Clinical Nutrition xxx (2014) 1e9

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Review

Alterations of gut barrier and gut microbiota in food restriction, food deprivation and protein-energy wasting L. Genton a, *, P.D. Cani b, J. Schrenzel c a

Clinical Nutrition, University Hospital, Geneva, Switzerland Universit e catholique de Louvain, Louvain Drug Research Institute, WELBIO (Walloon Excellence in Life Sciences and BIOtechnology), Metabolism and Nutrition Research Group, Brussels, Belgium c Service of Infectious Diseases, University Hospital, Geneva, Switzerland b

a r t i c l e i n f o

s u m m a r y

Article history: Received 9 April 2014 Accepted 6 October 2014

Increasing evidence shows that gut microbiota composition is related to changes of gut barrier function including gut permeability and immune function. Gut microbiota is different in obese compared to lean subjects, suggesting that gut microbes are also involved in energy metabolism and subsequent nutritional state. While research on gut microbiota and gut barrier has presently mostly focused on intestinal inflammatory bowel diseases and more recently on obesity and type 2 diabetes, this review aims at summarizing the present knowledge regarding the impact, in vivo, of depleted nutritional states on structure and function of the gut epithelium, the gut-associated lymphoid tissue (GALT), the gut microbiota and the enteric nervous system. It highlights the complex interactions between the components of gut barrier in depleted states due to food deprivation, food restriction and protein energy wasting and shows that these interactions are multidirectional, implying the existence of feedbacks. © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism.

Keywords: Gut barrier Gut microbiota Protein energy wasting

1. Introduction Protein energy wasting (PEW), also termed protein energy malnutrition, occurs in 20 to 50% of hospitalized patients [1]. It refers to a “state of decreased body stores of protein and energy fuels (body protein and fat masses)”, which is generally accompanied by decreased functional capacity [2]. PEW is associated with chronic diseases, its pathophysiological mechanism involves not only anorexia and the subsequent decrease of energy intakes, but also inflammation, insulin resistance and hypogonadism, and its hallmarks are body weight loss in adults and growth failure in children [2]. It should be differentiated from depleted states due solely to food restriction or food deprivation. With the emergence of techniques measuring gut microbiota composition and function, such as 16S rDNA high-throughput sequencing and shotgun sequencing, there is a growing interest in understanding the relationship between gut microbiota and nutritional state. Nowadays, it is accepted that the gut microbiota composition differs between obese and lean subjects [3] and varies

* Corresponding author. Clinical Nutrition Rue Gabrielle Perret-Gentil 4, Geneva University Hospital, 1211 Geneva 14, Switzerland. Tel.: þ41 22 3729 344; fax: þ41 22 3729 363. E-mail address: [email protected] (L. Genton).

with weight changes [4]. Furthermore, several studies have highlighted that gut microbiota composition is associated with gut barrier function [5]. These findings suggest that gut microbiota is involved in energy metabolism and subsequent nutritional state and it is likely that, just as obesity, PEW is associated with changes of the gut barrier including gut microbiota. This review aims at summarizing the present knowledge regarding the impact, in vivo, of depleted nutritional states due to food restriction, food deprivation and PEW, on structure and function of the gut epithelium, gut-associated lymphoid tissue (GALT), gut microbiota and enteric nervous system (ENS). Figure 1 summarizes the speculated links of PEW with gut barrier, which will be discussed in this article. 2. Structure and function of the gut barrier The gut barrier is secured by the epithelium, the tight junction proteins, which include mainly occludin, claudins, zonulaoccludens 1 and the junctional adhesion molecule, the overlying mucus, the GALT, the gut microbiota, and very likely the ENS. The gut barrier modulates the transfer of molecules as nutrients, electrolytes, water, toxins, microbes and microbial byproducts, from the intestinal lumen to the mucosa. These molecules can use either the transcellular pathway and cross the apical and basolateral membranes of enterocytes, or the paracellular pathway sealed by tight

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Fig. 1. This figure highlights the speculated mechanisms underlying the worsening of nutritional state at the gut barrier level. We speculate that food deprivation or restriction are associated with alterations at the level of epithelial gut barrier, GALT, gut microbiota and ENS, which closely interact with each other. These alterations in turn may contribute to worsening of nutritional state.

