Abstract MicroRNAs (miRNAs) are small noncoding RNA molecules that modulate gene expression at the posttranscriptional level. Numerous reports have elucidated the importance of miRNAs in the regulation of a wide array of biological processes including metabolism and energy homeostasis. miRNAs in the endocrine pancreas have been intensively studied over the last 15 years and linked to pancreatic islet development and function. In comparison, knowledge of miRNAs in gut endocrine cells, or enteroendocrine cells (EECs), is severely lacking. EECs have important roles in systemic energy homeostasis, are highly relevant to type 2 diabetes etiology, and may be critical to the mechanisms that underlie the rapid positive metabolic effects of bariatric surgery. Very recent studies reveal that several miRNAs are highly enriched in mature EECs and/or in intestinal stem cells that are primed to the EEC lineage. Moreover, functional experiments in enteroids/intestinal organoids suggest that some of these miRNAs may be important for the regulation of EEC differentiation and function. Another report has raised the possibility that EECs secrete miRNAs into circulation. These intriguing findings merit further investigation, particularly as it pertains to EEC miRNAs as novel therapeutic targets in type 2 diabetes and related diseases. MicroRNAs (miRNAs) are small, noncoding RNA molecules (18 to 24 nucleotides) that regulate many biological processes by modulating gene expression at the posttranscriptional level. The critical role of miRNAs in the mammalian endocrine system was discovered in studies of pancreatic islets in 2004 (1). Since then, numerous studies have been published that highlight miRNAs as important regulators of the development and function of the endocrine pancreas, etiological factors in islet dysfunction, and potential therapeutic targets for diabetes. In striking contrast to the extensive studies of miRNAs in the endocrine pancreas, the role of miRNAs in the gut endocrine lineage is woefully underexplored. Gut endocrine cells, usually termed enteroendocrine cells (EECs), sense nutrient/metabolic cues and respond by secreting peptide hormones that carry out important paracrine and endocrine functions. EECs are derived from intestinal stem cells (ISCs) located in the intestinal crypt and constitute only ∼1% of intestinal epithelial cells. The plasticity of EECs in early stages of differentiation (2) and the heterogeneity of EECs in terms of biogeographic location, molecular profile, and cellular function make the study of EECs rather challenging. Recent work is generating excitement about the potential role of miRNAs in the EEC lineage. Here, we review each of the three major publications on the topic and then provide a summary of the key open areas of research. miRNAs in the EEC Lineage The original study that offered a glimpse of miRNAs in the EEC lineage was published in 2015 by Knudsen et al. (3). They profiled miRNAs in sorted murine EECs and found that several miRNAs, notably miR-375, are highly enriched in EECs. Through in situ hybridization, they confirmed colocalization of miR-375 and EEC peptide hormones. They further showed via functional studies in ex vivo mouse enteroids that miR-375 suppresses the EEC fate, demonstrating the functional relevance of miRNAs in EEC development. The next major study came from Peck et al. (4) in early 2017. These authors used female Sox9–enhanced green fluorescent protein (EGFP) mice to sort distinct subpopulations of intestinal epithelial cells. By small RNA sequencing analysis, they identified an over-representation of several miRNAs, including miR-375, miR-7, miR-183, miR-182, and miR-672, in the subpopulation enriched for EECs. They also showed that miR-375 is robustly expressed in cells enriched for ISCs and highly sensitive to the presence of microbes. Through follow-up loss-of-function studies, they demonstrated that suppression of miR-375 promotes mouse enteroid proliferation. These findings expanded the functional relevance of miR-375 in the EEC lineage. A very recent study from Yan et al. (5) in late 2017 confirmed the findings from Peck et al. (4), this time in male Sox9-EGFP mice. In addition, the authors reported that miR-375 levels are elevated in circulation and correlated with plasma levels of gut hormones in human subjects only upon oral ingestion of glucose and not intravenous administration. This raises an intriguing idea that gut epithelial cells may be a primary source for the miR-375 present in circulation. A previous report showed that the pancreatic β cell, where miR-375 is highly expressed, likely contributes nominally to plasma levels of miR-375 (6). Whether the circulating miR-375 is mainly derived from EECs and whether it exerts functions beyond its proposed role in regulating EEC differentiation and function merit further investigation. miRNA regulation of EEC fate and function has emerged as an important area of research, and there remain several key open areas of investigation, four of which are described here. Key Open Areas of Research What is the miRNA landscape across EECs? The studies mentioned previously are limited by the fairly crude methods currently available for EEC isolation. For example, the Sox9-high subpopulation used in Peck et al. (4) is highly enriched for EECs but also contains other cell types, such as quiescent ISCs. Furthermore, the sorting of cells positive for specific peptide hormones, such as Cck, as done by Knudsen et al. (3), ensures only the isolation of a slice of the full spectrum of EECs, as not all EECs express the same cadre of hormones. Herein lies a major challenge in the field: how does one define EECs sensitively enough to capture all of the subtypes but specifically enough to avoid neighboring cells with limited-to-no endocrine functionality? Efforts similar to those reported by a recent study from the Kuo laboratory (2), in which high-resolution molecular characterization (single-cell sequencing) of EEC lineage cells was performed, will help provide more granular definitions of EEC subtypes and facilitate future studies to profile the miRNA landscape across EECs. Which miRNAs are most relevant to EEC functions? No study, to date, has explicitly linked miRNAs to mature EEC functions. Several in vitro models, such as murine EEC-like lines (STC-1 and GLUTag) and human EEC-like lines (NCI-H716), are currently available for such studies. Specifically, by transfecting nucleic acid miRNA mimics and inhibitors or applying clustered regularly interspaced short palindromic repeats–associated protein 9–based genetic manipulations of miRNAs, gain- and loss-of-function studies can be performed in these cell lines to evaluate the effects of certain miRNAs on hormone production and secretion from EECs. Another option is to use three-dimensional mouse or human enteroids or intestinal organoids (7), which are considered more physiologically relevant. However, these models are highly heterogeneous in terms of cellular composition and may require additional strategies for restricting miRNA knockout or overexpression only to EECs. Which miRNAs are most critical for EEC differentiation? A recent study by Yan et al. (2) suggests that the priming of ISCs to the EEC lineage may occur early. Specifically, they identified a class of cells with stem-cell potential in the crypts that expresses the marker Prospero homeobox 1 and likely represents secretory progenitors. Future studies of miRNA profiling in these cells may help reveal miRNAs critical for EEC differentiation. Another appealing approach to study EEC maturation is to leverage the models of de novo intestinal formation by directed differentiation of human pluripotent stem cells or by transdifferentiation of mouse embryonic fibroblasts. Do gut-derived circulating miRNAs act as endocrine molecules? Emerging evidence suggests that certain miRNAs may be messengers in cross-organ communication. At this point, it is unknown if the gut is also involved in such miRNA-mediated tissue crosstalk. Whereas there remain challenges associated with studies of circulating miRNAs (8), future studies are warranted to investigate the following: the (1) possibility of miRNA secretion from EECs; (2) mechanism(s) of transport of EEC-secreted miRNAs; (3) relationship between the miRNAs and peptide hormones secreted from the same EECs; and (4) key target tissues (if any) to which these miRNAs are delivered with function integrity. Conclusion EECs play an important role in regulating energy homeostasis. In light of the emerging evidence of miRNA-mediated control of EEC fate, it is clear that miRNAs merit continued investigation with regard to EEC maturation, plasticity, and function. Future work will help realize the potential of EEC miRNAs as pharmacologic targets in type 2 diabetes and related metabolic conditions. Abbreviations: EEC enteroendocrine cell EGFP enhanced green fluorescent protein ISC intestinal stem cell miRNA microRNA. Acknowledgments The authors thank Dr. Bailey Peck (University of Michigan) and members of the P.S. laboratory, including Dr. Ajeet Singh, Dr. Michael Shanahan, and Rowan Beck, for helpful comments on the article. Financial Support: The authors are grateful for support from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (R01DK105965, awarded to P.S.), American Diabetes Association Pathway Program (1-16-ACE-47, awarded to P.S.), and Empire State Stem Cell Fund through New York State Department of Health (Contract #C30293GG, awarded to Y.-H.H.). Disclosure Summary: Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Board, New York State Department of Health, or state of New York. References 1. Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M. A pancreatic islet-specific microRNA regulates insulin secretion. Nature . 2004; 432( 7014): 226– 230. Google Scholar CrossRef Search ADS PubMed 2. Yan KS, Gevaert O, Zheng GXY, Anchang B, Probert CS, Larkin KA, Davies PS, Cheng ZF, Kaddis JS, Han A, Roelf K, Calderon RI, Cynn E, Hu X, Mandleywala K, Wilhelmy J, Grimes SM, Corney DC, Boutet SC, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Wang F, von Furstenberg RJ, Smith NR, Chandrakesan P, May R, Chrissy MAS, Jain R, Cartwright CA, Niland JC, Hong YK, Carrington J, Breault DT, Epstein J, Houchen CW, Lynch JP, Martin MG, Plevritis SK, Curtis C, Ji HP, Li L, Henning SJ, Wong MH, Kuo CJ. Intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell 2017; 21( 1): 78– 90.e76. 3. Knudsen LA, Petersen N, Schwartz TW, Egerod KL. The microRNA repertoire in enteroendocrine cells: identification of miR-375 as a potential regulator of the enteroendocrine lineage. Endocrinology . 2015; 156( 11): 3971– 3983. Google Scholar CrossRef Search ADS PubMed 4. Peck BC, Mah AT, Pitman WA, Ding S, Lund PK, Sethupathy P. Functional transcriptomics in diverse intestinal epithelial cell types reveals robust microRNA sensitivity in intestinal stem cells to microbial status. J Biol Chem . 2017; 292( 7): 2586– 2600. Google Scholar CrossRef Search ADS PubMed 5. Yan X, Wang Z, Westberg-Rasmussen S, Tarbier M, Rathjen T, Tattikota SG, Peck BCE, Kanke M, Oxvig C, Frystyk J, Starup-Linde J, Sethupathy P, Friedländer MR, Gregersen S, Poy MN. Differential impact of glucose administered intravenously and orally on circulating miR-375 levels in human subjects. J Clin Endocrinol Metab . 2017; 102( 10): 3749– 3755. Google Scholar CrossRef Search ADS PubMed 6. Latreille M, Herrmanns K, Renwick N, Tuschl T, Malecki MT, McCarthy MI, Owen KR, Rülicke T, Stoffel M. miR-375 gene dosage in pancreatic β-cells: implications for regulation of β-cell mass and biomarker development. 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Endocrinology – Oxford University Press
Published: Feb 1, 2018
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