Tacrolimus reversibly reduces insulin secretion in paediatric renal transplant recipients
Guido Filler1,,,
Ines Neuschulz1,
Ilka Vollmer1,
Peter Amendt2 and
Berthold Hocher3
1 Department of Paediatric Nephrology,
2 Division of Paediatric Endocrinology, Charité Children's Hospital and
3 Department of Nephrology, Humboldt University, Berlin, Germany
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Abstract
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Background. Conflicting reports exist about the mechanism of tacrolimus-induced post-transplant diabetes mellitus.
Methods. We analysed intravenous glucose tolerance tests (IVGTT) of 14 paediatric renal transplant recipients on cyclosporin (CsA) microemulsion and 15 patients on tacrolimus (FK506). The groups were similar in age (13.2±4.2 vs 13.0±3.7 years), body mass index, serum creatinine concentrations (96±60 vs 97±44 µmol/l), time after renal transplantation, and cumulative steroid dose over 12 weeks prior to the test (3.4 vs 3.5 mg/m2/day, NS, MannWhitney). Parameters of glucose tolerance included glucose, insulin, C-peptide concentrations, and HbA1c. The mean concentrations of the primary immunosuppressant were similar to treatments employed in other centres (CsA 165±59 ng ml and FK506 7.5±2.2 ng ml).
Results. Baseline glucose concentrations were significantly higher on FK506 therapy compared with CsA microemulsion therapy. Baseline insulin concentrations and C-peptide concentrations were identical in both treatment groups. FK506 trough levels correlated negatively with k values (glucose constant decay) in the FK506 group. There was a significant reduction of the insulin first-phase concentrations, both after 1 min and after 3 min in the FK506 group compared with the CsA group (112±17 vs 237±57 µU/ml, P=0.034). In patients with repetitive IVGTTs, glucose constant decay and insulin production improved after lowering FK506 whole-blood trough levels.
Conclusions. We conclude that post-transplant glucose intolerance could be due to a dose-dependent, direct effect of FK506 on the pancreatic beta cell function, which can be controlled by dose reduction.
Keywords: calcineurin inhibitors; cyclosporin microemulsion; glucose tolerance; insulin production; renal transplantation; tacrolimus
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Introduction
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Tacrolimus (FK506) is a new immunosuppressant in organ transplantation; however, there have been reports about a possible diabetogenic effect [1,2]. In an American adult renal transplant population, an incidence of chronic new-onset insulin dependence of 8% was reported in a large single-centre trial [3]. There are few reports of post-transplant diabetes mellitus following renal transplantation and FK506-based immunosuppression in children [4,5]. The mechanism responsible for the development of post-transplant diabetes mellitus with FK506 treatment remains unclear [6]. Apart from drug effects, the underlying disease itself may also increase the risk of developing diabetes [7,8]. In addition, peripheral insulin resistance may be responsible for impaired glucose tolerance [9]. Finally, the use of recombinant human growth hormone in children may induce diabetes mellitus [10,11]. In order to investigate the possible effect of FK506 on the development of impaired glucose tolerance in paediatric renal transplant recipients, we analysed the results of intravenous glucose tolerance tests (IVGTTs) of patients receiving either cyclosporin (CsA) microemulsion or FK506. This investigation was undertaken to provide insights into insulin early and second-phase secretion under immunosuppressive therapy.
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Subjects and methods
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Twenty-nine paediatric renal transplant patients were included in this evaluation, 14 patients received CsA microemulsion and 15 patients received FK506. Patients with infantile cystinosis or on treatment with growth hormone were excluded from the study, as were patients with obviously impaired glucose tolerance. Data on age, time of the test after transplantation, height, body mass index (BMI), cumulative steroid dose over 12 weeks prior to the study expressed as mg/m2/day, mean CsA or FK506 trough concentrations, as well as serum creatinine, glomerular filtration rate (GFR), and parameters of carbohydrate tolerance are given in Table 1
. The groups did not differ significantly with respect to age, cumulative steroid dose, BMI, and concomitant medication.
