Prolonged Depletion of Guanosine Triphosphate Induces Death of Insulin-Secreting Cells by Apoptosis*

• DOI: 10.1210/endo.139.9.6207
• Publication Date: 2014-01-08T16:09:28Z
• Authors: Guodong Li; Venkatesh Babu G....; Mary E. Rabaglia; Rui-Hua Luo; Anjaneyulu Kowluru; Stewart A. Metz
• Keywords: 0301 basic medicine; 570; 0303 health sciences; Time Factors; Bacterial Toxins; 610; Mitosis; Apoptosis; Mycophenolic Acid; Rats; 3. Good health; Rats, Sprague-Dawley; Islets of Langerhans; 03 medical and health sciences; Bacterial Proteins; GTP-Binding Proteins; Insulin Secretion; Animals; Insulin; Guanosine Triphosphate; Lovastatin

AbstractInhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting β-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by[ 3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 μg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 μg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10–100μ m for 2–3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces β-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the β-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.