Aspects Of Intestinal Physiology Research Articles

Effect of dietary l-glutamine supplementation on the intestinal physiology and growth during Solea senegalensis larval development

The maturation of the intestinal digestive and absorptive functions might limit the amount of absorbed nutrients to fulfil the high requirements of the fast-growing marine fish larva. Glutamine (Gln) has been described to improve intestinal epithelium functions, due to its involvement in energy metabolism and protein synthesis. The purpose of this study was to evaluate dietary 0.2% Gln supplementation on aspects of intestinal physiology, protein metabolism and growth-related genes expression in Senegalese sole larvae. Experiment was carried out between 12 and 33 days post hatching (DPH) and fish were divided into two experimental groups, one fed Artemia spp. (CTRL) and the other fed Artemia spp. supplemented with Gln (GLN). GLN diet had two times more Gln than the CTRL diet. Samples were collected at 15, 19, 26 and 33 DPH for biometry, histology, and digestive enzymes activity, and at 33 DPH for gene expression, protein metabolism and AA content determination. Growth was significantly higher for Senegalese sole fed GLN diet, supported by differences on protein metabolism and growth-related gene expression. Slight differences were observed between treatments regarding the intestinal physiology. Overall, GLN diet seems to be directed to enhance protein metabolism leading to higher larval growth.

Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells.

The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that β-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration.

Molecular profiling of enteric nervous system cell lineages.

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

Symbioflor2® Escherichia coli Genotypes Enhance Ileal and Colonic Gene Expression Associated with Mucosal Defense in Gnotobiotic Mice.

Symbioflor2® is a probiotic product composed of six Escherichia coli genotypes, which has a beneficial effect on irritable bowel syndrome. Our objective was to understand the individual impact of each of the six genotypes on the host, together with the combined impact of the six in the compound Symbioflor2®. Gnotobiotic mice were mono-associated with one of the six genotypes or associated with the compound product. Ileal and colonic gene expression profiling was carried out, and data were compared between the different groups of gnotobiotic mice, along with that obtained from conventional (CV) mice and mice colonized with the probiotic E. coli Nissle 1917. We show that Symbioflor2® genotypes induce intestinal transcriptional responses involved in defense and immune mechanisms. Using mice associated with Symbioflor2®, we reveal that the product elicits a balanced response from the host without any predominance of a single genotype. The Nissle strain and the six bacterial genotypes have different effects on the intestinal gene expression, suggesting that the impacts of these probiotics are not redundant. Our data show the effect of the Symbioflor2® genotypes at the molecular level in the digestive tract, which further highlights their beneficial action on several aspects of intestinal physiology.

Neutraceutical Targeting of the Bile Acid Receptor, Farnesoid X Receptor, for Intestinal Disease

