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2'-Fucosyllactose (2'-FL) is one of the predominant oligosaccharides found in human milk and has several well-established beneficial effects in the host. It has previously been shown that 2'-FL can improve the metabolic phenotype in high-fat (HF)-fed mice. Here we investigated whether dietary supplementation with 2'-FL was associated with improved intestinal barrier integrity, signaling in the vagal afferent pathway and cognitive function. Mice were fed either a low-fat (LF, 10% fat per kcal) or HF (45% fat per kcal) diet with or without supplementation of 2'-FL (10% w/w) in the diet for 8 weeks. Body weight, energy intake, fat and lean mass, intestinal permeability ( ex vivo in Ussing chambers), lipid profiles, gut microbiome and microbial metabolites, and cognitive functions were measured. Vagal afferent activity was measured via immunohistochemical detection of c-Fos protein in the brainstem in response to peripheral administration of cholecystokinin (CCK). 2'-FL significantly attenuated the HF-induced increase in fat mass and energy intake. 2'-FL significantly reduced intestinal permeability and significantly increased expression of interleukin (IL)-22, a cytokine known for its protective role in the intestine. Additionally, 2'-FL led to changes in the gut microbiota composition and in the associated microbial metabolites. Signaling in the vagal afferent pathway was improved but there was no effect on cognitive function. In conclusion, 2'-FL supplementation improved the metabolic profiles, gut barrier integrity, lipid metabolism and signaling in the vagal afferent pathway. These findings support the utility of 2'-FL in the control of gut barrier function and metabolic homeostasis under a metabolic challenge.

A causal relationship between the pathophysiological changes in the gut epithelium and altered gut microbiota with the onset of obesity have been suggested but not defined. The aim of this study was to determine the temporal relationship between impaired intestinal barrier function and microbial dysbiosis in the small and large intestine in rodent high-fat (HF) diet-induced obesity. Rats were fed HF diet (45% fat) or normal chow (C, 10% fat) for 1, 3, or 6 wk; food intake, body weight, and adiposity were measured. Barrier function ex vivo using FITC-labeled dextran (4,000 Da, FD-4) and horseradish peroxidase (HRP) probes in Ussing chambers, gene expression, and gut microbial communities was assessed. After 1 wk, there was an immediate but reversible increase in paracellular permeability, decrease in IL-10 expression, and decrease in abundance of genera within the class Clostridia in the ileum. In the large intestine, HRP flux and abundance of genera within the order Bacteroidales increased with time on the HF diet and correlated with the onset of increased body weight and adiposity. The data show immediate insults in the ileum in response to ingestion of a HF diet, which were rapidly restored and preceded increased passage of large molecules across the large intestinal epithelium. This study provides an understanding of microbiota dysbiosis and gut pathophysiology in diet-induced obesity and has identified IL-10 and Oscillospira in the ileum and transcellular flux in the large intestine as potential early impairments in the gut that might lead to obesity and metabolic disorders., (Copyright © 2015 the American Physiological Society.)

Objectives: Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in selected bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of selected bifidobacteria on HMO stimulates the intestinal epithelium., Methods: Caco-2 and HT-29 cells were incubated with lactose (LAC)- or HMO-grown Bifidobacterium longum subsp infantis (B infantis) or B bifidum. Bacterial adhesion and translocation were measured by real-time quantitative polymerase chain reaction. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real-time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy., Results: We showed that HMO-grown B infantis had a significantly higher rate of adhesion to HT-29 cells compared with B bifidum. B infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand-1. Both B infantis and B bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin-10 compared with LAC-grown bacteria in Caco-2 cells. B bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cells and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation., Conclusions: The study provides evidence for the specific relation between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction.

The gut epithelium is a barrier between the 'outside' and 'inside' world. The major function of the epithelium is to absorb nutrients, ions and water, yet it must balance these functions with that of protecting the 'inside' world from potentially harmful toxins, irritants, bacteria and other pathogens that also exist in the gut lumen. The health of an individual depends upon the efficient digestion and absorption of all required nutrients from the diet. This requires sensing of meal components by gut enteroendocrine cells, activation of neural and humoral pathways to regulate gastrointestinal motor, secretory and absorptive functions, and also to regulate food intake and plasma levels of glucose. In this way, there is a balance between the delivery of food and the digestive and absorptive capacity of the intestine. Maintenance of the mucosal barrier likewise requires sensory detection of pathogens, toxins and irritants; breakdown of the epithelial barrier is associated with gut inflammation and may ultimately lead to inflammatory bowel disease. However, disruption of the barrier alone is not sufficient to cause frank inflammatory bowel disease. Several recent studies have provided compelling new evidence to suggest that changes in epithelial barrier function and inflammation are associated with and may even lead to altered regulation of body weight and glucose homeostasis. This article provides a brief review of some recent evidence to support the hypothesis that changes in the gut microbiota and alteration of gut epithelial function will perturb the homeostatic humoral and neural pathways controlling food intake and body weight.

