The role of peripheral benzodiazepine receptors on the function and survival of isolated human pancreatic islets.
OBJECTIVE
Peripheral benzodiazepine receptors (PBRs) are part of the mitochondrial permeability transition pore, and their activation may induce cell death. PBRs are expressed in human pancreatic islets, and cytokine-induced damage is accompanied by changes in their properties. We hypothesized that PBRs can have a role in human islet physiopathology, and evaluated the effects of prolonged exposure to two specific PBR ligands, PK11195 and Ro5-4864 on the function and survival of isolated human islets.


METHODS
Isolated human islets were prepared from the pancreas of 25 multiorgan cadaveric donors and incubated for 12 h in the presence of PK11195 or Ro5-4864. Insulin secretion studies and apoptosis experiments were then performed, together with assessment of intracellular pathways involved in islet cell function and survival.


METHODS
Islets were prepared by enzymatic digestion and density gradient purification. Insulin secretion was assessed by the batch incubation method, and glucose oxidation was evaluated by the use of D-[U-(14)C]glucose. Apoptosis was studied using the TUNEL technique, ELISA methods, and electron microscopy evaluation. PCR experiments were performed by the use of specific primers.


RESULTS
Glucose-stimulated insulin release was significantly lower after exposure to PK11195 than after exposure to Ro5-4864. This was accompanied by reduced glucose oxidation and no major change of insulin or GLUT-1 mRNA expression. Apoptosis was higher in PK11195-exposed islets, and electron microscopy demonstrated the involvement of beta-cells. The apoptotic effects were prevented by bongkrekic acid and low-dose cyclosporin A, which stabilize the mitochondrial membrane, and were associated with no evident change of inducible nitric oxide synthase (iNOS), B-cell leukemia/lymphoma-2 (Bcl-2) or Bcl-2-associated X protein (Bax) expression. Caspase inhibition markedly reduced the amount of apoptosis, and the role of these proteases was confirmed by the increased activity of caspase-3 and caspase-9.


CONCLUSIONS
Prolonged binding to PBRs may cause human beta-cells functional damage and apoptosis, a phenomenon which is prevented by stabilizing the mitochondrial membrane; occurs without changes of iNOS, Bax and Bcl-2 mRNA expression; and involves caspase activation. These results suggest an involvement of PBRs in human pancreatic beta-cell function and survival.
Dipartimento di Endocrinologia e Metabolismo, Sezione Metabolismo, Universitá di Pisa, via Paradisa 2, Ospedale Cisanello, 56100 Pisa, Italy.
Lorella
L
Marselli
Letizia
L
Trincavelli
Carmela
C
Santangelo
Roberto
R
Lupi
Silvia
S
Del Guerra
Ugo
U
Boggi
Alessandra
A
Falleni
Vittorio
V
Gremigni
Franco
F
Mosca
Claudia
C
Martini
Francesco
F
Dotta
Umberto
U
Di Mario
Stefano
S
Del Prato
Piero
P
Marchetti
D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001570:Benzodiazepinones / Q000378:metabolism / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D000960:Hypolipidemic Agents / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D008928:Mitochondria / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011963:Receptors, GABA-A / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; D001570:Benzodiazepinones; D061945:Caspase Inhibitors; D027342:Excitatory Amino Acid Transporter 2; D000960:Hypolipidemic Agents; D007328:Insulin; D007546:Isoquinolines; D008024:Ligands; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011963:Receptors, GABA-A; D051028:bcl-2-Associated X Protein; C028513:4'-chlorodiazepam; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C037850:PK 11195