New Findings r What is the central question of this study?Oxidative stress may play a role in compromising intestinal epithelial barrier integrity in pigs subjected to heat stress, but it is unknown whether an increase of dietary antioxidants (selenium and vitamin E) could alleviate gut leakiness in heat-stressed pigs. r What is the main finding and its importance? Levels of dietary selenium (1.0 p.p.m.) and vitamin E (200 IU kg −1 ) greater than those usually recommended for pigs reduced intestinal leakiness caused by heat stress. This finding suggests that oxidative stress plays a role in compromising intestinal epithelial barrier integrity in heat-stressed pigs and also provides a nutritional strategy for mitigating these effects.Heat stress compromises the intestinal epithelial barrier integrity of mammals through mechanisms that may include oxidative stress. Our objective was to test whether dietary supplementation with antioxidants, selenium (Se) and vitamin E (VE), protects intestinal epithelial barrier integrity in heat-stressed pigs. Female growing pigs (n = 48) were randomly assigned to four diets containing from 0.2 p.p.m. Se and 17 IU kg −1 VE (control, National Research Council recommended) to 1.0 p.p.m. Se and 200 IU kg −1 VE for 14 days. Six pigs from each dietary treatment were then exposed to either thermoneutral (20°C) or heat-stress conditions (35°C 09.00-17.00 h and 28°C overnight) for 2 days. Transepithelial electrical resistance and fluorescein isothiocyanate-dextran (4 kDa; FD4) permeability were measured in isolated jejunum and ileum using Ussing chambers. Rectal temperature, respiratory rate and intestinal HSP70 mRNA abundance increased (all P < 0.001), and respiratory alkalosis occurred, suggesting that pigs were heat stressed. Heat stress also increased FD4 permeability and decreased transepithelial electrical resistance (both P < 0.01). These changes were associated with changes indicative of oxidative stress, a decreased glutathione peroxidase (GPX) activity and an increased glutathione disulfide (GSSG)-to-glutathione (GSH) ratio (both P < 0.05). With increasing dosage of Se and VE, GPX-2 mRNA (P = 0.003) and GPX activity (P = 0.049) F. Liu and others increased linearly, the GSSG:GSH ratio decreased linearly (P = 0.037), and the impacts of heat stress on intestinal barrier function were reduced (P < 0.05 for both transepithelial electrical resistance and FD4 permeability). In conclusion, in pigs an increase of dietary Se and VE mitigated the impacts of heat stress on intestinal barrier integrity, associated with a reduction in oxidative stress.

Enteroendocrine cells were historically classified by a letter code, each linked to a single hormone, deduced to be the only hormone produced by the cell. One type, the L cell, was recognised to store and secrete two products, peptide YY (PYY) and glucagon-related peptides. Many other exceptions to the one cell – one hormone classifications have been reported over the last 40 years or so, and yet the one hormone dogma has persisted. In the last 6 years, a plethora of data has appeared that makes the concept unviable. Here we describe the evidence that multiple hormone transcripts and their products reside in single cells and evidence that the hormones are often, but not always, processed into separate storage vesicles. It has become clear that most enteroendocrine cells contain multiple hormones. For example, most secretin cells contain 5-hydroxytryptamine (5-HT), and in mouse many of these also contain cholecystokinin (CCK). Furthermore, CCK cells also commonly store ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), neurotensin, and PYY. Several hormones, for example secretin and 5-HT, are in separate storage vesicles at a subcellular level. Hormone patterns can differ considerably between species. Another complication is that relative levels of expression vary substantially. This means that data are significantly influenced by the sensitivities of detection techniques. For example, a hormone that can be detected in storage vesicles by super-resolution microscopy may not be above threshold for detection by conventional fluorescence microscopy. New nomenclature for cell clusters with common attributes will need to be devised and old classifications abandoned.

