Figure 1. Organoids of the human intestine, enriched for hormone producing enteroendocrine cells (EECs). Different hormones are colored in blue, green and cyan. Credit: Joep Beumer, copyright: Hubrecht Institute. 8 November 2022 New insights into the processing of hormones in the human gut Back to news The human gut is the largest mammalian organ to produce hormones. In response to food, the hormone-producing cells in this organ secrete dozens of peptides. Now, researchers from the Organoid group (Hubrecht Institute) and the group of Wei Wu (Utrecht University) have defined the broad spectrum of these peptides and the way they are processed. These data will help characterize potentially new hormones that the gut produces to control key aspects of human physiology, including appetite and bowel movement. The results were published in PNAS on 7 November 2022. The intestine is the largest organ in the human body to produce hormones. So-called enteroendocrine cells (EECs) are responsible for secreting these hormones. While EECs only comprise 1% of the surface of the intestine, they secrete dozens of peptides A molecule that is comprised of a small number of amino acids. in response to food intake. These hormones regulate important physiological responses, including appetite, bowel movement and insulin secretion. Hormone processing EECs produce large proteins termed prohormones, from which small peptides that are bioactive Substances that have a specific physiological or biological activity or function. are cleaved. The precise role of the enzymes (i.e. proteases) involved in this process, is not understood. Notably, mutations An error in the DNA. Mutations can, among other things, arise if the DNA is copied incorrectly or through external influences. For example, tumor cells often contain mutations that are beneficial for their growth. in these enzymes can cause endocrine diseases that result from the lack of some functional hormones. Researchers from the Organoid group teamed up with the group of Wei Wu at Utrecht University to study the role of these enzymes in gut hormone processing and identify potentially novel gut hormones. Organoids as model system EECs are rare in human tissue and have therefore been difficult to study. The researchers made use of human intestinal organoids Miniature organs that can be cultured in the laboratory. Organoids mimic the shape and function of an actual organ. Researchers use the structures to, for example, study the effects of medication on diseased organs. to greatly enrich the number of EECs, allowing them to study these in greater detail. EECs in human organoids resemble their tissue counterparts, and are therefore a great model to study how they function. Moreover, EEC’s in the human gut are in a different state depending on their precise anatomical location. For example, an EEC is in a different state in the crypts The lowest parts of the folded tissue of the intestines. This place contains, among other thing, the stem cells that continuously produce new intestinal cells. than in the villi The protrusions on the folded tissue of the intestines. The function of these structures is to increase the surface, to increase the uptake of nutrients out of the food we eat.. In the organoid models, the team was able to reproduce these different states of the EEC’s. Figure 1. Organoids of the human intestine, enriched for hormone producing enteroendocrine cells (EECs). Different hormones are colored in blue, green and cyan. Credit: Joep Beumer, copyright: Hubrecht Institute. Hormone factories The researchers turned the organoids into large hormone factories. At will, they were able to induce secretion of hormones and collect them to allow characterization (Figure 1). Joep Beumer explains: ‘’In tissues of humans or animals it is difficult to detect the hormones due to their low abundance. You will never be sure if what you measure is derived from EECs. The massive amounts of hormones that we obtain in organoids make it possible to almost entirely bypass this problem.” Figure 2. Left: organoid with healthy EECs. Right: Organoid with mutations in protease. Green indicates the peptidase that is lost in the mutant organoid. Cell nuclei in blue (DAPI) and actin in white (Phallloidin). Credit: Joep Beumer, copyright: Hubrecht Institute. Mutating hormone proteases Next, the team employed CRISPR-Cas9 A technique that researchers can use to cut the DNA in a very specific place, to make a change there. This way, researchers can study the effect of a specific change in the DNA. to delete the enzymes, or proteases, that could be involved in hormone processing, and that are found to be mutated in endocrine diseases (Figure 2). With an advanced technique (mass spectrometry-based peptidomics), they characterized the peptide hormones produced by healthy EECs and the ones produced by EECs with mutations in the processing enzymes. ‘’This technique allows us to accurately map what kind of hormones healthy and mutant EECs produce’’, explains co-first author Julia Bauzá-Martinez. ‘’Moreover, if specific peptides are depleted in the background of a mutant protease, one can assume it is an important hormone and not a random artefact’’. Glucagon in the human gut The researchers succeeded in performing a broad characterization of peptide hormones in normal EECs, as well as in EECs where specific proteases were missing. Against all expectations, the team could measure substantial amounts of glucagon from human EECs. Glucagon is a well-known hormone produced by the human pancreas, but whether the human gut can produce it too is subject of debate. ‘’Glucagon was particularly found when cells were stimulated with specific signals, mimicking the intestinal villus environment,” says Joep Beumer, other co-first author. The enzyme that can generate this glucagon – called PCSK2 – was indeed stimulated by the very same signals Future directions Previous work by the same research groups indicated that EECs can change the hormones they produce when migrating from crypt to villus. The current work suggests EECs could potentially also change how they process prohormones into bioactive peptides depending on their location in the gut. Future work could focus on the physiological relevance of the changes in the hormones that EECs produce. “In the future, a more complete understanding of hormone processing could contribute to better therapeutic interventions for endocrine diseases,” Beumer concludes. Publication Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models. Joep Beumer, Julia Bauzá-Martinez, Tim S. Veth, Veerle Geurts, Charelle Boot, Hannah Gilliam-Vigh, Steen S. Poulsen, Filip Knop, Wei Wu & Hans Clevers. PNAS, 2022. The Organoid group, previously Clevers group, studies the biology of Wnt signaling in tissue turnover and in cancer.