INTRODUCTIONBile acids, classically known for their roles in fat digestion, are now recognised as important regulators of many aspects of intestinal physiology, including epithelial proliferation/apoptosis, transport and barrier function. Thus, the nuclear bile acid receptor, farnesoid X receptor (FXR), represents a promising therapeutic target for intestinal disorders, such as inflammatory bowel disease and colorectal cancer. Previous studies suggest dietary plant‐derived phytochemicals may modulate FXR activity.AIMSTo investigate the effects of a phytochemical, denoted KFS1, and a KFS1 rich plant extract, denoted as QE1, on FXR signalling in colonic epithelial cells.METHODST84 colonic epithelial cells, grown as polarised monolayers, were treated bilaterally with the FXR agonist, GW4064 (5 μM), KFS1 (1 – 100 μM) or QE1 (30 – 100 μg/ml) for 24 hr. Expression of FXR and FGF‐19, an index of FXR activation, were measured by qRT‐PCR, western blotting and ELISA. Transepithelial electrical resistance (TEER) was measured as an index of epithelial barrier function. Lactate dehydrogenase (LDH) release and PARP cleavage were used as measurements of necrosis and apoptosis, respectively. FXR activity was also studied using an FXR/luciferase reporter HepG2 cell line. Data are expressed as the mean ± SEM for a series of n experiments.RESULTSGW4064 (5 μM) induced a 719.8 ± 183.3 fold increase in FGF‐19 mRNA expression (n = 20; p<0.001) and a 10.4 ± 4.9 fold increase in FGF‐19 protein release (n = 5; p<0.01). KFS1 (5 – 100 μM) did not alter FGF‐19 protein expression and at higher concentrations (50 – 100 μM) reduced TEER. Similarly, GW4064 (5 μM) significantly induced luciferase activity in FXR HepG2 reporter cells by 1580.7 ± 195.7 counts per second (p<0.001; n = 3), whereas treatment with KFS1 (1 – 100 μM) was without effect. Lower concentrations of KFS1 (5 μM), which alone did not alter TEER, increased FXR mRNA and protein expression by 4.2 ± 0.4 (p<0.05; n = 4) and 1.5 ± 0.04 fold (p<0.05; n = 4), respectively. Moreover, pre‐treatment of T84 cells with KFS1 (1, 5, or 10 μM) for 1 hr prior to treatment with GW4064 (5 μM; 24 hr) potentiated GW4064‐induced FGF‐19 mRNA expression by 2.5 ± 0.8, 2.9 ± 0.2, and 4.6 ± 0.5 fold, respectively (p<0.05; n = 6) and FGF‐19 protein expression by 2.5 ± 0.4, 4.1 ± 0.5, and 4.6 ± 0.5 fold, respectively (p<0.05, p<0.001; n = 7). Combined treatment with KFS1 (1, 5, 10 μM) and GW4064 (5 μM) also significantly reduced TEER to 49.8 ± 7.4%, 50.6 ± 8.3%, and 40.6 ± 6.6% of control values, respectively (n = 7). TEER reductions in response to such co‐treatments were not associated with significant LDH release or PARP cleavage (n = 3). Finally, a methanolic extract of plant material known to be rich in KFS1, QE1 (100 μg/ml), increased GW4064‐induced FGF‐19 release in T84 cells by 2.1 ± 0.3 fold (n = 6; p<0.01).CONCLUSIONThese data demonstrate that KFS1, a common dietary phytochemical, induces FXR expression in colonic epithelial cells and prime the cells for agonist‐induced FXR activation. These findings suggest that foods, or food supplements, rich in such plant‐derived phytochemicals have potential for development as FXR‐targeted neutraceuticals.Support or Funding InformationStAR PhD Scholarship from RCSI & Science Foundation Ireland (SFI)

Gut organoids: mini-tissues in culture to study intestinal physiology and disease.

In vitro, cell cultures are essential tools in the study of intestinal function and disease. For the past few decades, monolayer cellular cultures, such as cancer cell lines or immortalized cell lines, have been widely applied in gastrointestinal research. Recently, the development of three-dimensional cultures known as organoids has permitted the growth of normal crypt-villus units that recapitulate many aspects of intestinal physiology. Organoid culturing has also been applied to study gastrointestinal diseases, intestinal-microbe interactions, and colorectal cancer. These models are amenable to CRISPR gene editing and drug treatments, including high-throughput small-molecule testing. Three-dimensional intestinal cultures have been transplanted into mice to develop versatile in vivo models of intestinal disease, particularly cancer. Limitations of currently available organoid models include cost and challenges in modeling nonepithelial intestinal cells, such as immune cells and the microbiota. Here, we describe the development of organoid models of intestinal biology and the applications of organoids for study of the pathophysiology of intestinal diseases and cancer.

Gut Microbial Influences on the Mammalian Intestinal Stem Cell Niche.

The mammalian intestinal epithelial stem cell (IESC) niche is comprised of diverse epithelial, immune, and stromal cells, which together respond to environmental changes within the lumen and exert coordinated regulation of IESC behavior. There is growing appreciation for the role of the gut microbiota in modulating intestinal proliferation and differentiation, as well as other aspects of intestinal physiology. In this review, we evaluate the diverse roles of known niche cells in responding to gut microbiota and supporting IESCs. Furthermore, we discuss the potential mechanisms by which microbiota may exert their influence on niche cells and possibly on IESCs directly. Finally, we present an overview of the benefits and limitations of available tools to study niche-microbe interactions and provide our recommendations regarding their use and standardization. The study of host-microbe interactions in the gut is a rapidly growing field, and the IESC niche is at the forefront of host-microbe activity to control nutrient absorption, endocrine signaling, energy homeostasis, immune response, and systemic health.

Cannabinoid Receptors in Regulating the GI Tract: Experimental Evidence and Therapeutic Relevance.

