Naouel Tennoune

Gut Commensal E. coli Proteins Activate Host Satiety Pathways following Nutrient-Induced Bacterial Growth

The composition of gut microbiota has been associated with host metabolic phenotypes, but it is not known if gut bacteria may influence host appetite. Here we show that regular nutrient provision stabilizes exponential growth of E.xa0coli, with the stationary phase occurring 20xa0min after nutrient supply accompanied by bacterial proteome changes, suggesting involvement of bacterial proteins in host satiety. Indeed, intestinal infusions of E.xa0coli stationary phase proteins increased plasma PYY and their intraperitoneal injections suppressed acutely food intake and activated c-Fos in hypothalamic POMC neurons, while their repeated administrations reduced meal size. ClpB, a bacterial protein mimetic of α-MSH, was upregulated in the E.xa0coli stationary phase, was detected in plasma proportional to ClpB DNA in feces, and stimulated firing rate of hypothalamic POMC neurons. Thus, these data show that bacterialxa0proteins produced after nutrient-induced E.xa0coli growth may signal meal termination. Furthermore, continuous exposure to E.xa0coli proteins may influence long-term meal pattern.

Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide α-MSH, at the origin of eating disorders

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with α-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of α-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with α-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and α-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with α-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for α-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with α-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs.

Anti-ghrelin immunoglobulins modulate ghrelin stability and its orexigenic effect in obese mice and humans.

Obese individuals often have increased appetite despite normal plasma levels of the main orexigenic hormone ghrelin. Here we show that ghrelin degradation in the plasma is inhibited by ghrelin-reactive IgG immunoglobulins, which display increased binding affinity to ghrelin in obese patients and mice. Co-administration of ghrelin together with IgG from obese individuals, but not with IgG from anorectic or control patients, increases food intake in rats. Similarly, chronic injections of ghrelin together with IgG from ob/ob mice increase food intake, meal frequency and total lean body mass of mice. These data reveal that in both obese humans and mice, IgG with increased affinity for ghrelin enhances ghrelin’s orexigenic effect, which may contribute to increased appetite and overeating.

Glutamine supplementation, but not combined glutamine and arginine supplementation, improves gut barrier function during chemotherapy-induced intestinal mucositis in rats

BACKGROUND & AIMSnIncreased intestinal permeability occurs during chemotherapy-induced intestinal mucositis. Previous data suggest that glutamine and arginine may have additive or synergic effects to limit intestinal damage. The present study aimed to evaluate the effects of glutamine and arginine, each alone or in combination, on gut barrier function during methotrexate (MTX)-induced mucositis in rats.nnnMETHODSnEighty Sprague Dawley rats received during 7 days (d) standard chow supplemented with protein powder (PP), glutamine (G, 2%), arginine (A, 1.2%) or glutamine plus arginine (GA). All diets were isonitrogenous. Rats received subcutaneous injections of MTX (2.5 mg/kg) from d0 to d2. The intestinal permeability and tight junction proteins were assessed at d4 and d9 in the jejunum by FITC-dextran and by western blot and immunohistochemistry, respectively.nnnRESULTSnAt d4, intestinal permeability was increased in MTX-PP, MTX-A and MTX-GA rats compared with controls but not in MTX-G rats. The expression of claudin-1, occludin and ZO-1 was decreased in MTX-PP group compared with controls but was restored in MTX-G and MTX-A rats. In MTX-GA rats, occludin expression remained decreased. These effects could be explained by an increase of erk phosphorylation and a decrease of IκBα expression in MTX-PP and MTX-GA rats. At d9, Intestinal permeability remained higher only in MTX-GA rats. This was associated with a persistent decrease of occludin expression.nnnCONCLUSIONSnGlutamine prevents MTX-induced gut barrier disruption by regulating occludin and claudin-1 probably through erk and NF-κB pathways. In contrast, combined glutamine and arginine has no protective effect in this model.

Sex-related effects of nutritional supplementation of Escherichia coli: Relevance to eating disorders

OBJECTIVESnThe biological background of sex-related differences in the development of eating disorders (EDs) is unknown. Recent data showed that gut bacteria Escherichia coli induce autoantibodies against anorexigenic α-melanocyte-stimulating hormone (α-MSH) associated with psychopathology in ED. The aim of this study was to compare the effects of E. coli on feeding and autoantibodies against α-MSH and adrenocorticotropic hormone (ACTH), between female and male rats.nnnMETHODSnCommensal E. coli K12 were given in a culture medium daily to adult Wistar rats by intragastric gavage over a 3-wk period; control rats received culture medium only.nnnRESULTSnBefore gavage, E. coli K12 DNA was detected in feces of female but not male rats. E. coli provision was accompanied by an increase in body weight gain in females, but a decrease in body weight gain and food intake in males. Independent of E. coli treatment, plasma levels of anti-α-MSH and ACTH immunoglobulin (Ig)G were higher in female than male rats. Females responded to E. coli by increasing α-MSH IgG levels and affinity, but males by increasing α-MSH IgM levels. Affinity of IgG for ACTH was increased in both E. coli-treated females and males, although with different kinetics. IgG from females stimulated more efficiently α-MSH-induced cyclic adenosine monophosphate production by melanocortin 4 receptor-expressing cells compared with IgG from males.nnnDISCUSSIONnSex-related response to how E. coli affects feeding and anti-melanocortin hormone antibody production may depend on the presence of these bacteria in the gut before E. coli supplementation. These data suggest that sex-related presence of certain gut bacteria may represent a risk factor for ED development.

