Background

The intestinal wall of the GI tract is comprised of several different types of cells, each with specific and, sometimes exclusive functions (Allaire et al., 2018). In vitro testing of small molecule therapeutics or drug candidates has traditionally relied on mono-enteric cell lines. These in vitro systems do not determine the absorptive properties of the small molecule being tested, nor do they predict the behavior of these compounds in the gut. To some extent, enteroids recapitulate the complexity and diversity of the intestine, but they are an expensive system and do not always provide reproducible results due to variability in the reagents required (Blutt et al., 2018). There is an unmet need for simple experimental systems that mimic the gut epithelial layer to aid in the development of new chemical probes and drugs. Here, we focus on developing cell-based protocols to assay one intestinal signaling system, the small molecule-based activation of the G protein-coupled receptor TGR5 on intestinal enteroendocrine cells.

The study of small molecule agonists of TGR5 has gained significant importance in recent years due to the potential therapeutic benefits of these compounds, primarily in diabetes control (Duboc et al., 2014). TGR5 is expressed on enteroendocrine L-cells in the lower gastrointestinal (GI) tract, and when activated, results in secretion of the anti-diabetic hormone GLP-1 into the bloodstream (Eissele et al., 1992; Harach et al., 2012). Activation of this gut-initiated TGR5-GLP1 cascade shows promise as a potential target for type 2 diabetes therapeutics (Hodge and Nunez, 2016). However, activation of TGR5 outside of the gut can cause unwanted side effects (Cao et al., 2016; Hodge and Nunez, 2016). Therefore, a unified in vitro screening method that can be used to evaluate the effects of small molecules on TGR5 activation and GLP-1 secretion, as well as the interaction of these compounds with the gut epithelial barrier, will aid in the development of successful new TGR5-targeted therapies.

Here we describe a simple in vitro protocol from Chaudhari et al. (2021) to co-differentiate human intestinal epithelial cells with human enteroendocrine cells into a polarized monolayer in transwells. We then describe how to utilize the resulting epithelial layer to test the TGR5 agonism and gut restricted-properties of small molecules. Our system incorporates GLP-1-secreting NCI-H716 L-cells, which have been the predominant cell line used to test TGR5 activation and GLP-1 secretion (Kim et al., 2017; Sato et al., 2008), into an epithelial monolayer formed by surrounding human intestinal colon carcinoma Caco-2 epithelial cells. This monolayer forms a physical and biochemical barrier to small molecules.

Through electron microscopy imaging, we show that the enteroendocrine cells intercalate with the colon epithelial cells and are polarized, with distinct apical and basolateral faces. Mass spectrometry-based quantification of the passage of small molecules from the apical to the basolateral chamber through the differentiated monolayer allows for evaluation of the gut-restricted properties of the compounds (Chaudhari et al., 2021). Using this system, we can also perform RNA interference knockdowns, harvest cells to extract RNA and quantify TGR5 expression, and measure GLP-1 secretion. Importantly, because small molecules can be added to either face of the differentiated monolayer, information about whether compounds can induce TGR5 activation and expression and GLP-1 secretion from the apical face, basolateral face, or both faces, can be determined. Lastly, we describe how this system can be used to study toxic effects of small molecules on intestinal cells as well as the effects of these compounds on epithelial barrier integrity. This in vitro co-culture transwell system will enable the identification of small molecules that are both TGR5 agonists and possess limited off-targeted effects and will thus facilitate the development of novel type 2 diabetes therapies.