Background: Lumenal glucose initiates changes in gastrointestinal (GI) function, including inhibition of gastric emptying, stimulation of pancreatic exocrine and endocrine secretion, and intestinal fluid secretion. Glucose stimulates the release of GI hormones and 5-hydroxytryptamine (5-HT), and activates intrinsic and extrinsic neuronal pathways to initiate changes in GI function. The precise mechanisms involved in luminal glucose-sensing are not clear; studying gut endocrine cells is difficult due to their sparse and irregular localization within the epithelium., Methods: Here we show a technique to determine activation of gut epithelial cells and the gut-brain pathway in vivo in rats using immunohistochemical detection of the activated, phosphorylated, form of calcium-calmodulin kinase II (pCaMKII)., Key Results: Perfusion of the gut with glucose (60 mg) increased pCaMKII immunoreactivity in 5-HT-expressing enterochromaffin (EC) cells, cytokeratin-18 immunopositive brush cells, but not in enterocytes or cholecystokinin-expressing cells. Lumenal glucose increased pCaMKII in neurons in the myenteric plexus and nodose ganglion, nucleus of the solitary tract, dorsal motor nucleus of the vagus and the arcuate nucleus. pCaMKII expression in neurons, but not in EC cells, was significantly attenuated by pretreatment with the 5-HT(3) R antagonist ondansetron. Deoxynojirimycin, a selective agonist for the putative glucose sensor, sodium-glucose cotransporter-3 (SGLT-3), mimicked the effects of glucose with increased pCaMKII in ECs and neurons; galactose had no effect., Conclusions & Inferences: The data suggest that native EC cells in situ respond to glucose, possibly via SGLT-3, to activate intrinsic and extrinsic neurons and thereby regulate GI function., (© 2011 Blackwell Publishing Ltd.)

Background: Immune cells and inflammatory mediators are released from the gastrointestinal tract into the mesenteric lymph during sepsis causing distant organ dysfunction. Recently, it was demonstrated that macrophages in the gut wall are controlled by the vagus nerve, the so-called cholinergic anti-inflammatory pathway., Aim: This study aims to investigate whether an enteral diet with lipid prevents the activation of leukocytes in the gut wall., Methods: Mesenteric lymph was obtained from rats, receiving an enteral infusion of glucose or glucose + lipid before and after lipopolysaccharide (LPS) injection. Immune cells in mesenteric lymph were analyzed with fluorescence-activated cell sorting before and after LPS injection. Mesenteric lymph leukocytes from rats receiving enteral glucose with or without lipid were stimulated in vitro with LPS and tumor necrosis factor (TNF)α was measured in the supernatant., Results: The release of macrophages from the gut during sepsis was not significantly different in animals enterally treated with glucose or lipid. However, the release of TNFα from mesenteric lymph leukocytes after in vitro LPS stimulation was more than 3-fold higher in the glucose group compared to the lipid-treated group., Conclusions: During sepsis, activated macrophages are released from the gut into mesenteric lymph. However, an enteral diet with lipid is able to suppress the inflammatory cytokine release from mesenteric lymph leukocytes.

Background & Aims: Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells., Methods: We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice., Results: Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice., Conclusions: Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The presence of glucose in the intestinal lumen elicits a number of changes in gastrointestinal function, including inhibition of gastric emptying and food intake and stimulation of pancreatic and intestinal secretion. The present study tested the hypothesis that Na(+)-glucose cotransporter (SGLT)-3, a member of the SGLT family of transport proteins, is involved in detection of luminal glucose in the intestine. Gastric emptying, measured in awake rats, was significantly inhibited by perfusion of the intestine with glucose (60 and 90 mg); this effect was mimicked by alpha-methyl glucose (nonmetabolizable substrate of SGLT-1 and -3) but not 2-deoxy-d-glucose (substrate for GLUT-2) or isoosmotic mannitol. Gastric motility and intestinal fluid secretion, measured in anesthetised rats, were significantly inhibited and stimulated, respectively, by duodenal glucose but not galactose, which has a much lower affinity for SGLT-3 than glucose. Duodenal glucose but not galactose stimulated the release of 5-HT into mesenteric lymph and stimulated the discharge of duodenal vagal afferent fibers. mRNA for SGLT-3 was identified in the duodenal mucosa. Together these data suggest that detection of glucose in the intestine may involve SGLT-3, possibly expressed by enterochromaffin cells in the intestinal mucosa, and release of 5-HT.