junction proteins. As a consequence, the gut barrier may affect energy balance, water homeostasis, tolerance to food antigens and mucosal inflammation. Intestinal permeability refers to the property of unmediated passive diffusion across the intestinal wall. It refers to the paracellular pathway, which is mostly regulated by tight junction proteins and allows the passage of molecules smaller than 600 Da [6]. Intestinal permeability is higher in the small bowel than in the colon [7]. It can be evaluated in vivo by the flux of fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight  1000 Da) across the gut epithelium and recovery in the blood. However, this method cannot be applied to humans due to potential serious side effects of dextran. In humans, intestinal permeability is generally assessed by ingesting oral probes such as sugars, Cr-labeled EDTA, polyethylene glycols and water-soluble contrast medium, and by measuring their urinary recovery [8]. The most commonly used probes are sugars. Small bowel permeability is classically expressed as the ratio of the fractional urinary excretion of a large-size sugar like lactulose (342 Da) to a small-size sugar like mannitol (182 Da) or L-rhamnose (164 Da) [9]. The higher this ratio is, the higher is the small bowel permeability. However, it should be noted that permeability depends not only on the intrinsic characteristics of gut barrier but also on the concentration gradients across the gut epithelium, the surface area of the epithelium

and the transit time [9]. Interestingly, the lactulose/mannitol (L:M) ratio does not change with aging because the urinary excretion of both sugars decreases [10]. The transcellular pathway ensures the transport of molecules from the apical to the basolateral membrane of enterocytes through transcellular diffusion (e.g. water), transcytosis (e.g. food antigens) and carrier-mediated transport (e.g. glucose), and the transport of microbes through M cells or dendritic cells. The mechanisms of transcellular transport of bacteria and food antigens are described in details elsewhere [11]. In animals, the transcellular pathway can be tested with enteral administration of large molecules as ovalbumin (45 kDa), horseradish peroxidase (40 kDa) and b-lactoglobulin (18 kDa), and measuring their recovery in the mesenteric or portal blood [11]. In humans, this pathway can be evaluated by oral intake of D-xylose or by the D-xylose/3-O-methyl-D-glucose ratio and measuring their recovery in the urine or venous blood. Surrogate in vivo markers of intestinal permeability include plasma levels of lipopolysaccharides (LPS) and D-Lactate, and urinary levels of claudin-3. The often associated intestinal inflammation can be tested with neutrophil-derived proteins as fecal levels of calprotectin, lactoferrin, elastase, while enterocytic damage may be evaluated through plasma fatty acid binding proteins [12]. 3. Structural alterations of gut barrier in food deprivation, food restriction and PEW The structural alterations occurring with food restriction and deprivation and subsequent weight loss have been characterized mostly in rodents and are summarized in Table 1 [13e28]. A few animal studies focused specifically on the effects of protein restriction, as compared to calorie restriction. Belmonte et al. fed rats with a control diet containing 23% of protein or an isocaloric protein-free diet for 2 weeks. The protein-free diet decreased body weight, jejunal villi and lamina propria heights, and hepatic and jejunal levels of glutathione compared to the control diet, and led to some positive cultures in lymph nodes homogenates, defined as over 100 colony forming units/g tissue after 48 h [29]. Protein restriction also results in decreased secretory IgA levels [30] as well as absolute and relative amounts of intraepithelial lymphocytes

Table 1 Structural intestinal changes occurring with food deprivation (D) and restriction (R), in animals. Structure

Condition

Whole gut

D D D D D D D D D D D D D D D D R R R R R R R R

Gut mucosa

Mucus GALT

þR

þR þR þR

Impact

References

Y DNA, Y total protein content Y Intestinal weight Y RNA Y Proteins involved in glycolysis and energy metabolism Y Proteins involved in protein synthesis and amino acid metabolism [ Oxidized glutathion Y Villous volumes, number of villi, villous height and crypt depth, villous height-to crypt depth ratio Y Crypt cell production rate, proliferation of small intestine epithelial cells [ Apoptosis of small intestine epithelial cells Y Mucosal weight, mucosal surface areas and villous volumes [ Microvilli Y Microvilli area No difference in small intestine morphology, villus length and crypt depth [ Goblet cells Y Total protein content Y mRNA expression of paneth cells antimicrobials No difference in number of goblet cells Y n-3 and n-6 fatty acid concentrations of gut mucosa [ Claudin-3 expression in jejunal crypts, no difference in ZO-1, occludin, claudin-1 tight junction proteins Y Mucin in small bowel Y CD4þcells, CD8þ cells, dendritic cells, macrophages, lymphocytes Y Cells producing IL-2, IL-12, TNF-alpha, IFN-gamma, IL-6, IL-4 and IL-10 in the lamina propria Y Phagocytic activity of spleen and peritoneal macrophages No difference in number of IgAþcells

