However, in spite of these early findings, the role of 1,25-dihydroxyvitamin D 3 and calbindin in β cell function is not yet clear ( 10). It had been suggested that vitamin D may be acting in the pancreatic β cell by modulating intracellular calcium, perhaps by mechanisms involving calbindin. In the pancreas, early autoradiographic and immunocytochemical studies have localized 1,25-dihydroxyvitamin D 3 receptors ( 8) and calbindin ( 9), respectively, to the β cells. Various functions have been proposed for calbindin based on its calcium-binding properties, i.e., facilitation of the diffusional flux of calcium in the intestinal enterocyte ( 4), protection of neurons against excitatory calcium toxicity ( 5), and action as a mobile calcium buffer restricting evoked calcium signals in nerve synapses and hair cells ( 6, 7). However, in brain calbindin is not influenced by vitamin D status. In intestine and kidney, calbindin is regulated by 1,25-dihydroxyvitamin D 3. Calbindin-D 28k is present in the highest concentrations in avian intestine and in avian and mammalian kidney, brain, and pancreas ( 2, 3). These studies provide the first direct evidence (to our knowledge) for a role for calbindin in β cell function.Ĭalbindin-D 28k belongs to a family of high affinity calcium-binding proteins that includes calmodulin, parvalbumin, troponin C, and S100 protein ( 1). Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (−345/+1) or five copies of the Far-FLAT minienhancer (−247/−198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 μM calcium ionophore. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. To determine a role for calbindin-D 28k in the β cell, rat calbindin-D 28k was overexpressed in the pancreatic β cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Calbindin-D 28k is also present in pancreatic β cells, but its function in these cells is not known. Calbindin-D 28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues.
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