Interplay Between Gut Microbiota and Gastrointestinal Peptides: Potential Outcomes on the Regulation of Glucose Control

Journal Pre-proof The interplay between the gut microbiota and gastrointestinal peptides: potential outcomes on the regulation of glucose control Joseph Lupien-Meilleur, David E. Andrich, Samantha Quinn, Clément Micaelli-Baret, Roxane St-Amand, Denis Roy, David H. St-Pierre PII:

S1499-2671(19)30681-1

DOI:

https://doi.org/10.1016/j.jcjd.2019.10.006

Reference:

JCJD 1236

To appear in:

Canadian journal of Diabetics

Received Date: 10 August 2019 Revised Date:

11 October 2019

Accepted Date: 16 October 2019

Please cite this article as: Lupien-Meilleur J, Andrich DE, Quinn S, Micaelli-Baret C, St-Amand R, Roy D, St-Pierre DH, The interplay between the gut microbiota and gastrointestinal peptides: potential outcomes on the regulation of glucose control, Canadian Journal of Diabetes (2019), doi: https://doi.org/10.1016/ j.jcjd.2019.10.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Canadian Diabetes Association.

The interplay between the gut microbiota and gastrointestinal peptides: potential outcomes on the regulation of glucose control

Joseph Lupien-Meilleur1,2,3, David E Andrich1, Samantha Quinn1,4, Clément MicaelliBaret1, Roxane St-Amand1,5, Denis Roy2,3 and David H St-Pierre 1,2,3,6 1

Department of Exercise Sciences, Université du Québec à Montréal (UQAM); 2Department of Food Science, Université Laval; 3Institut de Nutrition et des Aliments Fonctionnels (INAF), Université Laval; 4Department of Kinesiology, McGill University; 5 Department of Kinesiology, Université de Montréal; 6 CHU Sainte-Justine Research Center

Running Head: Gut microbiota and gastrointestinal peptides; potential outcomes on glucose control Keywords: Gut microbiota, gastrointestinal peptides, inflammation, diets, prebiotics, probiotics, germ-free animals, fecal transfer, antibiotics, microbial metabolites

Grants, sponsors, and funding sources David H St-Pierre is funded by a NSERC Discovery Grant #418509-2012 and his salary support is provided by a FQRS Chercheur Boursier Junior 1 Award.

Correspondence: David H St-Pierre, Department of Exercise Science, 141 President-Kennedy Ave, Montreal, Quebec, Canada, H3C 3P8, Telephone: +1 (514) 987-3000 ext 5150# Fax: +1 (514) 987-6616 Email: [email protected] 1

ABSTRACT

A host of gastrointestinal (GI) peptides influence the regulation of vital functions such as growth, appetite, stress, gut motility, energy expenditure, digestion and inflammation as well as glucose and lipid homeostasis. Hence, impairments in the synthesis/secretion of glucagon-like peptide 1 (GLP-1), leptin, nesfatin-1, glucosedependent insulinotropic peptide (GIP), ghrelin (acylated/AG and unacylated/UAG forms), oxyntomodulin, vasoactive intestinal peptide (VIP), somatostatin, cholecystokinin (CCK), peptide tyrosine-tyrosine (PYY), GLP2, pancreatic polypeptide (PP) were previously associated with the development of obesity-related disorders. It is currently emphasized that the beneficial metabolic outcomes associated with the normalization of the gut microbiota (GM) is influenced by increases in GLP-1 and PYY secretion as well as by decreases in AG production. These effects are associated with reductions in body weight and adiposity in combination with the normalization of glucose and lipid metabolism. However, important questions remain unanswered regarding how GLP-1, PYY, AG and other metabolically-relevant GI peptides interact with the GM to modulate the host’s metabolic functions. In addition, it is likely that the GM and other biologically active GI peptides influence metabolic functions such as glucose control, although the mechanisms remain ill-defined. The present article will review how GM and GI peptides influence glucose metabolism in experimental models such as germ-free (GF) animals and dietary interventions. Emphasis will be placed on pathways through which GM and GI peptides could modulate intestinal permeability, nutrient absorption, short-chain fatty acid (SCFA) production, metabolic endotoxemia, oxidative stress and low-grade inflammation.

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1.

