Lipides, Nutrition, Cancer
EA 7293 Vascular and Tissue Stress in Transplantation – Federation of Translational medicine University of Strasbourg, France
Study of cell cross-talk between exocrine and endocrine pancreas for the understanding of insulin cell alteration: significance in cystic fibrosis-related diabetes and new onset diabetes
Background: Cystic fibrosis (CF) is due to the mutation of the chloride calcium-dependent channel CFTR. Patients undergo recurrent lung infection and chronic inflammation owing to viscous pulmonary mucus that traps pathogens in the bronchoalveolar fluid. In the obstructed pancreas ducts the accumulation of pro-inflammatory digestive exocrine enzymes is followed by endocrine dysfunction and the loss of insulin-producing ß cells at later stage leading to CF-related diabetes. In adulthood, all patients present P.aeruginosa chronic infection and 60% diabetes. Diabetes is a co-morbidity factor in those who need lung transplantation. To prevent a worsened outcome, combined pancreatic islet and lung transplantation is performed at the University Hospital of Strasbourg. Another form of diabetes is linked to immunosuppressive drugs. It can occur as early as 1-year after the beginning of immunosuppression and reaches 10 to 30 % of transplanted patients treated by Tacrolimus (TAC), an inhibitor of calcineurin or Sirolimus (SIR), another immunosuppressive drug that targets mTOR, Molecular mechanisms leading to both types of diabetes are poorly understood. Microparticles (MPs) are plasma membrane fragments shed from stimulated or damaged cells that act as cellular effectors. They bear active proteins and lipids from their parental cell and expose membrane antigens. Their properties vary with the cell lineage and with the stress that was at the origin of their release. Our aim was to study the contribution of MPs to pancreatic exocrine and endocrine cell cross-talk in CF and to understand how immunosuppressive drugs modify cell fate and function in the exocrine and endocrine pancreas. In a first set of experiments, human exocrine pancreatic cells with CFTRΔF508 mutation (CFPAC-1) and their normal counterparts (PANC-1) were stimulated by LPS from P.aeruginosa to produce MPLPS. RIN-m5F rat ß cells were targeted by 10 nM MPLPS or submitted to 25 µM CFTR-172 inhibitor (CFTRi) to mimic CFTR dysfunction at cell surface. Secreted insulin was measured by ELISA, cell apoptosis by cytometry. MP-mediated NF-κB activation via TLR-4 transfer was confirmed in HEK-Blue reporter cells. Treatment of RIN-m5F by CFTRi induced a 9 fold increase in apoptosis and a 70% decrease in insulin secretion. While LPS alone had no effect on endocrine cell apoptosis or insulin secretion, treatment with MPLPS from CFPAC-1 cells generated a 40% decrease in insulin secretion. Importantly, MPs from LPS-treated PANC-1 cells did not alter insulin secretion nor lysosomal activity, by neutral red. Similarly, MPLPS from CFPAC-1 induced a dose-dependent activation of NF-kB, while MPLPS from PANC-1 did not. However, all types of MPs induced RIN-m5F apoptosis. Our data demonstrate that CFTR activity is a key regulator in the endocrine cell fate and function and that infections might contribute to CF-related diabetes via new MPLPS-mediated exchanges between pancreatic cells. Taking advantage of our model, we examined in a second set of experiments the mechanisms by which immunosuppressive drugs could alter the exocrine and endocrine cell fate and function and possibly favor new onset diabetes mellitus (NODM). In this work, normal exocrine PANC-1 and endocrine RIB-m5F cells were submitted to 24 h-treatment by 100 nM TAC or SIR. The expression of Bax, active Caspase3 (actCasp3), p53 and p21 was assessed by Western blot. Mitochondrial membrane potential (ΔΨm) and mitochondrial ROS were measured by flow cytometry using the fluorescent DiOC6 and MitoSOX™ probes. In exocrine PANC-1 cells, TAC and SIR increased the expression of Bax and active actCasp3, raised mitochondrial permeability, prompted p53 and p21 overexpression, but only TAC induced apoptosis. Cell cycle arrest in G1 was induced only by SIR. In endocrine RIN-m5F cells, TAC and SIR decreased Bax and actCasp3 expression, and had no effect on ΔΨm or mitochondrial ROS. Conversely, apoptosis was enhanced by TAC and SIR treatment concomitantly with the increase of p53 and p21 expression. Only SIR induced RIN-m5F cell cycle arrest. Insulin secretion was decreased only by TAC. The two immunosuppressive drugs differentially act on endocrine and exocrine pancreatic cells and may promote cell dysfunction through mitochondrial-dependent apoptosis pathways eventually in association with the induction of senescence. Altogether, we have evidenced a new MP-mediated cross-talk between exocrine and endocrine pancreatic cells. Our data underline the crucial role of the plasma membrane and remodeling in the decline of the insulin-secreting cell function and fate.
1. Leclercq A, Gauthier B, Rosner V, Weiss L, Moreau F, Constantinescu AA, Kessler R, Kessler L.,Early assessment of glucose abnormalities during continuous glucose monitoring associated with lung function impairment in cystic fibrosis patients. J Cyst Fibros. 2014 Jul;13(4):478-84.
2. Constantinescu AA, Gleizes C, Alhosin M, Yala E, Zobairi F, Leclercq A, Stoian G, Mitrea IL, Prévost G, Toti F, Kessler L, Exocrine cell-derived microparticles in response to lipopolysaccharide promote endocrine dysfunction in cystic fibrosis.J Cyst Fibros 2014 Mar;13(2):219-26.
Invitation: Olivier Micheau - UMR866
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