Molecular and Cellular Biochemistry

, Volume 418, Issue 1–2, pp 91–102 | Cite as

Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells

  • Andrei Alexandru ConstantinescuEmail author
  • Malak Abbas
  • Mohamad Kassem
  • Céline Gleizes
  • Guillaume Kreutter
  • Valerie Schini-Kerth
  • Ioan Liviu Mitrea
  • Florence Toti
  • Laurence KesslerEmail author


To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine β-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1–100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-β-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-β-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype.


Endocrine β-cells Exocrine cells Tacrolimus Sirolimus Mitochondria-related apoptosis Cellular premature senescence 



Mitochondrial membrane potential


Annexin 5


Arbitrary units


5-Dodecanoylaminofluorescein Di-β-D-galactopyranoside


3,3′-Dihexyloxacarbocyanine iodide


Dulbecco’s modified Eagle’s medium


Enzyme-linked immunosorbent assay


Fetal bovine serum


Fluorescein diacetate


Fluorescein isothiocyanate


Hank’s balanced salt solution


Mammalian target of rapamycin


mTOR complex 1


Phosphate-buffered saline


Propidium iodide


Reactive oxygen species


Roswell Park Memorial Institute medium


Senescence-associated beta-galactosidase


Sodium dodecyl sulfate







We are grateful to Dr. C. Muller and Mr. D. Nabergoj from the eBIOCYT cytometry platform of Strasbourg University, and to Mr. R. Vauchelles from the PIQ quantitative imaging platform of Strasbourg University. We appreciate the kind assistance in isolation of rat islets by Prof. D. Bosco from Laboratoire d’Isolement et Transplantation Cellulaire, Hôpital Universitaire, Genève, Suisse. We thank the European Doctoral College, part of Strasbourg University, for its support during this scientific achievement.


