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

Podsumowanie

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.

Streszczenie

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}.

Wprowadzenie

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 progression and pancreatic pathology showing amyloid deposits as the dysmetabolic disease progresses ¹¹.

Here we report on our standard method of performing an IVGTT in NHPs as part of our colony characterization of metabolic status in these animals. This method is easy to perform relative to other, more time consuming and costly techniques ². The IVGTT is useful for characterizing a large colony of animals rapidly and frequently. When taken into consideration with the level of glycated hemoglobin (HbA1C), the animal's diet and food intake history, as well as their percent lean mass and body fat, the IVGTT is normally sufficient for characterizing an animal's metabolic status and progression toward overt diabetes ^{6, 8}.

HbA1C represents the average glycemic level over the life of a red blood cell, providing a reliable measure of glucose levels over the previous six weeks to three months. When measured from the fasted baseline blood sample of the IVGTT, this value provides a window into glycemic control during the months between procedures. If the animal has transitioned from dysmetabolic to diabetic since their last IVGTT, an HbA1C value much higher than their previous value would indicate that the transition began soon after their last IVGTT, whereas, an HbA1C value closer to their previous value would indicate that they have only recently transitioned. In general, in rhesus macaques, HbA1C values greater than 6% are considered abnormal, and indicate poor glycemic control ^{10, 23}.

Glycemic levels should be interpreted within the context of the behavior and general health of the animal as a whole. Diabetic macaques — like humans — exhibit hyperphagia, polydipsia, and polyuria. Group housing of animals provides significant challenges to measurement of these indicators and the individual care required for dysmetabolic and diabetic monkeys. We recommend singly housing the animals in order that more personalized care may be provided, and behavioral markers of the health of the monkey more easily be monitored ⁸. Additionally, diabetic macaques will exhibit weight loss, as well as an elevated lipid profile (increased cholesterol, hypertriglyceridemia) and disturbed mineral metabolism in serum chemistry. It is important to measure markers of liver and kidney function in serum chemistry, as damage to these organs are often complications of advancing metabolic disorder/diabetes, and may be co-determinants of glycemic, lipid and mineral imbalances ^{9, 11, 18, 24}.

When using this method, the historic values generated from multiple, frequent characterizations over the life of a monkey are of particular value. If other procedures, such as a glucose clamp or graded glucose infusion (GGI), are needed to fully assess an animal's health, it is commonly upon initial characterization when their history is unavailable. However, once a baseline has been established, repeated IVGTTs of a frequency of every three months are normally sufficient to track an animal's progress. This is particularly important when the animals are enrolled into multiple studies throughout a calendar year based upon their metabolic status. While their health may remain relatively stable for years at a time, when the metabolic status of an animal worsens, a dramatic increase in insulin resistance and glucose intolerance can occur very rapidly. HbA1C values allow for some interpolation of the decline or improvement of the health status of the animal between procedures scheduled three months apart. For this reason, this method is ideal for characterizing animals used in multiple, longitudinal studies over the course of their natural lifespan.

Protokół

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 animals (> 5 - 6 yr), do not select animals whose body weights between consecutive months differ by more than 10% without first examining the monkey and ruling out causes other than changing metabolic status for the dramatic change in weight.
2. For glucose, insulin and c-peptide analytes, prepare K₂EDTA sample collection tubes with protease inhibitor (Aprotinin and DPP4I).
   NOTE: The DPP4I + Aprotinin cocktail provides a broad spectrum of protease inhibition should this be required for additional analytes (glucagon, GLP-1). In the event that additional analytes are not collected, the blood tubes should be prepared in the same way to maintain consistency in sample collection. Samples for assays not validated for this method should be collected separately, according to the recommendations of the manufacturer.
   1. Prepare the protease inhibitor cocktail by mixing 100 mg lyophilized Aprotinin with 10 ml of DPP4I. Add 10 µl of the Aprotinin + DPP4I mixture to each blood tube for every milliliter of blood collected.
   2. Add an additional 10 µl of the protease inhibitor cocktail to each tube for possible collection overage. Store the treated-blood tubes at -20 °C until use. Keep the tubes on wet ice during the procedure and spin at 4 °C.
   3. Use a serum separator tube to collect a blood sample at baseline for standard serum chemistry analysis. For the Complete Blood Cell count (CBC), use a standard K₂EDTA without protease inhibitors. Use cryovials to aliquot plasma and serum after the blood samples have been spun down.
   4. Label the blood tubes and the cryovials appropriately with the animal identification, date, procedure, timepoint, and sample volume. Label the cryovials with the analyte(s) in plasma for appropriate assay.
3. Prepare the heparinized saline flush by injecting 0.15 ml of 1,000 USP units per ml heparin into a 250 ml bag of normal saline. Obtain a solution of 0.06 mg heparin/ml. Draw 40 - 60 ml of this solution into a saline lock for flushing between samples. Draw an additional 1 ml and 5 ml into separate syringes for flushing the dextrose infusion port before and after the infusion, respectively.