INTRODUCTION

Under obesogenic conditions, lipids accumulate in non-adipose tissues. This leads to the formation of ectopic fat that alters vital functions in the liver, skeletal muscle, intestinal tissue, pancreas, heart, kidney and bone marrow through the promotion of lipotoxicity. Lipotoxicity is considered a major driving force in the development of low-grade inflammation and metabolic disorders such as insulin resistance (IR), type 2 diabetes (T2D), dyslipidemia, non-alcoholic fatty liver disease and atherosclerosis. Recent advances in high-throughput microbial profiling has linked altered gut microbiota (GM; dysbiosis, see Figure 1) to a number of pathological conditions, which has enabled the detection and characterization of the biological relevance for previously unknown microbial species in the GI tract. Maternal obesity, delivery mode (vaginal vs. cesarean), breast feeding, diet, antibiotic treatments and living environment are determining factors for the establishment of eubiosis (normal GM) or dysbiosis. Dysbiosis is often associated with excessive body weight in children and adults. It is a condition that alters short-chain and branched-chain fatty acids (SCFA; BCFA; e.g. acetate, butyrate, propionate, methylbutyrate, isovalerate and isobutyrate) production and amino acid degradation while promoting excessive nutrient absorption, intestinal permeability, bacterial translocation, metabolic endotoxemia (e.g. lipopolysaccharides or LPS from Gram-negative bacteria),

oxidative stress and low-grade

inflammation[1,2]. Accordingly, dietary patterns exert a strong influence on the GM. Western diets (WD) are low in GM-accessible carbohydrates and cause a detrimental loss in bacterial diversity that is difficult to restore thereafter[3]. The associations between GM, diets and the regulation of metabolic functions were abundantly reported over the recent years. However, intermediate steps through which dysbiosis, oxidative stress and lowgrade inflammation lead to the onset of insulin resistance and T2D remain ill-defined.

The gastrointestinal (GI) tract plays a critical role in the development of obesity and metabolic disorders. Dysbiosis is associated with impairments in the secretion of several GI peptides that act as key mediators of metabolic functions such as appetite, insulin secretion and nutrient absorption, storage and metabolism [4]. As a result, a close relationship between the development of obesity-related disturbances and gut-derived hormonal dysregulation has been established[5]. Gut-derived peptides such as cholecystokinin (CCK), peptide YY (PYY), 3

glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), secretin and oxyntomodulin are neuroendocrine factors that promote satiety, insulin secretion and glucose disposal[4]. The acylated (AG) form of ghrelin is another gut-derived peptide which stimulates food intake, lipid buildup in adipose tissues and insulin resistance[4]. The beneficial effects of GLP-1 on glucose control are widely documented, but the biological functions of most other GI peptides remain overlooked. It was previously reported that the GM influences ghrelin, GLP-1 and PYY secretions[6-8]. Eubiosis is associated with increased GLP-1 and PYY secretion as well as decreased AG levels[9]. This is intimately linked with lower body weight and adiposity, along with improved glucose and lipid metabolism in humans and animal models. Although their metabolic relevance was previously evidenced, the interplay between the GM and other GI peptides such as oxyntomodulin, CCK, GLP-2, leptin, GIP, vasoactive intestinal peptide (VIP), secretin, pituitary adenylate cyclase-activating polypeptide (PACAP), nesfatin-1 and pancreatic polypeptide (PP) needs to be clarified. This article reviews the interactions between GM and GI hormones and their influence on the mechanisms regulating glucose metabolism.

2.

THE INFLAMMATORY SYSTEM

Throughout the digestive system (see Figure 1), both GI peptides and the GM is proposed to exert critical roles on the regulation of inflammation. We have previously reviewed how obesogenic diets alter the GM, increase oxidative stress and promote GI mucosal inflammation, intestinal permeability, endotoxin and bacterial translocation as well as systemic low-grade inflammation[2]. Those events were then suggested to play a critical role in the etiology of obesity-related disorders. This section mainly focuses on the role of altered GM and GI peptide secretion on the development of insulin resistance and T2D. Gut microbial ecology, the secretion of GI peptides and the activation of inflammatory mechanisms are all proposed to regulate one another. For instance, obesity is suggested to alter vagal afferent neuronal activities and therefore the transmission of signals involved in the control of vital functions such as appetite, satiation, satiety and energy metabolism. The alteration of the GM increases the translocation of micro-organisms and the transport of microbial components (e.g. LPS production from Gram-negative bacteria) into the system while 4

impairing leptin’s vagal afferent signaling[10]. This could be modulated by alterations in the neuronal integration of critical neuroendocrine signals such as CCK, serotonin and leptin through increases in the expression of the endocannabinoid receptor 1 (CB1) and melanin-concentrating hormone receptor 1 (MCH1R), as well as by the down-regulation of Y2 and leptin receptors[11-13]. Furthermore, our previous work also suggested that LPS can affect the secretion of ghrelin, an orexigenic hormone with an influence on the regulation of metabolic functions. Interestingly, an acute administration of LPS inhibited ghrelin secretion through mechanisms involving IL-1β and prostaglandins in rats[14]. Further, ghrelin administration was able to circumvent the digestive impairments caused by LPS.

