Long-chain fatty acid-induced intracellular signaling in GPR120-expressing brush cells at the limiting ridge of the murine stomach.

Brush cells at the gastric groove have been proposed to operate as sensory cells capable of sensing constituents of ingested food. Recent studies have indicated that these cells express GPR120 (also known as FFAR4), the G protein-coupled receptor for long-chain fatty acids (LCFAs). However, functional implications of this receptor in brush cells have remained elusive. Here, we show that a great proportion of brush cells express GPR120. We used phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) as a readout to monitor brush cell responses to the LCFAs oleic acid and α-linolenic acid. Our results demonstrate that ERK1/2 phosphorylation is increased upon exposure to both fatty acids. Increased ERK1/2 phosphorylation is accompanied by upregulated mRNA and protein levels of cyclooxygenase 2 (COX-2), a key enzyme for prostaglandin biosynthesis. Immunohistochemical experiments confirmed that oleic acid caused ERK1/2 phosphorylation and induced COX-2 expression in brush cells. Our results indicate that LCFA sensing elicits a signaling process in brush cells that may be relevant for a local regulation of gastric functions.

Gustatory sensory cells express a receptor responsive to protein breakdown products (GPR92).

The ingestion of dietary protein is of vital importance for the maintenance of fundamental physiological processes. The taste modality umami, with its prototype stimulus, glutamate, is considered to signal the protein content of food. Umami was thought to be mediated by the heterodimeric amino acid receptor, T1R1 + T1R3. Based on knockout studies, additional umami receptors are likely to exist. In addition to amino acids, certain peptides can also elicit and enhance umami taste suggesting that protein breakdown products may contribute to umami taste. The recently deorphanized peptone receptor, GPR92 (also named GPR93; LPAR5), is expressed in gastric enteroendocrine cells where it responds to protein hydrolysates. Therefore, it was of immediate interest to investigate if the receptor GPR92 is expressed in gustatory sensory cells. Using immunohistochemical approaches we found that a large population of cells in murine taste buds was labeled with an GPR92 antibody. A molecular phenotyping of GPR92 cells revealed that the vast majority of GPR92-immunoreactive cells express PLCß2 and can therefore be classified as type II cells. More detailed analyses have shown that GPR92 is expressed in the majority of T1R1-positive taste cells. These results indicate that umami cells may respond not only to amino acids but also to peptides in protein hydrolysates.

Short-term high fat feeding induces inflammatory responses of tuft cells and mucosal barrier cells in the murine stomach.

Feeding mice with a high fat diet (HFD) induces inflammation and results in changes of gene expression and cellular composition in various tissues throughout the body, including the gastrointestinal tract. In the stomach, tuft cells expressing the receptor GPR120 are capable of sensing saturated long chain fatty acids (LCFAs) and thus may be involved in initiating mechanisms of mucosal inflammation. In this study, we assessed which cell types may additionally be affected by high fat feeding and which candidate molecular mediators might contribute to mucosa-protective immune responses. A high fat dietary intervention for 3 weeks caused an expansion of tuft cells that was accompanied by a higher frequency of mucosal mast cells and surface mucous cells which are a known source of the insult-associated cytokine interleukin 33 (IL-33). Our data demonstrate that both brush and mucosal mast cells comprise the enzyme ALOX5 and its activating protein FLAP and thus have the capacity for synthesizing leukotriene (LT). In HFD mice, several tuft cells showed a perinuclear colocalization of ALOX5 with FLAP which is indicative of an active LT synthesis. Monitoring changes in the expression of genes encoding elements of LT synthesis and signaling revealed that transcript levels of the leukotriene C4 synthase, LTC4S, catalyzing the first step in the biosynthesis of cysteinyl (cys) LTs, and the cysLT receptors, cysLTR2 and cysLTR3, were upregulated in mice on HFD. These mice also showed an increased expression level of IL-33 receptors, the membrane-bound ST2L and soluble isoform sST2, as well as the mast cell-specific protease MCPT1. Based on these findings it is conceivable that upon sensing saturated LCFAs tuft cells may elicit inflammatory responses which result in the production of cysLTs and activation of surface mucous cells as well as mucosal mast cells regulating gastric mucosal function and integrity.

Candidate chemosensory cells in the porcine stomach.

