Proglucagon processing in an islet cell line: effects of PC1 overexpression and PC2 depletion.
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.
Dhanvantari
S
S
Department of Physiology, University of Toronto, Ontario, Canada.
Brubaker
P L
PL
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D016373:DNA, Antisense; D015870:Gene Expression; D053771:Glicentin; D005934:Glucagon / Q000096:biosynthesis / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1; D053767:Glucagon-Like Peptide 2; D004763:Glucagon-Like Peptides / Q000096:biosynthesis; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D053772:Oxyntomodulin; D010446:Peptide Fragments / Q000096:biosynthesis; D010455:Peptides / Q000378:metabolism; D052336:Proglucagon; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D016373:DNA, Antisense; D053767:Glucagon-Like Peptide 2; D053772:Oxyntomodulin; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D012333:RNA, Messenger; D052336:Proglucagon; D004763:Glucagon-Like Peptides; D053771:Glicentin; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases