Butyrylcholinesterase distribution in the mouse gastrointestinal tract: An immunohistochemical study

Abstract Butyrylcholinesterase (BChE) is a hydrolytic enzyme that together with acetylcholinesterase (AChE) belongs to the cholinesterase family. Whereas AChE has a well‐established role in regulating cholinergic neurotransmission in central and peripheral synapses, the physiological role of BChE remains elusive. In this morphological immunohistochemical and double‐label confocal microscopy study we investigated the distribution of BChE in the mouse gastrointestinal tract. BChE‐positive cells were detected in the liver (both in hepatocytes and cholangiocytes), in the keratinised layers of the squamous epithelium of the oesophagus and forestomach, in the oxyntic mucosa of the stomach, in the mucus‐secreting cells of duodenal Brunner glands and the small and large intestinal mucosa. Interestingly, BChE‐positive cells were often detected close to gastrointestinal proliferative niches. In the oxyntic mucosa, the close proximity of ghrelin‐producing and BChE‐positive parietal cells suggests that BChE may be involved in ghrelin hydrolysation through paracrine action. To our knowledge, this is the first comprehensive morphological study performed to gain insight into the physiological role of BChE in the gastrointestinal tract.

suggests that mammals are endowed with non-neuronal cholinergic systems that are characterised by multiple albeit poorly characterised physiological functions (Reichrath et al., 2016). However, in mammalian organs this powerful molecule needs to be confined to the area where it is released, to prevent noxious hormone-like actions. This posits the existence of an enzyme system capable of its rapid hydrolysation.
BChE expression is especially high in the liver, which is the main source of plasma BChE (Jbilo et al., 1994). Together, AChE and BChE activities determine and regulate the action of ACh in peripheral organs and the central nervous system (Vaknine & Soreq, 2020).
BChE is considered a non-specific cholinesterase due to its ability to hydrolyse as substrates for other choline and non-choline esters (Massoulié et al., 1993). However, unlike AChE, its physiological function is still debated and numerous hypotheses have been advanced.
As a bioscavenger molecule, BChE appears to be one of the enzymes involved in the detoxification of organophosphates, drugs and dietary compounds (Hou et al., 2013;Johnson & Moore, 2012).
Moreover, plasma and liver BChE activity are elevated in patients with obesity and diabetes and rodent models of metabolic disorders (Chen et al., 2017;Perelman et al., 1990;Randell et al., 2005), whereas it is low in individuals suffering from malnutrition or inflammatory states (Lampon et al., 2012). Despite the clinical importance of these discoveries, few studies have tried to establish a causal relationship between BChE and obesity. Findings from two recent studies point to a role for it in hydrolysing ghrelin, the appetite-promoting hormone produced by the stomach (Brimijoin et al., 2016;Chen et al., 2017). These studies have found that BChE hydrolyses ghrelin at a rate that strongly affects weight gain and fat metabolism (Brimijoin et al., 2016) and that BChE knockout mice fed a high-fat diet rapidly become obese and show 50% higher plasma ghrelin levels (Chen et al., 2017).
The role of BChE in ghrelin regulation and its altered expression in patients with metabolic dysfunction suggests that it plays a physiological role in the gastrointestinal (GI) tract. Evidence for its distribution in the GI tract mostly comes from biochemical studies; of these, few have examined its presence in the rodent and human stomach and intestine (Cheng et al., 2008;L'Hermite et al., 1996;Roivainen et al., 2004;Sine et al., 1988Sine et al., , 1991Wang et al., 2004).
In this study, we investigated the presence and distribution of BChE in the mouse GI tract using peroxidase immunohistochemistry and confocal microscopy.
Collectively, our findings show that BChE is widely expressed in epithelial secretory cells; in particular, BChE-positive ( + ) cells were detected in the keratinised epithelium of the oesophagus and the forestomach, in the glandular portion of the stomach and the small and large intestinal epithelium. This distribution suggests a dual role for BChE against the compounds passing through the gastric and intestinal lumina and/or in the physiological processes that take place in the mucosal layer of the GI tract.

| Animals and tissue processing
Adult male C57BL/6 mice purchased from Charles River Laboratories (Calco, Italy) were housed in plastic cages in constant environmental conditions in a 12 h light/dark cycle at 22°C with free access to standard chow and water. Handling was limited to cage cleaning. Their care was according to Council Directive 2010/63/ EU. All experiments were approved by the Italian Health Ministry (Authorisation no. 405/2018-PR). After a week on the normal diet, animals were divided into 2 groups of 6 with similar body weight and fed for 10 weeks either the normal diet (Control group) or a highfat diet (HFD; Charles River; 50 kJ% from fat, 30 kJ% from carbohydrates and 20 kJ% from proteins; HFD group). At 12-14 weeks all mice were sacrificed. The livers of both mouse groups were analysed to test the specificity of the primary BChE antibody. All the other analyses were performed only in Control mice.
The animals used for morphological analyses were anaesthetised with an overdose of 2,2,2-tribromoethanol (Sigma-Aldrich) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The oesophagus, stomach, small and large intestine and liver were dissected and postfixed with the same fixative solution for 24 h at 4°C before being washed in PB. Mice used for western blotting were anaesthetised and euthanised by cervical dislocation; the livers were rapidly removed, snap-frozen in liquid nitrogen and stored at −80°C until use.

| Antibodies
The primary and secondary antibodies used in the study are listed in Table 1.
Total protein extracts (20-15 μg) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane using Bio-Rad's Trans-Blot Turbo TM Transfer system. Loading and transfer efficiency were checked by Ponceau staining (Santa Cruz Biotechnology).
Membranes were then blocked for 1 h at room temperature (RT) in TBS-Tween 20 containing 5% non-fat dried milk and subsequently incubated overnight with primary antibodies (Table 1a). Finally, they were incubated with a secondary antibody conjugated to horseradish peroxidase (Vector Laboratories;

| Peroxidase immunohistochemistry
Serial paraffin sections (3 μm thick) were obtained with a PFM slide microtome (Bio-Optica), dried and stored until use. In brief, after dewaxing, paraffin sections were reacted with H 2 O 2 (3% in deionised H 2 O; 5 min) to block endogenous peroxidase, rinsed twice in phosphate-buffered saline (PBS) and treated with normal serum blocking solution (2% in PBS; 20 min). Next, they were incubated with the polyclonal goat anti-BChE antibody (Table 1a) Laboratories, 5 min). Sections were finally counterstained with haematoxylin, dehydrated, cleared with xylene and mounted in Eukitt medium (Sigma-Aldrich). Staining was never observed when the primary antibody was omitted.

| Immunofluorescence and confocal microscopy
For immunofluorescence, paraffin sections were dewaxed, washed in 0.1% PBS-Tween and incubated with an antigen retrieval solution (Histo-VT-One, PH 9, Nacalai Tesque) for 40 min at 70°C. After a thorough rinse in 0.1% PBS-Tween, they were incubated with blocking solution (Blocking One Histo, Nacalai Tesque) at RT for 40 min. Next, they were incubated with a mixture of two primary antibodies diluted in 1% bovine serum TA B L E 1 Antibodies used in the study. Images (1024 × 1024 pixels) were obtained sequentially from two channels using a confocal pinhole of 1.1200 and stored as TIFF files. The brightness and contrast of the final images were adjusted using Photoshop 6 (Adobe Systems).

| Statistical analysis
All values are mean ± standard error of the mean (SEM). Data were analysed for significance with GraphPad Prism (version 8).
Differences between groups were analysed using an unpaired student's t-test. The threshold for significance was p < 0.05.

| Liver BChE expression and distribution in the liver from mice fed a normal diet or the HFD
Since BChE is mainly synthesised in the liver and its levels are abnormally high in plasma and liver of HFD mice (Chent e al., 2017), the specificity of the primary BChE antibody was tested in the liver samples from Control and HFD mice using Western blot analysis and immunohistochemistry. All the other morphological analyses of GI tract organs, involved in the study, were performed only in Control mice. We used an affinity-purified goat polyclonal anti-BChE antibody (Table 1a) that yields a single band of 75 kDa.

| BChE distribution in the oesophagus
Immunohistochemical analysis disclosed that the cells of the 4-6 layers of the keratinised squamous epithelium and the smooth muscle cells of the muscularis mucosa were positive for BChE (Figure 2a,b).
Moreover, scattered BChE + cells were detected in the muscular layers of the muscularis externa ( Figure 2b). The elongated shape of these BChE + cells is reminiscent of the endothelial cells or smooth muscle cells that are found in the muscularis externa of adult mouse oesophagus (Rishniw et al., 2007). In particular, these sections were near the stomach, in the mid-lower portion of the oesophagus.
Notably, BChE staining seemed to be found not only in the keratinised epithelial layer but also in the oesophageal lumen (Figure 2a), possibly as a result of the desquamation of keratinised cells and/or secretory processes.