A continuous chemosensory monitoring of the ingested food is of vital importance for adjusting digestive processes according to diet composition. Although any dysfunction of this surveillance system may be the cause of severe gastrointestinal disorders, information about the cellular and molecular basis of chemosensation in the gastrointestinal tract is limited. The porcine alimentary canal is considered as an appropriate model for the human gastrointestinal tract. Therefore, in this study we have investigated the gastric mucosa of swine for cells which express gustatory transduction elements such as TRPM5 or PLCß2, and thus may represent candidate "chemosensors". It was found that the porcine stomach indeed contains cells expressing gustatory marker molecules; however, the morphology and topographic distribution of putative chemosensory cells varied significantly from that in mice. Whereas in the murine stomach these cells were clustered at a distinct region near the gastric entrance, no such compact cell cluster was found in the pig stomach. These results indicate substantial differences regarding the phenotype of candidate chemosensory cells of mice and swine and underline the importance of choosing the most suitable model organisms.

Distinct Cell Types With the Bitter Receptor Tas2r126 in Different Compartments of the Stomach.

Cells expressing bitter taste receptors (T2Rs or Tas2rs) in extraoral tissues are considered to be chemosensory cells mediating protective responses to potentially harmful or even antiinflammatory or antimicrobial compounds. In a previous study the activity of the Tas2R143/Tas2R135/Tas2r126 cluster promoter in the stomach was monitored using a Cre-reporter mouse line. Reporter gene expression and Tas2r126 mRNA were found in brush cells located at the distal wall of the gastric groove. In this study, we explored whether brush cells and epithelial cells of the stomach in fact contain the Tas2r126 receptor protein. Using immunohistochemistry, we demonstrate the presence of Tas2r126 immunoreactivity in different cell populations in the glandular stomach, in a subset of brush cells at the gastric groove and in unique glandular units as well as in certain enteroendocrine cells. In brush cells at the gastric groove, a strong immunofluorescence signal for the Tas2r126 receptor was observed at the most apical region of the cells, i.e., the microvillar tuft. In addition, we found a high density of Tas2r126-positive brush cells in the unique glandular units. These invaginations are located distally to the groove, open directly into the furrow and are enwrapped by smoothelin-immunoreactive muscles. In the corpus, Tas2r126 immunoreactivity was found in histamine-producing ECL cells and in ghrelin-producing X/A-like cells, the main enteroendcrine cells of this compartment. In the antrum, Tas2r126 labeling was observed in serotonin-storing EC cells and ghrelin cells, both representing only minor populations of enteroendocrine cells in this compartment. In conclusion, our data provide evidence for the presence of the Tas2r126 receptor protein in distinct cell types in the epithelium lining the mouse stomach which render the stomach responsive to agonists for bitter receptors.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition.

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice.

High fat feeding affects the number of GPR120 cells and enteroendocrine cells in the mouse stomach.

Long-term intake of dietary fat is supposed to be associated with adaptive reactions of the organism and it is assumptive that this is particularly true for fat responsive epithelial cells in the mucosa of the gastrointestinal tract. Recent studies suggest that epithelial cells expressing the receptor for medium and long chain fatty acids, GPR120 (FFAR4), may operate as fat sensors. Changes in expression level and/or cell density are supposed to be accompanied with a consumption of high fat (HF) diet. To assess whether feeding a HF diet might impact on the expression of fatty acid receptors or the number of lipid sensing cells as well as enteroendocrine cell populations, gastric tissue samples of non-obese and obese mice were compared using a real time PCR and immunohistochemical approach. In this study, we have identified GPR120 cells in the corpus region of the mouse stomach which appeared to be brush cells. Monitoring the effect of HF diet on the expression of GPR120 revealed that after 3 weeks and 6 months the level of mRNA for GPR120 in the tissue was significantly increased which coincided with and probably reflected a significant increase in the number of GPR120 positive cells in the corpus region; in contrast, within the antrum region, the number of GPR120 cells decreased. Furthermore, dietary fat intake also led to changes in the number of enteroendocrine cells producing either ghrelin or gastrin. After 3 weeks and even more pronounced after 6 months the number of ghrelin cells and gastrin cells was significantly increased. These results imply that a HF diet leads to significant changes in the cellular repertoire of the stomach mucosa. Whether these changes are a consequence of the direct exposure to HF in the luminal content or a physiological response to the high level of fat in the body remains elusive.

