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Proglucagon Processing in an Islet Cell Line: Effects of PC1 Overexpression and PC2 Depletion¹

Departments of Physiology (S.D., P.L.B.) and Medicine (P.L.B.), University of Toronto, Toronto, Ontario M5S 1A8, Canada

Address all correspondence and requests for reprints to: Dr. P. L. Brubaker, Room 3366, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada. E-mail: p.brubaker{at}utoronto.ca

Proglucagon (proG) is differentially processed in the A cells of the pancreas to yield glucagon, and in the L cells of the intestine to generate glicentin, oxyntomodulin, the incretin glucagon-like peptide (GLP)-1^7–36NH2 and the intestinotropin GLP-2. To establish roles for the prohormone convertases PC1 and PC2 in proG processing within the context of a physiological model, we created stable cell lines from an islet-derived cell line, InR1-G9. These cells express proG and PC2, but not PC1, messenger RNA (mRNA). InR1-G9 cells were stably transfected with PC1 or antisense PC2. Selection was carried out in G418 (InR1-G9/PC1) or Zeocin (InR1-G9/ASPC2). Both PC1 mRNA and protein were highly expressed in InR1-G9/PC1 cells (P < 0.01–0.001) compared with wild-type (WT) cells. Cells transfected with ASPC2 demonstrated significant decreases in both PC2 mRNA (P < 0.001) and protein (P < 0.05) levels. ProG-derived peptides in WT, control, InR1-G9/PC1, and InR1-G9/ASPC2 cells were identified by HPLC and RIA. Overexpression of PC1 in InR1-G9 cells resulted in increased processing to glicentin (P < 0.01), oxyntomodulin (P < 0.05), and GLP-2 (P < 0.05). Interestingly, processing to GLP-1^7–36NH2 did not increase upon transfection of PC1. Transfection of InR1-G9 cells with ASPC2 resulted in the disappearance of glicentin (P < 0.05). However, production of glucagon was not altered by antisense deletion of PC2. Surprisingly, GLP-1^7–36NH2 production appeared to be augmented (P < 0.05) in InR1-G9/ASPC2 cells, whereas GLP-2 production was not altered. In conclusion, these studies establish the role of PC1 in the processing of proG to the intestinal proG-derived peptides. This study also establishes a role for PC2 in the production of glicentin; however, the liberation of glucagon appears to be mediated by another, yet to be identified, convertase.

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