| BChE distribution in the stomach
The mouse stomach can be divided into forestomach, characterised by a keratinised squamous mucosa resembling the oesophageal mucosa, and glandular stomach, which in turn can be divided into the squamocolumnar junction, corpus and pyloric region (Ghoshal & Bal, 1989). At the junction of the forestomach and glandular stomach, the mucosa forms a "limiting ridge" (Luciano & Reale, 1992) covered by a thick, keratinised, stratified squamous epithelium. In the glandular stomach, the oxyntic glands of the corpus are clearly distinct from mucus-secreting pyloric glands (Engevik et al., 2020). Some BChE + cells negative for H + ,K + -ATPase were occasionally detected at the gland base.
In contrast, BChE + cells in mouse oxyntic glands were negative for gastric intrinsic factor (GIF; Figure 4g-i) and chromogranin-A (CgA; Figure 4j-l), respectively, a chief cell and an enteroendocrine cell marker (Boass & Wilson, 1964;Dieckgraefe et al., 1988;Engelstoft et al., 2015). GIF is a major secreted protein of human parietal cells which in mice and rats is found in zymogenic chief cells (Lorenz & Gordon, 1993), whereas CgA is an acidic protein found in large densecore secretory vesicles that are believed to be expressed in all cells of the mouse enteroendocrine GI tract (Engelstoft et al., 2015).
Since BChE plays a role in ghrelin hydrolysis by cleaving the octanoyl group (Brimijoin et al., 2016;Chen et al., 2017), doublelabel immunofluorescence experiments were conducted to establish whether BChE + cells also produce ghrelin. Although BChE + cells were detected in close proximity to ghrelin-producing cells in the corpus oxyntic mucosa, BChE staining was never observed in ghrelin-producing cells ( Figure S1, enlarged in Figure S1).

| BChE distribution in the small intestine
The mouse small intestine can be divided into three functionally  it may be secreted by enterocytes and/or crypt cells (Figure 5a,b).

Double-label experiments, conducted to identify BChE + cells in
the crypts, showed that BChE + cells at the crypt bottom also contained lysozyme (Figure 5g-i), the marker commonly used to identify Paneth cells (Clevers & Bevins, 2013).
Given the presence of BChE + cells in the crypts and the fact that crypts contain functional stem cells scattered amongst Paneth cells, double-label experiments were also performed to test the expression of olfactomedin-4 (Olfm4), which has recently emerged as a robust marker of murine intestinal stem cells (Schuijers et al., 2014).
Although BChE + cells were detected in close proximity to Olfm4 + cells, thus suggesting a role for BChE in stem cell physiology, none expressed both markers (Figure 5j-l).

| BChE distribution in the large intestine
The mouse colonic epithelium consists of the surface epithelium (columnar and goblet cells) and the crypts of Lieberkühn (McGraw-Hill, 1966). The mucosal wall is thick, with a thin muscularis mucosa and no discernible submucosa (Nguyen et al., 2015). Our immunohistochemical experiments documented BChE + cells in the epithelial

F I G U R E 4 BChE + mouse gastric cells. (a-c) Representative confocal microscopy images of the glandular stomach in the corpus region
showing BChE + cells (red) that also express H + ,K + -ATPase, a widely used marker of mouse gastric parietal cells (green). (d-f) At higher magnification, nearly all BChE + cells (red) are also H + ,K + -ATPase-positive (green). (g-i) Representative confocal images from the glandular stomach in the corpus region showing BChE + cells (red) that are negative for, but very close, to cells positive for GIF, a widely used marker of mouse gastric chief cells (green). (j-l) Representative confocal images of the glandular stomach in the corpus region showing BChE + cells (red) that are negative for, but in close proximity to, cells positive for CgA, a widely used marker of mouse gastric neuroendocrine cells (green).
mucosal layer in clusters of goblet cells (Figure 5e,f). Intense BChE staining was detected in the cytoplasm of some cells (arrows, Figure 5f), whereas in most cells only the mucous content seemed to show BChE positivity. These data suggest that BChE is secreted by goblet cells, like mucins and other factors that are released into the intestinal lumen.

| Proximity of BChE + cells to the proliferative niche in the mouse GI tract
To establish whether BChE + cells are found near progenitor cells, we examined the distribution of Ki67 + proliferative cells. In the liver, BChE + cells resembling bile duct epithelial cells were often found near Ki67 + cells (Figure 6a-c). Notably, in mice, the hepatic stem cell niche has been described in the canals of Hering and the space of Disse (Kordes & Häussinger, 2013). In the stomach, the proliferative zone is in the isthmus, a region between the gland and the pit (Barker et al., 2010), where BChE + cells were detected near Ki67 + cells (Figure 6d-f). In the intestine, progenitor cells are found with Paneth cells in the crypts (Sato et al., 2011).
Interestingly, BChE + Paneth cells showed a close spatial relationship with Ki67 + cells in the crypts (Figure 6g-i). Collectively, BChE + cells were present close to the proliferative niches of the liver, stomach and intestine.

