Guillaume de Lartigue

Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function

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.

Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance.

Cocaine- and Amphetamine-Regulated Transcript: Stimulation of Expression in Rat Vagal Afferent Neurons by Cholecystokinin and Suppression by Ghrelin

The neuropeptide transmitter cocaine- and amphetamine-regulated transcript (CART) inhibits food intake and is expressed by both vagal afferent and hypothalamic neurons. Here we report that cholecystokinin (CCK) regulates CART expression in rat vagal afferent neurons. Thus, CART was virtually undetectable after energy restriction for 24 h, but administration of CCK to fasted rats increased CART immunoreactivity, and refeeding of fasted animals promptly increased CART by a mechanism sensitive to a CCK-1 receptor antagonist. In vagal afferent neurons incubated in serum-free medium, CART was virtually undetectable, whereas the orexigenic peptide melanin-concentrating hormone (MCH) was readily detected. The addition of CCK rapidly induced CART expression and downregulated MCH. Using a CART promoter–luciferase reporter vector transfected into cultured vagal afferent neurons, we showed that CCK stimulation of CART transcription was mediated by activation of protein kinase C and cAMP response element-binding protein (CREB). The action of CCK on CART expression was inhibited by the orexigenic peptide ghrelin, through a mechanism that involved exclusion of phosphorylated CREB from the nucleus. Thus, CCK reciprocally regulates expression of CART and MCH within the same vagal afferent neuron; ghrelin inhibits the effect of CCK at least in part through control of the nuclear localization of phosphoCREB, revealing previously unsuspected modulation of gut–brain signals implicated in control of food intake.

Cholecystokinin Regulates Expression of Y2 Receptors in Vagal Afferent Neurons Serving the Stomach

The intestinal hormones CCK and PYY3–36 inhibit gastric emptying and food intake via vagal afferent neurons. Here we report that CCK regulates the expression of Y2R, at which PYY3–36 acts. In nodose ganglia from rats fasted up to 48 h, there was a fivefold decrease of Y2R mRNA compared with rats fed ad libitum; Y2R mRNA in fasted rats was increased by administration of CCK, and by refeeding through a mechanism sensitive to the CCK1R antagonist lorglumide. Antibodies to Y2R revealed expression in both neurons and satellite cells; most of the former (89 ± 4%) also expressed CCK1R. With fasting there was loss of Y2R immunoreactivity in CCK1R-expressing neurons many of which projected to the stomach, but not in satellite cells or neurons projecting to the ileum or proximal colon. Expression of a Y2R promoter-luciferase reporter (Y2R-luc) in cultured vagal afferent neurons was increased in response to CCK by 12.3 ± 0.1-fold and by phorbol ester (16.2 ± 0.4-fold); the response to both was abolished by the protein kinase C inhibitor Ro-32,0432. PYY3–36 stimulated CREB phosphorylation in rat nodose neurons after priming with CCK; in wild-type mice PYY3–36 increased Fos labeling in brainstem neurons but in mice null for CCK1R this response was abolished. Thus Y2R is expressed by functionally distinct subsets of nodose ganglion neurons projecting to the stomach and ileum/colon; in the former expression is dependent on stimulation by CCK, and there is evidence that PYY3–36 effects on vagal afferent neurons are CCK dependent.

Leptin resistance in vagal afferent neurons inhibits cholecystokinin signaling and satiation in diet induced obese rats

Background and Aims The gastrointestinal hormone cholecystokinin (CCK) plays an important role in regulating meal size and duration by activating CCK1 receptors on vagal afferent neurons (VAN). Leptin enhances CCK signaling in VAN via an early growth response 1 (EGR1) dependent pathway thereby increasing their sensitivity to CCK. In response to a chronic ingestion of a high fat diet, VAN develop leptin resistance and the satiating effects of CCK are reduced. We tested the hypothesis that leptin resistance in VAN is responsible for reducing CCK signaling and satiation. Results Lean Zucker rats sensitive to leptin signaling, significantly reduced their food intake following administration of CCK8S (0.22 nmol/kg, i.p.), while obese Zucker rats, insensitive to leptin, did not. CCK signaling in VAN of obese Zucker rats was reduced, preventing CCK-induced up-regulation of Y2 receptor and down-regulation of melanin concentrating hormone 1 receptor (MCH1R) and cannabinoid receptor (CB1). In VAN from diet-induced obese (DIO) Sprague Dawley rats, previously shown to become leptin resistant, we demonstrated that the reduction in EGR1 expression resulted in decreased sensitivity of VAN to CCK and reduced CCK-induced inhibition of food intake. The lowered sensitivity of VAN to CCK in DIO rats resulted in a decrease in Y2 expression and increased CB1 and MCH1R expression. These effects coincided with the onset of hyperphagia in DIO rats. Conclusions Leptin signaling in VAN is required for appropriate CCK signaling and satiation. In response to high fat feeding, the onset of leptin resistance reduces the sensitivity of VAN to CCK thus reducing the satiating effects of CCK.

