Abstract
Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.
Highlights
Islet amyloid polypeptide (IAPP) and insulin are expressed in the -cells of the islets of Langerhans
Administration of dexamethasone to low dose streptozotocin-treated rats resulted in an increase in IAPP mRNA levels, whereas insulin mRNA levels were markedly reduced [30]
In human islets of Langerhans, following treatment with high glucose for relatively long periods, there was a greater stimulation of IAPP than insulin mRNA levels [27]
Summary
Cell Culture—NES2Y cells were derived from islets of Langerhans isolated from the pancreas of a patient with persistent hyperinsulinemic hypoglycemia of infancy as described previously [21]. Isolated intact human islets were prepared and maintained as described previously [24]. Northern blot analysis was performed as described previously [23] using, as probe, a full-length human IAPP cDNA University of Oxford, Oxford, United Kingdom) and a human insulin cDNA. Cells were incubated for 15 min at room temperature in blocking buffer containing 0.7% (v/v) glycerol, 0.2% (v/v) Tween 20, and 2% (w/v) bovine serum albumin. Primary antibodies were added to each chamber in blocking buffer, and the samples were left overnight at 4 °C. Cells were washed in 0.4% Tween 20, 0.7% glycerol, and 2% bovine serum albumin for 1–2 h with gentle rotation at room temperature in the dark before mounting medium containing 4,6-diamidino2-phenylindole was added, and coverslips were affixed. Control samples containing primary antibody only, secondary antibody only, and nonimmune mouse or rabbit serum all stained negative
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