New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

Abstract

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal's blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.

New-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.

History

The intact male captive-bred cynomolgus macaque in this case report was approximately 5.5-y old at presentation and was housed at Massachusetts General Hospital (Boston, MA). Prior to arrival, he was tested and found negative for Macacine herpesvirus 1, simian retrovirus, SIV, and simian T-cell leukemia virus type 1. He was individually housed for study purposes in an AAALAC-accredited facility and used in accordance with an IACUC-approved protocol. He had visual access to conspecifics and was provided with various enrichment devices and foodstuffs according to institutional guidelines on environmental enrichment for NHP. The macaque was housed with a 12:12-h light:dark cycle, at controlled and monitored temperature (18 to 29 °C) and 10 to 15 air changes hourly. He was fed a commercial primate chow (Monkey Diet 5038 Lab Diet, PMI Nutritional International, St Louis, MO), with daily supplementation of a variety of fresh fruits and vegetables. Animal Biosafety Laboratory level 2 practices were followed in the animal facility and during handling of NHP, and quarterly intradermal tuberculin testing was performed on all animals not currently on an immunosuppressive regimen.

The macaque was enrolled in a heart and thymus transplantation protocol. Preprocedural blood tests showed normoglycemia as well as normal values of CBC (HemaTrue Veterinary Analyzer, HESKA, Loveland, CO), liver and kidney function, and serum electrolytes. He underwent preemptive host thymectomy on transplantation day –28, and a heterotopic en-bloc heart–thymus transplantation from an allogeneic donor was performed 28 d later (day 0). On the day of transplantation, the macaque's body weight was 6.7 kg. Immunosuppressive therapy was initiated according to protocol on day –2 and comprised intravenous antithymocyte globulin (Atgam, Pfizer, New York, NY) and a short course of intravenous antiIL6 receptor monoclonal antibody (aIL6R, Actemra, Genentech, San Francisco, CA), along with oral mycophenolate mofetil (CellCept, Genentech), intramuscular methylprednisolone (Solu-Medrol, Pfizer), and intramuscular tacrolimus (Prograf, Astellas Pharma, Northbrook, IL) to prevent transplant rejection. Tacrolimus, mycophenolate mofetil, and methylprednisolone were continued daily, and dosages were titrated as needed and to achieve therapeutic blood levels of 20 to 30 ng/mL for tacrolimus (Figure 1).

Figure 1.

Tacrolimus levels (ng/mL). Tacrolimus therapy was discontinued on PT day 208.

After transplantation, the macaques's appetite and body weight declined. On posttransplantation day 19, he was bright and alert with a body weight of 5.56 kg, an approximately 17% decrease in body weight since the day of transplantation. Because treatment with tacrolimus has been associated with side effects leading to inappetence,⁵ tacrolimus levels were monitored, and the levels did not exceed the target range. In addition, mycophenolate mofetil has been associated with gastrointestinal side effects such as vomiting, diarrhea, as well as loss of appetite;⁵ therefore the dose was decreased from 200 mg to 150 mg PO once daily. In addition, supplemental feeding by oral gavage (Ensure, Abbott Laboratories, Abbott Park, IL) during sedated exams performed 3 times weekly was initiated. Because the macaque's appetite and body weight did not improve significantly, the dose of mycophenolate mofetil was further decreased to 100 mg on day 26. Subsequently, although the macaque's appetite remained poor, he began to gain weight and remained bright and alert, with no signs of transplant rejection. On day 56, the macaque was noted as having a distended abdomen; therefore, on day 57, he was fasted and sedated with ketamine (30 mg IM; Ketaset, Zoetis, Florham Park, NJ) and dexmeditomidine (0.1 mg IM; Dexdomitor, Zoetis, Florham Park, NJ) for a complete physical exam, blood collection, and abdominal radiographs. The exam revealed a body condition score of 2.5 on a scale of 5, stable but low body weight, and a strong heartbeat from the transplanted heart; the remainder of the exam revealed no significant findings. Abdominal radiographs were within normal limits. Blood was submitted for a CBC and serum chemistry panel.