13e16 13 15 17 17 16 15, 17, 19, 22, 23 20, 21 22,26 13, 18 27 24 20 27 16 20 23 28 22 14 23 23 23 23

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[31] but does not change mucosal protein content [32]. Thus, not only the amount of calories but also the amount of protein influences the gut barrier morphology. Human studies focused on the impact of PEW compared to a good nutritional state [33e38]. They showed contradictory results on gut mucosa structure, at least partly related to different primary diseases, diagnostic criteria, duration of PEW, and age of the patients (Table 2). 4. Gut epithelium in food deprivation, food restriction and PEW This chapter focused on the impact of depleted nutritional states on intestinal permeability, and absorption and digestion of nutrients. 4.1. Function of gut epithelium in depleted nutritional states in animals In animals, calorie deprivation and restriction increase small bowel permeability determined by the FITC-dextran fluxes [20], and increase whole bowel intestinal permeability assessed by serum levels of ovalbumin [16]. Similarly, the progression to cachexia, an advanced state of PEW, in a mouse model of cancer, was related to increased whole gut permeability to FITC-dextran [39]. Calorie deprivation and restriction have also been related to a decreased activity of brush border enzymes in animals [28], including alkaline phosphatase, which can detoxify LPS by dephosphorylation [40]. However, their impact on protein and glucose absorption is controversial [25,41]. These animal studies agree that depleted states induce alterations of intestinal permeability and digestive ability. 4.2. Function of gut epithelium in depleted nutritional states in humans In humans, starvation for 36 h does not influence intestinal permeability assessed by the lactulose/rhamnose ratio [42]. However, it leads to a reduced intestinal absorption evaluated through a 60 min D-xylose/3-O-methyl-D glucose ratio, compared to baseline, in healthy subjects starved over 36 h and obese subjects starved

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over 10 and 11 days [42]. Elia et al. found a decreased intestinal uptake of mannitol after 4 days of starvation already, confirming the impact of food deprivation on intestinal absorption [43]. Interestingly, they also demonstrated that 300 kcal in severely obese subjects were sufficient to maintain mannitol absorption [44]. Surprisingly, in anorexia nervosa, which could be compared to a state of chronic calorie restriction, the L:M ratio was not increased compared to controls [45]. The authors found a significant reduction of urinary lactulose excretion and a non-significant decrease of urinary mannitol excretion. These findings suggest that long-term food restriction tightens the paracellular pathway, and highlights the difficulty of interpreting sugar ratios and their metabolism at the intestinal level. Regarding the impact of PEW on human gut function, most studies have been performed in children. In 1968 already, James et al. described that PEW in children was associated with poor glucose absorption, which was reversed with high protein feeding [46]. Furthermore, growth failure, one hallmark of PEW in children, has been associated with increased gut permeability assessed by the L:M ratio in Gambian [35], Malawian [47] and Indian children [48], and infants from Bangladesh [49]. These results may have been influenced by the presence of enteric parasites, especially Giardia lamblia and Cryptosporidium, whose byproducts are associated with epithelial gut barrier breakdown and immunological responses [50]. Plasma Giardia-specific-IgM were only measured in the infants from Bangladesh, and occurred in 98% of them. PEW was also associated with decreased small bowel permeability (L:M ratio), in hospitalized adults [33] and patients with liver cirrhosis [51]. Interestingly, Hulsewe et al. could not reproduce these results in 26 patients requiring preoperative total parenteral nutrition (TPN) [52]. They showed that intestinal inflammation based on high erythrocyte sedimentation rate was correlated with increased intestinal permeability, but not PEW, defined by 6% IWL in 6 months BMI < 19 kg/m2 or >6% IWL in 6 months

