EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: The mechanism responsible for the development of posttransplant diabetes mellitus associated with tacrolimus treatment remains unclear. To investigate the possible effect of tacrolimus on the development of impaired glucose tolerance in transplant recipients, this study focused on early and second phase insulin secretion, which may be affected by reactive oxygen species under tacrolimus therapy.

Materials and Methods: We measured the antioxidant status and reactive oxygen species levels before and after tacrolimus treatment. The pro-duction of reactive oxygen species was determined by flow cytometry. Total antioxidant status was measured using total antioxidant status kits.

Results: The antioxidant status decreased while reactive oxygen species level increased significantly (P = .032)

Conclusions: Tacrolimus treatment resulted in dose- and time-dependent increases in the production of reactive oxygen species by beta cells. The antioxidant status decreased in beta cells after tacrolimus treatment. Increased production of reactive oxygen species and decreased antioxidant status by tacrolimus in beta cells may lead to some adverse events.

Key words : Calcineurin inhibitor, Free radical, Diabetes, Transplant

Solid-organ transplant is the optimal treatment for patients with end-stage organ failure. Posttransplant immunosuppressive regimens consist of one of the calcineurin inhibitors (cyclosporine and tacrolimus) in combination with mycophenolate mofetil or azathioprine, and steroids. Tacrolimus was approved to prevent graft rejection for liver transplant recipients, and in 1997 it was used for kidney transplant.¹

During T-cell receptor activation by antigens, a calcium signal is induced with increased intracellular calcium level. This activates the serine/threonine protein phosphatase and calcineurin. The calcineurin with cyclophilin/FK-binding protein dephosphory-lates nuclear factor of activated T-cell, which increase the synthesis of interleukin (IL-2) gene in the nucleus. The tacrolimus, as an immunosuppressive drug, binds with FK-binding protein and interrupts this pathway by inhibiting phosphatase activity. Therefore T-cell mediated immune responses and transcription of IL-2 is blocked,^2,3 However, using calcineurin inhibitors induce posttransplant diabetes mellitus, a form of type 2 diabetes mellitus, which is an independent risk factor for cardiovascular events. The posttransplant diabetes mellitus develops in about 11% to 16% of liver, kidney, or heart/lung transplant recipients which in turn significantly decreases the graft and patient survival.^4,5

Clinical investigations have shown that tacrolimus has greater potential to induce posttransplant diabetes mellitus than cyclosporine and improved glucose tolerance abnormalities have been seen when a conversion from tacrolimus to cyclosporine occurs.⁴

Tacrolimus induces posttransplant diabetes melli-tus by several mechanisms, including increased insulin resistance, and effects insulin secretion with a direct toxic effect on the pancreatic beta cells. Recent in vitro studies stress an effect on the increased apoptosis of beta cells when exposed to these drugs.

It also has been demonstrated that a high dose of tacrolimus decreases transcription of the insulin gene.^5-7 Tacrolimus-binding protein 12 (tacrolimus-BP) is not only expressed by T cells, but also found at high levels in Langerhans beta cells.⁸ Tacrolimus has a significant effect on the intracellular calcium signaling pathway and affects the exocytosis of insulin vesicles from pancreatic beta cells. Furthermore, tacrolimus also can inhibit insulin secretion by beta cells.^5-7 Therefore, tacrolimus influences several aspects of beta cell function and insulin secretion, from mRNA transcription to its degranulation.^7,8

Increase in insulin resistance is also suggested as an additional mechanism in the development of posttransplant diabetes mellitus.^8-10 Therefore, the exact mechanism is not fully known.⁴

Reactive oxygen species (ROS) are chemically reactive molecules which contains oxygen. The examples are superoxide anion, hydrogen peroxide, and the hydroxyl radical. The ROS are the natural byproduct of the normal metabolism of oxygen during respiration. The macrophages and neutrophils also generate ROS used for the killing of bacteria after phagocytosis. During environmental stress such as ultraviolet exposure or ionizing radiation, ROS levels increase and affect cell function with subsequent damage to the structure of DNA, RNA, oxidation of polyunsaturated fatty acids (lipid peroxidation), and amino acid oxidation of proteins.¹¹

Normally, the cells defend against harmful ROS damage with enzymes such as superoxide dismutases, catalases, and glutathione peroxidases. Superoxide dismutases and catalase convert the 2 reactive products of hydrogen peroxide and superoxide into oxygen and water. Other small molecules such as ascorbic acid, tocopherol, and glutathione also are important antioxidants by scavenging free radicals. An imbalance between ROS production and scavenging systems is usually called oxidative stress.^10,11 Glutathione peroxidase requires glutathione as substrate and converts hydrogen peroxide to 2H₂O.¹¹

Recently, much attention has focused on the role of ROS and oxidative stress in the pathogenesis of diabetes.¹² The Langerhans islet cells in the pancreas express relatively low levels of antioxidant defense enzymes such as catalase and glutathione peroxidase,¹³ the glutathione antioxidant seems to be the most important enzyme to protect those cells against oxidative damage. Therefore, the beta cells of the pancreas are considered to be particularly susceptible to oxidative damage.¹³ Oxidative stress has been suggested as a mechanism causing diabetes and diabetic complications.¹⁴ There are experimental and clinical evidences prove that production of reactive oxygen species is increased in both types of diabetes mellitus and the onset of the disease is closely associated with oxidative stress. It is caused by more generation of oxygen free-radical as well as alteration in antioxidant enzymes. An imbalance of antioxidant status and ROS production causes damages.^15-17 Therefore, ROS and free radicals might be responsible for damage to beta cells after drug therapy during transplant.