2. Animal Sedation and Preparation

1. Remove food from the animal's cage no less than 14 hr before the procedure, and no more than 18 hr.
   NOTE: It is important that animals be fasted for the procedure to avoid any post-prandial variation in glycemic values. It is also a precaution to avoid regurgitation and aspiration of stomach contents while anesthetized.
2. Sedate animals for the duration of the IVGTT procedure with ketamine given intramuscularly as a general anesthetic, at 10 mg/kg. Administer additional ketamine (5 - 10 mg/kg) at intervals of 20 - 30 min, or as needed, during the procedure.
   1. Weigh the sedated animal. Place the animal in a laterally recumbent position on a heated procedure table.
   2. Monitor clinical parameters every 15 to 20 min to ensure the animal is in a stable plane of anesthesia. Measure heart rate (100 - 200 bpm) and SPO₂ (> 92%) with a pulse oximeter. Measure respiratory rate (20 - 50 breaths/min) with a stopwatch, counting respirations visually or by hand over fifteen seconds and multiplying by four. Measure temperature (> 97 °F) rectally. Monitor the color of the mucous membrane around the gum and lips (moist, pink).
3. Prepare two cannula sites. Use hair clippers to trim the hair from the area of interest where the catheter will be inserted, and sterilize the entire region with alternating scrubs of chlorhexidine and 70% alcohol.
   1. Place one catheter in the region of the left or right cephalic or saphenous veins and attach it to a heparin saline flush (0.06 mg heparin/ml) with a three-way stopcock. This is the sampling blood draw site.
   2. Place the second catheter in another leg or arm in the region of the cephalic or saphenous veins and attach a port. Use this site for the dextrose infusion. Use a small, 1 ml flush of heparinized saline to keep the cannula patent prior to dextrose infusion.

3. IVGTT Procedure

NOTE: The IVGTT procedure consists of 8 blood draw sampling time-points (Table 1).

1. Take the baseline sample and use a hand held glucometer to measure the fasting blood glucose level. Obtain a serum sample for standard chemistry analysis, as well as a whole blood sample for a CBC in order to assess the overall health of the animal. Collect plasma samples to assay glucose and insulin levels from the baseline sample using a kit validated for use with macaques as per the manufacturer's instructions ^{8, 9}.
   NOTE: It is important to have a pre-draw of 0.5 ml taken from the cannula prior to taking any sample for blood collection to remove residual blood or heparin in the dead-space of the cannula.
2. After obtaining the baseline sample, infuse the dose of 50% dextrose (250 mg/kg) over 30 sec into the dextrose infusion port.
   NOTE: Higher dose models (500 mg/kg) can be used, though the dose should be fixed across procedures in order to make longitudinal comparisons.
   1. Flush the infusion port with 5 ml of heparinized saline to make sure that there is no dextrose left in the port. The end of the infusion is the T0. Have the technician replace the gloves, as residual dextrose from the infusion may contaminate subsequent blood samples.
3. The first post-infusion sample time point is at T3 min, from the end of dextrose infusion, followed by T5 min, T7 min, T10 min, T15 min, T20 min, and the last sample time point is at T30 min. Collect plasma from each timepoint to assay glucose and insulin levels with the baseline sample (see step 3.1).
   1. At the T3 min time point, use the hand held glucometer to once again check the blood glucose level.
      NOTE: The glucometer readings at baseline and T3 are only to confirm the infusion of the dextrose. The blood glucose level at the T3 min time point should be ~100 mg/dl higher compared to the fasting baseline plasma glucose level.

4. Animal Recovery and Sample Processing

1. Remove the cannulas and apply pressure to the catheterized sites for hemostasis after the T30 min time-point. Monitor the animal until it has regained consciousness and is sitting up. Offer feed once the animal is fully recovered.
2. Immediately place each whole blood sample into K₂EDTA tubes on ice. Centrifuge at 3,000 rpm at a temperature of 4 °C within 10 min of collection. Aliquot plasma samples into cryovials, freeze and store at -80 °C until analysis.
   1. Allow the blood into the serum tube for standard serum chemistry analysis to sit at room temperature for no less than 20 min and no more than 30 min before centrifugation at 3,000 rpm at RT. Freeze the serum samples until assayed within 48 hr of collection.
      NOTE: Refrigerate the whole blood samples collected for CBC analysis until assayed within 24 hr of collection.

5. Data Treatment

1. After establishing the plasma insulin and glucose curves, determine the glucose clearance rate K from the slope of the natural log of glucose values above baseline ^{16, 17}.
   NOTE: A healthy NHP may be expected to have a glucose clearance rate K well above 1, often greater than 2 or more, as a healthy animal will often return to their baseline glucose values within 30 min. As insulin production drops off, the glucose clearance rate K will drop more dramatically, falling below 1.
2. Calculate the AUC as a whole, as the sum of totals of the area of the trapezoids representing the area under the curve of each line segment between time-points, through T30 ^{16, 17}.
   NOTE: Traditionally, the AUC of the first ten minutes of the procedure is considered the acute insulin response to glucose (AIR), while the AUC from the last 20 min of the procedure is considered the late insulin response (LIR). As the animal becomes more dysmetabolic, the insulin AUC will increase, reflecting compensation for increasing insulin insensitivity. As the animal transitions to overt diabetes, however, the AUC will decrease, often initially in the acute insulin phase, captured by the AIR.

Wyniki

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.

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Allegra X-15R Centrifuge			plasma: 4C @3000 rpm for 10 min
Sorvall ST16R Centrifuge			serum: 22C @3000 rpm for 10 min
Thermo Scientific -86C Freezer, Forma 88000 Series		Model: 88500A
Dextrose 50% (D50)	Webster	07-8008986	I.V. glucose infusate
3mL Luer Lock Syringe	Midwest Veterinary Suppy		serial blood draws
5ml Luer Lock Syringe	Midwest Veterinary Suppy		heparinized saline flush
10mL Luer Lock Syringe	Midwest Veterinary Suppy		delivery of I.V. D50
Gauze sponges 2x2	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, 2mL	VWR	95057-239	blood collection tubes
Aprotinin, 100mg	Sigma	A1153-100MG	blood collection tube protease additive
22g 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 10yd	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) 100mg/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