| DISCUSS ION
BChE is a hydrolytic enzyme of the carboxylesterase family that is found in all mammals. Although a clear physiological function has not been identified for it, abnormal BChE serum levels are found in patients with conditions such as neurological disorders (especially Alzheimer's disease), obesity, type 2 diabetes and elevated cardiovascular risk (Darvesh et al., 2003). Interestingly, BChE hydrolyses the appetite-promoting hormone ghrelin (Chen et al., 2015).
The bulk of BChE is synthesised in the liver and secreted into the circulation; however, a fraction is also expressed in numerous neuronal and non-neuronal cells, especially cells with a barrier function such as epithelial and immune cells (Wessler et al., 2003). With regard to BChE distribution, most studies have investigated cholinergic enzyme activity by biochemical assays aimed at detecting the cells that are capable of producing and releasing it. They have found that BChE activity exceeds AChE activity in mouse liver, intestine, heart, lung and serum (Li et al., 2000). However, these investigations, which have quantified total BChE by homogenisation and enzyme isolation procedures, may have underestimated its levels in peripheral organs and may have been unable to identify its cellular localisation with precision (Darvesh et al., 2003). Notably, BChE distribution in the GI tract, where it would be able to perform several of its putative metabolic functions, has never been investigated by morphological techniques.

F I G U R E 6
Close proximity of BChE + cells to the proliferative niche in the mouse GI tract. (a-c) Representative confocal microscopy images of the liver showing BChE + cells (red) that are negative for, but in close proximity to, cells positive for Ki67, a widely used marker of mouse proliferative cells (green). (d-f) Representative confocal images of the stomach showing BChE + cells (red) negative for, but in close proximity to, Ki67 + cells. (g-i) Representative confocal images of the small intestine showing BChE + cells (red) that are negative for, but in close proximity to, Ki67 + cells. Upper right corner of (a-i) enlargement of the areas framed in each panel.
The present study was devised to examine BChE distribution in the mouse GI tract by immunohistochemistry and double-label confocal microscopy. To validate the antibody, we investigated BChE presence and distribution in the GI tract and its liver expression in mice fed an HFD (Chen et al., 2017). By immunohistochemistry,  et al., 1996); accordingly, we also detected BChE + cells close to Ki67 + cells in other stem cell niches, such as the isthmus of the stomach and the intestinal crypts (Barker et al., 2010;Sato et al., 2011). Since in the gut endogenous ACh released from the intestinal epithelium is involved in cell renewal via nicotinic receptors in Paneth cells (Takahashi et al., 2018), the presence of BChE in these cells could indicate its involvement in the renewal processes of intestinal mucosal cells.
Our experiments also revealed a spatial relationship between ghrelin-producing cells and BChE + parietal cells in the oxyntic mucosa of the stomach. The role of BChE in hydrolysing the hunger hormone ghrelin (Chen et al., 2017;De Vriese et al., 2004)  documented in this study, and its presence in "the major organs of entry", such as lung, liver and serum (Jbilo et al., 1994), suggest a role for it as a detoxifier, acting as a first-line barrier against compounds introduced with the diet. The notion is consistent with the hypothesis that it acts as a detoxification enzyme, which Ziegler (Ziegler, 1991) based on three criteria: the presence of BChE in the main organs of entry, its broad substrate range and its different concentrations in different species in relation to their diets (Johnson & Moore, 2012). Notably, the large acyl pocket of BChE accommodates a wide range of substrates like traditional organophosphates and carbamates as well as compounds such as cocaine (Xie et al., 1999) and heroin (Qiao et al., 2014). Interestingly, numerous BChE substrates derive from natural plant metabolism and are used by the plants themselves as protection against herbivores (Johnson & Moore, 2012).
Lastly, foods are natural sources of substances that may exert crucial effects on the nervous system, such as neurotransmitters (Briguglio et al., 2018). In particular, ACh is found in ingested meat as well as in more than 50 plant species belonging to all the major systematic groups, whilst Lactobacillus species in the gut microbiota have been demonstrated to produce ACh (Lyte, 2011;Smallman & Maneckjee, 1981). Therefore, BChE might act as a detoxifier against ACh released into the gastric and intestinal lumina in response to food ingestion. Accordingly, BChE released from gastric and intestinal secretory cells by exocrine processes or from oesophageal endothelial cells would be able to exert a direct action on the concentration of this powerful molecule in the GI lumen.
Altogether, the ubiquitous distribution of BChE along the mouse GI tract, emerging from our study, suggests that it is involved in a wide range of major physiological functions. These functions, which include bile formation and liver homeostasis, appetite and fat regulation, mucosal renewal processes and resistance to GI toxicity, warrant further detailed investigation with obesity and calorie restriction mouse models and in vitro culture systems that mimic the effects of BChE treatment as well as analysis of human samples from donors with normal and high and low body mass index.

AUTH O R CO NTR I B UTI O N S
IS designed the study, performed experiments, data analysis and interpretation and wrote the manuscript. SA, JP, EDM and MS performed experiments, data analysis and interpretation. AG designed the study, provided lab facilities and critically revised the manuscript.