33

Adults

NRI < 98.5

Cancer in 80% of malnourished patients

33

Adults

NRI < 98.5

33

Adults

NRI < 98.5

33 38 38

Adults Adults Adults

NRI < 98.5 NRI < 83.5 NRI < 83.5

Chronic heart failure

Cancer in 75% of malnourished patients Cancer in 75% of malnourished patients

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and absorptive capacity. However, it remains unclear whether these alterations affect similarly all segments of the gastrointestinal tract, whether they occur only after a certain amount of weight loss, whether they are related to a deficient intake of calories, protein or both and whether they are aggravated by other components of PEW than nutritional intake, such as inflammation. 4.3. Alterations of gut epithelium and role of feeding route The importance of enteral stimulation for epithelial gut barrier function has been demonstrated in humans undergoing food deprivation or fed by TPN. Both lead to a decreased activity of brush border enzymes in humans [57]. Additionally, TPN has been associated with a decreased number of tight junction proteins [58] and a higher L:M ratio [59] in animals. Thus, both parenteral nutrition and food deprivation impair the function of gut epithelium. 5. GALT in food deprivation, food restriction and PEW The GALT, considered as the largest mass of immune cells in the human body, comprises lymphocytes scattered in the Peyer's patches, lamina propria and intestinal epithelium, M cells, granulocytes, macrophages and mast cells. The gut barrier controls the passage of gut microbiota, byproducts of gut microbiota and food antigens across the gut mucosa. These elements may act as antigens and elicit an immune response which can lead to inflammation. This chapter will cover the relationship of depleted states with intestinal immune response, evaluated through levels of cytokines, acute phase proteins, immunoglobulins (Ig), and immune cell functions. 5.1. Function of GALT in depleted nutritional states in animals Food restriction and PEW stimulate bacterial translocation to the spleen, liver and intestinal tissue in rodents, which received Salmonella enterica subsp. Typhimurium intragastrically [23] or underwent liver transplantation [60], and increase serum levels of endotoxin [60]. Regarding cytokine secretion, food restriction leads to increased serum levels of tumor necrosis factor-alpha (TNF-a) and low levels of interleukin-2 in vitro [61]. When considering specifically PEW, cancer cachexia in mice results in hypertrophy of mesenteric lymph nodes and increased plasma concentrations of endotoxins and interleukin-6 (IL-6) [39]. Besides bacterial translocation and cytokine modulation, food restriction has been related with a diminished phagocytic activity of spleen and peritoneal macrophages [23] and decreased T cell proliferation, blood ratio of CD4:CD8 cells and natural killer cell activity [62]. Again, not only the amount of enteral calories is essential for GALT function. Protein restriction increases bacterial translocation in mice [63], the relative amount of intraepithelial lymphocytes crossing the basement membrane of enterocytes [31], the plasma anti-ß-lactoglobulin IgG in response to cow-milk intake and the intestinal anaphylactic responses to b-lactoglobulin [64]. It decreases secretory IgA levels [30], which may be due to a decreased expression of the polymeric immunoglobulin receptor transporting IgA from the basolateral membrane to the intestinal lumen [65]. These animal studies suggest that calorie or solely protein restriction associated with inflammation, as in PEW, influence both humoral and cellular immunity. 5.2. Function of GALT in depleted nutritional states in humans In humans, PEW has been related with an inflammatory state as shown by high serum levels of IL-6 and C-reactive protein (CRP), low expression of mucosal interleukin-10 (IL-10), and low

