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

Scientific Reportsvolume 7, Article number: 15823 (2017) | Download Citation


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.


Post-transplant diabetes mellitus represents a major adverse effect of immunosuppressive drugs1,2,3,4 and is associated with high cumulative incidence of cardiac events, vascular disease, and overall impaired survival rates5. Sirolimus (rapamycin) was introduced in the Edmonton immunosuppression protocol in islet transplant recipients6,7, attempting to minimize the diabetogenic effects observed with corticosteroids and other immunosuppressive regimens. Despite the initial enthusiasm, 5-year results of this clinical trial revealed that only ~10% of patients maintained insulin independence1,8, endorsing the detrimental role of sirolimus in glucose homeostasis.

A randomized trial of immunosuppressive drugs in kidney transplantation, the Efficacy Limiting Toxicity Elimination (ELITE) – Symphony study9, identified sirolimus as the one with the highest incidence of hyperglycemia, even higher than calcineurin inhibitors9. Since then, several investigators sought to determine the mechanisms underlying new-onset diabetes mellitus after transplantation10,11,12,13. The effects of sirolimus in vivo are quite complex, as confirmed by numerous controversial findings: indeed, albeit several studies demonstrate that its administration causes glucose intolerance14,15,16, there are also reports showing that it does improve insulin sensitivity in diabetic mice17, protects against obesity18,19, reduces atherosclerosis20,21 and cardiac or renal fibrosis22,23, and extends lifespan24.

We decided to test the effects of sirolimus in pancreatic β cells. Our hypothesis is that one of the mechanisms underlying the diabetogenic action of sirolimus is the impairment of metabolism-secretion coupling in β cells. We focused on the effect of sirolimus on the key organelle in metabolism-secretion coupling, i.e. the mitochondrion25,26,27,28. Indeed, such organelle is considered the main responsible for coupling different fuel secretagogues to insulin exocytosis, through a process that includes oxidation of nutrients within the mitochondrial matrix and subsequent ATP generation, increasing intracellular calcium (Ca2+) via closure of ATP-sensitive K+ channels and depolarization of the plasma membrane27,28,29,30,31,32.


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 recipients33 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.

Figure 1
Figure 1

Sirolimus impairs glucose-stimulated insulin secretion from pancreatic β cells. Evaluation of the effect of sirolimus on clonal rat β cells (ac), 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%.

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 metabolism34, 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.

Figure 2
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
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.

Sirolimus decreases mitochondrial Ca2+ uptake in β cells

Mounting evidence indicates that Ca2+ 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 types35,36,37,38,39. Thus, we assessed mitochondrial Ca2+ 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 Ca2+ in β cell function31,40,41,42,43,44,45. Since sirolimus has been reported to modulate intracellular Ca2+ fluxes in different tissues46,47,48,49, we assessed ER Ca2+ stores and we found that sirolimus-treated β cells exhibited depleted intracellular Ca2+ stores and increased Ca2+ leak (Fig. 4c,d). When measuring cytosolic Ca2+ levels, we also observed a slightly reduced response to glucose in sirolimus-treated β cells (Figure S2), further supporting our data on decreased GSIS.

Figure 4
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.

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 activity50,51,52, we tested its effect on the expression levels of key players in Ca2+ handling, namely inositol 1,4,5-trisphosphate receptor (IP3R)53, ryanodine receptor (RyR)31,54 and sarco/endoplasmic reticulum Ca2+-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.

Figure 5
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.


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 action3,9,55, including β cell failure4,13,56,57 and alteration of metabolic parameters controlled by insulin signaling58,59,60,61. Here we show that sirolimus has a detrimental effect on Ca2+ 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 patients33 – 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 proliferation49,56,62, without affecting overall insulin content56. 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 cells63.

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 Ca2+ 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 reduced number of interactions between ER and mitochondria64. Additionally, Madec and colleagues had shown that exposing pancreatic islets to high glucose concentrations led to increased levels of IP3R65. Also, mutations in the gene encoding for IP3R have been associated to perturbations in glucose homeostasis and enhanced susceptibility to diet-induced diabetes mellitus66. Further studies are necessary to better delineate the exact role of IP3Rs in the regulation of Ca2+ 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 disorders67,68, in which the drug has been shown to be overall protective, inducing autophagy and enhancing lysosomal activation in order to remove damaged mitochondria69. 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 secretion57. 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 cells56. The exact mechanisms underlying such different pharmacologic responses need to be characterized in future studies.