EGR1 Is a Target for Cooperative Interactions between Cholecystokinin and Leptin, and Inhibition by Ghrelin, in Vagal Afferent Neurons

Food intake is regulated by signals from peripheral organs, but the way these are integrated remains uncertain. Cholecystokinin (CCK) from the intestine and leptin from adipocytes interact to inhibit food intake. Our aim was to examine the hypothesis that these interactions occur at the level of vagal afferent neurons via control of the immediate early gene EGR1. We now report that CCK stimulates redistribution to the nucleus of early growth response factor-1 (EGR1) in these neurons in vivo and in culture, and these effects are not dependent on EGR1 synthesis. Leptin stimulates EGR1 expression; leptin alone does not stimulate nuclear translocation, but it strongly potentiates the action of CCK. Ghrelin inhibits CCK-stimulated nuclear translocation of EGR1 and leptin-stimulated EGR1 expression. Expression of the gene encoding the satiety peptide cocaine- and amphetamine-regulated transcript (CARTp) is stimulated by CCK via an EGR1-dependent mechanism, and this is strongly potentiated by leptin. Leptin potentiated inhibition of food intake by endogenous CCK in the rat in conditions reflecting changes in EGR1 activation. The data indicate that by separately regulating EGR1 activation and synthesis, CCK and leptin interact cooperatively to define the capacity for satiety signaling by vagal afferent neurons; manipulation of these interactions may be therapeutically beneficial.

Vagal afferent neurons in high fat diet-induced obesity; intestinal microflora, gut inflammation and cholecystokinin.

The vagal afferent pathway is the major neural pathway by which information about ingested nutrients reaches the CNS and influences both GI function and feeding behavior. Vagal afferent neurons (VAN) express receptors for many of the regulatory peptides and molecules released from the intestinal wall, pancreas, and adipocytes that influence GI function, glucose homeostasis, and regulate food intake and body weight. As such, they play a critical role in both physiology and pathophysiology, such as obesity, where there is evidence that vagal afferent function is altered. This review will summarize recent findings on changes in vagal afferent function in response to ingestion of high fat diets and explore the hypothesis that changes in gut microbiota and integrity of the epithelium may not only be important in inducing these changes but may be the initial events that lead to dysregulation of food intake and body weight in response to high fat, high energy diets.

Deletion of leptin signaling in vagal afferent neurons results in hyperphagia and obesity.

The vagal afferent pathway senses hormones released from the gut in response to nutritional cues and relays these signals to the brain. We tested the hypothesis that leptin resistance in vagal afferent neurons (VAN) is responsible for the onset of hyperphagia by developing a novel conditional knockout mouse to delete leptin receptor selectively in sensory neurons (Nav1.8/LepRfl/fl mice). Chow fed Nav1.8/LepRfl/fl mice weighed significantly more and had increased adiposity compared with wildtype mice. Cumulative food intake, meal size, and meal duration in the dark phase were increased in Nav1.8/LepRfl/fl mice; energy expenditure was unaltered. Reduced satiation in Nav1.8/LepRfl/fl mice is in part due to reduced sensitivity of VAN to CCK and the subsequent loss of VAN plasticity. Crucially Nav1.8/LepRl/fl mice did not gain further weight in response to a high fat diet. We conclude that disruption of leptin signaling in VAN is sufficient and necessary to promote hyperphagia and obesity.

Cocaine- and Amphetamine-Regulated Transcript Mediates the Actions of Cholecystokinin on Rat Vagal Afferent Neurons

BACKGROUND & AIMS Cholecystokinin (CCK) acts on vagal afferent neurons to inhibit food intake and gastric emptying; it also increases expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART), but the significance of this is unknown. We investigated the role of CARTp in vagal afferent neurons. METHODS Release of CART peptide (CARTp) from cultured vagal afferent neurons was determined by enzyme-linked immunosorbent assay. Expression of receptors and neuropeptides in rat vagal afferent neurons in response to CARTp was studied using immunohistochemistry and luciferase promoter reporter constructs. Effects of CARTp and CCK were studied on food intake. RESULTS CCK stimulated CARTp release from cultured nodose neurons. CARTp replicated the effect of CCK in stimulating expression of Y2R and of CART itself in these neurons in vivo and in vitro, but not in inhibiting cannabinoid-1, melanin-concentrating hormone, and melanin-concentrating hormone-1 receptor expression. Effects of CCK on Y2R and CART expression were reduced by CART small interfering RNA or brefeldin A. Exposure of rats to CARTp increased the inhibitory action of CCK on food intake after short-, but not long-duration, fasting. CONCLUSIONS The actions of CCK in stimulating CART and Y2R expression in vagal afferent neurons and in inhibiting food intake are augmented by CARTp; CARTp is released by CCK from these neurons, indicating that it acts as an autocrine excitatory mediator.

Chronic exposure to low dose bacterial lipopolysaccharide inhibits leptin signaling in vagal afferent neurons.

Bacterially derived factors are implicated in the causation and persistence of obesity. Ingestion of a high fat diet in rodents and obesity in human subjects is associated with chronic elevation of low plasma levels of lipopolysaccharide (LPS), a breakdown product of Gram-negative bacteria. The terminals of vagal afferent neurons are positioned within the gut mucosa to convey information from the gut to the brain to regulate food intake and are responsive to LPS. We hypothesized that chronic elevation of LPS could alter vagal afferent signaling. We surgically implanted osmotic mini-pumps that delivered a constant, low-dose of LPS into the intraperitoneal cavity of rats (12.5 μg/kg/hr for 6 weeks). LPS-treated rats developed hyperphagia and showed marked changes in vagal afferent neuron function. Chronic LPS treatment reduced vagal afferent leptin signaling, characterized by a decrease in leptin-induced STAT3 phosphorylation. In addition, LPS treatment decreased cholecystokinin-induced satiety. There was no alteration in leptin signaling in the hypothalamus. These findings offer a mechanism by which a change in gut microflora can promote hyperphagia, possibly leading to obesity.