Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2 (proprotein convertaseyneuroendocrine precursorygene disruption)

The prohormone convertase SPC2 (PC2) par- ticipates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core gran- ule convertase SPC3 (PC3yPC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neo r ) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physi- ological roles of many neuropeptides and peptide hormones. SPC2 and SPC3 are members of a larger mammalian family of serine proteases that are related to the yeast convertase kexin and to the bacterial protease subtilisin (1-5). SPC2 and SPC3 are expressed almost exclusively in neuroendocrine cells throughout the brain, gut, pancreatic islets, and other endocrine tissues of the body (6-9). Their acidic pH optima, requirement for calcium ions, and ability to be sorted into dense-core vesicles of the regulated secretory pathway are consistent with their putative role as the major endoproteases responsible for the maturation of a large number of hormone and neuropeptide precursors throughout the organism (10-15). In the islets of Langerhans, both SPC2 and SPC3 are expressed in the beta cells and are believed to act together to process proinsulin into insulin by cleavage at the Lys-Arg and Arg-Arg sites that flank the C peptide, followed by excision of these basic residues from the products by the exopeptidase carboxypeptidase E (7, 16-18). SPC2 is also expressed at high levels in the islet alpha cells, where it selectively processes proglucagon to liberate only glucagon as the major active hormone from this large multihor- mone precursor (19). On the other hand, in the neuroendocrine L cells of the gut, proglucagon is processed at different sites by SPC3 to liberate mainly two glucagon-like peptides—GLP1 and GLP2 (20-22). GLP17-37 enhances insulin secretion from the beta cells in response to glucose, whereas glucagon counters the hypoglycemic action of insulin by triggering hepatic glycogenol- ysis and enhancing hepatic gluconeogenesis to raise blood glucose levels (23-25). Thus, under some circumstances, SPC2 and SPC3 may act together to promote precursor maturation (e.g., in the case of proinsulin; refs. 3 and 7), whereas in other instances these enzymes are expressed differentially in certain cell populations, leading to tissue-specific processing of multifunctional precursors such as proglucagon (19, 20) and proopiomelanocortin, the precursor of corticotropin, melanocyte-stimulating hormone, and b-endorphin (10, 26, 27). The widespread expression of these two convertases in the central nervous system is consistent with suggestions that they play important roles in processing a wide variety of neuropeptide precursors (28-30). Here we report the effects of disruption of the mSPC2 gene in the processing of three important prohormones of the islets of Langerhans: proinsulin, proglucagon, and prosomatostatin. The results unequivocally establish SPC2 as a major convertase in neuroendocrine and hormone precursor processing and support the hypothesis that SPC2 and SPC3 represent the core set of secretory granule processing endoproteases in neuroendocrine cells (10).