This research was supported by the Sectorial Operational Programme Development of Human Resources 20072013 (POSDRU) through the Financial Agreement POSDRU/107/1.5/S/76888 of the Government of Romania, recurrent founding, by the Association Vaincre la Mucoviscidose (VLM) and by the Association d’Aide aux Insuffisants Respiratoires Alsace-Lorraine (ADIRAL).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Peddi VR, Wiseman A, Chavin K, Slakey D (2013) Review of combination therapy with mTOR inhibitors and tacrolimus minimization after transplantation. Transplant Rev 27(4):97–107. doi: 10.1016/j.trre.2013.06.001 CrossRefGoogle Scholar
  2. 2.
    Dong M, Parsaik AK, Eberhardt NL, Basu A, Cosio FG, Kudva YC (2012) Cellular and physiological mechanisms of new-onset diabetes mellitus after solid organ transplantation. Diabet Med J Br Diabet Assoc 29(7):e1–e12. doi: 10.1111/j.1464-5491.2012.03617.x CrossRefGoogle Scholar
  3. 3.
    Redmon JB, Olson LK, Armstrong MB, Greene MJ, Robertson RP (1996) Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells. J Clin Investig 98(12):2786–2793. doi: 10.1172/JCI119105 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Carroll PB, Boschero AC, Li MY, Tzakis AG, Starzl TE, Atwater I (1991) Effect of the immunosuppressant FK506 on glucose-induced insulin secretion from adult rat islets of Langerhans. Transplantation 51(1):275–278CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hirano Y, Fujihira S, Ohara K, Katsuki S, Noguchi H (1992) Morphological and functional changes of islets of Langerhans in FK506-treated rats. Transplantation 53(4):889–894CrossRefPubMedGoogle Scholar
  6. 6.
    Shimodahira M, Fujimoto S, Mukai E, Nakamura Y, Nishi Y, Sasaki M, Sato Y, Sato H, Hosokawa M, Nagashima K, Seino Y, Inagaki N (2010) Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism. J Endocrinol 204(1):37–46. doi: 10.1677/JOE-09-0216 CrossRefPubMedGoogle Scholar
  7. 7.
    Shivaswamy V, Boerner B, Larsen J (2016) Post-transplant diabetes mellitus: causes, treatment, and impact on outcomes. Endocr Rev 37(1):37–61. doi: 10.1210/er.2015-1084 CrossRefPubMedGoogle Scholar
  8. 8.
    Moreira M, Matias JE, Souza CJ, Nicoluzzi JE, Caron PE, Repka JC (2011) Action of tacrolimus in arginine induced experimental acute pancreatitis. Revista do Colegio Brasileiro de Cirurgioes 38(4):260–265CrossRefPubMedGoogle Scholar
  9. 9.
    Meirelles Junior RF, Salvalaggio P, Pacheco-Silva A (2015) Pancreas transplantation: review. Einstein 13(2):305–309. doi: 10.1590/S1679-45082015RW3163 CrossRefPubMedGoogle Scholar
  10. 10.
    Sastry J, Young S, Shaw PJ (2004) Acute pancreatitis due to tacrolimus in a case of allogeneic bone marrow transplantation. Bone Marrow Transplant 33(8):867–868. doi: 10.1038/sj.bmt.1704429 CrossRefPubMedGoogle Scholar
  11. 11.
    Doi R, Inoue K, Chowdhury P, Kaji H, Rayford PL (1993) Structural and functional changes of exocrine pancreas induced by FK506 in rats. Gastroenterology 104(4):1153–1164CrossRefPubMedGoogle Scholar
  12. 12.
    Jin S, Orabi AI, Le T, Javed TA, Sah S, Eisses JF, Bottino R, Molkentin JD, Husain SZ (2015) Exposure to radiocontrast agents induces pancreatic inflammation by activation of nuclear factor-kappaB, calcium signaling, and calcineurin. Gastroenterology 149(3):753–764. doi: 10.1053/j.gastro.2015.05.004 CrossRefPubMedGoogle Scholar
  13. 13.
    Muili KA, Wang D, Orabi AI, Sarwar S, Luo Y, Javed TA, Eisses JF, Mahmood SM, Jin S, Singh VP, Ananthanaravanan M, Perides G, Williams JA, Molkentin JD, Husain SZ (2013) Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin. J Biol Chem 288(1):570–580. doi: 10.1074/jbc.M112.428896 CrossRefPubMedGoogle Scholar
  14. 14.
    Liu C, Dou K, Dou C, Liu J, Zhao Q (2010) Anti-inflammatory effects of tacrolimus in a rat model of acute pancreatitis. Med Chem 6(1):37–43CrossRefPubMedGoogle Scholar
  15. 15.
