Sirolimus induces depletion of intracellular calcium stores and mitochondrial dysfunction in pancreatic beta cells

Abstract

Sirolimus (rapamycin) is an immunosuppressive drug used in transplantation. One of its major side effects is the increased risk of diabetes mellitus; however, the exact mechanisms underlying such association have not been elucidated. Here we show that sirolimus impairs glucose-stimulated insulin secretion both in human and murine pancreatic islets and in clonal β cells in a dose- and time-dependent manner. Importantly, we demonstrate that sirolimus markedly depletes calcium (Ca²⁺) content in the endoplasmic reticulum and significantly decreases glucose-stimulated mitochondrial Ca²⁺ uptake. Crucially, the reduced mitochondrial Ca²⁺ uptake is mirrored by a significant impairment in mitochondrial respiration. Taken together, our findings indicate that sirolimus causes depletion of intracellular Ca²⁺ stores and alters mitochondrial fitness, eventually leading to decreased insulin release. Our results provide a novel molecular mechanism underlying the increased incidence of diabetes mellitus in patients treated with this drug.

Figure 1

Sirolimus impairs glucose-stimulated insulin secretion from pancreatic β cells. Evaluation of the effect of sirolimus on clonal rat β cells (a–c), murine islets (d,e) and human islets (f,g). INS-1 β cells were treated for 24 h with vehicle or sirolimus at the indicated doses (a). INS-1 β cells were treated with vehicle or sirolimus (25 nM) for the indicated times (b). INS-1 β cells were treated for 24 h with 25 nM sirolimus (c). Effect of 25 nM sirolimus (24 h) on insulin release and cell viability in murine (d,e) and human islets (f,g). Data are presented as mean ± s.e.m of at least 5 experiments (clonal β cells and murine islets) or at least 3 experiments (human islets) performed in triplicate. *p < 0.05 vs vehicle. In panel c, data are expressed as percentage of the responses determined following treatment with vehicle, taken as 100%.

Figure 2

Sirolimus compromises insulin secretion from β cells in response to fuel secretagogues. INS-1 β cells (a,b), murine islets (c,d) and human islets (e,f) were incubated for 24 h with vehicle or 25 nM sirolimus and then stimulated with leucine (Leu) and glutamine (Gln, panels a,c,e) or with KCl (panels b,d,f). Data are presented as mean ± s.e.m of at least 3 experiments performed in triplicate. *p < 0.05 vs vehicle.

Figure 3

Sirolimus impairs mitochondrial respiration in pancreatic β cells. The time course of oxygen consumption rate (OCR) was measured using the Extracellular Flux Analyzer in β cells incubated for 24 h with vehicle or 25 nM sirolimus and then treated with glucose, oligomycin, phenylhydrazone (FCCP), antimycin A and rotenone (panel a); see methods for further details. The maximal respiratory capacity is quantified in panel b, in which whiskers represent 5% to 95% spread of the data. Data represent mean ± s.e.m. of 4 independent experiments, each performed in at least 7 replicates. *p < 0.05 vs vehicle.

Figure 4

Effects of sirolimus on Ca²⁺ dynamics in mitochondria and endoplasmic reticulum (ER). Mitochondrial Ca²⁺ uptake was measured in clonal β cells incubated for 24 h with vehicle or 25 nM sirolimus and then stimulated with glucose (16.7 mM, panel a). Amplitude of mitochondrial response was calculated as the level of Rhod-2 F1/F ₀ at the peak (b). ER Ca²⁺ stores (c) and ER Ca²⁺ leak (d) were assessed following 24 h incubation with vehicle or 25 nM sirolimus. In panel c, the green arrowhead indicates thapsigargin (1 μm). Data are presented as mean ± s.e.m of at least 4 experiments performed in triplicate. *p < 0.05 vs vehicle. In panel b, whiskers represent 5% to 95% spread of the data.

Figure 5

Effects of sirolimus on the expression of IP3Rs, RyR2 and SERCA in clonal β cells and murine and human islets. The effects of sirolimus (25 nM, 24 h) on mRNA levels of IP3Rs, RyR2, and SERCA in rat β cells (a) and murine (b) and human (c) islets were evaluated by real-time RT-qPCR analysis of total RNA, relative to vehicle-treated samples (horizontal dashed line), using GAPDH as internal standard. Primer sequences are reported in Supplementary Table 2. Each bar represents mean ± s.e.m. of at least 3 independent experiments in each of which reactions were performed in triplicate. *P < 0.05 vs vehicle.