Cannabinoid receptors are fundamentally involved in all aspects of intestinal physiology, such as motility, secretion, and epithelial barrier function. They are part of a broader entity, the so-called endocannabinoid system which also includes their endocannabinoid ligands and the ligands' synthesizing/degrading enzymes. The system has a strong impact on the pathophysiology of the gastrointestinal tract and is believed to maintain homeostasis in the gut by controlling hypercontractility and by promoting regeneration after injury. For instance, genetic knockout of cannabinoid receptor 1 leads to inflammation and cancer of the intestines. Derivatives of Δ9-tetrahydrocannabinol, such as nabilone and dronabinol, activate cannabinoid receptors and have been introduced into the clinic to treat chemotherapy-induced emesis and loss of appetite; however, they may cause many psychotropic side effects. New drugs that interfere with endocannabinoid degradation to raise endocannabinoid levels circumvent this obstacle and could be used in the future to treat emesis, intestinal inflammation, and functional disorders associated with visceral hyperalgesia.

The bile acid receptor, TGR5, regulates basal and cholinergic‐induced secretory responses in rat colon

Bile acids (BA) are becoming increasingly appreciated as enteric hormones that regulate many aspects of intestinal physiology. The BA receptor, TGR5, has been recently shown to be expressed on enteric nerves and enterochromaffin cells (ECs), where its activation regulates small intestinal and colonic motility. Here, we show that TGR5 is also expressed on colonic epithelial cells and that its activation decreases basal secretory tone and inhibits cholinergic-induced secretory responses. Our data demonstrate a new role for TGR5 in regulating colonic fluid and electrolyte transport and suggest that the receptor represents a good therapeutic target for intestinal transport disorders.

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

Orazipone, a locally acting immunomodulator, ameliorates intestinal radiation injury: A preclinical study in a novel rat model

Intestinal radiation injury (radiation enteropathy) is relevant to cancer treatment, as well as to radiation accidents and radiation terrorism scenarios. This study assessed the protective efficacy of orazipone, a locally-acting small molecule immunomodulator. Male rats were orchiectomized, a 4-cm segment of small bowel was sutured to the inside of the scrotum, a proximal anteperistaltic ileostomy was created for intraluminal drug administration, and intestinal continuity was re-established by end-to-side anastomosis. After three weeks postoperative recovery, the intestine in the "scrotal hernia" was exposed locally to single-dose or fractionated X-radiation. Orazipone (30 mg/kg/day) or vehicle was administered daily through the ileostomy, either during and after irradiation, or only after irradiation. Structural, cellular, and molecular aspects of intestinal radiation toxicity were assessed two weeks after irradiation. Orazipone significantly ameliorated histologic injury and transforming growth factor-beta immunoreactivity levels, both after single-dose and fractionated irradiation. Intestinal wall thickness was significantly reduced after single-dose and nonsignificantly after fractionated irradiation. Mucosal surface area and numbers of mast cells were partially restored by orazipone after single-dose irradiation. This work (1) demonstrates the utility of the ileostomy rat model for intraluminal administration of response modifiers in single-dose and fractionated radiation studies; (2) shows that mucosal immunomodulation during and/or after irradiation ameliorates intestinal toxicity; and (3) highlights important differences between single-dose and fractionated radiation regimens.

Sugar‐dependent selective induction of mouse jejunal disaccharidase activities.

1. Sugar-containing diets chosen not to affect intestinal structure or enterocyte turnover have been fed to mice previously maintained on a low carbohydrate diet in order to determine their ability to induce disaccharidase enzymes in the small intestine. 2. Glucose-, fructose- and 3-O-methyl-glucose-containing diets increased sucrase and maltase but not lactase activities in mouse jejunal homogenates. These effects were either absent or negligible in more distal regions of the small intestine. 3. Placing mice on glucose-, fructose- or 3-O-methyl-glucose-containing diets was further shown, by quantitative cytochemistry, to cause a 1.6-, 2.6- and 3.2-fold increase in the initial rate at which alpha-glucosidase activity (sucrase + maltase) appeared in the brush-border membrane of developing enterocytes. 4. The time during which alpha-glucosidase activity increased in enterocyte brush-border membranes fell from 30 h for low carbohydrate fed mice to 21, 19 and 17 h in mice fed glucose, fructose and 3-O-methyl-glucose respectively. Change of diet had no effect on the kinetics of lactase expression by developing enterocytes. 5. Maximal alpha-glucosidase activity detected in enterocyte brush-border membranes is equal to RT, where R is the initial rate of enzyme appearance and T is the time during which this rate operates. The ability of sugars to increase R selectively, but only at the expense of T, defines unexpected limits to the capacity of enterocytes to adapt to changes in luminal nutrition. 6. The above results are discussed in relation to other aspects of enterocyte differentiation recently subjected to quantitative analysis. The need to standardize other aspects of intestinal physiology and redefine the energy content of diets containing non-metabolizable substrates in this type of work is also emphasized.