    Rau BM, Kruger CM, Hasel C, Oliveira V, Rubie C, Beger HG, Schilling MK (2006) Effects of immunosuppressive and immunostimulative treatment on pancreatic injury and mortality in severe acute experimental pancreatitis. Pancreas 33(2):174–183. doi: 10.1097/01.mpa.0000226895.16817.a1 CrossRefPubMedGoogle Scholar
  16. 16.
    Bussiere CT, Lakey JR, Shapiro AM, Korbutt GS (2006) The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells. Diabetologia 49(10):2341–2349. doi: 10.1007/s00125-006-0374-5 CrossRefPubMedGoogle Scholar
  17. 17.
    Muller CA, Belyaev O, Burr W, Munding J, McArthur N, Bergmann U, Werner J, Tannapfel A, Uhl W (2012) Effects of FTY720 and rapamycin on inflammation in taurocholate-induced acute pancreatitis in the rat. Pancreas 41(7):1086–1091. doi: 10.1097/MPA.0b013e3182496fd7 CrossRefPubMedGoogle Scholar
  18. 18.
    Mayer JM, Kolodziej S, Jukka Laine V, Kahl S (2012) Immunomodulation in a novel model of experimental chronic pancreatitis. Minerva Gastroenterol Dietol 58(4):347–354PubMedGoogle Scholar
  19. 19.
    Chen P, Huang L, Zhang Y, Qiao M, Yao W, Yuan Y (2011) The antagonist of the JAK-1/STAT-1 signaling pathway improves the severity of cerulein-stimulated pancreatic injury via inhibition of NF-kappaB activity. Int J Mol Med 27(5):731–738. doi: 10.3892/ijmm.2011.632 PubMedGoogle Scholar
  20. 20.
    Rostambeigi N, Lanza IR, Dzeja PP, Deeds MC, Irving BA, Reddi HV, Madde P, Zhang S, Asmann YW, Anderson JM, Schimke JM, Nair KS, Eberhardt NL, Kudva YC (2011) Unique cellular and mitochondrial defects mediate FK506-induced islet beta-cell dysfunction. Transplantation 91(6):615–623. doi: 10.1097/TP.0b013e3182094a33 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wang Y, Mendoza-Elias JE, Qi M, Harvat TA, Ahn SJ, Lee D, Gutierrez D, Jeon H, Paushter D, Oberholzer J (2012) Implication of mitochondrial cytoprotection in human islet isolation and transplantation. Biochem Res Int 2012:395974. doi: 10.1155/2012/395974 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Schieke SM, Phillips D, McCoy JP Jr, Aponte AM, Shen RF, Balaban RS, Finkel T (2006) The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity. J Biol Chem 281(37):27643–27652. doi: 10.1074/jbc.M603536200 CrossRefPubMedGoogle Scholar
  23. 23.
    Erin N, Lehman RA, Boyer PJ, Billingsley ML (2003) In vitro hypoxia and excitotoxicity in human brain induce calcineurin-Bcl-2 interactions. Neuroscience 117(3):557–565CrossRefPubMedGoogle Scholar
  24. 24.
    Furuichi Y, Noto T, Li JY, Oku T, Ishiye M, Moriguchi A, Aramori I, Matsuoka N, Mutoh S, Yanagihara T (2004) Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats. Brain Res 1014(1–2):120–130. doi: 10.1016/j.brainres.2004.04.031 CrossRefPubMedGoogle Scholar
  25. 25.
    Dai ZJ, Gao J, Ma XB, Kang HF, Wang BF, Lu WF, Lin S, Wang XJ, Wu WY (2012) Antitumor effects of rapamycin in pancreatic cancer cells by inducing apoptosis and autophagy. Int J Mol Sci 14(1):273–285. doi: 10.3390/ijms14010273 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Bosco D, Armanet M, Morel P, Niclauss N, Sgroi A, Muller YD, Giovannoni L, Parnaud G, Berney T (2010) Unique arrangement of alpha- and beta-cells in human islets of Langerhans. Diabetes 59(5):1202–1210. doi: 10.2337/db09-1177 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50(2):98–115. doi: 10.2144/000113610 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12):1798–1806. doi: 10.1038/nprot.2009.191 CrossRefPubMedGoogle Scholar
  29. 29.
    Khoo KH, Verma CS, Lane DP (2014) Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov 13(3):217–236. doi: 10.1038/nrd4236 CrossRefPubMedGoogle Scholar
  30. 30.
    Janjic D, Wollheim CB (1992) Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay. Diabetologia 35(5):482–485CrossRefPubMedGoogle Scholar
  31. 31.
