Name: characterization of metabolic status in nonhuman primates with the intravenous glucose tolerance test
Uploaded: 2016-11-13T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test at JoVE.com

The authors would like to acknowledge the strong support of the DHMRI CLAS animal care staff, Facility Manager Mr. Daniel Peralta and attending veterinarian, Dr. Glicerio Ignacio, DVM MRCVS.

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
<ol>
	<li>Select diet and weight stable animals based on monthly food intake and body weight records. 
	NOTE: A...

<ol>
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Invest. 79, 790-800 (1987).</li><li>Hovorka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+glucose+distribution/transport,+disposal,+and+endogenous+production+during+IVGTT.">Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT.</a> Am. J. Physiol. Endocrinol. Metab. 282, E992-E1007 (2002).</li><li>Salinari, S., Guidone, C., Bertuzzi, A., Manco, M., Asnaghi, S., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-phase+insulin+secretion+restoration+and+differential+response+to+glucose+load+depending+on+the+route+of+administration+in+type+2+diabetic+subjects+after+beriatric+surgery.">First-phase insulin secretion restoration and differential response to glucose load depending on the route of administration in type 2 diabetic subjects after beriatric surgery.</a> Diabetes Care. 32 (3), 375-380 (2009).</li><li>Roden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Diabetes Research: Methods and Techniques. , (2007).</li><li>Cobelli, C., Pacini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+and+hepatic+extraction+in+humans+by+minimal+modeling+of+c-peptide+and+insulin+kinetics.">Insulin secretion and hepatic extraction in humans by minimal modeling of c-peptide and insulin kinetics.</a> Diabetes. 37, 223-231 (1988).</li><li>Lorenzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposition+index,+glucose+effectiveness,+and+conversion+to+type+2+diabetes:+the+insulin+resistance+atherosclerosis+study.">Disposition index, glucose effectiveness, and conversion to type 2 diabetes: the insulin resistance atherosclerosis study.</a> Diabetes Care. 33, 2098-2103 (2010).</li><li>Hansen, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+and+treatment+of+type+2+diabetes+in+nonhuman+primates.">Investigation and treatment of type 2 diabetes in nonhuman primates.</a> Methods Mol Biol. 933, 177-185 (2012).</li><li>Hansen, B. C., Bodkin, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+IVGTT.+Importance+of+method+used+to+calculate+glucose+disappearance.">Standardization of IVGTT. Importance of method used to calculate glucose disappearance.</a> Diabetes Care. 16 (5), 847 (1993).</li><li>Hardwood, J. H., Listrani, P., Wagner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhuman+primates+and+other+animal+models+in+diabetes+research.">Nonhuman primates and other animal models in diabetes research.</a> J Diabetes Sci Tech. 3, 503-514 (2012).</li><li>De Koning, E. J., Bodkin, N. L., Hansen, B. C., Clark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+in+Macaca+mulatta+monkeys+is+characterized+by+islet+amyloidosis+and+reduction+in+beta-cell+population.">Diabetes mellitus in Macaca mulatta monkeys is characterized by islet amyloidosis and reduction in beta-cell population.</a> Diabetologia. 36, 378-384 (1993).</li><li>Letiexhe, M. R., Scheen, A. J., Gerard, P. L., Desaive, C., Lefebvre, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion,+clearance+and+action+before+and+after+gastroplasty+in+severely+obese+subjects.">Insulin secretion, clearance and action before and after gastroplasty in severely obese subjects.</a> Int J Obes Relat Metab Disord. 18, 295-300 (1994).</li><li>Letiexhe, M. R., Scheen, A. J., Gerard, P. L., Desaive, C., Lefebvre, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postgastroplasty+recovery+of+ideal+body+weight+normalizes+glucose+and+insulin+metabolism+in+obese+women.">Postgastroplasty recovery of ideal body weight normalizes glucose and insulin metabolism in obese women.</a> J Clin Endocrinol Metab. 80, 364-369 (1995).</li><li>Kim, S. H., Abbasi, F., Chu, J. W., McLaughlin, T. L., Lamendola, C., Polonsky, K. S., Reaven, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosiglitazone+reduces+glucose-stimulated+insulin+secretion+rate+and+increases+insulin+clearance+in+nondiabetic,+insulin-resistant+individuals.">Rosiglitazone reduces glucose-stimulated insulin secretion rate and increases insulin clearance in nondiabetic, insulin-resistant individuals.</a> Diabetes. 54, 2447-2452 (2005).</li><li>Toffolo, G., Breda, E., Cavaghan, M. K., Ehrmann, D. A., Polonsky, K. S., Cobelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+indexes+of+beta-cell+function+during+graded+up+and+down+glucose+infusion+from+C-peptide+minimal+models.">Quantitative indexes of beta-cell function during graded up and down glucose infusion from C-peptide minimal models.</a> Am J Physiol Endocrinol Metab. 280, E2-E10 (2001).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+beta-cell+insulin+secretory+function+using+a+graded+glucose-infusion+with+C-peptide+deconvolution+in+dysmetabolic,+and+diabetic+cynomolgus+monkeys.">Quantification of beta-cell insulin secretory function using a graded glucose-infusion with C-peptide deconvolution in dysmetabolic, and diabetic cynomolgus monkeys.</a> Diabetology and Metabolic Syn. 5, 40 (2013).</li><li>Xiao, Y. F., Wang, B., Wang, X., Du, F., Benzinou, M., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylazine-induced+reduction+of+tissue+sensitivity+to+insulin+leads+to+acute+hyperglycemia+in+diabetic+and+normoglycemic+monkeys.">Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys.</a> Anesthesiology. 13 (33), (2013).</li><li>Porte, D., Kahn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-cell+dysfunction+and+failure+in+type+2+diabetes+potential+mechanisms.">&#946;-cell dysfunction and failure in type 2 diabetes potential mechanisms.</a> Diabetes. 50, S160-S163 (2001).</li><li>DeFronzo, R. A., Tobin, J. D., Andres, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+clamp+technique:+a+method+for+quantifying+insulin+secretion+and+resistance.">Glucose clamp technique: a method for quantifying insulin secretion and resistance.</a> American Journal of Physiology. 237 (3), G214-G223 (1979).</li><li>Ferrannini, E., Gastaldelli, A., Miyazaki, Y., Matsuda, M., Mari, A., DeFronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-cell+function+in+subjects+spanning+the+range+from+normal+glucose+tolerance+to+overt+diabetes:+a+new+analysis.">&#946;-cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis.</a> J Clin Endocrinol Metab. 90 (1), 493-500 (2005).</li><li>Vaughan, K. L., Szarowicz, M. D., Herbert, R. L., Mattison, J. 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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&#39;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...

The IVGTT is a convenient functional assay that is routinely used to determine the &#946;-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&#39;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&#39;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 &#8212; like humans &#8212; 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&#39;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&#39;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.

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			<td style="width:188px;">plasma: 4C @3000 rpm for 10 min</td>
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			<td style="width:188px;">serum: 22C @3000 rpm for 10 min</td>
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characterization of metabolic status in nonhuman primates with the intravenous glucose tolerance test

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.

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

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.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

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.

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