Effects of passage through the digestive tract on incretin secretion: Before and after birth.

AIMS/INTRODUCTION: It was reported that fetuses secrete endogenous incretin; however, the stimulants of fetal incretin secretion are not fully understood. To investigate the association between the passage of amniotic fluid through the intestinal tract and fetal secretion of incretin, we analyzed umbilical cord incretin levels of infants with duodenum atresia. MATERIALS AND METHODS: Infants born from July 2017 to July 2019 (infants with duodenum atresia and normal term or preterm infants) were enrolled. We measured and compared the concentrations of glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide/glucose-dependent insulinotropic polypeptide (GIP) in the umbilical vein and preprandial blood samples after birth. RESULTS: A total of 98 infants (47 term, 46 preterm and 5 with duodenum atresia) were included. In patients with duodenum atresia, umbilical vein GLP-1 and GIP levels were the same as those in normal infants. In postnatal samples, there were positive correlations between the amount of enteral feeding and preprandial serum concentrations of GLP-1 (r = 0.47) or GIP (r = 0.49). CONCLUSIONS: Our results show that enteral feeding is important for secretion of GLP-1 and GIP in postnatal infants, whereas the passage of amniotic fluid is not important for fetal secretion of GLP-1 and GIP. The effect of ingested material passing through the digestive tract on incretin secretion might change before and after birth. Other factors might stimulate secretion of GLP-1 and GIP during the fetal period.

Discovery of a novel chalcone derivative inhibiting CFTR chloride channel via AMPK activation and its anti-diarrheal application.

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl- channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 µM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl- secretion in T84 cells with an IC50 of ∼1.5 µM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl- secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl- channel activity via CaMKKß-AMPK pathways.

Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Bile acid diarrhea (BAD) is common with ileal resection, Crohn's disease, and diarrhea-predominant irritable bowel syndrome. Here, we demonstrate the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (R)-benzopyrimido-pyrrolo-oxazine-dione-27 (BPO-27) in reducing bile acid-induced fluid and electrolyte secretion in colon. Short-circuit current measurements in human T84 colonic epithelial cells and planar colonic enteroid cultures showed a robust secretory response following mucosal but not serosal addition of chenodeoxycholic acid (CDCA) or its taurine conjugate, which was fully blocked by CFTR inhibitors, including (R)-BPO-27. (R)-BPO-27 also fully blocked CDCA-induced secretory current in murine colon. CFTR activation by CDCA primarily involved Ca2+ signaling. In closed colonic loops in vivo, luminal CDCA produced a robust secretory response, which was reduced by ∼70% by (R)-BPO-27 or in CFTR-deficient mice. In a rat model of BAD produced by intracolonic infusion of CDCA, (R)-BPO-27 reduced the elevation in stool water content by >55%. These results implicate CFTR activation in the colon as a major prosecretory mechanism of CDCA, a bile acid implicated in BAD, and support the potential therapeutic efficacy of CFTR inhibition in bile acid-associated diarrheas.-Duan, T., Cil, O., Tse, C. M., Sarker, R., Lin, R., Donowitz, M., Verkman, A. S. Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Involvement of butyrate in electrogenic K+ secretion in rat rectal colon.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (ISC) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted ISC in a negative direction, but did not attenuate the activity of epithelial Na+ channels (ENaC). The application of bumetanide, a Na+-K+-2Cl- cotransporter inhibitor, to the basolateral side reduced the negative ISC shift induced by butyrate. The application of XE991, a KCNQ-type K+ channel inhibitor, to the apical side decreased the ISC shift induced by butyrate in a dose-dependent manner. The ISC shift was independent of HCO3- and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H+-coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H+-coupled monocarboxylate transporters, activates K+ secretion through KCNQ-type K+ channels on the apical membrane in rat rectal colon. KCNQ-type K+ channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.

Co-storage and release of insulin-like peptide-5, glucagon-like peptide-1 and peptideYY from murine and human colonic enteroendocrine cells.

