Cellular specificity of proexendin-4 processing in mammalian cells in vitro and in vivo.
Glucagon-like peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Exendin-4(1-39) (Ex-4), isolated from Gila monster venom, is a highly specific GLP-1 receptor agonist that exhibits a prolonged duration of action in vivo. Although the processing mechanisms underlying liberation of GLP-1 from its prohormone have been elucidated, those for Ex-4 remain unknown. To examine the requirements for proEx-4 processing in mammalian cells, BHK fibroblasts, InR1-G9 islet A cells, and AtT-20 corticotropes, which express different prohormone convertases (furin, prohormone convertase 2, and prohormone convertase 1, respectively) were transfected with full-length lizard proEx-4, and the processing of proexendin was examined by HPLC and RIA (n = 3). All of the transfected cell lines exhibited Ex-4-like immunoreactivity in the media, and Ex-4-like immunoreactivity was detected in extracts of InR1-G9 and AtT-20 cells. However, only media and extracts from AtT-20 cells (not InR1-G9 and BHK cells) contained a single peak by HPLC corresponding to synthetic Ex-4. To establish whether proEx-4 can be processed to Ex-4 in nonimmortalized mammalian cells in vivo, the molecular forms of exendin-4 were examined in mice expressing a metallothionein-proEx-4 transgene (n = 3-6 for both males and females). ProEx4 mRNA transcripts were detected by RT-PCR in a broad range of both endocrine and nonendocrine tissues. Ex-4-like immunoreactivity was detected in pituitary, fat, adrenals, and testes; however HPLC analyses demonstrated that processed Ex-4 was found only in adrenals and testes. These results indicate that lizard proEx-4 is processed to mature bioactive Ex-4 in both rodent endocrine and nonendocrine mammalian cell types in vitro and in murine tissues in vivo. These findings may be useful for engineering cells that express a lizard pro-Ex4 transgene for the treatment of type 2 diabetes.
Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
F A
FA
Adatia
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Baggio
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Xiao
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Drucker
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Brubaker
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D000077270:Exenatide; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism; D052216:Glucagon-Like Peptide 1; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009928:Organ Specificity; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011863:Radioimmunoassay; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014688:Venoms
D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D014688:Venoms; D052216:Glucagon-Like Peptide 1; D000324:Adrenocorticotropic Hormone; D005934:Glucagon; D000077270:Exenatide