This study aimed to evaluate the function of tacrolimus in beta cells. Tacrolimus treatment may result in the production of ROS in beta cells. Evaluation of ROS levels and antioxidant status before and after tacrolimus treatment could demonstrate the effect of tacrolimus.

Materials and Methods

The study was approved by the research committee of Shiraz University of Medical Sciences. All of the protocols conformed with the ethical guidelines of the 1975 Helsinki Declaration.

The beta cell lines (CRI-D2) were supplied by national cell bank of Iran, Pasteur Institute (Tehran, Iran). The cells were cultured in RPMI supplemented with 10% fetal bovine serum; (Life Technologies, Grand Island, NY, USA) and 1% penicillin 100 U/mL, streptomycin 100 μg/mL (Life Technologies), and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Upon reaching 70% to 80% confluence, adherent cells were harvested with trypsinization (0.05% trypsin- EDTA; Life Techno-logies) and single cell suspension was used for subsequent experiments.

The cells were grown in culture plates, seeded at (5000 cells/well) in 96-cell plates, and treated with tacrolimus (Fujisawa Pharmaceutical; Osaka, Japan) with final concentrations of 0.001 to 100 μmol.

Evaluation of diphenyltetrazolium
Cell viability was evaluated by the diphenylte-trazolium (MTT) assay to measure the degree of cell survival against tacrolimus cytotoxicity. MTT3-(4,5-dimethyl-2-thiazolyl)-2,5-(MTT) assay (3-(4,5-simethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), is reduced to purple formazan in living cells. A soluble solution (usually dimethyl sulfoxide) is added to dissolve the purple Formosan product into a colored solution. Then, by a spectrophotometer at a wavelength usually between 500 and 600 nm, the absorbance of this colored solution can be quantified. Beta cells which were seeded at a density of 5000 cells/well in a flat bottomed 96-well plate (SPL Life Sciences, Gyeong-gido, South Korea), and were incubated at 37°C in a 5% humidified CO₂. After 24 hours, 48 hours, and 72 hours posttreatment with tacrolimus (0.001-100 μmol), the MTT assay was done. Diphenyltetrazolium solution (1 mg/mL) was added into each well and incubated for 3 hours at 37°C in 5% CO₂ incubator. Media was totally removed and the dark blue formazan crystal was dissolved in dimethyl sulfoxide (0.5 mg /mL). Then the optical density was measured at 570 nm using an ELISA reader. The reference wavelength was 690 nm. The mean optical density ± SD for each group was calculated.

Determination of insulin level
Before and after treatment with tacrolimus (after 48 h), the beta cells were treated with low and high glucose according to Merglen and associates protocol12 to cause insulin secretion. Cells were treated with low glucose 3.3 mmol and high glucose 16.7 mmol.

First, the cells were washed and incubated for 1 hour in Krebs Ringer buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), which contains 137 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO⁴, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 5 mM NaHCO₃, 0.2% (w/v) bovine serum albumin, and 16 mM HEPES including 0.754 NaCl, 0.0358 KCl, 0.0296 MgSO₄, 0.0163 KH₂PO₄, 0.0277 CaCl₂, 0.0420 NaHCO₃, 0.2383 for fixing pH to 7.4. The in vitro insulin secretion was then assessed by incubations of the cells in 7 mL of Krebs Ringer buffer HEPES containing 3.3 mM glucose for low glucose solution and 16.7 mM glucose for high glucose solution for 1 hour at 37°C in humidified air and 5% CO₂ for the duration of the incubation and stimulation. Then the cells were washed with HEPES buffer in between 2 incubations to remove the residual insulin. At the end of each incubation, 1000 mL of the Krebs Ringer buffer HEPES solution was removed and frozen at -20°C for storage before insulin assay. Insulin concentrations were determined using Ultrasensitive EIA assay kits (ALPCO Diagnostics, Salem, NH, USA).

Determination of reactive oxygen species level
Flow cytometry procedure has been used to identify ROS by using the dyes 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as ROS sub-strates.^13-15 Each plate had 5 × 104 cells. About 1.5 mL media was discarded and 1.5 mL tacrolimus with 30 μmol concentration was added.