    Deer EL, Gonzalez-Hernandez J, Coursen JD, Shea JE, Ngatia J, Scaife CL, Firpo MA, Mulvihill SJ (2010) Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39(4):425–435. doi: 10.1097/MPA.0b013e3181c15963 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Skelin M, Rupnik M, Cencic A (2010) Pancreatic beta cell lines and their applications in diabetes mellitus research. Altex 27(2):105–113PubMedGoogle Scholar
  33. 33.
    Hoshino A, Ariyoshi M, Okawa Y, Kaimoto S, Uchihashi M, Fukai K, Iwai-Kanai E, Ikeda K, Ueyama T, Ogata T, Matoba S (2014) Inhibition of p53 preserves Parkin-mediated mitophagy and pancreatic beta-cell function in diabetes. Proc Natl Acad Sci USA 111(8):3116–3121. doi: 10.1073/pnas.1318951111 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Nam SY, Lee MK, Sabapathy K (2008) The tumour-suppressor p53 is not required for pancreatic beta cell death during diabetes and upon irradiation. J Physiol 586(2):407–417. doi: 10.1113/jphysiol.2007.142612 CrossRefPubMedGoogle Scholar
  35. 35.
    Hernandez AM, Colvin ES, Chen YC, Geiss SL, Eller LE, Fueger PT (2013) Upregulation of p21 activates the intrinsic apoptotic pathway in beta-cells. Am J Physiol Endocrinol Metab 304(12):E1281–E1290. doi: 10.1152/ajpendo.00663.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mihailidou C, Chatzistamou I, Papavassiliou AG, Kiaris H (2015) Regulation of P21 during diabetes-associated stress of the endoplasmic reticulum. Endocr Relat Cancer 22(2):217–228. doi: 10.1530/ERC-15-0018 CrossRefPubMedGoogle Scholar
  37. 37.
    Kloster-Jensen K, Sahraoui A, Vethe NT, Korsgren O, Bergan S, Foss A, Scholz H (2016) Treatment with tacrolimus and sirolimus reveals no additional adverse effects on human islets in vitro compared to each drug alone but they are reduced by adding glucocorticoids. J Diabetes Res 2016:4196460. doi: 10.1155/2016/4196460 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Gross A, Jockel J, Wei MC, Korsmeyer SJ (1998) Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 17(14):3878–3885. doi: 10.1093/emboj/17.14.3878 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Munoz-Espin D, Serrano M (2014) Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 15(7):482–496. doi: 10.1038/nrm3823 CrossRefPubMedGoogle Scholar
  40. 40.
    Helman A, Klochendler A, Azazmeh N, Gabai Y, Horwitz E, Anzi S, Swisa A, Condiotti R, Granit RZ, Nevo Y, Fixler Y, Shreibman D, Zamir A, Tornovsky-Babeay S, Dai C, Glaser B, Powers AC, Shapiro AM, Magnuson MA, Dor Y, Ben-Porath I (2016) p16-induced senescence of pancreatic beta cells enhances insulin secretion. Nat Med. doi: 10.1038/nm.4054 PubMedGoogle Scholar
  41. 41.
    Castedo M, Ferri KF, Kroemer G (2002) Mammalian target of rapamycin (mTOR): pro- and anti-apoptotic. Cell Death Differ 9(2):99–100. doi: 10.1038/sj.cdd.4400978 CrossRefPubMedGoogle Scholar
  42. 42.
    Newmeyer DD, Ferguson-Miller S (2003) Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112(4):481–490CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Andrei Alexandru Constantinescu
    • 1
    • 2
    • 6
    Email author
  • Malak Abbas
    • 1
    • 3
  • Mohamad Kassem
    • 1
  • Céline Gleizes
    • 1
  • Guillaume Kreutter
    • 1
  • Valerie Schini-Kerth
    • 4
  • Ioan Liviu Mitrea
    • 2
  • Florence Toti
    • 4
  • Laurence Kessler
    • 1
    • 5
    Email author
  1. 1.EA7293, Vascular and Tissular Stress in Transplantation, Federation of Translational Medicine of Strasbourg, Faculty of MedicineUniversity of StrasbourgIllkirch, StrasbourgFrance
  2. 2.Department of Parasitology and Parasitic Diseases and Animal Biology, Faculty of Veterinary MedicineUniversity of Agronomical Sciences and Veterinary MedicineBucharestRomania
  3. 3.Ecole Doctorale de Sciences et TechnologiesUniversité LibanaiseHadathLebanon
  4. 4.UMR7213 CNRSLaboratory of Biophotonics and PharmacologyIllkirchFrance
  5. 5.Department of DiabetologyUniversity HospitalStrasbourg CedexFrance
  6. 6.INSERM, UMR866«Equipe labellisée Ligue contre le Cancer » and Laboratoire d’Excellence LipSTICDijonFrance

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