The CBC showed a mild-to-moderate leukopenia and mild anemia, as expected given the current immunosuppressive regimen. Blood chemistry analysis (Dri-Chem 7000, Heska, Loveland, CO) revealed the following abnormalities: BUN, 33.1 mg/dL (reference range, 5 to 25 mg/dL); albumin, 4.2 g/dL (2.6 to 3.4), and glucose 727 mg/dL (42 to 111); a serum chemistry panel performed during quarantine after arrival revealed no abnormalities and a serum glucose of 50 mg/dL. The elevated BUN and albumin are suggestive of dehydration, and the markedly elevated blood glucose is highly suggestive of diabetes mellitus. Analysis of glycated hemoglobin (HbA1c, DCA Vantage Analyzer, Siemens, Malvern, PA) yielded a result of 10.1% (normal for our lab is 4.0% ± 0.6%). Urine was collected via a clean pan the following day and submitted for urinalysis. The urine was concentrated (specific gravity, 1.028), and marked glucosuria (4+) and mild ketonuria (1+) were present. Given the markedly elevated blood glucose level, the glucosuria and ketonuria, a diagnosis of idiopathic diabetes mellitus was made.

Insulin therapy was initiated with a 1:3 mixture of regular insulin (Humulin R, Lilly, Indianapolis, IN) and NPH insulin (Humulin N, Lilly, Indianapolis, IN) once to twice daily and glargine (Lantus, Sanofi, Bridgewater, NJ) once daily, with doses given subcutaneously and titrated to effect. The macaque had been trained to present his tail for regular conscious blood glucose monitoring by using a hand-held glucometer (OneTouch Ultra 2, LifeScan, Milpitas, CA). Blood glucose and HbA1c were monitored regularly to evaluate response to therapy (Figures 2 and 3). After blood glucose monitoring and insulin therapy began, the macaque's condition steadily improved with an improved appetite, increased body weight, and increased activity level by day 64. The dosage of mycophenolate mofetil was increased to the previous level, and the macaque remained clinically stable, with a good appetite and steady weight gain (Figure 4). With the introduction of diabetes as a study variable, on day 167, the decision was made to wean the macaque off immunosuppressive therapy and to use this macaque as a control subject. Doses of immunosuppressive drugs were decreased gradually, and methylprednisolone, mycophenolate mofetil, and tacrolimus were discontinued on days 178, 193 and 207, respectively. Insulin therapy had been discontinued on day 197 due to the discontinuation of the immunosuppressant therapy but was reinitiated on day 212 in light of increasing blood glucose and HbA1c levels. Insulin therapy was discontinued once again on day 281, 73 d after stopping all immunosuppressive therapy. The animal's blood glucose and HbA1c levels have remained stable without further insulin treatment since this time, and the macaque is no longer considered to be diabetic.

Figure 2.

Blood glucose levels (mg/dL) with corresponding insulin dose (units/day). Insulin therapy was discontinued on day 198 after transplantation in view of the stable blood glucose level and discontinuation of the immunosuppressant therapy. Insulin therapy was reinitiated on day 212 and maintained until discontinuation on day 282.

Figure 3.

HbA1c levels (%). Insulin therapy was discontinued on day 282 after transplantation.

Figure 4.

Body weight (kg). Thymectomy occurred 28 d before en-bloc heart and thymus transplantation (day 0). Insulin therapy was initiated on day 58 after therapy and ultimately was discontinued on day 282.

Discussion

A common complication of transplantation, NODAT is caused by immunosuppressive drugs in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} In one report, the frequency of NODAT within the first 12 mo in human transplantion patients was estimated to be between 2% and 50%.³⁰ However, NODAT is not reported frequently in macaques used in transplantation research; information regarding this condition is widely taken from human medicine. The American Diabetes Association defines diabetes mellitus in humans as the presence any of the following: 1) HbA1c of 6.5% or greater; 2) fasting plasma glucose of 126 mg/dL or greater; 3) 2-h plasma glucose level during an oral glucose tolerance test of 200 mg/dL or greater; or 4) classic symptoms of hyperglycemia or a hyperglycemic crisis with a random plasma glucose level of 200 mg/dL.^1,2 According to this definition, this macaque was diagnosed with diabetes mellitus after 59 d of immunosuppressive therapy in light of an HbA1c level of 10.1% and a fasting plasma glucose concentration of 727 mg/dL. Because the macaque had no symptoms or hematologic evidence of diabetes prior to drug therapy and because the disease resolved after withdrawal of drug therapy, we suspect that the immunosuppressive agents caused the diabetes mellitus in this animal.