Due to the age dependency of insulin concentration [12], i.e. the higher insulin concentrations of pubertal children and the strict age dependency of 1 min+3 min first-phase insulin secretion, it was important that the groups compared well in this respect. The proportion of prepubertal children compared to pubertal children was similar in each group (3/11 CsA group, 4/11 FK506 group). Because of the small number of patients in each group, no distinction between pubertal and prepubertal children was made.
All patients received a normal carbohydrate-rich diet 3 days prior to the investigation and, after a fasting period of 12 h, attended the outpatient clinic for testing. Dosing of CsA and FK506 was not changed in any of the patients for at least an 8-day period. Twelve-hour post-dose trough concentrations were measured on the morning of the glucose tolerance test. IVGTTs were performed according to the method described by Srikanta et al. [13]. Glucose (20%) was injected intravenously at a dose of 0.5 g/kg body weight (max. 25 g) over 120 s after measurement of two consecutive basal whole blood glucose (BG) concentrations, immunoreactive insulin (IRI), and human C-peptide (HCP) concentrations. Consecutive samples of BG, IRI and HCP were taken via a second intravenous line at 1, 3, 5, 10, 20, 30, 40, 50 and 60 min after injection. Blood glucose was analysed using a standard glucose-oxidase method (Hitachi-autoanalyser). k Values were calculated according to the method of Marble and Ferguson [14]. Immunoreactive insulin IRI and HCP were measured using commercially available radioimmunological methods as previously described [8]. Impaired glucose tolerance was defined in this study as a k value below 1.2%/min.
For a case report, a 17-year-old non-cystinotic renal transplant recipient on FK506 with a period of impaired glucose tolerance was investigated repetitively 19, 46, and 177 days after renal transplantation.
Statistical analysis was performed using the commercially available computer program GraphPad Prism 2.01 for Windows 95. For statistical evaluation of cross-tabulated contingency tables, Fisher's exact test was used. Deviations from Gaussian distribution were tested using the KolmogorovSmirnov test, and for comparison, the unpaired t-test was used in the case of a Gaussian distribution, whereas the MannWhitney test was used in case of a non-Gaussian distribution. Where applicable, data were stated and plotted as mean±1 standard deviation or as median and range. For statistical analysis, P values of two-tailed or two-sided tests are given.
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Results
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The two treatment groups did not differ with regard to age, time after renal transplantation, height, weight (data not shown), body mass index, serum creatinine, and GFR estimate according to Schwartz et al. [15]. More importantly, the cumulative steroid dose was identical in both groups (Table 1
). In contrast, mean fasting blood glucose in the CsA group was significantly lower than in the FK506 group (4.8±0.4 vs 5.5±0.5 mmol/l, P=0.0007, unpaired t-test). There was a tendency towards a higher proportion of HbA1c in the FK506 group (5.8±0.5 vs 5.2±0.1% in the CsA group, P=0.2087, unpaired t-test).
On IVGTT, 27 out of a total of 29 patients had a k value (glucose constant decay) of >1.2%/min; 14/14 patients in the CsA group and 13/15 patients in the FK506 group (P=0.4828, NS, Fisher's exact test). The mean k value in the CsA group was 1.9±0.4%/min compared with 1.7±0.5%/min in the FK506 group (P=0.2584, unpaired t-test). Results of the blood glucose measurements are summarized in Figure 1a
.

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Fig. 1. (a) Whole venous blood glucose concentration before and during IVGTT. Fasting baseline blood glucose concentrations were significantly higher in the FK506 group (n=15) when compared to the CsA group (n=14). Data are given as mean±1 standard deviation (SD). (b) Insulin concentrations before and during IVGTT. Data are given as mean±1 standard deviation (SD). Insulin concentrations at 1 and 3 min were significantly lower in the FK506 group.