activation of lamina propria lymphocytes and enterocytes [33]. In the latter study, PEW did not influence mucosal expression of TNFa and mucosal sIgA. The impact of PEW on inflammatory and immune parameters was also shown in Gambian children with growth failure [35]. They had evidence of elevated circulating levels of leucocytes, lymphocytes, IgG, IgA, IgM, endotoxins and antiendotoxins antibody. The plasma levels of Ig and IgG endotoxincore antibody were negatively correlated with growth rates and intestinal permeability [35]. In Bangladeshi children, growth failure and skin test anergy were both predictors of chronic diarrhea in a logistic regression, showing the link between PEW, cell-mediated immunity and clinical enteropathy [66]. None of the children included in the latter two studies were checked for Giardia and Cryptosporidium, associated per se with inflammatory and immune responses [50]. Finally, Sandek et al. studied the immune response of patients with chronic heart failure, a population at risk for PEW [67]. They described in these patients an increased bacterial adherence to the gut mucosa, higher serum levels of IgA anti-LPS antibodies, IL-6 and blood leucocytes compared to controls, but similar levels of endotoxins [54]. Thus, food restriction and PEW both seem to favor bacterial translocation, potentially due to increased epithelial gut permeability. However, while food restriction alone rather impairs the immune response, PEW, by definition associated with inflammation, stimulates a systemic and intestinal immune response. 5.3. Relationship between intestinal inflammation and gut epithelium in depleted nutritional states As mentioned previously, depleted nutritional states have been related to increased gut permeability. Muscle wasting, a hallmark of PEW, has been associated with increased levels of proinflammatory cytokines, such as TNF-a, interferon-gamma (IFNg), interleukin-1 and IL-6 [68]. IFN-g and TNF-a seem to be essential modulators of gut barrier function as they decrease transepithelial resistance [69], increase gut permeability [70] and affect tight junction proteins. The detailed mechanisms of IFN-g and TNF-a on tight junction proteins are detailed elsewhere [71,72]. Thus, PEW may affect gut permeability through pro-inflammatory cytokines, as speculated on Fig. 2. 5.4. Alterations of GALT and role of feeding route Like food deprivation, the absence of enteral stimulation affects the GALT. TPN decreases the GALT mass, the IgA levels in intestinal washes, the levels of two IgA-stimulating cytokine levels which are interleukin-4 and IL-10, and has been related to increased levels of TNF-a and IFN-g in animals [73]. Similarly, infants and neonates under long-term parenteral nutrition presenting with sepsis were shown to experience often a translocation of enteric bacteria [74]. These results suggest that parenteral nutrition affects the GALT, but it is unknown whether this effect is similar to food deprivation in humans. 6. Gut microbiota in food deprivation, food restriction and PEW Ileal digesta contain up to 107e108 bacteria/ml and the colon contains up to 1013e1014 total bacteria [75]. In ileostomy patients, the prominent bacterial phyla are among the Gram-negative, the Bacteroidetes and Proteobacteria, and among the Gram-positive, the Firmicutes [76]. These phyla are comparable to the ones encountered in the large intestine. The interaction of commensal gut microbiota with epithelial gut barrier and the immune system has been detailed elsewhere [77].

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Enterobacteriaceae, total Lactobacillus spp. and total anaerobic bacteria [23], and a lower bacterial adherence of Gram negative bacteria to the intestinal mucosa [83]. In rats treated with dexamethasone to induce stress, starvation resulted also in higher bacterial adherence to the gut mucosa [84]. A recent animal study linked muscle atrophy, a hallmark of PEW, to gut microbiota [85]. In a mouse model of leukemia, the authors found a decrease in Lactobacillus reuteri and Lactobacillus gasseri. Reversing the dysbiosis with oral supplementation of this deficient Lactobacillus population reduced the expression of muscular atrophy markers and the plasma levels of pro-inflammatory-cytokines. As a consequence, depleted nutritional states seem to be associated with changes in gut microbiota composition and function, which in turn may affect muscle mass, suggesting the presence of a gut-muscle axis [86]. 6.2. Gut microbiota in depleted nutritional states in humans

Fig. 2. This figure represents the view of the authors on speculated mechanisms and consequences of chronic diseases on epithelial gut barrier, GALT and gut microbiota, whose alterations could lead to PEW. These interactions are multidirectional, implying the existence of feedbacks represented by dashed lines.

Briefly, colonic gut microbiota ferments non-digested carbohydrates into short chain fatty acids (SCFA). SCFA have been shown to regulate proliferation and differentiation of enterocytes, generation of reactive oxygen species and gastrointestinal motility, and decrease gut permeability [78,79]. Commensal gut microbes also stimulate the production of antimicrobial peptides by epithelial and immune cells in the small bowel and the colon [80]. Antimicrobial peptides are components of the innate immune system and are small cationic peptides identified as defensins and cathelicidins [74]. Finally, gut microbiota influences the GALT. For example, germ-free animals have smaller and fewer Peyer's patches, decreased lymphocytes in the intestinal lamina propria and the mesenteric lymph nodes, and a lower level of circulating immunoglobulins, compared to animals displaying microbiota [74]. Recent animal work showed that gut microbiota stimulated antibody production by mucosal cells [81] and that in turn intestinal IgA deficiency lead to modification of gut microbiota [82]. These studies illustrate the potential relationship between gut microbiota, epithelial gut barrier and GALT. 6.1. Gut microbiota in depleted nutritional states in animals Only a few studies have explored the interaction of gut microbiota and gut barrier in nutritionally depleted states and this interaction likely depends on gut microbiota composition and function. Compared to well-nourished rodents, food restricted rodents show a lower bacterial count in the ileum and cecum [83], a lower colonic count of Bifidobacterium spp. but a similar count of