Increased insulin resistance or defective insulin production due to immunosuppressive regimens is thought to be the cause of new-onset diabetes mellitus (type 2).⁴¹ Multiple drugs have been implicated in NODAT, including calcineurin inhibitors (tacrolimus, cyclosporin), glucocorticoids, and antimTOR (the mechanistic target of rapamycin) drugs (sirolimus, everolimus).^{3,8,9,15,17-19,21,22,29-31,33,34,37,39,40,42} The macaque we present here was treated with 2 drugs that have been linked to the development of diabetes mellitus, tacrolimus and methylprednisolone. The enzyme calcineurin has a role both in T-cell activation and pancreatic β -cell growth and regulation.^16,35 Tacrolimus, a calcineurin inhibitor, is believed to lead to diabetes mellitus primarily by means of impaired insulin secretion due to direct β-cell toxicity^10,15,20,32. Additional reports include peripheral insulin resistance as a contributing factor.^{21,24,29,31,38,44} Glucocorticoids, such as methylprednisolone, affect glucose metabolism primarily by increasing insulin resistance in tissues, decreasing insulin's action to suppress hepatic glucose production and stimulate peripheral glucose use.^6,10,12 However, glucocorticoids have been reported to have a direct inhibitory effect of insulin release in β cells.^11,23,32 Serum from this macaque was submitted for insulin and C-peptide levels to determine whether his disease was due to decreased insulin secretion compared with increased insulin resistance. Serum taken at the diagnosis of diabetes (day 58) revealed a blood glucose level of 677 mg/dL, an insulin level of less than 5.0 µU/mL, and a C-peptide level of less than 0.1 ng/dL. Serum collected after the resolution of diabetes (day 432) revealed a blood glucose value of 67 mg/dL, insulin of 6.3 µU/mL, and C-peptide of less than 0.1 ng/dL. The low insulin and C-peptide levels in concert with elevated blood glucose on day 58 suggest impaired insulin secretion as the primary cause of this macaque's diabetes; however, increased insulin resistance might be a contributing factor, given the published effects of both tacrolimus and glucocorticoids leading to insulin resistance.^{11,21,23,29,31,32} Multiple studies have shown that the diabetogenic effects of tacrolimus^{4,13,36,38,44} and glucocorticoids²³ are dose-dependent and reversible, thus perhaps accounting for the slow return to euglycemia after cessation of the immunosuppressant therapies.

Information regarding NODAT is largely taken from human medicine. Immunosuppressive therapy accounts for approximately 74% of the risk for NODAT in human patients,^9,21,34 and we suspect that immunosuppressive therapy was the cause of the disease in this macaque. Whether tacrolimus or methylprednisolone individually led to the macaque's diabetes cannot be determined, but we surmise that the diabetes was likely due to the combination of the therapies. Additional risk factors in humans (including older age, nonwhite ethnicity, obesity, and hepatitis C infection)^{9,10,21,29,34,39} are not applicable to our macaque, thus supporting immunosuppressive therapy as the cause of his diabetes. Whether anything might be done to prevent NODAT from occurring in future animals on this protocol is unclear, although several reports suggest that lowering steroid and tacrolimus doses can decrease incidence in humans without increasing the risk of rejection.^4,5,18,22,40 Lowering immunosuppressive drug dosages, particularly of glucocorticoids and tacrolimus, may decrease the incidence in the cynomolgus macaque transplantation model.

Diabetes mellitus in human patients is associated with considerable complications after transplantation, including cardiovascular disease and graft rejection.^9,10,15,19 NODAT in human kidney transplant patients has been associated with a 2- to 3-fold increase of cardiovascular disease events when compared with nondiabetic patients.³⁴ No evidence of cardiovascular disease or other pathology has been observed in this macaque.

Why this particular animal became diabetic whereas countless other macaques treated with comparable immunosuppressive regimens have not is unknown. Although the risk of NODAT in human transplant patients is 2% to 50%, perhaps cynomolgus macaques are more resistant to diabetes due to immunosuppressive therapy, leading to a much lower percentage of NODAT in the macaque model. We noted no significant differences when we compared this animal with other macaques (that is, age, sex, organ transplanted, clinical pathology, length of treatment, and so forth) on this or similar transplant protocols. Additional testing is not plausible in this macaque, because it is no longer diabetic and because reintroducing the immunosuppressive regimen to reinduce diabetes would interfere with the current protocol. Any future macaques found to be diabetic will undergo additional testing to help determine a more definitive cause for the diabetes. In light of the clinical information and our search of the literature, this case is the first reported incidence of NODAT in a cynomolgus macaque.