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No correlation of the k value and BMI or the cumulative steroid dose was observed in either the FK506 or the CsA group. Furthermore, within the CsA group, there was no correlation between CsA trough concentrations and the k value. Basal insulin and C-peptide concentrations in peripheral blood did not differ in patients on CsA compared to patients who received FK506. Since the renal function was equal in both groups, differences in C-peptide could not be attributed to a difference of GFR, and therefore this could not be attributed as direct evidence for peripheral insulin resistance in either group. In contrast, there was a significant reduction of both the insulin and reactive insulin (above baseline, referred to as delta insulin) concentrations in the FK506 group compared with the CsA group. The early-phase insulin secretion (sum of 1 and 3 min) was 237±57 µU/ml in the CsA group and 112±17 µU/ml in the FK506 group (P=0.0342, unpaired t-test, Figure 1b
), and the reactive (i.e. above baseline) insulin early-phase concentration (1+3 min) was 200±55 µU/ml in the CsA group and 85±14 µU/ml in the FK506 group (P=0.0407, unpaired t-test, Figure 2a
). There was also a reduction of the C-peptide concentrations in the FK506 group; however, the difference in total C-peptide area did not reach statistical significance (CsA group 551±355, FK506 group 458±142, Figure 2b
).

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Fig. 2. (a) Age-independent reactive insulin concentrations above baseline ( insulin) before and during IVGTT. Data are given as mean±1 standard deviation (SD). Insulin concentrations at 1 and 3 min were significantly lower in the FK506 group. (b) C peptide concentrations before and during IVGTT. Data are given as mean±1 standard deviation (SD). C-peptide concentrations at 1 and 3 min were significantly lower in the FK506 group.
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These data point to a decreased early-phase insulin concentration in children and adolescents receiving therapy with FK506 following renal transplantation. In order to investigate whether FK506 trough levels might be related for these findings, we performed a regression analysis between FK506 trough concentration and k value. There was a tendency towards lower k values in patients with higher FK506 trough levels in 15 patients. However, using data from these available 35 tests in 28 patients, we could demonstrate a weak but significant correlation between FK506 trough levels and the glucose constant decay or the insulin 3-min concentration above baseline (Figure 3a
and b
).

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Fig. 3. (a) Correlation between FK506 trough concentration and k value. (b) Correlation between FK506 trough concentrations and 3-min insulin.
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These data could be interpreted as a dose-dependent influence of FK506 on the k value and insulin secretion. This observation was supported by an analysis of a single patient with repetitive IVGTTs following substantial FK506 dose reduction with unaltered steroid dosing. This is described in the following case report.
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Case
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A 17-year-old patient received a cadaveric renal transplant because of end-stage renal failure due to tuberous sclerosis and associated polycystic kidney disease. Immunosuppression consisted of FK506, azathioprine and steroids. A glucose tolerance test was performed while the patient received prednisolone at a dose of 30 mg/m2, revealing a k value of 1.14 on day 19 after renal transplantation. Despite steroid reduction to 4 mg/m2, glucose tolerance deteriorated, and the patient developed a severely decreased k value of 0.61 on day 46 after renal transplantation with decreased insulin and C-peptide concentrations. At that time, the patient received FK506 at a dose of 6.2 mg/m2/day and had a trough level of 9.7 ng/ml. FK506 area under the time concentration curve (AUC) was 126 ng h/ml, which is in the expected range. Following conversion to triple daily FK506 dosing because of a high Cmax (28.8 ng/ml), sharp FK506 dose reduction to 2.8 mg/m2/day as well as reduced AUC of 85 ng h/ml, the k value increased to 1.16 and even reached 1.65 after 269 days without further dose reduction of FK506 (Figure 4a
). Following FK506 dose reduction, the insulin secretion also improved (Figure 4b
).

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Fig. 4. (a and b) Repetitive IVGTTs performed in a single patient on days 19, 46, and 177 after renal transplantation. Glucose concentration is plotted on the y axis of (a) and insulin is plotted on the y-axis of (b). At the time of the worst k value (day 46) insulin concentrations were lowest, and were highest following substantial dose reduction on day 177.
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Discussion
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The diabetogenic effect of immunosuppressive therapies after organ transplantation is well recognized [1]. However, it is difficult to determine whether insulin resistance or a possible effect on the pancreatic ß-cell is responsible for this effect. In order to differentiate between the two mechanisms, we analysed IVGTTs in patients on FK506 therapy and compared their data with a comparable group of patients on CsA microemulsion therapy. A word of caution is necessary considering the k values obtained from the IVGTTs, since it is not known if it depends on insulin secretion or on insulin sensitivity. Because the investigations were performed in children, we refrained from using the minimal model [16] requiring large number of blood samples, which would have helped to overcome this problem.