Receptors for short-chain fatty acids in brush cells at the "gastric groove".

In the stomach of rodents clusters of brush cells are arranged at the "gastric groove," immediately at the transition zone from the non-glandular reservoir compartment to the glandular digestive compartment. Based on their taste cell-like molecular phenotype it has been speculated that the cells may be capable to sense constituents of the ingested food, however, searches for nutrient receptors have not been successful. In this study, it was hypothesized that the cells may express receptors for short-chain fatty acids, metabolites generated by microorganisms during the storage of ingested food in the murine forestomach, which lacks the acidic milieu of more posterior regions of the stomach and is colonized with numerous microbiota. Experimental approaches, including RT-PCR analysis and immunohistochemical studies, revealed that the majority of these brush cells express the G-protein coupled receptor types GPR41 (FFAR3) and GPR43 (FFAR2), which are activated by short-chain fatty acids. Both, the GPR41 receptor proteins as well as an appropriate G-protein, α-gustducin, were found to be segregated at the apical brush border of the cells, indicating a direct contact with the luminal content of this gastric region. The exposure of microvillar processes with appropriate receptors and signaling elements to the gastric lumen suggests that the brush cells may in fact be capable to sense the short-chain fatty acids which originate from fermentation processes during the retention of ingested food in the anterior part of the stomach.

Surgical technique of a vertical sleeve gastrectomy in mice.

BACKGROUND/AIMS: The prevalence of morbid obesity is increasing. In failing of conservative methods to weight reduction, one effective surgical option is the sleeve gastrectomy. Aim of this study was to show the feasibility of simplified surgical techniques of sleeve gastrectomy in normal weight mice as base model for surgery in super-obese mice. METHODS: In an animal study, 15 male C57/Bl6 mice were randomized into two groups of 5 (1) and 10 animals (2) to undergo sleeve gastrectomy with antidromic suture course. The sutures of the tubular stomach were performed from aborally to orally in group 1 and from orally to aborally in group 2. Mean body weight was 20.8 ± 0.6 g. Body weight was recorded daily for 14 days after surgery and weekly for further 10 weeks. RESULTS: In our study, 12 of 15 animals survived the procedure and follow-up period. Out of group 1, two mice died because of leakage of the gastric sleeve with diffuse peritonitis. Out of group 2, one animal died seven weeks after surgery due to an abscess in the abdominal wall. Regarding the weight charts, there was a decrease until the third postoperative day with continuous increase thereafter. CONCLUSION: The presented model of sleeve gastrectomy is feasible in mice with low mortality and tolerable morbidity. The simplified model enables short operation times, which is decisive especially in obese mice.

Band-like arrangement of taste-like sensory cells at the gastric groove: evidence for paracrine communication.

The discovery of taste-related elements within the gastrointestinal tract has led to a growing interest in the mechanisms and physiological significance of chemosensory monitoring of chymus composition. Previous work suggests that brush cells located in the "gastric groove," which parallels the "limiting ridge," a structure in rodents that divides the fundus from the corpus, are candidate sensory cells. A novel sectioning technique revealed that these cells are arranged in a palisade-like manner forming a band which borders the whole length of the corpus epithelium. Using transgenic PLCß2 promoter-GFP mice and specific antibodies, we have demonstrated that most of these cells express gustducin, PLCß2, and TRPM5; typical signaling proteins of gustatory sensory "type II" cells. These molecular features strongly suggest that the cells may be capable of sensing nutrient or non-nutrient constituents of the ingested food. Since there is no evidence that brush cells are endocrine cells, attempts were made to explore how such putative chemosensory cells might transmit the information to "effector" cells. It was found that most of the cells express the neuronal nitric oxide synthase (NOS) suggesting some paracrine interaction with adjacent cells. Moreover, they also express choline acetyltransferase (ChAT) as well as the vesicular protein SNAP25, indicating the potential for cholinergic transmission, possibly with subjacent enteric nerve fibers.

Putative interaction of brush cells with bicarbonate secreting cells in the proximal corpus mucosa.

The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the "gastric groove," the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII), the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na(+)/H(+) exchanger (NHE1). However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracrine interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1) and the neighboring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution.