Maternal periodontitis was recently associated with insulin resistance and inflammation in adult rodent offsprings[15,16]. However, the mechanisms underlying these effects remain largely uncharacterized. In the oral cavity, the presence of the periodontopathogen Fusobacterium nucleatum is reported to upregulate the expression of the ghrelin receptor (GHSR-1) in vitro in cultured periodontal cells, and in vivo in human and rat gingival fibroblasts of the lamina priora and gingival epithelial cells[17]. In addition, after infecting cultured periodontal cells with F. nucleatum for 24 h, expression of pro-inflammatory CCL2, IL-6 and IL-8 was increased while the combined treatment with ghrelin upregulated GHSR-1 transcription and significantly reduced the expression of each cytokines. Conversely, both 2-day (cultured periodontal cells) and prolonged (biopsies from the oral cavity of rats infected with F. nucleatum for 12 days, or humans with periodontitis) infections were associated with a downregulation of GHSR-1 expression. This indicates that the antiinflammatory activity associated with ghrelin is both acutely stimulated and chronically inhibited by F. nucleatum in the oral cavity. In cultured adipocytes, LPS from the periodontal pathogen Porphyromonas gingivalis activates TLR2- and TLR4-mediated signaling, which potentiates the production of leptin, IL-6, MCP1 and resistin[18]. While the definite mechanism is yet to be determined, this indicates that the GM influences the activity of important hormonal regulators of metabolic functions, and their disturbance is associated with the development of diabetes. Surprisingly, TLR activation is also reported to increase PYY expression (but not proglucagon) through NFκB signaling in L-cells[19]. Although not exhaustively clarified, this effect could be mediated through the increased expression of TLR-4 and NFκB in response to butyrate treatment. In humans 5

with high post-prandial endotoxemia, a treatment with Bacillus indicus, Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis and Bacillus clausii was shown to decrease endotoxemia as well as circulating levels of ghrelin, IL-12p70 and IL-1β [20]. In addition, children with short stature, and carriers of Candida albicans or Helicobacter pylori display an increased risk (2 to 3 times) of developing autoantibodies against neuroendocrine peptides such as ghrelin, leptin, orexin A and α-MSH[21]. These elements clearly indicate an interplay between GM, GI peptides and the inflammatory system; however, the mechanisms through which they interact in order to impair glucose control remain ill-defined.

3.

GERM-FREE ANIMALS, ANTIBIOTICS AND FECAL TRANSFER

Experimental models using animals lacking GM (germ-free or GF), treated with antibiotics or submitted to fecal transplants have provided milestones in the recognition of the relevance of GM in the regulation of vital functions. However, there is still very limited information regarding the mechanistic pathways through which the GM exerts its effects on metabolic functions in these models. One appealing hypothesis is that the absence/modulation of the GM influences the regulation of metabolically-relevant GI peptides. Two of the best candidates remain two hormones mainly co-secreted from L-cells; GLP-1 and PYY. A large number of GLP-1 receptor agonists were developed by the pharmaceutical industry to control insulin resistance and type 2 diabetes over the last decades. In addition, due to its anorexigenic properties, PYY (more specifically its 3-36 form) is considered to be a candidate against obesity and ensuing metabolic disorders.

