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

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.


Sirolimus impairs glucose-induced insulin secretion in pancreatic β cells.
To test the effect of sirolimus on pancreatic β cell function, we evaluated the response to glucose in INS-1 β cells. We first performed a dose-response assay, and we found that increasing doses of sirolimus progressively reduce glucose-stimulated insulin secretion (GSIS, Fig. 1a). Then, we performed a time-course experiment using the dose of sirolimus (25 nM) that has been measured in the blood of transplant recipients 33 and we observed that a 24-hour incubation significantly decreased GSIS (Fig. 1b). Importantly, we did not detect any significant effect of sirolimus on cell viability (Fig. 1c). These results were also confirmed in murine (Fig. 1d,e) and in human (Fig. 1f,g) islets.
Sirolimus reduces mitochondrial respiration in pancreatic β cells. When testing the effect of sirolimus on insulin release in response to the fuel secretagogues leucine and glutamine, which are known to stimulate insulin exocytosis through increased mitochondrial metabolism 34 , we found a significantly impaired response in sirolimus-treated cells (Fig. 2a), whereas cells from both groups were similarly responsive to KCl-mediated depolarization (Fig. 2b). We obtained similar findings in murine (Fig. 2c,d) and in human (Fig. 1e,f) islets, thereby suggesting an action of the immunosuppressant drug on the mechanisms underlying metabolism-secretion coupling. Therefore, we tested the effect of sirolimus on mitochondrial respiration, observing a significant decrease in oxygen consumption rate (OCR) in clonal β cells (Fig. 3) and in islets isolated from mice and humans ( Figure S1) treated with sirolimus.
Sirolimus decreases mitochondrial Ca 2+ uptake in β cells. Mounting evidence indicates that Ca 2+ represents a major regulator of mitochondrial function, and a decreased uptake of this ion by this organelle has been functionally linked to reduced mitochondrial respiration in various cell types [35][36][37][38][39] . Thus, we assessed mitochondrial Ca 2+ uptake in pancreatic β cells following incubation with sirolimus and we observed a significantly decreased uptake compared with vehicle-treated cells (Fig. 4a,b). We and others have demonstrated the importance of ER Ca 2+ in β cell function 31,[40][41][42][43][44][45] . Since sirolimus has been reported to modulate intracellular Ca 2+ fluxes in different tissues [46][47][48][49] , we assessed ER Ca 2+ stores and we found that sirolimus-treated β cells exhibited depleted intracellular Ca 2+ stores and increased Ca 2+ leak (Fig. 4c,d). When measuring cytosolic Ca 2+ levels, we also observed a slightly reduced response to glucose in sirolimus-treated β cells ( Figure S2), further supporting our data on decreased GSIS. Sirolimus regulates the expression of inositol 1,4,5-trisphosphate receptor in clonal β cells and human and murine islets. Since sirolimus is known to modulate transcriptional activity 50-52 , we tested its effect on the expression levels of key players in Ca 2+ handling, namely inositol 1,4,5-trisphosphate receptor (IP3R) 53 , ryanodine receptor (RyR) 31,54 and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) 44 in clonal β cells and human and murine islets. We observed a significant upregulation of all three IP3R isoforms in sirolimus-treated compared with vehicle-treated cells, a result that was consistent in all of the tested species (rat, mouse, human, Fig. 5), strongly suggesting that the modulation of IP3R is one of the mechanisms underlying the effects of sirolimus on pancreatic β cells.

Discussion
Immunosuppressive therapy has been shown to be associated with glucose intolerance and post-transplantation diabetes mellitus; such a diabetogenic effect is common to immune-modulating agents acting via different mechanisms of action 3,9,55 , including β cell failure 4,13,56,57 and alteration of metabolic parameters controlled by insulin signaling [58][59][60][61] . Here we show that sirolimus has a detrimental effect on Ca 2+ handling and GSIS both in human and murine islets and in rat insulinoma cell line INS-1.
We also demonstrate that at 25 nM -the dose measured in the blood of transplanted patients 33 -sirolimus does not significantly affect cell vitality, consistent with previous experiments performed in various cell types revealing that only supra-therapeutic doses of sirolimus lead to increased apoptosis and reduced cell proliferation 49,56,62 , without affecting overall insulin content 56 . Furthermore, earlier investigations had established that immunosuppressant drugs, when used at concentrations comparable with therapeutic levels in humans, do not cause apoptosis in pancreatic β cells 63 .
We show here for the first time that sirolimus significantly compromises mitochondrial respiration, directly assessed by measuring oxygen consumption, both in pancreatic islets and clonal β cells. The direct modulation of intracellular Ca 2+ release channels on the ER offers a novel mechanistic insight on the diabetogenic effect of sirolimus. Intriguingly, the upregulation of IP3R observed in pancreatic β cells following sirolimus treatment is consistent with the recent observation that IP3R levels are increased in islets from diabetic patients, mirrored by a  64 . Additionally, Madec and colleagues had shown that exposing pancreatic islets to high glucose concentrations led to increased levels of IP3R 65 . Also, mutations in the gene encoding for IP3R have been associated to perturbations in glucose homeostasis and enhanced susceptibility to diet-induced diabetes mellitus 66 . Further studies are necessary to better delineate the exact role of IP3Rs in the regulation of Ca 2+ fluxes in β cells and to identify other potential mechanisms.
Interestingly, the properties of sirolimus observed in β cells are cell-specific and seem to be in contrast with its effects seen in models of neurodegenerative and ischemic disorders 67,68 , in which the drug has been shown to be overall protective, inducing autophagy and enhancing lysosomal activation in order to remove damaged mitochondria 69 . The complexity of the pathways induced by sirolimus is further confirmed by the experimental findings of Fuhrer and colleagues, who observed that, despite sirolimus significantly suppresses β cell response to glucose (in agreement with our findings), the incubation of RIN-5F cells with high doses of sirolimus in absence of glucose can instead increase insulin secretion 57 . However, opposite to their results, Barlow and colleagues found that 200 nM sirolimus caused a significant reduction in both basal and glucose-stimulated insulin release in Min-6 cells 56 . The exact mechanisms underlying such different pharmacologic responses need to be characterized in future studies.

