From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept

Abstract

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Calcineurin inhibitors (CNIs) are the cornerstone of immunosuppressive regimen to prevent rejection. CNIs, and especially tacrolimus, increase the risk of post-transplant diabetes (PTD). It was recently shown that late conversion from CNI regimen to belatacept regimen (a costimulation blocker) in kidney transplant recipients led to improvement in HbA1c (>0.5%) in case of PTD or de novo diabetes after transplantation ¹ . Based on this observation, we assessed if the effect on PTD was due to CNI withdrawal only or if belatacept has its own mechanism of action.

We tested the effect of tacrolimus and belatacept on a beta cell line and rat islets. MIN6 cells were obtained from AddexBio (SanDiego, CA, USA) and rat islets were isolated as previously described ² . Both were exposed to tacrolimus 100 µg/ml or belatacept 1 mg/ml for 24 h as previously tested in the laboratory (data not shown). Analysis was performed immediately after drug exposition. MIN6 viability was performed using flow cytometer with annexin V and propidium iodiure (PI) labeling, and viability of islets was performed using confocal microscopy with Syto 13 and PI labeling. Glucose-stimulated insulin secretion was performed using static incubation in Krebs–Ringer buffer medium at 2.8- and 16.7-mM glucose. Results were obtained using an enzyme-linked immunosorbent assay and expressed as the insulin stimulation index (SI): 100 × (insulin secreted in high glucose / insulin secreted in low glucose).

No alteration of viability was observed upon exposure to tacrolimus compared to control for both MIN6 cells (86.1 ± 3.2% vs control 87.0 ± 2.5%, P = 0.90, Fig. 1A) and islets (97.9 ± 1.1% vs control 97.9 ± 1.1%, P = 0.85, Fig. 1B).

Figure 1.

Viability and insulin secretion of MIN6 and islets exposed to tacrolimus and belatacept. MIN6 and rat islets are exposed to tacrolimus (100 µg/ml) or belatacept (1 mg/ml) for 24 h. (A) Effect of tacrolimus and belatacept on MIN6 cells’ viability. (B) Effect of tacrolimus and belatacept on rat islets’ viability. (C) Effect of tacrolimus and belatacept on MIN6 cells’ insulin stimulation index. (D) Effect of tacrolimus and belatacept on rat islets’ insulin stimulation index. Data are expressed as the mean ± SEM, n = 5, one-way ANOVA Fisher’s with Tukey’s post hoc test. SEM: standard error of the mean; ANOVA: analysis of variance. P-values are denoted as follows: *P < 0.05; ***P < 0.001.

No alteration of viability was observed upon exposure to belatacept compared to control for both MIN6 cells (86.5 ± 2.9% vs control 87.0 ± 2.5%, P = 0.90, Fig. 1A) and islets (97.5 ± 1.2% vs control 97.9 ± 1.1%, P = 0.85, Fig. 1B).

A decrease in the insulin SI was observed upon exposure to tacrolimus compared to the control for both MIN6 cells (0.91 ± 0.17 vs control 1.30 ± 0.17, P = 0.015, Fig. 1C) and islets (0.80 ± 0.32 vs control 2.33 ± 0.35, P < 0.001, Fig. 1D).

The insulin SI remains unchanged upon exposure to belatacept compared to the control for both MIN6 cells (1.52 ± 0.39 vs control 1.30 ± 0.17, P = 0.52, Fig. 1C) and islets (1.98 ± 0.44 vs control 2.33 ± 0.35, P = 0.33, Fig. 1D).

We demonstrated that tacrolimus alters insulin secretion from beta cells and rat pancreatic islets without affecting cell viability, whereas belatacept had no deleterious effects on cell viability or insulin secretion ability. These preliminary findings suggest a preservation of the secretory function of the islets under belatacept. This sustains the clinical observation of HbA1c improvement when switching from tacrolimus to belatacept. The use of belatacept could also be beneficial in islet or pancreas transplantation, indicated in patients with unstable diabetes. Recently, a series of five cases of islet-transplanted patients successfully treated with belatacept (without sever hypoglycemia) was reported ³ .

Further research is needed to understand the metabolic changes occurring in humans during the switch from CNI to belatacept, but preservation of insulin secretion appears to be a line of research.