Materials and Methods


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 described70. Murine islets of Langerhans were isolated as previously described31. 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 group71. Insulin levels were determined as described and validated31,71,72,73. In some experiments the cells were treated with glucose (5.5 and 16.7 mM, Bio-Techne, Abingdon, UK), sirolimus (LC Laboratories, Woburn, MA, dissolved in dymethylsulfoxide), or L-leucine (10 mM, MyBioSource, San Diego, CA, USA) and glutamine (2 mM, MyBioSource). Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT colorimetric assay, spectophotometrically (570 nm) measuring the ability of metabolically active cells to reduce MTT.

Extracellular flux analyses

Extracellular flux analyses were performed using the Extracellular Flux 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.

Ca2+ dynamics in cytosol, ER and mitochondria

Ca2+ dynamics were evaluated as previously described and validated74,75,76,77. 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 described31: changes in intracellular Ca2+ were expressed as the ratio of fluorescence emission acquired above 510 nm in response to excitation at 340 nm and 380 nm. ER Ca2+ was assessed in cells transfected with the luminal Ca2+ sensor D1ER (Addgene, Cambridge, MA), as described78,79,80 and the rate of Ca2+ leak was measured as function of [Ca2+]ER following the addition of thapsigargin (1 μM). To evaluate mitochondrial Ca2+, 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-esterification31,77,81. 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 treatment82,83, 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.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Shapiro, A. M. et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 355, 1318–1330 (2006).

  2. 2.

    Krentz, A. J. & Wheeler, D. C. New-onset diabetes after transplantation: a threat to graft and patient survival. Lancet 365, 640–642 (2005).

  3. 3.

    Shivaswamy, V., Boerner, B. & Larsen, J. Post-Transplant Diabetes Mellitus: Causes, Treatment, and Impact on Outcomes. Endocr Rev 37, 37–61 (2016).

  4. 4.

    D’Amico, E., Hui, H., Khoury, N., Di Mario, U. & Perfetti, R. Pancreatic beta-cells expressing GLP-1 are resistant to the toxic effects of immunosuppressive drugs. J Mol Endocrinol 34, 377–390 (2005).

  5. 5.

    Li, L. C. et al. Proteinuria and baseline renal function predict mortality and renal outcomes after sirolimus therapy in liver transplantation recipients. BMC Gastroenterol 17, 58 (2017).

  6. 6.

    Shapiro, A. M. et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230–238 (2000).

  7. 7.

    Manning, B.D. Game of TOR - The Target of Rapamycin Rules Four Kingdoms. N Engl J Med (2017).

  8. 8.

    Ryan, E. A. et al. Five-year follow-up after clinical islet transplantation. Diabetes 54, 2060–2069 (2005).

  9. 9.

    Ekberg, H. et al. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med 357, 2562–2575 (2007).

  10. 10.

    Lamming, D. W. et al. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science 335, 1638–1643 (2012).

  11. 11.

    Hjelmesaeth, J., Midtvedt, K., Jenssen, T. & Hartmann, A. Insulin resistance after renal transplantation: impact of immunosuppressive and antihypertensive therapy. Diabetes Care 24, 2121–2126 (2001).

  12. 12.

    Houde, V. P. et al. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes 59, 1338–1348 (2010).

  13. 13.

    Paty, B. W., Harmon, J. S., Marsh, C. L. & Robertson, R. P. Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets. Transplantation 73, 353–357 (2002).

  14. 14.

    Whiting, P. H. et al. Toxicity of rapamycin–a comparative and combination study with cyclosporine at immunotherapeutic dosage in the rat. Transplantation 52, 203–208 (1991).

  15. 15.

    Zhang, N. et al. Sirolimus is associated with reduced islet engraftment and impaired beta-cell function. Diabetes 55, 2429–2436 (2006).

  16. 16.

    Yang, S. B. et al. Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity. J Mol Med (Berl) 90, 575–585 (2012).

  17. 17.

    Deepa, S. S. et al. Rapamycin Modulates Markers of Mitochondrial Biogenesis and Fatty Acid Oxidation in the Adipose Tissue of db/db Mice. J Biochem Pharmacol Res 1, 114–123 (2013).

  18. 18.

    Chang, G. R. et al. Rapamycin protects against high fat diet-induced obesity in C57BL/6J mice. J Pharmacol Sci 109, 496–503 (2009).

  19. 19.

    Yang, S. B. et al. Rapamycin ameliorates age-dependent obesity associated with increased mTOR signaling in hypothalamic POMC neurons. Neuron 75, 425–436 (2012).

  20. 20.

    Castro, C. et al. Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27 Kip1 -independent pathway. Atherosclerosis 172, 31–38 (2004).

  21. 21.

