Prolonged Exposure to Free Fatty Acids Has Cytostatic and Pro-Apoptotic Effects on Human Pancreatic Islets
Evidence that β-Cell Death Is Caspase Mediated, Partially Dependent on Ceramide Pathway, and Bcl-2 Regulated
- Roberto Lupi1,
- Francesco Dotta2,
- Lorella Marselli1,
- Silvia Del Guerra1,
- Matilde Masini1,
- Carmela Santangelo2,
- Giovanni Patané3,
- Ugo Boggi4,
- Salvatore Piro3,
- Marcello Anello3,
- Ettore Bergamini1,
- Franco Mosca4,
- Umberto Di Mario2,
- Stefano Del Prato1 and
- Piero Marchetti1
- 1Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy
- 2Department of Clinical Sciences-Endocrinology, “La Sapienza” University, Rome, Italy
- 3Department of Endocrinology, University of Catania, Catania, Italy
- 4Department of Oncology, University of Pisa, Pisa, Italy
Abstract
In an effort to better understand the phenomenon of lipotoxicity in human β-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of β-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic β-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.
Footnotes
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Address correspondence and reprint requests to Dr. Piero Marchetti, Department of Endocrinology and Metabolism, Metabolic Unit, Ospedale Cisanello, Via Paradisa 2, 56100 Pisa, Italy. E-mail: marchant{at}immr.med.unipi.it.
Received for publication 6 December 2000 and accepted in revised form 11 February 2002.
DCI, 3,4-dichlorolsocoumarin; ELISA, enzyme-linked immunosorbent assay; FFA, free fatty acid; HBSS, Hanks’ balanced salt solution; KRB, Krebs-Ringer bicarbonate; n-NAME, Nω-nitro-l-arginine methyl ester; NF-κB, nuclear factor κB; NO, nitric oxide; OD, optical density; PPAR, peroxisome proliferator–activated receptor; TUNEL, transferase-mediated dUTP nick-end labeling; VAD-FMK, valine alanine aspartic acid-fluoromethylketone.
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