Background & Aims: Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs., Methods: Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder., Results: Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment., Conclusions: Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.

It is well recognized that lipid in the intestine is a potent inhibitor of gastric secretomotor function. Progress has been made in the identification of the "sensor" for lipid in the intestinal wall. Long-chain free fatty acids are the stimulus both for release of CCK and for the production of functional effects. Long-chain triglyceride requires chylomicron formation for absorption, and there is strong evidence that the postabsorptive products of long-chain triglyceride absorption, including chylomicrons and apolipoproteins, are involved in sensory transduction in the intestinal wall.

Results from functional studies point to the importance of chemoreceptive endings in the duodenum innervated by vagal afferents in the regulation of gastrointestinal functions such as gastric emptying and acid secretion, as well as in the process of satiation. In order to visualize the vagal sensory innervation of this gut segment, vagal afferents were selectively labeled in vivo by injecting the lipophilic carbocyanine dye DiI into either the left or the right nodose ganglion of young adult rats. Thick cryostat sections or whole-mounted peels of muscularis externa or submucosa of formalin-fixed tissue were analyzed with conventional and/or confocal microscopy. In the mucosa, many DiI-labeled vagal afferent fibers were found with terminal arborizations mainly between the crypts and the villous lamina propria. In both areas, vagal terminal branches came in close contact with the basal lamina, but did not appear to penetrate it so as to make direct contact with epithelial cells. Labeled vagal afferent fibers in the villous and cryptic lamina propria were found to be in intimate anatomical contact with fibrocyte-like cells that may belong to the class of interstitial cells of Cajal, and with small granular cells that might be granulocytes or histiocytes. Although our analysis was not quantitative, and considering that labeling was unilateral and not complete, it appears that the overall density of vagal afferent mucosal innervation was variable; many villi showed no evidence for innervation while other areas had quite dense networks of arborizing terminal fibers in several neighboring villi. Analysis of separate whole-mounted muscularis externa and submucosa peels revealed the presence of large bundles of labeled afferent fibers running within the myenteric plexus along the mesenteric attachment primarily in an aboral direction, with individual fibers turning towards the antimesenteric pole, and either penetrating into the submucosa or forming the characteristic intraganglionic laminar endings (IGLEs). Although the possibility of individual fibers issuing collaterals to myenteric IGLEs and at the same time to mucosal terminals was not demonstrated, it cannot be ruled out. These anatomical findings are discussed in the context of absorptive mechanisms for the different macronutrients and the implication of enteroendocrine cells such as CCK-containing cells that may function as intestinal "taste cells".

The mechanism by which lipid in the duodenum inhibits gastric emptying was investigated in awake rats fitted with chronic gastric and duodenal cannulas. Perfusion of the duodenum with lipid (Intralipid, 5 and 10%; total amount 50 and 100 mg) caused a significant inhibition (26 and 78%, respectively) of gastric emptying of a nonnutrient liquid (0.9% saline). Functional ablation of the capsaicin-sensitive vagal, but not the spinal, sensory innervation to the upper gastrointestinal tract significantly attenuated by 57% lipid-induced inhibition of gastric emptying. In intact rats, administration of a specific cholecystokinin (CCK)-A receptor antagonist, devazepide, significantly attenuated by 66% the response to lipid. Administration of devazepide in perivagal capsaicin-treated rats did not further reduce the response to lipid. These results suggest that lipid in the duodenum inhibits gastric emptying via a mechanism involving an action of CCK at type A receptors and capsaicin-sensitive vagal afferents.

The role of the vagal and spinal afferent innervation in the inhibition of gastric emptying induced by duodenal perfusion with a disaccharide (maltose 300 mM) was investigated in awake rats fitted with gastric and duodenal cannulas. Perfusion of the duodenum with maltose inhibited gastric emptying by 44%. Maltose-induced inhibition of gastric emptying was reversed by 46% and 100% by functional ablation of the vagal or spinal capsaicin-sensitive afferent innervation, respectively. Pretreatment with the specific CCK 'A' receptor antagonist, MK329, completely abolished the effects of maltose on gastric emptying. These results suggest that disaccharides inhibit gastric emptying via activation of capsaicin-sensitive afferents in the duodenal mucosa via a mechanism involving an action of CCK at type 'A' receptors, possibly located on afferent fibers.