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Phosphatase inhibitors induce defective hormone secretion in insulin-secreting cells and entry into apoptosis
A Krautheim1, I Rustenbeck, H J Steinfelder
1Institute of Pharmacology and Toxicology, University of Göttingen, Germany.
Abstract:
A long-term (> or =24 h) exposure of insulin-secreting HIT T15 cells to the phosphatase inhibitor, okadaic acid (OA), at concentrations inhibiting serine/threonine phosphatases 1 (PP1) and 2A (PP2A) reduced proliferation and insulin secretion. The reduced proliferation was related to the induction of apoptosis as evidenced by morphological criteria and the occurrence of internucleosomal DNA fragmentation after 15 h in 50 nM OA. The compromised insulin secretion was not simply a consequence of a lowered hormone content and cell growth, but comprised also a complete suppression of secretion stimulated by K+ depolarisation and forskolin. K+ depolarisation of HIT cells cultured for 24 h in 50 nM OA resulted in a nearly unimpaired influx of Ca2+, but did not induce secretion. These observations suggest that the secretory defect may be localised distal to Ca2+ influx in stimulus secretion coupling of insulin-secreting cells.
Insights
Okadaic acid (OA) inhibits serine/threonine phosphatases (PP1 and PP2A), leading to apoptosis and reduced insulin secretion in HIT T15 cells. The secretory defect occurs downstream of calcium influx, impacting stimulus-secretion coupling.
Area of Science:
- Cell Biology
- Endocrinology
- Biochemistry
Background:
- Insulin secretion is crucial for glucose homeostasis.
- Serine/threonine phosphatases, protein phosphatase 1 (PP1) and 2A (PP2A), regulate cellular functions.
- Okadaic acid (OA) is a potent inhibitor of PP1 and PP2A.
Purpose of the Study:
- To investigate the effects of long-term okadaic acid (OA) exposure on insulin-secreting HIT T15 cells.
- To elucidate the mechanisms underlying OA-induced alterations in cell proliferation and insulin secretion.
Main Methods:
- HIT T15 cells were exposed to okadaic acid (OA) for > or = 24 hours.
- Cell proliferation was assessed using morphological criteria and DNA fragmentation assays.
- Insulin secretion was measured following stimulation with K+ depolarization and forskolin.
- Calcium (Ca2+) influx was monitored during K+ depolarization.
Main Results:
- Long-term OA exposure reduced HIT T15 cell proliferation and insulin secretion.
- Apoptosis, characterized by DNA fragmentation, was induced by 50 nM OA after 15 hours.
- Insulin secretion stimulated by K+ and forskolin was completely suppressed.
- K+ depolarization in OA-treated cells showed unimpaired Ca2+ influx but failed to trigger secretion.
Conclusions:
- Okadaic acid (OA) induces apoptosis and impairs insulin secretion in HIT T15 cells.
- The secretory defect in OA-treated cells is localized downstream of calcium influx.
- These findings suggest a critical role for phosphatases PP1 and PP2A in insulin secretion regulation and stimulus-secretion coupling.