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, 25and50ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50ng/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 25and50ng/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 25and50ng/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.