OBJECTIVE: Insulin-like peptide-5 (INSL5) is an orexigenic gut hormone found in a subset of colonic and rectal enteroendocrine L-cells together with the anorexigenic hormones glucagon-like peptide-1 (GLP-1) and peptideYY (PYY). Unlike GLP-1 and PYY, INSL5 levels are elevated by calorie restriction, raising questions about how these hormones respond to different stimuli when they arise from the same cell type. The aim of the current study was to identify whether and how INSL5, GLP-1 and PYY are co-secreted or differentially secreted from colonic L-cells. METHODS: An inducible reporter mouse (Insl5-rtTA) was created to enable selective characterisation of Insl5-expressing cells. Expression profiling and Ca2+-dynamics were assessed using TET-reporter mice. Secretion of INSL5, PYY, and GLP-1 from murine and human colonic crypt cultures was quantified by tandem mass spectrometry. Vesicular co-localisation of the three hormones was analysed in 3D-SIM images of immunofluorescently-labelled murine colonic primary cultures and tissue sections. RESULTS: INSL5-producing cells expressed a range of G-protein coupled receptors previously identified in GLP-1 expressing L-cells, including Ffar1, Gpbar1, and Agtr1a. Pharmacological or physiological agonists for these receptors triggered Ca2+ transients in INSL5-producing cells and stimulated INSL5 secretion. INSL5 secretory responses strongly correlated with those of PYY and GLP-1 across a range of stimuli. The majority (>80%) of secretory vesicles co-labelled for INSL5, PYY and GLP-1. CONCLUSIONS: INSL5 is largely co-stored with PYY and GLP-1 and all three hormones are co-secreted when INSL5-positive cells are stimulated. Opposing hormonal profiles observed in vivo likely reflect differential stimulation of L-cells in the proximal and distal gut.

Intestinal secretion of indoxyl sulfate as a possible compensatory excretion pathway in chronic kidney disease.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that progressively accumulates in plasma during chronic kidney disease (CKD), and its accumulation is associated with the progression of CKD. This study examined the intestinal secretion of IS using in situ single-pass intestinal perfusion in a rat model of renal insufficiency, MRP2- and BCRP-overexpressing Sf9 membrane vesicles, and Caco-2 cell monolayers. An in situ single-pass perfusion study in CKD model rats demonstrated that a small amount of IS is secreted into intestinal lumen after iv administration of IS, and the clearance increased AUC-dependently. An excess amount of IS (3 mm) partially inhibited the MRP2- and BCRP-mediated uptake of specific fluorescent substrates, CDCF and Lucifer yellow, respectively, into the membrane vesicles, although IS was not taken up at a physiological concentration, 10 µm. In the Caco-2 cell monolayers, the IS transport was higher in the absorptive direction than in the secretory direction (p < 0.05). p-Aminohippuric acid (PAH) strongly inhibited IS transport in both directions (absorptive, p = 0.142; secretory, p < 0.01). Given that the blood IS levels are much higher than those in the intestinal lumen, it is possible that this unknown PAH-sensitive system contributes to the intestinal IS secretion. Although in situ inhibition study is needed to confirm that this unknown transporter mediates the in vivo intestinal secretion of IS, we speculate that this unknown active efflux system works as a compensatory excretion pathway for excess organic anions such as IS especially in end-stage renal disease.

Antidiarrheal activity of a novel sulfated polysaccharide from the red seaweed Gracilaria cervicornis.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of marine seaweeds as a source of natural compounds with medicinal purposes is increasing in Western countries in the last decades, becoming an important alternative in the traditional medicine of many developing countries, where diarrhea still remains a severe public health problem, with high rates of mortality and morbidity. Sulfated polysaccharides (PLS) extracted from red seaweeds can exhibit therapeutic effects for the treatment of gastrointestinal disorders. Thus, the pharmacological properties of the PLS from Gracilaria cervicornis, an endemic seaweed found in the Brazilian northeast coast, was evaluated as an alternative natural medication for diarrhea. AIM OF THE STUDY: This study aimed to evaluate the antidiarrheal activity of sulfated polysaccharides (PLS) extracted from the red seaweed G. cervicornis in Swiss mice pre-treated with castor oil or cholera toxin. MATERIALS AND METHODS: The seaweed Gracilaria cervicornis was collected at Flecheiras beach (city of Trairí, State of Ceará, Brazil) and the PLS was obtained through enzymatic extraction and administered in mice (25-30 g) before diarrhea induction with castor oil or cholera toxin. For the evaluation of the total number of fecal output and diarrheal feces, the animals were placed in cages lined with adsorbent material. The evaluation of intestinal fluid accumulation (enteropooling) on castor oil-induced diarrhea in mice occurred by dissecting the small intestine and measuring its volume. The determination of Na+/K+-ATPase activity was measured in the small intestine supernatants by colorimetry, using commercial biochemistry kits. The gastrointestinal motility was evaluated utilizing an activated charcoal as a food tracer. The intestinal fluid secretion and chloride ion concentration were evaluated in intestinal closed loops in mice with cholera toxin-induced secretory diarrhea. The binding ability of PLS with GM1 and/or cholera toxin was evaluated by an Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The G. cervicornis PLS showed antidiarrheal effects in both acute and secretory diarrhea, reducing the total number of fecal output, diarrheic stools, intestinal fluid accumulation, and increasing small intestine Na+/K+-ATPase activity on castor oil-induced diarrhea. However, the PLS did not affect gastrointestinal motility, indicating that this compound has a different action mechanism than loperamide. In secretory diarrhea, the PLS decreased intestinal fluid secretion and small intestine chloride excretion, binding with GM1 and/or cholera toxin and blocking their attachment to the enterocyte cell surface. CONCLUSIONS: In conclusion, PLS has a significant antidiarrheal effect in acute and secretory diarrhea. Further investigation is needed towards its use as a natural medicine to treat diarrhea.