Cultured cells were exposed to 2,7-dichloro-dihydrofluorescein diacetate (H2DCFDA) to detect ROS production. After H2DCFDA were added to the culture media with final concentrations of 10 μM, the cells were incubated at 37°C for 30 minutes; then, they were washed twice with fresh medium. By adding trypsin, the cells were separated from the plate, washed twice with phosphate buffered saline, and finally suspended in 0.5 cc phosphate buffered saline. After pipetting, the samples were evaluated by flow cytometry using FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Determination of total antioxidant status
Each plate had 5 × 10⁴ cells. Total antioxidant status (TAS) was measured using TAS kits (RANDOX Laboratories, London, UK) The ABTS (2.2 = azino- di- [3-thybenzthiazolinesulfate] is incubated with a peroxidase (metmyoglobin) and hydrogen peroxide to produce the radical action ABTS, which has stable blue-green color measured at the wavelength of 600 nm. Antioxidants in the sample cause creation of this blue-green color to a degree that is proportional to their concentration. At first, the initial absorbance was recorded; and after 3 minutes, it was performed again for each sample at 37°C and at a wavelength of 600 nm.

Statistical analysis
All the results are presented as means ± SD. Data for cell viability, ROS, and TAS were derived from 3 to 8 independent experiments. Statistical evaluation of the significance of differences between control and treated groups was determined by Mann-Whitney U test. P values less than .05 were considered significant. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 15.0, IBM Corporation, Armonk, NY, USA).

Tacrolimus resulted in dose- and time-dependent increases in the production of ROS by beta cells. The antioxidant status decreased significantly (P = .032) in the beta cells after tacrolimus treatment. Increased production of ROS species and decreased antioxidant status by tacrolimus in beta cells may contribute to several adverse effects. The cell viability was evaluated by MTT assay. Cell viability was significantly reduced after 48 hours exposure to tacrolimus with 30 μmol concentration (Figure 1).

This toxicity was increased in a time- and dose-dependent manner (Figure 2). After 48 hours treatment with tacrolimus, beta cells were exposed to high and low concentrations of glucose (16.7 mmol and 3.3 mmol).

After tacrolimus treatment by either high and low glucose stimulation, the insulin concentration was ≤ 0.2 which is a considerable difference as compared to untreated cells with insulin concentrations of 1.5 mLU/mL.

After treatment with tacrolimus, the antioxidant level showed significant differences in treated cells compared to controls (P = .032). Beta cells, after 48 hours treatment with tacrolimus, were exposed to DCF and ROS concentration was evaluated by flow cytometry. As shown in Figure 3, after 48 hours treatment with tacrolimus the area under curve in treated cells was more than that in the control groups.

Posttransplant diabetes mellitus is a form of type 2 diabetes mellitus, and an important complication after a solid-organ transplant associated with relative insulin deficiency resulting from increased insulin resistance or impaired insulin production, or a combination of both.^18-20 Sustained chronic hyperglycemia induces excessive formation of reactive oxygen species and reactive nitrogen species, which leads to apoptosis of insulin secreting beta cells related to oxidative stress.^11,13

Experiments on isolated pancreatic islets or beta cell lines revealed that sustained high glucose, which mimics the diabetic conditions, results to significant impairment of beta cell function.²¹ Although ROS species are generally necessary for proper cell function, excessive ROS production is harmful. Early therapeutic managements in patients with type 1 as well as type 2 diabetes mellitus are associated with favorable outcomes on recovery and maintenance of beta cell function with prevention of beta cell loss.²¹

The macrolide antibiotic tacrolimus or tacrolimus is produced by the Streptomyces tsukubaensis and is used as a calcineurin inhibitor in posttransplant immunosuppressive regimens.²²

The diabetogenicity of tacrolimus after organ transplant is well recognized,^1,22,23 and related to the adverse effect on islet cells.²³ The islet cells toxicity and decrease of insulin secretion is reversible when tacrolimus is discontinued. Reduction of tacrolimus blood level causes an increase in both insulin and C-peptide secretion by 24% and 36%. The effect of tacrolimus on insulin secretion is dose related.^23-25 It has been reported that tacrolimus tends to reduce insulin release, while the use of corticosteroids, which is used in transplants, increases the insulin resistance.^26,27

However, it is still not known whether during tacrolimus treatment insulin resistance or a possible effect on the pancreatic beta cell is responsible for the negative effect. Therefore, to differentiate between these 2 mechanisms, we evaluated ROS and TAS levels in beta cells before and after treatment with tacrolimus.

Oxidative stress is viewed by excessive ROS production as well as by low antioxidant enzyme activities. As tacrolimus resulted in production of ROS in the beta cells, it was increased with the time of exposure. Our findings suggested that tacrolimus might cause diabetes mellitus by ROS production as well.

On the other hand, the islet cells express relatively low levels of antioxidant defense enzymes and are more susceptible to oxidative damage and subse-quently cell dysfunction and/or beta cell death.

In conclusion, we determined that treatment of beta cells with tacrolimus might cause cytotoxicity and change in the antioxidant level. Further studies on the detailed mechanism and protection of these cells with antioxidants are recommended.

Volume: 13 Issue: 6 December 2015

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