Effects of rabbit anti-α-melanocyte-stimulating hormone (α-MSH) immunoglobulins on α-MSH signaling related to food intake control.

Anti-α-melanocyte-stimulating hormone (α-MSH) polyclonal antibodies have been used for α-MSH neutralization in functional studies, but the results are sometime inconsistent with the antibody expected blocking properties. The present study aimed to determine if rabbit (Rb) anti-α-MSH immunoglobulins (Ig) may inhibit or enhance α-MSH signaling on melanocortin receptor type 4 (MC4R) and α-MSH-induced anorexigenic effect if presented as immune complexes with α-MSH. Polyclonal Rb anti-α-MSH IgG were commercially available and their ability to bind α-MSH has been confirmed by the immunohistochemical detection of α-MSH neurons in the rat hypothalamus. In vitro assay of the cyclic-adenosine mono-phosphate (cAMP) secreted by cells transfected with MC4R was performed to analyze effect of Rb IgG on α-MSH-induced cAMP production. We found that adding Rb IgG to α-MSH resulted in stimulation of cAMP detected at lower peptide concentrations as compared to α-MSH alone. To determine effects of Rb IgG on food intake, rats were injected into the arcuate hypothalamic nucleus with either α-MSH, Rb IgG alone or Rb IgG preincubated with α-MSH. During 2 days after injections, food intake was increased in both groups of rats receiving Rb IgG. However, during following 4 days when food was restricted to 1h/day, only the Rb IgG group displayed higher food intake. Furthermore, after the refeeding, 24h food intake was lower in rats receiving Rb IgG - α-MSH immune complexes. This group of rats was also characterized by higher number of immunopositive neurons in the arcuate nucleus expressing α-MSH and agouti-related protein but not tyrosine hydroxylase. Taken together, these results show that Rb anti-α-MSH antisera, although efficient for immunohistochemical detection of α-MSH, does not always display α-MSH blocking properties but, in contrast, may enhance α-MSH binding to MC4R and increase α-MSH anorexigenic effects when presented as immune complexes with the peptide.

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation.

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.

Increased Ghrelin but Low Ghrelin-Reactive Immunoglobulins in a Rat Model of Methotrexate Chemotherapy-Induced Anorexia.

Background and aims Cancer chemotherapy is commonly accompanied by mucositis, anorexia, weight loss, and anxiety independently from cancer-induced anorexia–cachexia, further aggravating clinical outcome. Ghrelin is a peptide hormone produced in gastric mucosa that reaches the brain to stimulate appetite. In plasma, ghrelin is protected from degradation by ghrelin-reactive immunoglobulins (Ig). To analyze possible involvement of ghrelin in the chemotherapy-induced anorexia and anxiety, gastric ghrelin expression, plasma levels of ghrelin, and ghrelin-reactive IgG were studied in rats treated with methotrexate (MTX). Methods Rats received MTX (2.5u2009mg/kg, subcutaneously) for three consecutive days and were killed 3u2009days later, at the peak of anorexia and weight loss. Control rats received phosphate-buffered saline. Preproghrelin mRNA expression in the stomach was analyzed by in situ hybridization. Plasma levels of ghrelin and ghrelin-reactive IgG were measured by immunoenzymatic assays and IgG affinity kinetics by surface plasmon resonance. Anxiety- and depression-like behaviors in MTX-treated anorectic and in control rats were evaluated in the elevated plus-maze and the forced-swim test, respectively. Results In MTX-treated anorectic rats, the number of preproghrelin mRNA-producing cells was found increased (by 51.3%, pu2009<u20090.001) as well were plasma concentrations of both ghrelin and des-acyl-ghrelin (by 70.4%, pu2009<u20090.05 and 98.3%, pu2009<u20090.01, respectively). In contrast, plasma levels of total IgG reactive with ghrelin and des-acyl-ghrelin were drastically decreased (by 87.2 and 88.4%, respectively, both pu2009<u20090.001), and affinity kinetics of these IgG were characterized by increased small and big Kd, respectively. MTX-treated rats displayed increased anxiety- but not depression-like behavior. Conclusion MTX-induced anorexia, weight loss, and anxiety are accompanied by increased ghrelin production and by a decrease of ghrelin-reactive IgG levels and affinity binding properties. Such changes of ghrelin-reactive IgG may underlie their decreased ghrelin-transporting capacities compromising ghrelin orexigenic and anxiolytic effects and contributing to chemotherapy-induced loss of appetite.