Human studies focused rather on obesity. However, Monira et al. showed that children with PEW displayed a higher fecal proportion of Proteobacteria but a lower fecal proportion of Bacteroidetes, than healthy children [87]. One study investigated whether gut microbiota differed between obese, lean and anorectic patients. Anorectic patients had a higher proportion of Methanobrevibacter smithii spp, but comparable proportions to lean subjects of Firmicutes, Bacteroidetes and Lactobacillus [88]. The authors suggested that the proliferation of M. smithii, a reductor of CO2 and H2 to methane, could increase fermentation efficiency by accelerating the removal of H2, and thus energy harvesting [89]. Recently, Smith et al. performed an exciting research in twin children discordant for Kwashiorkor, a type of PEW [90]. They transplanted the gut microbiota of these children, which was not significantly different regarding enteropathogens including protozoan, into gnotobiotic mice. When fed a Malawian diet for 3 weeks, the mice transplanted with the Kwashiorkor microbiome experienced more weight loss and increased the cecal proportions of Bilophila wadswortia spp (phylum Proteobacteria) and Clostridium innoccum spp (phylum Firmicutes). The mice were then switched, for 2 weeks, to a ready to use therapeutic food made of peanut paste, milk, vegetable oil, sugar, vitamins and minerals. With this diet, both groups of mice increased weight, cecal concentrations of Bifidobacterium spp, Lactobacillus spp and Ruminococcus spp, cecal and fecal levels of SCFA and of essential amino acids, but reduced their levels of cecal and fecal mono- and disaccharides, especially in mice with the Kwashiorkor microbiome [90]. In summary, it seems that gut microbiota composition and function changes in nutritional depleted states and regulates energy metabolism. 6.3. Relationship between intestinal inflammation and gut microbiota in depleted nutritional states The mechanisms linking PEW to intestinal inflammation was recently investigated and may be related to gut microbiota. Hashimoto et al. showed that essential amino acids are absorbed via a neutral amino acid transporter whose expression requires angiotensin I converting 2 enzyme (ACE2), a regulator of the reninangiotensin system [91]. Once absorbed into the enterocytes, amino acids stimulate the expression of antimicrobial peptides in ileal enterocytes, which in turn could modify the composition of gut microbiota. The authors hypothesize that the loss of ACE2 and the subsequent inhibition of antimicrobial peptide expression explains the intestinal inflammation in PEW. On the basis of the available literature, we suggest a link between gut barrier alterations including gut microbiota and the occurrence of PEW (Fig. 2).

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6.4. Gut microbiota and role of feeding route

7.2. Relationship between ENS and epithelial gut barrier

As for gut epithelium and GALT, the absence of enteral stimulation affects gut microbiota. Indeed, TPN promotes a predominance of Firmicutes over Proteobacteria [92]. Other studies report a decreased ratio of Firmicutes to Bacteroidetes and a higher mRNA expression of lysozyme, a-defensin 5 and a-defensin 8, which are antimicriobial proteins produced by Paneth cells [93]. Clayburg et al. suggested that parenteral nutrition modifies gut microbiota, which in turn stimulates inflammation and the secretion of proinflammatory cytokines [94].

Animal studies demonstrated that glucagon-like peptide 2 (GLP-2), synthesized by enteroendocrine L cells, increases enterocyte proliferation, expression of tight junction proteins and decreases enterocyte apoptosis and gut permeability [97,99]. Bombesin, an analog to mammalian gastrin-releasing peptide, decreased intestinal permeability in burned rats [100] and increased the number of GALT lymphocytes and intestinal IgA levels in mice [101]. Other neuropeptides and gastro-intestinal hormones can also enhance epithelial gut barrier in vitro or in animal models, as NPY [102], cholecystokinin [103] and ghrelin [104]. An interesting study looked at the impact of cecal denervation on gut trophicity in rats [105]. The authors first demonstrated that SCFA, which can be produced by commensal gut microbiota, stimulated jejunal trophicity when infused in the cecum, as shown by increases in jejunal DNA, villous height, surface area and crypt depth and gastrin. With cecal denervation, this trophic jejunal effect of SCFA was abrogated. Thus, the ENS and gut hormones regulate epithelial gut barrier.

