Targeted overexpression of an inactive calmodulin that binds Ca2+ to the mouse pancreatic beta-cell results in impaired secretion and chronic hyperglycemia.
We have previously reported that elevated levels of calmodulin in pancreatic beta-cells of mice resulted in a unique secretory defect. To determine if this effect was due to Ca2+ buffering, a mutant form of calmodulin that has an eight-amino acid deletion in the central helix (CaM-8) was used. The mutated calmodulin binds Ca2+ normally, but alters the ability to interact with known Ca2+/calmodulin-activated enzymes. In vitro competition analysis using HIT cell extracts verified that in the presence of Ca2+, CaM-8 exhibited at least a 100-fold lower affinity for calmodulin-binding proteins than did normal CaM in this model beta-cell. Transgenic mice were then generated by targeting the CaM-8 to pancreatic beta-cells. The CaM-8 mice were normoglycemic at birth, but developed a hyperglycemic condition starting at about 6 days of age. This condition was progressive and characterized by elevated blood glucose that coincided with reduced levels of pancreatic insulin and low circulating serum insulin levels. Hormone measurements and immunohistochemical analysis revealed that islets exhibited a nonimmune reduction of insulin immunoreactive beta-cells, reduced amounts of insulin, and a 5-fold higher level of CaM-8 protein relative to normal CaM protein. Perifusion assays were used to test the secretion response to glucose. CaM-8 islets demonstrated a reduction in first and second phase insulin secretion, which became progressively worse with age. Depolarization of the membrane with 50 mM K+ in the presence of high glucose did not significantly improve secretion. Carbachol, which is thought to act in beta-cells through the release of intracellular Ca2+ stores and activation of protein kinase-C, restored both phases of secretion to normal levels. These results suggest that disruption of intracellular Ca2+ homeostasis alone is sufficient to interfere with the insulin secretion pathway.
Ribar, TJ; Epstein, PN; Overbeek, PA; Means, AR
Volume / Issue
Start / End Page
Pubmed Central ID
Electronic International Standard Serial Number (EISSN)
International Standard Serial Number (ISSN)
Digital Object Identifier (DOI)