Enterochromaffin cells were the first endocrine cells of the gastrointestinal tract to be chemically distinguished, almost 150 years ago. It is now known that the chromaffin reaction of these cells was due to their content of the reactive aromatic amine, 5-hydroxytryptamine (5-HT, also known as serotonin). They have commonly been thought to be a special class of gut endocrine cells (enteroendocrine cells) that are distinct from the enteroendocrine cells that contain peptide hormones. The study by Martin et al in the current issue of this journal reveals that the patterns of expression of nutrient receptors and transporters differ considerably between chromaffin cells of the mouse duodenum and colon. However, even within regions chromaffin cells differ; in the duodenum there are chromaffin cells that contain both secretin and 5-HT, cholecystokinin and 5-HT, and all three of secretin, cholecystokinin and 5-HT. Moreover, the ratios of these different cell types differ substantially between species. And, in terms of function, 5-HT has many roles, including in appetite, motility, fluid secretion, release of digestive enzymes and bone metabolism. The paper thus emphasizes the need to define the many different classes of enterochromaffin cells and relate this to their roles.

TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relative potencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 μM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 μM), and were greatly diminished in Trpa1−/− duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.

The majority of 5-HT (serotonin) in the body is contained in enteroendocrine cells of the gastrointestinal mucosa. From the time of their discovery over 80 years ago, the 5-HT-containing cells have been regarded as a class of cell that is distinct from enteroendocrine cells that contain peptide hormones. However, recent studies have cast doubt on the concept of there being distinct classes of enteroendocrine cells, each containing a single hormone or occasionally more than one hormone. Instead, data are rapidly accumulating that there are complex patterns of colocalisation of hormones that identify multiple subclasses of enteroendocrine cells. In the present work, multiple labelling immunohistochemistry is used to investigate patterns of colocalisation of 5-HT with enteric peptide hormones. Over 95 % of 5-HT cells in the duodenum also contained cholecystokinin and about 40 % of them also contained secretin. In the jejunum, about 75 % of 5-HT cells contained cholecystokinin but not secretin and 25 % contained 5-HT plus both cholecystokinin and secretin. Small proportions of 5-HT cells contained gastrin or somatostatin in the stomach, PYY or GLP-1 in the small intestine and GLP-1 or somatostatin in the large intestine. Rare or very rare 5-HT cells contained ghrelin (stomach), neurotensin (small and large intestines), somatostatin (small intestine) and PYY (in the large intestine). It is concluded that 5-HT-containing enteroendocrine cells are heterogeneous in their chemical coding and by implication in their functions.

Recent studies reveal complex patterns of hormone coexpression within enteroendocrine cells (EECs), contrary to the traditional view that gut hormones are expressed individually in EECs. Moreover, different hormones have been found in separate subcellular vesicles. However, detailed analysis of relative expression of multiple hormones has not been made. Subcellular studies have been confined to peptide hormones, and have not included the indolamine 5-hydroxytryptamine (5-HT) or the neuroendocrine protein chromogranin A (CgA). In the present work, coexpression of 5-HT, CgA, secretin, cholecystokinin (CCK), ghrelin, and glucagonlike peptide (GLP)-1 in mouse duodenum was quantified at a cellular and subcellular level by semiautomated cell counting and quantitative vesicle measurements. We investigated whether relative numbers of cells with colocalized hormones analyzed at a cell level matched the numbers revealed by examination of individual storage vesicles within cells. CgA and 5-HT were frequently expressed in EECs that contained combinations of GLP-1, ghrelin, secretin, and CCK. Separate subcellular stores of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 were identified. In some cases, high-resolution analysis revealed small numbers of immunoreactive vesicles in cells dominated by a different hormone. Thus the observed incidence of cells with colocalized hormones is greater when analyzed at a subcellular, compared with a cellular, level. Subcellular analysis also showed that relative numbers of vesicles differ considerably between cells. Thus separate packaging of hormones that are colocalized is a general feature of EECs, and EECs exhibit substantial heterogeneity, including the colocalization of hormones that were formerly thought to be in cells of different lineages.

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