In GF animals, GLP-1 expression was recently shown to be increased. This was associated with reduced intestinal transit under caloric restriction (see Figure 1). Furthermore, the mechanism proposed involved SCFA production since butyrate, the primary energy source of colonocytes, cannot be synthesized by the GM in GF animals. The unavailability of butyrate would upregulate GLP-1 production and, in turn, slow down the 6

intestinal transit and nutrient systemic absorption while optimizing energy substrate utilization[22]. It is also observed that GF mice express more bile acid transporters in their ileum than mice with a conventional GM. Accordingly, GF mice display a higher rate of non-12α-OH bile acids transport towards the system. This effect would promote the activation of the Takeda G protein receptor 5 (TGR5) signaling, gallbladder size, and oleanolic acid (OA) production while upregulating GLP-1 and PYY levels[23,24]. Different mechanisms were proposed to explain how OA stimulates GLP-1 and PYY production, including the activation of the GαS protein subunit and cAMP production. Hence, this process could also involve phosphoinositol (PI) hydrolysis through phospholipase C-ɛ (PLC-ɛ) and increased intracellular Ca2+ levels[24]. The same authors also reported that H2S would reduce GLP-1 and PYY release through the inhibition of PI hydrolysis. In contrast, H2S-producing bacteria submitted to chondroitin sulfate were shown to promote GLP-1 and insulin secretion as well as glucose tolerance while decreasing food consumption in mice. These effects were associated with increased H2S production by Desulfovibrio piger in feces[25], indicating a potential role of H2S in the regulation of GLP-1. However, its outcomes and the mechanisms through which they are mediated need to be exhaustively clarified and characterized.

Alarming reports indicate that the over-use of antibiotics in humans and in the feedings of farm animals are closely related to the development of antibiotic resistance. This is critical for social health authorities since a number of multi-resistant bacterial strains have emerged over the last decades. This abusive use of antibiotics could also influence the development of metabolic disorders. Similar to GF/gnotobiotic animals, antibiotics facilitate the control of GM ecology in order to determine the impact of selective or global bacterial eradication on metabolic functions in experimental models. In response to a combined vancomycin and bacitracin (both specific antibiotics against Gram-positive bacteria) treatment, reduced Bacteroidetes and Firmicutes growth was observed in mice[26]. This treatment increased GLP-1 levels while normalizing glucose intolerance, hyperinsulinemia and insulin resistance. Cefazidime (selective for Gram-negative bacteria) treatment also induced weight loss, improvements in glucose control, increased PYY and GLP-1 secretion in HFD-fed mice and Zucker Diabetic rats supplemented with oligofructose[27]. Still, the chronic effects of antibiotic treatments on GM ecology and the alteration of metabolic functions need to be clarified. Experimental models using 7

antibiotics remain useful to study the role of the GM on the regulation of GI peptide secretion and metabolic functions. However, while using such models, it is critical to consider the antimicrobial spectrum and its role on GM ecology before drawing direct mechanistic conclusions. Hence, after determining the modes of action through which antibiotics influence metabolic functions, translational studies will be necessary to confirm their long-term outcomes in humans.

There is currently limited information regarding the role of fecal transfer on the regulation of GI peptide secretion and the modulation of metabolic functions. However, promising data indicate the potential of fecal transplant in patients with digestive disorders. For instance, after a fecal transfer from patients with short bowel syndrome to gnotobiotic rats, an increase in circulating GLP-1 and ghrelin levels was reported[28]. This is of particular interest since both GI peptides are known to be secreted in high concentrations in response to the human disease. While there is no direct information regarding their effect on the secretion of GI peptides, fecal transplants from rats submitted to a control diet to other rats fed a high-fat, high-glucose/fructose diet also improved insulin signaling[29]. This suggests the relevance of considering fecal transplants as a potential therapeutic intervention in humans. To do so, it will be critical to develop new models to understand the relevance of specific strains as well as the ecological relationships between bacterial species of the GM. This will clarify how these microorganisms promote metabolic disorders such as insulin resistance and the different types of diabetes. Of course, GI peptides are likely mediators of these effects since they were abundantly shown to regulate metabolic functions in the intestine as well as in central and peripheral tissues and organs.

4.

MICROBIAL METABOLITES

4.1 Short-Chain Fatty Acids Short-chain fatty acids such as acetate, butyrate, propionate and branched-chain fatty acids (BCFA) isovalerate, methylbutyrate and isobutyrate are important GM metabolites produced by Bifidobacterium, Lactobacillus and by a host of other species present in the intestine[30]. It is reported that SCFAs reduce gut permeability, 8

