Tacrolimus inhibits insulin release and promotes apoptosis of Min6 cells through the inhibition of the PI3K/Akt/mTOR pathway

Abstract

As a calcineurin inhibitor, tacrolimus is commonly used as a first-line immunosuppressant in organ transplant recipients. Post-transplantation diabetes mellitus (PTDM) is a common complication following kidney transplantation and is associated with immunosuppressant drugs, such as tacrolimus. PTDM caused by tacrolimus may be related to its influence on insulin secretion and insulin resistance. However, the specific mechanism has not been fully elucidated. The aim of the present study was to investigate whether the PI3K/Akt/mTOR signaling pathway served an important role in the pathogenesis of PTDM induced by tacrolimus. In the present study, the Cell Counting Kit-8 assay was used to measure the effect of tacrolimus on the viability of Min6 mouse insulinoma cells. The effects of tacrolimus on the insulin secretion and the activity of caspase-3 of Min6 cells stimulated by glucose exposure were measured by ELISA. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using WST-8 and thiobarbituric acid assays, respectively. The effects of tacrolimus on the mRNA expression levels of PI3K, Akt and mTOR were detected by reverse transcription-quantitative PCR (RT-qPCR), whereas the protein expression levels of PI3K, Akt, mTOR, phosphorylated (p)-AKT and p-mTOR in Min6 cells were assessed using western blotting. The present data indicated that, compared with the control group, 5, 25 and 50 ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50 ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50 ng/ml tacrolimus markedly increased the activity of caspase-3 by 175.1% (P<0.05), it also decreased the SOD activity (P<0.01) and increased MDA levels (P<0.05). The RT-qPCR results demonstrated that the mRNA expression levels of PI3K, Akt and mTOR were downregulated by 25 and 50 ng/ml tacrolimus (P<0.01). Furthermore, the western blotting results suggested that tacrolimus had no significant effects on the expression levels of total PI3K, Akt and mTOR proteins (P>0.05), but 25 and 50 ng/ml tacrolimus could significantly inhibit the expression levels of p-Akt and p-mTOR (P<0.01). In conclusion, tacrolimus decreased the activity and insulin secretion of pancreatic β cells and induced the apoptosis of islet β cells by inhibiting the mRNA expression levels of PI3K, Akt and mTOR and reducing the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway, which may ultimately lead to the occurrence of diabetes mellitus, and may be considered as one of the specific mechanisms of PTDM caused by tacrolimus.

Fast Tacrolimus Metabolism Does Not Promote Post-Transplant Diabetes Mellitus after Kidney Transplantation

“…Based on the knowledge that the underlying mechanism of Tac-associated PTDM is supposed to directly affect insulin secretion in pancreatic β-cells rather than inducing insulin resistance as an underlying mechanism for Tac-associated PTDM [ 9 , 10 ], we modeled the effects of slow and fast Tac metabolism on insulin-producing pancreatic cells (INS-1 cells) in vitro. The kinetics of Tac concentration used in cell culture experiments was determined in a former study showing that fast Tac metabolism is associated with increased nephrotoxicity in vitro and in vivo, respectively, and affected allograft function and allograft survival [ 4 , 14 ].…”

“…Because CNI toxicity is strongly related to the administered Tac dose relative to the trough level (C/D ratio) and thus to higher peak Tac levels, a higher risk of Tac renal toxicity in fast metabolizers is understandable [ 15 , 16 ]. However, the toxic action of Tac is not limited to renal cells and function, but, amongst others, is also apparent in pancreatic β-cells [ 10 , 12 ]. One mechanism for the diabetogenic effect of Tac might be the inhibition of the calcineurin-nuclear factor of activated T-cells (NFAT) pathway.…”

“…In rats, high-dose Tac treatment (10 mg/kg/day) decreased insulin mRNA transcription and reduced insulin production due to high levels of FK506-binding protein-12 in pancreatic β-cells [ 9 ]. Tong et al found that high Tac concentrations inhibited insulin release and induced apoptosis in ß-cells [ 10 ]. Furthermore, CNI-induced hypomagnesemia is associated with post-transplant diabetes mellitus (PTDM), although the underlying mechanisms are still unknown [ 11 ].…”

Translation suppression underlies the restrained COVID-19 mRNA vaccine response in the high-risk immunocompromised group

“…The ISs such as T, S, M, and P and their widely applied combination of TMP/TSP used in immunosuppressed populations can either directly or indirectly perturb mRNA translation (17)(18)(19)(20)(21)(22)42). However, no data exists whether these ISs either individually or in combination repress translation of Co-mV, which could be one of the potential reasons for the dampened Co-mV response seen in IC patients.…”

“…Although the current Co-mV is sequence optimized to increase mRNA stability and maximize protein translation, several determinants such as nutrient availability, genetics, cellular stress, ribosome quality, inclusion of modified nucleotides, mRNA secondary structure, and importantly, drugs/inhibitors present in the host system, can interfere with the process of translation and its kinetics. Among different types of inhibitors/drugs, certain immunosuppressants (ISs) such as glucocorticoids, tumor necrosis factor inhibitors, the combination of inhibitors of tacrolimus (T) (calcineurin inhibitor), mycophenolate (M) (antimetabolite), mammalian target of rapamycin (mTORi), including rapamycin/sirolimus (S), and prednisone (P) (glucocorticoids) (TMP and TSP combinations) that are commonly used to achieve and maintain disease response and remission in IC patients including SOT patients, have been individually or in combination (TMP/TSP) reported involve in translation process (protein synthesis) directly or indirectly by modulating the related physiological processes (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”