Effects of atrial natriuretic peptide on rat pancreatic islet beta-cells
Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is primarily expressed and stored in granules in the atria, and are released during atrial stretch due to volume overload or hypertension in the cardiovascular system. ANP has been well studied for various physiological effects in humans, including regulation of fluid retention and blood pressure, inhibition of renin secretion, reduction of aldosterone secretion, and relaxation of vascular smooth muscle. Since the pancreatic islet is exposed to elevated ANP level in patients with type 1 diabetes mellitus, an aim of this study was to determine the short- and long-term effects of ANP on pancreatic β-cell growth and insulin secretion. In the first part of the study, the expression of the natriuretic peptide receptor-A (NPR-A) was detected in INS-1E cells and rat pancreatic islet α- and β-cells by Western blot and immunofluorescent staining. NPR-B was not detectable in either INS-1E cells or pancreatic islets. In the second part of the study, ANP stimulated cell proliferation and [ 3 H]thymidine incorporation in DNA of INS-1E cells and islet. DNA biosynthesis stimulated by ANP in INS-1 cell was blocked by pre-incubation with LY294002, an inhibitor of phosphatidylinositol 3'-kinase (PI3K). An indicator of cell cycle progression, cyclin D2 mRNA was up-regulated by 2- to 3-fold in ANP-treated INS-1E cells and islets, and these responses were inhibited by LY294002. ANP stimulated the phosphorylation of Akt and forkhead box O1a (Foxo1a) in INS-1E cells and islets, and LY294002 inhibited these responses. The mRNA of both pancreas duodenum homeobox-1 (PDX-1) and glucokinase in islets and INS-1E cells were increased in response to ANP. The evidence suggests that stimulation of NPR-A results in activation of a growth promoting signaling pathway that includes PI3K/Akt/Foxo1a/cyclin D2 in pancreatic β-cells. These data support the conclusion that the activation of Akt by ANP or 8-br-cGMP promotes cyclin D2, PDX-1 and glucokinase transcription by phosphorylating and restricting Foxo1a activity. In the third part of the study, chronic (7 day) culture of islets with ANP negatively impacted glucose-stimulated insulin secretion compared to control islets. When ANP was washed away, the islet insulin secretory response to glucose was restored within 24 h of additional culture, demonstrating continued viability of the islets. Moreover, the insulin content of the islets was not changed following 7-day treatment with ANP. The insulin secretory response to other insulin secretagogues, including α-ketoisocaproic acid (KIC), forskolin, potassium chloride (KCl) and ionomycin, were also inhibited by chronic exposure to ANP. Even though the expression of NRP-A in isolated islets was reduced to 60% of control after 7-day incubation with ANP, the ANP-stimulated cGMP levels remained comparable to control levels, suggesting that the loss of cGMP biosynthesis was not responsible for the loss of secretory response or ATP generation in these cells. Elevated phospho-hormone-sensitive lipase (HSL) was observed in ANP-treated islets, as well as upregulated mRNA levels of uncoupling protein 2 (UCP2). Removal of ANP partially restored the phospho-HSL and UCP2 levels. Compared to control islets, in the presence or absence of glucose stimulation, the ATP levels were significantly reduced after 7-day ANP treatment. The ATP levels partially recovered after removal of ANP for 24 h. The effects of ANP on glycolysis did not show a marked difference in glucose utilization compared to control islets in the absence of ANP. These results support the conclusion that long-term exposure to ANP inhibits glucose-stimulated insulin secretion in part by effects on HSL/UCP2/ATP signaling. And it is not likely that ANP effects on glycolysis account for the changes in islet ATP production. In summary, this study investigated the expression of NPR-A and NPR-B on INS-1E cells and pancreatic islets. The novel findings of this study establish that islet α- and β-cells express NPR-A, and describe for the first time the mechanism of intracellular signal transduction mediating the effects of ANP and cGMP on pancreatic β-cell proliferation and insulin secretion.