Altered Proglucagon Processing in an α-Cell Line Derived from Prohormone Convertase 2 Null Mouse Islets

Abstract

The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic ␣-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel ␣-cell line (␣TC-⌬PC2) derived from PC2 homozygous null animals. The ␣TC-⌬PC2 cells are shown to be similar to the well characterized ␣TC1-6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in ␣TC-⌬PC2 leads to a complete block in the production of mature glucagon. Surprisingly, ␣TC-⌬PC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70 -71). Further analysis reveals that ␣TC-⌬PC2 cells, unlike ␣TC1-6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that ␣TC-⌬PC2 cells provide additional evidence for PC2 as the major convertase in ␣-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases.

Pax6 Regulates the Proglucagon Processing Enzyme PC2 and Its Chaperone 7B2

“…Indeed, PC1/3 mRNA was hardly detected and not altered in the Pax6-DN306 clone in agreement with data obtained in ␣ cells of PC2 knockout mice (15,66). The reported induction of PC1/3 in an ␣-cell line not expressing PC2 (␣TC⌬PC2 cells) is thus likely to be specific for this cell line (70,72).…”

A Switch From Prohormone Convertase (PC)-2 to PC1/3 Expression in Transplanted α-Cells Is Accompanied by Differential Processing of Proglucagon and Improved Glucose Homeostasis in Mice

“…As has been reported previously (18,19), we observed that ␣TC-1 cells express PC2 but not PC1/3 and secrete glucagon but not GLP-1. In contrast, ␣TC⌬PC2 cells, which lack bioactive PC2 (14), expressed abundant levels of PC1/3 and thus produced high levels of GLP-1 but very little glucagon. The upregulation of PC1/3 in the absence of PC2 seems to be specific to this cell line and not a more general feature of loss of function of PC2 in ␣-cells, since there is no apparent induction of PC1/3 expression in the ␣-cells of PC2 Ϫ/Ϫ mice (2,20).…”

“…Cell culture and analysis. ␣TC⌬PC2 cells are an ␣-cell line derived from mice lacking active PC2, precluding glucagon production, and have been described previously (14). ␣TC-1 and ␣TC⌬PC2 cells were cultured in high-glucose Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin.…”

TCF-4 Mediates Cell Type-specific Regulation of Proglucagon Gene Expression by β-Catenin and Glycogen Synthase Kinase-3β

“…Because the glu gene encodes three major peptide hormones that play critical roles in blood glucose homeostasis and in controlling satiety (11)(12)(13), extensive research has been conducted to elucidate the molecular mechanisms that underlie expression of this gene (29 -41). These studies have identified nearly a dozen transcription factors and signaling molecules.…”

“…Although the identical glu mRNA and prohormone are generated in these three tissues, post-translational processing leads to the cell/tissue-specific biosynthesis of three major peptide hormones, glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2. These three peptide hormones exert diametrically opposed or overlapping biological functions (11)(12)(13). Glucagon, synthesized in the pancreatic ␣ cells, is a counter-regulatory hormone to insulin.…”