    Chen, W. Q. et al. Oral rapamycin attenuates inflammation and enhances stability of atherosclerotic plaques in rabbits independent of serum lipid levels. Br J Pharmacol 156, 941–951 (2009).

  22. 22.

    Haller, S.T. et al. Rapamycin Attenuates Cardiac Fibrosis in Experimental Uremic Cardiomyopathy by Reducing Marinobufagenin Levels and Inhibiting Downstream Pro-Fibrotic Signaling. J Am Heart Assoc 5 (2016).

  23. 23.

    Rafehi, H. & El-Osta, A. HDAC Inhibition in Vascular Endothelial Cells Regulates the Expression of ncRNAs. Non-Coding RNA 2, 4 (2016).

  24. 24.

    Kennedy, B. K. & Lamming, D. W. The Mechanistic Target of Rapamycin: The Grand ConducTOR of Metabolism and Aging. Cell Metab 23, 990–1003 (2016).

  25. 25.

    Gauthier, B. R. et al. PDX1 deficiency causes mitochondrial dysfunction and defective insulin secretion through TFAM suppression. Cell Metab 10, 110–118 (2009).

  26. 26.

    Akhmedov, D. et al. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells. FASEB J 24, 4613–4626 (2010).

  27. 27.

    Kennedy, E. D. & Wollheim, C. B. Role of mitochondrial calcium in metabolism-secretion coupling in nutrient-stimulated insulin release. Diabetes Metab 24, 15–24 (1998).

  28. 28.

    Fujimoto, S. et al. Impaired metabolism-secretion coupling in pancreatic beta-cells: role of determinants of mitochondrial ATP production. Diabetes Res Clin Pract 77(Suppl 1), S2–10 (2007).

  29. 29.

    Aizawa, T. & Komatsu, M. Rab27a: a new face in beta cell metabolism-secretion coupling. The Journal of clinical investigation 115, 227–230 (2005).

  30. 30.

    Wang, H., Gauthier, B. R., Hagenfeldt-Johansson, K. A., Iezzi, M. & Wollheim, C. B. Foxa2 (HNF3beta) controls multiple genes implicated in metabolism-secretion coupling of glucose-induced insulin release. The Journal of biological chemistry 277, 17564–17570 (2002).

  31. 31.

    Santulli, G. et al. Calcium release channel RyR2 regulates insulin release and glucose homeostasis. The Journal of clinical investigation 125, 1968–1978 (2015).

  32. 32.

    Hughes, S. J. et al. Electrophysiological and metabolic characterization of single beta-cells and islets from diabetic GK rats. Diabetes 47, 73–81 (1998).

  33. 33.

    Desai, N. M. et al. Elevated portal vein drug levels of sirolimus and tacrolimus in islet transplant recipients: local immunosuppression or islet toxicity? Transplantation 76, 1623–1625 (2003).

  34. 34.

    Vetterli, L. et al. Delineation of glutamate pathways and secretory responses in pancreatic islets with beta-cell-specific abrogation of the glutamate dehydrogenase. Molecular biology of the cell 23, 3851–3862 (2012).

  35. 35.

    Perocchi, F. et al. MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake. Nature 467, 291–296 (2010).

  36. 36.

    Naghdi, S. et al. Mitochondrial Ca2+ uptake and not mitochondrial motility is required for STIM1-Orai1-dependent store-operated Ca2+ entry. J Cell Sci 123, 2553–2564 (2010).

  37. 37.

    Fu, A. et al. LKB1 couples glucose metabolism to insulin secretion in mice. Diabetologia 58, 1513–1522 (2015).

  38. 38.

    Santulli, G. & Marks, A. R. Essential roles of intracellular calcium release channels in muscle, brain, metabolism, and aging. Current Molecular Pharmacology 8, 206–222 (2015).

  39. 39.

    Bononi, A. et al. BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation. Nature 546, 549–553 (2017).

  40. 40.

    Clark, A. L. et al. Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death. Scientific reports 7, 5611 (2017).

  41. 41.

    Rutter, G. A. et al. Local and regional control of calcium dynamics in the pancreatic islet. Diabetes Obes Metab 19(Suppl 1), 30–41 (2017).

  42. 42.

    Kang, G. et al. A cAMP and Ca2+ coincidence detector in support of Ca2+ -induced Ca2+ release in mouse pancreatic beta cells. The Journal of physiology 566, 173–188 (2005).

  43. 43.

    Gwiazda, K. S., Yang, T. L., Lin, Y. & Johnson, J. D. Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells. Am J Physiol Endocrinol Metab 296, E690–701 (2009).

