Secretory Granules Tell (Almost) All in Enteroendocrine Cells

“By the help of microscopes, there is nothing so small, as to escape our attention.”—Robert Hooke, 1665, Micrographia Inside our cells are entire worlds of organelles responsible for cellular function. Some organelles confer unique functions to particular types of cells. In the case of endocrine cells, it is the content of the secretory granule, the marker of endocrine cells, that has traditionally been used to categorize these cell types, because their morphology can be characterized by electron microscopy, and the peptide hormones stored in granules can be readily visualized with immunofluorescence microscopy. Indeed, identification of enteroendocrine cells (EECs) that reside in the gastrointestinal tract has been based on the peptide hormone content of their secretory granules, leading to the hypothesis that EECs can be classified based on their distinct hormonal products. However, this dogma is being challenged by an increasing number of studies that have revealed much more complex patterns of hormone distribution among EECs in different locations in the gut. In 2012, two studies published in Endocrinology (1, 2) showed coexpression of multiple gut hormones within individual EECs by using cholecystokinin (CCK)-, proglucagon-, and gastric inhibitory polypeptide–promoter-driven green fluorescent protein expression to first label EECs. More recent reports using a similar transgene-based approach have also shown that single mature EECs throughout the mouse gut can express several peptide hormones (3) and that there are distinct populations of secretory granules within these cells that store these hormones (4). Can these granule populations be identified and, importantly, quantified? Do EEC secretory granules contain one peptide hormone product or several? What is the functional significance of peptide hormone costorage in EECs? These are the questions posed by Fothergill et al. (5) in this issue of Endocrinology. Why is it important to identify different granule populations in EECs? Secretory granules are the signature of the regulated secretory pathway, in which peptide hormones destined for secretion are stored until a stimulus triggers their release into the circulation. Secretory granules thus render EECs exquisitely sensitive to nutritional, hormonal, and neural stimuli. Of particular interest are the direct effects of products of nutrient digestion, such as glucose, amino acids, and long-chain fatty acids; short-chain fatty acids produced by gut microbiota; proteolytic products; and bile acids. Many of these metabolites activate various G protein–coupled receptors that act as chemosensors (6) that subsequently trigger membrane depolarization, leading to an influx of extracellular Ca2+ through voltage-gated Ca2+ channels. This cascade of events leads to the secretion of gut peptide hormones, which then can act in a paracrine manner on enterocytes, activate afferent enteric nerves, and function in an endocrine manner to regulate insulin secretion, appetite, and gastrointestinal motility. All these effects have generated tremendous interest in the manipulation of EEC function as a therapeutic target for diabetes and obesity (7). As the EEC becomes a pharmaceutical target, the questions of what EECs secrete which peptide hormones, and their collective effects on metabolism and nutrient homeostasis, become vitally important. To this end, Fothergill et al. (5) first set out to determine the patterns of hormone storage within EECs that expressed CCK, ghrelin, glucagonlike peptide-1 (GLP-1), peptide YY (PYY), secretin, and the bioactive amine 5-hydroxytryptamine using triple-labeling immunofluorescence confocal microscopy. The EECs were labeled in situ in sections of duodena obtained from 8- to 10-week-old male C57BL/6 mice that had not been fasted. In contrast to previous studies, these mice did not harbor a transgene to mark any particular type of EEC; rather, patterns of peptide hormone storage in EECs throughout the duodenum were characterized in an unbiased manner by simply selecting cells that were immunopositive for one hormone, regardless of other hormones that may have been coexpressed. Additionally, they profiled the storage of chromogranin A (CgA) in relation to the aforementioned hormones, because CgA is a commonly used marker of secretory granules in endocrine cells in general. In the first set of experiments, they quantified the hormone signature of EECs in two ways: first by the percentage of cells that showed overlap of immunofluorescence, and second by counting the number of immunofluorescent granules in a given area within each population of cells. This careful and diligent quantitative approach identified CgA-immunopositive granules in ghrelin-expressing EECs, where traditional cellular