Abstract
The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.
Highlights
Of dibasic amino acids with different efficiencies in vitro [3,4,5,6,7,8,9]
The results presented above indicate that PC1/3 is both essential and sufficient for the cleavage of pro-glucose-dependent insulinotropic polypeptide (proGIP) to glucose-dependent insulinotropic polypeptide (GIP)
An essential role for PC1/3 in proGIP processing was demonstrated by analysis of both acidic and neutral intestinal extracts applied to gel filtration
Summary
Animals—The PC1/3 and PC2 null mutant mice were generated by introducing a targeted mutation in 129sv embryonic stem cells as previously described [12, 15]. 3–5-Month-old PC1/3 and 5–9month-old PC2 null mice and age-matched controls on a C57BL/6j x 129sv F1 background were used in the study. Animals—The PC1/3 and PC2 null mutant mice were generated by introducing a targeted mutation in 129sv embryonic stem cells as previously described [12, 15]. 3–5-Month-old PC1/3 and 5–9month-old PC2 null mice and age-matched controls on a C57BL/6j x 129sv F1 background were used in the study. The mice were housed in a specific pathogen-free environment at the Howard Hughes Medical Institute, and all experiments were performed in accordance with institutional guidelines. Immunohistochemistry—Specimens of upper and lower jejunum and ileum from three wild-type mice and four PC1/3 null mice were fixed in 4% buffered paraformaldehyde and embedded in paraffin. The tissue sections were dewaxed and subjected to antigen-retrieval by microwave irradiation in citrate buffer (pH 6.0) before the immunohistochemical staining.
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