Intestinal lymph as a readout of meal-induced GLP-1 release in an unrestrained rat model.

Intestinal lymph supposedly provides a readout for the secretion of intestinal peptides. We here assessed how mesenteric lymph duct (MLD) lymph levels of glucagon-like peptide (GLP-1), insulin, and metabolites [glucose and triglycerides (TG)] evolve after isocaloric high- and low-fat diet (HFD and LFD) meals and how they compare with hepatic portal vein (HPV) plasma levels. Moreover, we examined the effects of intraperitoneally administered GLP-1 (1 or 10 nmol/kg) on these parameters. At 20 min after the HFD meal onset, GLP-1 levels were higher in MLD lymph than in HPV plasma. No such difference occurred with the LFD meal. Intraperitoneal injections of 10 nmol/kg GLP-1 before meals enhanced the meal-induced increases in MLD lymph and HPV plasma GLP-1 levels except for the MLD lymph levels after the HFD meal. Intraperitoneal injection of 1 nmol/kg GLP-1 only increased HPV plasma GLP-1 levels at 60 min after the HFD meal. GLP-1 injections did not increase the MLD lymph or HPV plasma GLP-1 concentrations beyond the physiological range, suggesting that intraperitoneal GLP-1 injections can recapitulate the short-term effects of endogenous GLP-1. Dipeptidyl peptidase IV (DPP-IV) activity in MLD lymph was lower than in HPV plasma, which presumably contributed to the higher levels of GLP-1 in lymph than in plasma. Insulin and glucose showed similar profiles in MLD lymph and HPV plasma, whereas TG levels were higher in lymph than in plasma. These results indicate that intestinal lymph provides a sensitive readout of intestinal peptide release and potential action, in particular when fat-rich diets are consumed.

The downregulation of sweet taste receptor signaling in enteroendocrine L-cells mediates 3-deoxyglucosone-induced attenuation of high glucose-stimulated GLP-1 secretion.

CONTEXT: Sweet taste receptors (STRs) involve in regulating the release of glucose-stimulated glucagon-like peptide-1 (GLP-1). Our in vivo and in vitro studies found that 3-deoxyglucosone (3DG) inhibited glucose-stimulated GLP-1 secretion. OBJECTIVE: This study investigated the role of STRs in 3DG-induced inhibition of high glucose-stimulated GLP-1 secretion. METHODS: STC-1 cells were incubated with lactisole or 3DG for 1 h under 25 mM glucose conditions. Western blotting was used to study the expression of STRs signaling molecules and ELISA was used to analyse GLP-1 and cyclic adenosine monophosphate (cAMP) levels. RESULTS: Lactisole inhibited GLP-1 secretion. Exposure to 25 mM glucose increased the expressions of STRs subunits when compared with 5.6 mM glucose. 3DG decreased GLP-1 secretion and STRs subunits expressions, with affecting other components of STRs pathway, including the downregulation of transient receptor potential cation channel subfamily M member 5 (TRPM5) expression and the reduction of intracellular cAMP levels. CONCLUSION: 3DG attenuates high glucose-stimulated GLP-1 secretion by reducing STR subunit expression and downstream signaling components.

Effect of T-2 toxin-injected shrimp muscle extracts on mouse macrophage cells (RAW264.7).

