtra Glucometer	Abbott	9381599728K7	Handheld glucometer