Materials and Methods
Cells. Human islets with >90% purity and viability were obtained from non-diabetic de-identified cadaveric donors through the Integrated Islet Distribution Program (IIDP). The characteristics of the donors are reported in Supplementary Table 1. Upon receipt, the islets were cultured as described 70 . Murine islets of Langerhans were isolated as previously described 31 . Procedures on rodents have been performed according to guidelines and regulations approved by the Einstein Animal Care and Use Committee. INS-1 cells were maintained in monolayer culture in RPMI-1640 medium, as previously described by our group 71 . Insulin levels were determined as described and validated 31,[71][72][73]   Analyzer (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer's instructions. Specifically, the following drugs were added to each well: glucose (16.7 mM, at minute 32) to determine response to high glucose; oligomycin (1 μM, at minute 104) to inhibit ATP synthase and assess coupling efficiency; carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.5 μM, at minute 176) to uncouple the mitochondrial oxidative phosphorylation and measure both maximum respiration and spare capacity; antimycin A and rotenone (both 1 μM, at minute 216) to inhibit the respiratory chain and measure non-mitochondrial respiration. After each assay, the cells were collected to quantify DNA via QuantiFluor dsDNA System (Promega, Madison, WI, USA), according to the manufacturer's instructions. Ca 2+ dynamics in cytosol, ER and mitochondria. Ca 2+ dynamics were evaluated as previously described and validated [74][75][76][77] . Briefly, cells attached on glass bottom culture dishes (MatTek Corporation, Ashland, MA) were loaded with Fura-2 acetoxymethyl (AM) ester (Thermo Fisher Scientific, Waltham, MA, USA, 5 μM, 15 min, 37 °C). Images were obtained using a dual excitation fluorescence imaging system, as described 31 : changes in intracellular Ca 2+ were expressed as the ratio of fluorescence emission acquired above 510 nm in response to excitation at 340 nm and 380 nm. ER Ca 2+ was assessed in cells transfected with the luminal Ca 2+ sensor D1ER (Addgene, Cambridge, MA), as described [78][79][80] and the rate of Ca 2+ leak was measured as function of [Ca 2+ ] ER following the addition of thapsigargin (1 μM). To evaluate mitochondrial Ca 2+ , the samples were loaded with rhod-2 AM (Thermo Fisher Scientific, 3 μM, 30 min, 37 °C), followed by washout and 1 hour rest at room temperature for de-esterification 31,77,81 . Fluorescence was detected using a pass-band filter of 545-625 nm in response to excitation at 542 nm.
Real-time RT-qPCR. Total RNA was isolated from β cells and islets using TRIzol reagent (Thermo Fisher Scientific) in combination with the RNeasy Mini kit (Qiagen, Hilden, Germany) followed by DNase treatment 82,83 , and cDNA was synthesized via a Thermo-Script RT-PCR System (Thermo Fisher Scientific). After reverse transcription, real-time quantitative PCR was performed on an AbiPRISM 7300 fast real-time cycler using the power SYBR Green real-time PCR master mix kit and quantified by built-in SYBR Green Analysis (Thermo Fisher Scientific) 77,84 . Samples were measured in triplicates and results were confirmed by at least three independent experiments. The relative amount of specific mRNA was normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of oligonucleotide primers (Merck KGaA, Darmstadt Germany) for gene analysis are listed in Supplementary Table 2.
Statistical analysis. All results are presented as mean ± s.e.m. Unless otherwise noted, experiments were performed in a blinded fashion at least three times. Statistical analysis was performed via Student's t test (for 2 groups) unless otherwise indicated, using Prism 7 software (GraphPad, San Diego, CA, USA). A value of P < 0.05 was considered statistically significant. Data Availability. All data generated or analyzed during this study are included in the present article.