immunofluorescence failed to show such coexpression. Therefore, superresolution microscopy can reveal hormone coexpression patterns at the level of granule populations that cannot be visualized by regular cellular immunofluorescence. Now, the question was, can an individual secretory granule contain more than one hormone? The power of superresolution microscopy methods such as photoactivated localization microscopy/stochastic optical reconstruction microscopy, stimulated emission depletion, and structured illumination microscopy may be able to reveal the presence of multiple cargoes within granules. Although another study showed the presence of neurotensin-, PYY- and GLP-1–positive granules in the same EEC in mouse distal ileum by using structured illumination microscopy (4), they were unable to show colocalization of different hormones within each granule. Fothergill et al. (5) recognized that the difficulty in determining the cargo of individual secretory granules lay in the manner in which they clustered within the cell. Although superresolution microscopy can clearly visualize the size of a secretory granule (150–300 nm in diameter), determining the peptide hormone content of individual secretory granules that are tightly clustered within an EEC pushes the limits of the resolving power of the microscope, because the distance between granules may be beyond the limit of resolution. Also, simultaneous acquisition of multiple fluorescence wavelengths, which is necessary to determine the presence of multiple peptide hormones within granules, is subject to chromatic aberration because of the different refractive indices of each fluorescent emission. As a result, different fluorescent wavelengths will not be brought into focus, and resulting blur will make it difficult to distinguish individual secretory granules. Fothergill et al. (5) confronted these technical problems by attempting to correct for chromatic aberration by imaging fluorescent beads 100 nm in diameter and by applying stringent thresholding at the same level across all images. Despite the use of beads as a positive control, chromatic aberration could not be completely overcome, and therefore areas of fluorescence overlap could not be attributed to multiple hormones within a granule. However, a large proportion of granules did appear to contain only a single hormone, a result that addressed another limitation: potential antibody cross-reactivity, a common problem in immunofluorescence microscopy. That granule populations segregated on the basis of immunofluorescence demonstrated that the antibodies did not cross-react. Altering the threshold to distinguish individual granules in areas of high granule density resulted only in losing signal with a high threshold and gaining nonspecific signal or signal from other focal planes with a low threshold. Similar limits in spatial resolution were observed to obscure the structure of newly characterized tubular matrices in the endoplasmic reticulum (8); the smallest average distance between tubules that could be detected was 150 nm, within the lower range of the diameter of a secretory granule. Therefore, the high-density arrangement of granules and other subcellular organelles is currently beneath the resolving power of superresolution microscopy. What is the functional significance of hormone costorage in EECs? If different hormones are stored in separate granule populations, are they cosecreted in response to nutrient ingestion? One study has shown that that a variety of metabolite, hormonal, and neural secretagogues that stimulate GLP-1 secretion from perfused intestinal preparations or crypt cell cultures also stimulated secretion of PYY and neurotensin, with similar temporal kinetics (4). This group also found that cosecretion of multiple hormones had synergistic effects on gastric emptying and food intake but divergent effects on glucose homeostasis. The big picture that emerges from such studies is one of multiple secretagogues acting on EECs, causing the simultaneous exocytosis of different granule populations whose peptide hormone contents have synergistic or complementary effects on metabolism, appetite regulation, and gastrointestinal motility. What Fothergill et al. (5) showed is that imaging at the level of the secretory granule, rather than simply quantifying the extent of immunofluorescence overlap at the cellular level, can more accurately and precisely reveal the complexity of hormone storage patterns in EECs. The diverse world of secretory granules inside the EEC is now getting our attention with the help of microscopes, just as Robert Hooke observed centuries ago. cholecystokinin chromogranin A enteroendocrine cell glucagonlike peptide-1 peptide YY. Disclosure Summary: The author has nothing to disclose.

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