7. ENS in food deprivation, food restriction and PEW The ENS is involved in appetite regulation. Appetite is modulated by the arcuate nucleus of the hypothalamus, which contains specific neurons involved in energy metabolism. For instance, anorexigenic neurons express pro-opiomelanocortin (i.e, a-melanocyte stimulating hormone) and cocaine and amphetamine regulated transcript, whereas orexigenic neurons express neuropeptide Y (NPY) and Agouti-related protein [95]. The arcuate nucleus regulates food intake by integrating signals from appetite mediators directly, via the brainstem or the vagal nerve. Since appetite loss may lead ultimately to a depleted nutritional state and since depleted states are related to gut barrier alterations, it seems logical to consider that mediators of appetite play a role in gut barrier integrity. 7.1. ENS in depleted nutritional states Mediators of appetite include neuropeptides and hormones, some of which are synthesized in the digestive tract (Table 3). Research is scarce regarding the impact of depleted nutritional state on appetite mediators of the digestive tract. In rats, food deprivation for three days resulted in decreased levels of plasma peptide YY (PYY), enteroglucagon and gastrin [96]. In anorexia nervosa, characterized by chronic food restriction, fasting plasma levels of leptin were low while plasma levels of ghrelin, and PYY were high compared to healthy subjects [97]. In hemodialysis, a disease often associated with PEW, anorexia has been associated with high levels of plasma ghrelin [98]. Thus, there are indications that the ENS and intestinal hormonal system are linked with the development of PEW and this may occur, at least partly, through modifications at the levels of the gut barrier.

Table 3 Appetite mediators and their place of synthesis. Orexigenic neuropeptides/ hormones

Anorexigenic neuropeptides/hormones

Neuropeptide Y (Hypothalamus) Ghrelin (Stomach) Agouti-related peptide (Hypothalamus) Endocannabinoids as arachidonoylethanolamide

Cholecystokinin (duodenum) Endocannabinoids as oleoylethanolamide Peptide YY (ileum and colon) Glucagon-like peptide-1 (ileum and colon) Leptin (adipocyte) Insulin (pancreas) a-melanocyte-stimulating hormone (pituitary gland) Corticotropin-releasing hormone (hypothalamus) Thyrotropin- releasing hormone (hypothalamus) Cocaine- and amphetamine-regulated transcript (hypothalamus) Serotonin (gut, central nervous system) Cytokines: TNF-alpha, IL-1b, IL-6 (immune cells)

7.3. Relationship between intestinal inflammation, gut microbiota and ENS in depleted nutritional states Interestingly, some of the neuropeptides implicated in increased appetite and intestinal epithelial permeability are related to inflammation. For instance, mice knock-out for NPY (-/-) show a lower release of TNF-a and a lower intestinal permeability [102]. Administration of anti-TNF antibodies decreases the expression of NPY and ameliorates inflammation. Microbiota likely also influence gut hormones. Food restriction for 5 days in rats led to numerous changes in gut microbiota composition. For instance, the number of Bacteroides and Prevotella was increased compared to non-restricted rats and positively correlated with serum ghrelin levels, while there was a decrease in the number of Bifidobabacterium and Lactobacillus, which correlated positively with serum leptin level [106]. Cani et al. found that prebiotics (inulin-type fructans) increase plasma and intestinal glucagon-like peptide-1 (GLP-1), GLP-2, PYY and decreases ghrelin in several animal models [107,108]. In healthy volunteers, they found that gut microbiota fermentation of prebiotics decreased appetite and increased satiety, a phenomenon associated with an increase in plasma GLP-1 and PYY [109]. Parnell et al. found similar results in obese subjects [110], and more recently, confirmed that inulin type-fructans fibers fed for 10 weeks upregulated cecal proglucagon, the precursor of GLP-2 and peptide YY mRNA but downregulated ileal ghrelin mRNA in lean rats [111]. Thus, the literature suggests that the ENS may interact with the intestinal epithelial cells, the GALT and the gut microbiota. 8. Conclusion Food deprivation, food restriction and PEW affect the epithelial gut barrier, GALT, gut microbiota and ENS. However, the mechanisms by which PEW leads to these alterations are difficult to untangle as these systems closely interact, often in a bidirectional way, and, in humans, are often tested in the context of co-morbidities, which blunts the impact of food deprivation and food restriction alone. Future research on nutritional depletion and refeeding should encompass simultaneous analysis of these different systems, instead of studying one at a time, as they are highly interrelated. This would allow gaining more insights into the mechanisms of PEW at the gut barrier level and open new areas for treatment of PEW.

