TNF-α acutely upregulates amylin expression in murine pancreatic beta cells

Aims/hypothesis Amylin, a secretory protein mainly produced by pancreatic beta cells, is elevated in the circulation of patients with diseases related to acute and chronic inflammation, including acute pancreatitis, pancreas graft rejection, obesity and insulin resistance. TNF-alpha is involved in these disorders. We investigated the effect of TNF-alpha on amylin levels and the underlying mechanisms, using murine pancreatic beta cell line MIN6 and pancreatic islets. Methods Amylin, proinsulin and prohormone convertase 1/3, 2 (Pc1/3, Pc2 [also known as Pcsk1/3 and Pcsk2, respectively]) mRNA levels, and amylin promoter and nuclear factor kappa B (NF-kappa B) activation were examined by real-time PCR and luciferase reporter assay, respectively. Amylin protein level and mitogen-activated protein kinase phosphorylation were detected by western blot. Activator protein 1 (AP1) activation was examined by electrophoretic mobility shift assay (EMSA). Results TNF-alpha acutely induced amylin expression at the transcriptional level and increased proamylin and the intermediate form of amylin in MIN6 cells and islets. However, it had no effect on proinsulin, Pc1/3 and Pc2 expression. Studies with (1) MIN6 cells treated with inhibitors of MEK1/2, c-Jun-N-terminal kinase (JNK) or protein kinase C zeta (PKC zeta), (2) MIN6 cells expressing a c-Jun-dominant negative construct and (3) islets from Fos knockout mice demonstrated that TNF-alpha induced amylin expression through the PKC zeta-extracellular signal-regulated kinase (ERK)/JNK pathways. EMSA showed that PKC zeta, JNK and ERK1/2 were involved in TNF-alpha-induced AP1 activation, suggesting that TNF-alpha induces murine amylin expression through the PKC zeta-ERK1/2-AP1 and PKC zeta- JNK-AP1 pathways. Further studies showed that TNF-alpha also induced murine amylin expression through the phosphatidylinositol 3 kinase-NF-kappa B signalling pathway and enhanced human amylin promoter activation through NF-kappa B and AP1. Conclusions/interpretation TNF-alpha acutely induces amylin gene expression in beta cells through multiple signalling pathways, possibly contributing to amylin elevation in acute inflammation-related pancreatic disorders.