MON-PP031: Development of a Plasma Assay for the Bacterial ClpB Protein as a Biomarker of Eating Disorders

compared with animal-derived proteins. However, there are few data to support this contention. We assessed postprandial plasma amino acid profiles and whole-body protein synthesis following the ingestion of 35 g wheat protein hydrolysate compared with casein and whey in healthy, older males. Methods: Healthy older males (73±1 y) received a primed continuous infusion of L-[C6]-phenylalanine and L-[H2]tyrosine and ingested 35 g wheat protein hydrolysate (WHEAT; n = 12), whey (WHEY; n = 12), or micellar casein (CAS; n = 12). Plasma and muscle samples were collected at regular intervals. Repeated measures ANOVA was used to identify differences between groups over time. Results: Plasma leucine concentrations increased following protein ingestion (P < 0.01), to a similar extent following WHEAT and CAS (P = 0.86) and to a greater extent following WHEY (P < 0.01). In agreement, peak leucine concentrations averaged 351±11, 316±24, and 580±18mM for WHEAT, CAS, and WHEY, respectively (WHEAT vs CAS P = 0.40; WHEY vs WHEAT and CAS P < 0.01). Whole-body protein synthesis increased following protein ingestion in all groups (P < 0.01), with no significant differences between groups (P = 0.13). Postprandial muscle protein synthesis rates will be assessed by determining the incorporation of L-[C6]-phenylalanine into myofibrillar protein. Conclusion: Ingesting wheat protein hydrolysate increases plasma amino acid availability and stimulates whole-body protein synthesis as effectively as casein in healthy, older males.

O15: Les effets des protéines d’E. coli K12 sur la prise alimentaire et les voies cérébrales de la satiété dépendent de l’état nutritionnel bactérien

Introduction et but de l’etude Le controle de la prise alimentaire implique des voies cerebrales regulees par des hormones de faim (ghreline), de satiete (CCK, PYY) et de stockage energetique (leptine). De nombreuses etudes associent le phenotype metabolique de l’hote, tel que l’obesite, a la composition du microbiote intestinal (e.g. ratio bacteroidetes/firmicutes). Pourtant, malgre ces donnees recentes, la comprehension des mecanismes d’action des bacteries sur le controle de la prise alimentaire de l’hote reste meconnue. Le but de cette etude a ete de tester si des proteines bacteriennes peuvent influencer le controle de la prise alimentaire de l’hote et si cette influence depend de l’etat nutritionnel des bacteries. Materiel et methodes La bacterie commensale E. coli K12 a recu un milieu riche en nutriments toutes les 12xa0h pendant 5 jours afin de mimer 2 repas/jour chez l’homme. Les proteines bacteriennes ont ete extraites en deux temps : lors de la phase exponentielle (Exp) et lors de la phase stationnaire (Stat) de croissance. Par la suite, ces fractions proteiques ont ete comparees par electropho-reses bidimensionnelles et spectrometrie de masse. Puis, les effets de l’administration intraperitoneale de ces proteines bacteriennes a la dose de 0,1xa0mg/Kg, ont ete etudies sur la prise alimentaire de rats Wistar. Des analyses immunohistochimiques, sur les coupes de cerveaux de rat ont ete realisees 2xa0h apres l’injection de proteines bacteriennes pour detecter l’expression de c-fos (marqueur d’activite neuronale) dans les neurones anorexigenes a proopiomelanocortine (POMC) du noyau arque de l’hypothalamus et dans les neurones a calcitonin gene-related peptide (CGRP) du noyau central de l’amygdale. Resultats et Analyse statistique Des la 5 e administration de nutriments, la bacterie E. coli K1 2 alterne de maniere reguliere des phases Exp de croissance (20xa0min) et des phases Stat apres chaque nouvel apport de nutriments. Les analyses proteomiques revelent la presence de proteines differentiellement exprimees entre les deux phases Exp et Stat. Seule l’injection intraperitoneale de proteines bacteriennes en phase Stat reduit la prise alimentaire (2,0 xa0±xa0 0,2 g vs 1,2 xa0±xa0 0,3 g, P 0,05, test t de student) des rats et augmente l’expression du c-fos dans les neurones POMC (45,3 xa0±xa0 3,8 cellules vs 58,4 xa0±xa0 1,4 cellules, P 0,05, post-test de Tukey, ANOVA) et CGRP (54 ±7 % vs 92 ± 4%, P Conclusion Nos donnees montrent que les proteines issues des bacteries intestinales peuvent influencer directement le controle central de la prise alimentaire, selon leur phase de croissance; elles-memes alternees par l’apport de nutriments. Dans cette etude, l’effet satietogene provoque par les proteines bacteriennes extraites en phase Stat de croissance, suggere une implication du microbiote dans l’induction de la satiete de l’hote apres un repas.