  44. 44.

    Tong, X. et al. SERCA2 Deficiency Impairs Pancreatic beta-Cell Function in Response to Diet-Induced Obesity. Diabetes 65, 3039–3052 (2016).

  45. 45.

    Bertram, R., Sherman, A. & Satin, L. S. Electrical bursting, calcium oscillations, and synchronization of pancreatic islets. Adv Exp Med Biol 654, 261–279 (2010).

  46. 46.

    Luik, R. M., Wang, B., Prakriya, M., Wu, M. M. & Lewis, R. S. Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature 454, 538–542 (2008).

  47. 47.

    Lamming, D. W., Ye, L., Sabatini, D. M. & Baur, J. A. Rapalogs and mTOR inhibitors as anti-aging therapeutics. J Clin Invest 123, 980–989 (2013).

  48. 48.

    Santulli, G. & Totary-Jain, H. Tailoring mTOR-based therapy: molecular evidence and clinical challenges. Pharmacogenomics 14, 1517–1526 (2013).

  49. 49.

    Li, J., Kim, S. G. & Blenis, J. Rapamycin: one drug, many effects. Cell Metab 19, 373–379 (2014).

  50. 50.

    Fielhaber, J. A. et al. Inactivation of mammalian target of rapamycin increases STAT1 nuclear content and transcriptional activity in alpha4- and protein phosphatase 2A-dependent fashion. The Journal of biological chemistry 284, 24341–24353 (2009).

  51. 51.

    Wang, Y. et al. Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival. Oncogene 27, 7106–7117 (2008).

  52. 52.

    Laberge, R. M. et al. MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nat Cell Biol 17, 1049–1061 (2015).

  53. 53.

    Luciani, D. S. et al. Roles of IP3R and RyR Ca2+ channels in endoplasmic reticulum stress and beta-cell death. Diabetes 58, 422–432 (2009).

  54. 54.

    Blodgett, D. M. et al. Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets. Diabetes 64, 3172–3181 (2015).

  55. 55.

    Han, E., Kim, M. S., Kim, Y. S. & Kang, E. S. Risk assessment and management of post-transplant diabetes mellitus. Metabolism 65, 1559–1569 (2016).

  56. 56.

    Barlow, A. D. et al. Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2). Diabetologia 55, 1355–1365 (2012).

  57. 57.

    Fuhrer, D. K., Kobayashi, M. & Jiang, H. Insulin release and suppression by tacrolimus, rapamycin and cyclosporin A are through regulation of the ATP-sensitive potassium channel. Diabetes Obes Metab 3, 393–402 (2001).

  58. 58.

    Bianchi, G., Marchesini, G., Marzocchi, R., Pinna, A. D. & Zoli, M. Metabolic syndrome in liver transplantation: relation to etiology and immunosuppression. Liver Transpl 14, 1648–1654 (2008).

  59. 59.

    Nagaraja, P., Ravindran, V., Morris-Stiff, G. & Baboolal, K. Role of insulin resistance indices in predicting new-onset diabetes after kidney transplantation. Transpl Int 26, 273–280 (2013).

  60. 60.

    Wyzgal, J. et al. Insulin resistance in kidney allograft recipients treated with calcineurin inhibitors. Ann Transplant 12, 26–29 (2007).

  61. 61.

    Bogan, J. S. Endocytic cycling of glucose transporters and insulin resistance due to immunosuppressive agents. J Clin Endocrinol Metab 99, 3622–3624 (2014).

  62. 62.

    Van de Velde, S., Hogan, M. F. & Montminy, M. mTOR links incretin signaling to HIF induction in pancreatic beta cells. Proceedings of the National Academy of Sciences of the United States of America 108, 16876–16882 (2011).

  63. 63.

    Hernandez-Fisac, I. et al. Tacrolimus-induced diabetes in rats courses with suppressed insulin gene expression in pancreatic islets. Am J Transplant 7, 2455–2462 (2007).

  64. 64.

    Thivolet, C., Vial, G., Cassel, R., Rieusset, J. & Madec, A. M. Reduction of endoplasmic reticulum- mitochondria interactions in beta cells from patients with type 2 diabetes. PLoS One 12, e0182027 (2017).

  65. 65.

    Madec, A. M. et al. Losartan, an angiotensin II type 1 receptor blocker, protects human islets from glucotoxicity through the phospholipase C pathway. FASEB J 27, 5122–5130 (2013).

  66. 66.

    Ye, R. et al. Inositol 1,4,5-trisphosphate receptor 1 mutation perturbs glucose homeostasis and enhances susceptibility to diet-induced diabetes. J Endocrinol 210, 209–217 (2011).