In this investigation, the incidence of impaired glucose tolerance, defined as a k value below 1.2%/min, was more frequent in patients on FK506 (13%) compared to patients who received CsA therapy (0%), although this did not reach statistical significance. k Values tended to be lower in patients on FK506 therapy, again, without reaching statistical significance. These findings are in accordance with a study comparing oral glucose tolerance test in liver-transplant recipients treated with either CsA or FK506 [17]. Both basal insulin and C-peptide concentrations were reported to be elevated after renal transplantation [18].
Immunosuppressants such as corticosteroids and CsA play an important role in altering glucose tolerance after renal transplantation and may induce insulin resistance [19,20]. In this study, cumulative steroid doses were identical in both treatment groups, and trough concentrations were in a medium range for both drugs. Despite these similarities, it is unfortunate that there was a tendency towards a shorter time after renal transplantation in the FK506 group. This difference of the time interval after renal transplantation and the IVGTT may introduce some bias. There was a tendency towards increased basal C-peptide concentrations in both the CsA therapy group and the FK506 therapy group when compared to the values obtained from adolescents with a similar degree of renal insufficiency without immunosuppressive therapy [5]. This slight increase could be attributed to renal insufficiency, which was similar in both groups, since C-peptide accumulates in renal failure. Under basal conditions, fasting insulin and C-peptide concentrations were also not different between patients receiving FK506 or CsA treatment respectively.
The fasting glucose concentrations were higher in patients on FK506 therapy. Following intravenous glucose load, there were significantly diminished insulin, reactive insulin, and reactive C-peptide early-phase concentrations in patients receiving FK506 compared to patients treated with CsA microemulsion. These data point to a reduced functional reserve of hormone production of the B cell in patients on FK506 therapy.
This study cannot elucidate whether a toxic effect on the pancreas is responsible for the phenomenon. In previous patients who had become diabetic during FK506 treatment, no anti-islet or anti-insulin antibodies could be identified, suggesting that diabetes mellitus was not due to autoantibodies. Furthermore, a weak correlation between FK506 trough levels and k values could be demonstrated, indicating a dose-dependent effect of FK506.
The repetitive IVGTTs performed in the patient with impaired glucose tolerance supported the notion of a dose-dependent influence of FK506 on pancreatic ß cells. These data correspond with in vitro data obtained in a study that investigated the effect of FK506 on human insulin secretion in a model system using the HIT-T15 ß-cell line [21]. In that study, a time-and dose-dependent decrease of HIT cell insulin secretion was demonstrated. The cell line was shown to contain calcineurin, and the authors suggested that FK506 might have direct effects to reversibly inhibit insulin gene transcription, leading to a decline of insulin mRNA levels, insulin synthesis, and insulin secretion. It has been shown that inhibition of insulin production by FK506 is caused at the transcriptional level in pancreatic beta cell when FK BP-12 content is relatively high [22]. Our in vivo study could not contribute to clarification of the mechanisms involved on a cellular level. However, the decreased insulin concentrations and the demonstrated dose dependency suggest a direct inhibition of pancreatic ß-cell function as reported by Redmon et al. [21]. This study gives a rationale for reducing the FK506 dose in patients in whom insulin resistance can be excluded and decreased early-phase insulin concentration can be demonstrated. We therefore recommend a regular surveillance of the parameters of glucose tolerance, insulin, and C-peptide concentrations in patients on FK506 therapy. It may be feasible to reduce FK506 dosing in case of impaired glucose tolerance because decreased insulin secretion must be considered as a direct inhibitory effect on ß-cell function.
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Acknowledgments
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We thank Prof. Rohde, Department of Experimental Endocrinology, Charité, Humboldt University, Berlin, for measuring all the insulin and C-peptide concentrations of all the tests evaluated in this study.
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Notes
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Correspondence and offprint requests to: Guido Filler MD FRCPC, Professor of Paediatrics, Head, Division of Paediatric Nephrology, Department of Paediatrics, University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1. 
Present address: Department of Paediatrics, University of Ottawa, Ottawa, Canada. 
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Received for publication: 15. 7.99
Revision received 14.12.99.