The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.

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A means of estimating human enteroglucagon (glucagon-like immunoreactivity of intestinal origin) in tissues and plasma is described, based on the subtraction of RIA values obtained with the C-terminal-directed glucagon antiserum RCS5 from the total glucagon-like immunoreactivity determined with the N-terminal- to midmolecule-directed glucagon antiserum R59. Gel filtration on Sephadex G-50 of human plasma and extracts of normal human intestine separated the R59 immunoreactivity into three peaks: a small peak of void volume material, a major peak coeluting with porcine glicentin, and a smaller peak coeluting with pancreatic glucagon. No RCS5 immunoreactivity was detected in the human gut, except for a small amount constituting less than 2% of the total glucagon-like immunoreactivity in the ileum and rectum only. In extracts of human pancreas, the chromatographic profiles obtained with RCS5 and R59 assays differed from the intestinal patterns, but were identical to each other, giving no evidence of a significant amount of pancreatic R59 immunoreactivity that was not also reactive with RCS5. Chromatography of plasmas from healthy subjects and patients with dumping syndrome, active coeliac disease, and tropical sprue showed that only the second major peak of R59 immunoreactivity reflected the basal or postnutrient increases in the plasma enteroglucagon concentration. In patients with exaggerated enteroglucagon release, the rise was again found to be entirely due to an increase in this peak of immunoreactivity. This major molecular form of human enteroglucagon, similar in size to porcine glicentin, is, thus, the form most likely to be of physiological and pathophysiological significance.

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