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Financial support None. Authors' contributions LG did the literature search and wrote the draft of this review. PDC and JS critically reviewed the document and added valuable information and references. Conflict of interest None of the authors has a conflict of interest. Acknowledgments PDC is a research associate at FRS-FNRS (Fonds de la Recherche Scientifique), recipient of grants from the Fonds De La Recherche dicale, Scientifique and FRSM (Fonds de la recherche scientifique me e). PDC is a Belgium) and ARC (Action de Recherche Concerte recipient of ERC Starting Grant 2013 (European Research Council, Starting grant 336452-ENIGMO). References [1] Norman K, Pichard C, Lochs H, Pirlich M. Prognostic impact of disease-related malnutrition. Clin Nutr 2008;27:5e15. [2] Evans WJ, Morley JE, Argiles J, Bales C, Baracos V, Guttridge D, et al. Cachexia: a new definition. Clin Nutr 2008;27:793e9. [3] Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027e31. [4] Jumpertz R, Le DS, Turnbaugh PJ, Trinidad C, Bogardus C, Gordon JI, et al. Energy-balance studies reveal associations between gut microbes, caloric load, and nutrient absorption in humans. Am J Clin Nutr 2011;94:58e65. [5] Tremaroli V, Backhed F. Functional interactions between the gut microbiota and host metabolism. Nature 2012;489:242e9. [6] Watson CJ, Rowland M, Warhurst G. Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers. Am J Physiol Cell Physiol 2001;281:C388e97. [7] Markov AG, Veshnyakova A, Fromm M, Amasheh M, Amasheh S. Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B 2010;180:591e8. [8] Teixeira TF, Collado MC, Ferreira CL, Bressan J, Peluzio Mdo C. Potential mechanisms for the emerging link between obesity and increased intestinal permeability. Nutr Res 2012;32:637e47. [9] Arrieta MC, Bistritz L, Meddings JB. Alterations in intestinal permeability. Gut 2006;55:1512e20. [10] Saltzman JR, Kowdley KV, Perrone G, Russell RM. Changes in small-intestine permeability with aging. J Am Geriatr Soc 1995;43:160e4. [11] Menard S, Cerf-Bensussan N, Heyman M. Multiple facets of intestinal permeability and epithelial handling of dietary antigens. Mucosal Immunol 2010;3:247e59. [12] Derikx JP, Luyer MD, Heineman E, Buurman WA. Non-invasive markers of gut wall integrity in health and disease. World J Gastroenterol 2010;16: 5272e9. [13] McManus JP, Isselbacher KJ. Effect of fasting versus feeding on the rat small intestine. Morphological, biochemical, and functional differences. Gastroenterology 1970;59:214e21. [14] Sherman P, Forstner J, Roomi N, Khatri I, Forstner G. Mucin depletion in the intestine of malnourished rats. Am J Physiol 1985;248:G418e23. [15] Steiner M, Bourges HR, Freedman LS, Gray SJ. Effect of starvation on the tissue composition of the small intestine in the rat. Am J Physiol 1968;215: 75e7. [16] Boza JJ, Moennoz D, Vuichoud J, Jarret AR, Gaudard-de-Weck D, Fritsche R, et al. Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats. J Nutr 1999;129:1340e6. [17] Lenaerts K, Sokolovic M, Bouwman FG, Lamers WH, Mariman EC, Renes J. Starvation induces phase-specific changes in the proteome of mouse small intestine. J Proteome Res 2006;5:2113e22. [18] Ross GA, Mayhew TM. Effects of fasting on mucosal dimensions in the duodenum, jejunum and ileum of the rat. J Anat 1985;142:191e200. [19] Botsios D, Economou L, Manthos A, Tsokali M, Sioga A, Agelopoulos S, et al. Ultrastructural alterations of the rat intestinal epithelium fed with polymeric, oligopeptidic or elementary full diet, following starvation. Histol Histopathol 1993;8:527e35.

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