  67. 67.

    Bove, J., Martinez-Vicente, M. & Vila, M. Fighting neurodegeneration with rapamycin: mechanistic insights. Nat Rev Neurosci 12, 437–452 (2011).

  68. 68.

    Li, Q. et al. Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke. Biochemical and biophysical research communications 444, 182–188 (2014).

  69. 69.

    Galluzzi, L. et al. Molecular definitions of autophagy and related processes. EMBO J (2017).

  70. 70.

    Lombardi, A. & Tomer, Y. Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress. J Autoimmun 80, 48–55 (2017).

  71. 71.

    Santulli, G. et al. Age-related impairment in insulin release: the essential role of beta(2)-adrenergic receptor. Diabetes 61, 692–701 (2012).

  72. 72.

    Lombardi, A. et al. Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway. Diabetologia 55, 141–153 (2012).

  73. 73.

    Fiory, F. et al. Methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line INS-1E. Diabetologia 54, 2941–2952 (2011).

  74. 74.

    Xie, W. et al. Imaging atrial arrhythmic intracellular calcium in intact heart. J Mol Cell Cardiol 64, 120–123 (2013).

  75. 75.

    Umanskaya, A. et al. Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging. Proc Natl Acad Sci USA 111, 15250–15255 (2014).

  76. 76.

    Gambardella, J., Trimarco, B., Iaccarino, G. & Santulli, G. New Insights in Cardiac Calcium Handling and Excitation-Contraction Coupling. Adv Exp Med Biol (2017).

  77. 77.

    Santulli, G., Xie, W., Reiken, S. R. & Marks, A. R. Mitochondrial calcium overload is a key determinant in heart failure. Proceedings of the National Academy of Sciences of the United States of America 112, 11389–11394 (2015).

  78. 78.

    Palmer, A. E., Jin, C., Reed, J. C. & Tsien, R. Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proceedings of the National Academy of Sciences of the United States of America 101, 17404–17409 (2004).

  79. 79.

    Carmosino, M. et al. The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca2+ handling. J Cell Mol Med 20, 2194–2207 (2016).

  80. 80.

    Yang, Y. H., Manning Fox, J. E., Zhang, K. L., MacDonald, P. E. & Johnson, J. D. Intraislet SLIT-ROBO signaling is required for beta-cell survival and potentiates insulin secretion. Proceedings of the National Academy of Sciences of the United States of America 110, 16480–16485 (2013).

  81. 81.

    Xie, W. et al. Mitochondrial oxidative stress promotes atrial fibrillation. Sci Rep 5, 11427 (2015).

  82. 82.

    Santulli, G. et al. A selective microRNA-based strategy inhibits restenosis while preserving endothelial function. J Clin Invest 124, 4102–4114 (2014).

  83. 83.

    Lombardi, A. et al. Endoplasmic reticulum stress as a novel mechanism in amiodarone-induced destructive thyroiditis. J Clin Endocrinol Metab 100, E1–10 (2015).

  84. 84.

    Sorriento, D. et al. Intracardiac injection of AdGRK5-NT reduces left ventricular hypertrophy by inhibiting NF-kappaB-dependent hypertrophic gene expression. Hypertension 56, 696–704 (2010).

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G.S. is supported by the NIH (K99DK107895) and ES-DRC P30DK20541. We thank Drs. Yaron Tomer, Richard N. Kitsis, Thomas V. McDonald, and Jeffrey E. Pessin (Einstein College of Medicine) for insightful discussion and precious assistance.

Author information


  1. Department of Medicine, Albert Einstein College of Medicine, New York, NY, USA

    • Angela Lombardi
    • , Xue-Liang Du
    • , Maurizio Mauro
    •  & Gaetano Santulli
  2. Department of Advanced Biomedical Sciences, “Federico II” University of Naples, Naples, Italy

    • Jessica Gambardella
    • , Daniela Sorriento
    • , Bruno Trimarco
    •  & Gaetano Santulli
  3. Department of Medicine, Surgery and Dentistry, “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy

    • Jessica Gambardella
    •  & Guido Iaccarino


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A.L. designed and performed experiments, analyzed data and wrote the paper; J.G. performed experiments and analyzed data; X.L.D. and M.M. performed experiments; D.S., G.I., and B.T. analyzed data and contributed to discussion; G.S. supervised the project, designed experiments, analyzed data, and wrote the paper.

Competing Interests

The authors declare that they have no competing interests.

Corresponding author

Correspondence to Gaetano Santulli.

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