Following intramuscular injections of 0.1 mL, 3 mg kg-1 BW-1(1/10 LD50) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t1/2) of T-2 in blood was 40.47 ± 0.24 min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471 ± 0.012 ng g-1 BW-1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70 ± 2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure > NaOH > trifluoroacetic acid > 0.02 M HCl > 0.2 M HCl > controls), and also artificial digestion (artificial intestinal juice > artificial gastric juice > N type intestinal juice > N type gastric liquid > controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis.

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.

cAMP-dependent secretagogues stimulate the NaHCO3 cotransporter in the villous epithelium of the brushtail possum, Trichosurus vulpecula.

In the ileum of the brushtail possum, Trichosurus vulpecula, fluid secretion appears to be driven by electrogenic HCO3- secretion. Consistent with this, the cystic fibrosis transmembrane conductance regulator is expressed in the apical membrane of the ileal epithelial cells and the pancreatic or secretory variant of the NaHCO3 cotransporter in the basolateral membrane. This suggests that in the possum ileum, electrogenic HCO3- secretion is driven by basolateral NaHCO3 cotransporter (NBC) activity. To determine if the NBC contributes to HCO3- secretion in the possum ileum, intracellular pH (pHi) measurements in isolated villi were used to demonstrate NBC activity in the ileal epithelial cells and investigate the effect of cAMP-dependent secretagogues. In CO2/HCO3--free solutions, recovery of the epithelial cells from an acid load was Na+-dependent and ≈80% inhibited by ethyl-isopropyl-amiloride (EIPA, 10 µmol L-1), indicative of the presence of an Na+/H+ exchanger, most likely NHE1. However, in the presence of CO2/HCO3-, EIPA only inhibited ≈ 50% of the recovery, the remainder was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 500 µmol L-1), indicative of NBC activity. Under steady-state conditions, NHE1 inhibition by EIPA had little effect on pHi in the presence or absence of secretagogues, but NBC inhibition with DIDS resulted in a rapid acidification of the cells, which was increased fivefold by secretagogues. These data demonstrate the functional activity of an NaHCO3 cotransporter in the ileal epithelial cells. Furthermore, the stimulation of NBC activity by secretagogues is consistent with the involvement of an NaHCO3 cotransporter in electrogenic HCO3- secretion.

Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells.

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing.NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

Transintestinal and Biliary Cholesterol Secretion Both Contribute to Macrophage Reverse Cholesterol Transport in Rats-Brief Report.

OBJECTIVE: Reverse cholesterol transport comprises efflux of cholesterol from macrophages and its subsequent removal from the body with the feces and thereby protects against formation of atherosclerotic plaques. Because of lack of suitable animal models that allow for evaluation of the respective contributions of biliary cholesterol secretion and transintestinal cholesterol excretion (TICE) to macrophage reverse cholesterol transport under physiological conditions, the relative importance of both pathways in this process has remained controversial. APPROACH AND RESULTS: To separate cholesterol traffic via the biliary route from TICE, bile flow was mutually diverted between rats, continuously, for 3 days. Groups of 2 weight-matched rats were designated as a pair, and both rats were equipped with cannulas in the bile duct and duodenum. Bile from rat 1 was diverted to the duodenum of rat 2, whereas bile from rat 2 was rerouted to the duodenum of rat 1. Next, rat 1 was injected with [3H]cholesterol-loaded macrophages. [3H]Cholesterol secreted via the biliary route was consequently diverted to rat 2 and could thus be quantified from the feces of that rat. On the other hand, [3H]cholesterol tracer in the feces of rat 1 reflected macrophage-derived cholesterol excreted via TICE. Using this setup, we found that 63% of the label secreted with the fecal neutral sterols had travelled via the biliary route, whereas 37% was excreted via TICE. CONCLUSIONS: TICE and biliary cholesterol secretion contribute to macrophage reverse cholesterol transport in rats. The majority of macrophage-derived cholesterol is however excreted via the hepatobiliary route.

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.

Beneficial effects of mucous fistula refeeding in necrotizing enterocolitis neonates with enterostomies.

