Costorage of Enteroendocrine Hormones Evaluated at the Cell and Subcellular Levels in Male Mice.

Linda J Fothergill, Brid Callaghan, Billie Hunne, David M Bravo, John B Furness

Recent studies reveal complex patterns of hormone co-expression within enteroendocrine cells (EEC), contrary to the traditional view that gut hormones are expressed individually in EEC. Moreover, different hormones have been found in separate subcellular vesicles. However, detailed analysis of relative expression of multiple hormones has not been made. Subcellular studies have been confined to peptide hormones, and have not included the indolamine, 5-HT, or the neuroendocrine protein, chromogranin A (CgA). In the present work, co-expression of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 in mouse duodenum was quantified at a cellular and subcellular level by semi-automated cell counting and quantitative vesicle measurements. We investigated whether relative numbers of cells with co-localised hormones analysed at a cell level matched the numbers revealed by examination of individual storage vesicles within cells. CgA and 5-HT were frequently expressed in EEC that contained combinations of GLP-1, ghrelin, secretin and CCK. Separate subcellular stores of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 were identified. In some cases, high resolution analysis revealed small numbers of immunoreactive vesicles in cells dominated by a different hormone. Thus the observed incidence of cells with co-localised hormones is greater when analysed at a subcellular, compared with a cell level. Subcellular analysis also showed that relative numbers of vesicles differ considerably between cells. Thus separate packaging of hormones that are co-localised is a general feature of EEC, and EEC exhibit substantial heterogeneity, including the co-localisation of hormones that were formerly thought to be in cells of different lineages.