Receptors responsive to protein breakdown products in g-cells and d-cells of mouse, swine and human.

Monitoring the luminal content in the stomach is of vital importance for adjusting the gastric activities, including the release of gastric hormones such as gastrin. Our previous studies have shown that in mice the gastrin-secreting G-cells express receptor types which are responsive to amino acids. Since the pig is considered as more suitable model for studying gastro-physiological aspects relevant for men, in this study we have analyzed the distribution of G-cells and D-cells in the gastric antrum of men, swine, and mouse and the expression of receptor types which may render these cells responsiveness to protein breakdown products. The results indicate that the number of G-cells per antral invagination was significantly higher in swine and human compared to mice and also the distribution pattern of G-cells differed between the species. The molecular phenotyping revealed that the receptors GPRC6A and CaSR were also expressed in G-cells and in a subpopulation of D-cells from swine and men. As an additional receptor type, the peptone-receptor GPR92, was found to be expressed in G-cells and a subpopulation of D-cells; this receptor type may be particular suitable for sensing protein breakdown products and thus be a key element to adjust the activity of G-cells and D-cells according to the progress of the digestive processes in the stomach. In search for elements of an intracellular signaling cascade it was found that G-cells express the G-protein subunit Gαq as well as the phospholipase C subtype PLCß3; in contrast, D-cells expressed the subtype PLCß2 and neither Gαq. These results indicate that there are significant species differences concerning the number and distribution pattern, but not concerning the molecular phenotype of the gastric endocrine cells. However, G-cells and D-cells significantly differ from each other regarding the repertoire of receptors and signaling elements.

Expression of Tas1 taste receptors in mammalian spermatozoa: functional role of Tas1r1 in regulating basal Ca²âº and cAMP concentrations in spermatozoa.

BACKGROUND: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. METHODOLOGY/PRINCIPAL FINDINGS: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca²âº ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca²âº levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. CONCLUSIONS/SIGNIFICANCE: Since Ca²âº and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract.

Nutrient sensing receptors in gastric endocrine cells.

Sensing protein breakdown products in the luminal content is of particular importance for the regulation of digestive activities in the stomach which are mainly governed by gastric hormones. The molecular basis for tuning the release of hormones according to the protein content is still elusive. In this study we have analysed the murine stomach for candidate nutrient receptors. As a promising candidate we have concentrated on the broadly tuned amino acid receptor GPRC6A. Expression of GPRC6A could be demonstrated in different regions of the murine stomach; especially in the gastric antrum. Using immunohistochemical approaches, a large cell population of GPRC6A-positive cells was visualized in the basal half of the antral gastric mucosa. Molecular phenotyping of GPRC6A-immunoreactive cells revealed that most of them contained the peptide hormone gastrin. A small population turned out to be immunoreactive for somatostatin. In search for additional amino acid receptors in antral gastric mucosa, we obtained evidence for expression of the gustatory amino acid receptor subunit T1R3 and the calcium-sensing receptor CaSR. Many CaSR-cells were found in the gastric antrum and most of them also contained gastrin; very similar to GPRC6A-cells. In contrast, T1R3 was found only in a small population of gastrin-negative cells. The finding that GPRC6A-and CaSR-receptors are both expressed in many if not all gastrin cells strongly suggests that both receptor types are co-expressed in the same cells, where they could form heterodimers providing a unique response spectrum of these cells.

Expression of the G-protein alpha-subunit gustducin in mammalian spermatozoa.

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit alpha-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether alpha-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-alpha-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the alpha-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether alpha-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for alpha-gustducin. Although a possible function for alpha-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein alpha-subunit may process intracellular signals controlling sperm motility.

MHC class I peptides as chemosensory signals in the vomeronasal organ.

The mammalian vomeronasal organ detects social information about gender, status, and individuality. The molecular cues carrying this information remain largely unknown. Here, we show that small peptides that serve as ligands for major histocompatibility complex (MHC) class I molecules function also as sensory stimuli for a subset of vomeronasal sensory neurons located in the basal Gao- and V2R receptor-expressing zone of the vomeronasal epithelium. In behaving mice, the same peptides function as individuality signals underlying mate recognition in the context of pregnancy block. MHC peptides constitute a previously unknown family of chemosensory stimuli by which MHC genotypic diversity can influence social behavior.