BACKGROUND: Necrotizing enterocolitis in premature neonates often results in bowel resection and stoma formation. One way to promote bowel adaptation before stoma closure is to introduce proximal loop effluents into the mucous fistula. In this study, we reviewed our experience with distal loop refeeding with respect to control group. METHODS: All patients with necrotizing enterocolitis between 2000 and 2014 necessitating initial diverting enterostomies and subsequent stoma closure in a tertiary referral center were included. Medical records were retrospectively reviewed. Demographic data, surgical procedures, and postoperative outcomes were analyzed. RESULTS: 92 patients were identified, with 77 patients receiving mucous fistula refeeding. The refeeding group showed less bowel ends size discrepancy (25 vs 53%, p=0.034) and less postoperative anastomotic leakage (3 vs 20%, p=0.029). Fewer refeeding group patients developed parenteral nutrition related cholestasis (42 vs 73%, p=0.045) and required shorter parenteral nutrition support (47 vs 135days, p=0.002). The mean peak bilirubin level was higher in the non-refeeding group (155 vs 275µmol/L, p<0.001). No major complication was associated with refeeding. CONCLUSIONS: Mucous fistula refeeding is safe and can decrease risk of anastomotic complication and parental nutrition related cholestasis. It provides both diagnostic and therapeutic value preoperatively and its use should be advocated. Level III Treatment Study in a Case Control Manner.

A novel guluronate oligomer improves intestinal transit and survival in cystic fibrosis mice.

BACKGROUND: Cystic fibrosis (CF) patients experience intestinal complications characterized by the accumulation of thick viscous mucus. CF mice were utilized to determine if a novel guluronate oligomer, OligoG, may be a potential therapy in reducing intestinal mucus and subsequent CF-related intestinal manifestations. METHODS: Intestinal transit, intestinal histology, survival and growth were examined in wildtype and CF mice on regular water and OligoG. CONCLUSIONS: OligoG improves intestinal transit and survival in CF mice by reducing the accumulation of intestinal mucus. OligoG's ability to directly bind mucin, disrupt mucin interaction and/or sequester calcium allowing for mucin expansion may explain the decrease in mucus accumulation.

NEW RENIN-INHIBITORS--STABILITY AND ACTIVITY DETERMINATION. PART III.

A series of new four potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-4) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of α-chymotrypsin was determined. Compound 1 was unstable, compounds 2, 3 were stable, compound 4 was partly unstable, (liver and kidney homogenates, (α-chymotrypsin solution). Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-4). Compound 1, 2, 3 and 4 showed inhibitory activity (1.7 x 10(-6), 9.6 x 10(-7), 1.05 x 10(-9) and 1.31 x 10(-7)M, respectively).

Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 µM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 µM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.

Cephalic phase secretion of insulin and other enteropancreatic hormones in humans.

Enteropancreatic hormone secretion is thought to include a cephalic phase, but the evidence in humans is ambiguous. We studied vagally induced gut hormone responses with and without muscarinic blockade in 10 glucose-clamped healthy men (age: 24.5 ± 0.6 yr, means ± SE; body mass index: 24.0 ± 0.5 kg/m(2); HbA1c: 5.1 ± 0.1%/31.4 ± 0.5 mmol/mol). Cephalic activation was elicited by modified sham feeding (MSF, aka "chew and spit") with or without atropine (1 mg bolus 45 min before MSF + 80 ng·kg(-1)·min(-1) for 2 h). To mimic incipient prandial glucose excursions, glucose levels were clamped at 6 mmol/l on all days. The meal stimulus for the MSF consisted of an appetizing breakfast. Participants (9/10) also had a 6 mmol/l glucose clamp without MSF. Pancreatic polypeptide (PP) levels rose from 6.3 ± 1.1 to 19.9 ± 6.8 pmol/l (means ± SE) in response to MSF and atropine lowered basal PP levels and abolished the MSF response. Neither insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP), nor glucagon-like peptide-1 (GLP-1) levels changed in response to MSF or atropine. Glucagon and ghrelin levels were markedly attenuated by atropine prior to and during the clamp: at t = 105 min on the atropine (ATR) + clamp (CLA) + MSF compared with the saline (SAL) + CLA and SAL + CLA + MSF days; baseline-subtracted glucagon levels were -10.7 ± 1.1 vs. -4.0 ± 1.1 and -4.7 ± 1.9 pmol/l (means ± SE), P < 0.0001, respectively; corresponding baseline-subtracted ghrelin levels were 303 ± 36 vs. 39 ± 38 and 3.7 ± 21 pg/ml (means ± SE), P < 0.0001. Glucagon and ghrelin levels were unaffected by MSF. Despite adequate PP responses, a cephalic phase response was absent for insulin, glucagon, GLP-1, GIP, and ghrelin.