metabolic endotoxemia, oxidative stress and inflammation while improving overall metabolic status (see Figure 1). These effects are mediated through the activation of free fatty acid receptors 2 (GPR43) and 3 (GPR41) by the various SCFAs[31]. One proposed mechanism through which SCFAs exert their biological activities involve the regulation of GI peptide secretion. For instance, acetate production by the GM was reported to promote acylated ghrelin production in rodents[7]. Butyrate and propionate were proposed to stimulate GLP-1[32] and PYY[33] secretion by L-cells in rodent models. Interestingly, the deletion of GPR41 or GPR43 hindered the stimulatory effect of SCFA on PYY and GLP-1 production in intestinal L-cells of mice[32]. This suggests that GPR43 is necessary to promote SCFA-induced L-cell proliferation in the colon[34]. Among rats supplemented with dietary-resistant starch, increases in caecal butyrate-producing bacteria and SCFA levels as well as in circulating GLP-1 concentrations, β-cell mass, insulin sensitivity and pancreatic insulin content were also observed[35]. However, GLP-1 deletion inhibited the beneficial effects of prebiotics on weight gain, inflammation and glucose metabolism in mice[36]. Capsaicin supplementation also improved insulin signaling, butyrate production, GLP-1 levels, Firmicutes/Bacteroidetes ratio and Roseburia richness while decreasing plasma total ghrelin, TNF-α, IL-1β and IL-6 as well as Bacteroides and Parabacteroides abundance[37].

In pigs fed with alfalfa (enriched in insoluble fibers), increases in caecal butyrate levels as well as in the expression of PYY, proglucagon, GPR41 and SCFA transporters sodium monocarboxylate transporter 1 (SMCT1/SLC5A8 responsible for butyrate transport) and monocarboxylate transporter 1 (MCT1) indicate that SCFAs display intestinal and systemic activities[38]. In humans, propionate increased GLP-1 and PYY levels, which was associated with reductions in appetite, weight gain, intra-abdominal adipose tissue and hepatic steatosis as well as with improvements in glucose control[39]. Further, in response to fermentable dietary fibers, patients with type 2 diabetes who displayed the greatest improvements in glycated hemoglobin (HbA1c) had the largest increases in SCFA microbial producers[40]. These results were also associated with a decrease in microbial producers of indole and H2S. The production of SCFAs by the GM regulates the secretion of GI peptides through mechanisms that need to be thoroughly characterized. These GI peptides are known to influence metabolic functions. However, it is also suggested that the microbial production of SCFAs directly influences glucose homeostasis[41]. 9

Obesity is associated with an increased production of leptin by adipose tissues and the stomach. In pregnant mice submitted to caloric restriction or a high fat diet, low leptin levels were associated with increases in the overall microbial abundance of Allobaculum specie ID4 and Trichodesmium erythraeum[42]. Allobaculum are strict Gram-positive anaerobes using acetate to produce butyrate[43] while T. erythraeum micro-organisms require high iron levels to fix nitrogen and protect against oxidative stress[44]. These properties could explain how low leptin levels are associated with reduced inflammation and adipokines production[45]. In turn, it suggests that low acetate, butyrate or iron levels could reduce leptin secretion. On the other hand, decreased leptin levels were observed in HFD rats supplemented with Bifidobacterium longum, an acetate-producing bacterium. After acetate is synthesized by B. longum, it can be utilized by other bacteria by cross-feeding to produce other forms of SCFA (butyrate and propionate)[46]. Moreover, among diabetic mice with an inactive form of the leptin receptor, Clostridium butyricum supplementation improved glucose homeostasis, GLP-1 secretion and the abundance of SCFA (butyrate)-producing micro-organisms and reduced the number of Allobaculum in the intestine[47]. This suggested that butyrate inhibited leptin secretion while C. butyricum used an important proportion of the acetate to decrease its availability for other bacteria such as Allobaculum. In turn, these reduced acetate levels impaired Allobaculum growth in the intestine. In HFD mice treated with streptozotocin, Lactobacillus casei improved glucose control and lipid metabolism and increased the abundance of butyrateproducing Allobaculum and Bacteroides[48].

4.2 Other mechanisms Other GM products could influence metabolic functions such as glucose control. For instance, fatty acids produced by lactic acid bacteria were shown to increase CCK production in a mouse enteroendocrine cell line through a GPR40-dependant mechanism[49]. If this effect is confirmed in vivo, the anorexigenic effects of CCK could then prevent obesity and the onset of metabolic disorders. Another pathway is through bacterial species causing periodontitis, P. gingivalis, Tannerella forsythia and Prevotella intermedia which produce enzymes with DPP-IV activity known to degrade the active form of GLP-1[50]. In turn, these enzymes impair levels of released

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insulin when administered in mice. This suggests that even at the extreme proximal end of the GI tract, the GM could influence glucose homeostasis by disrupting the beneficial effects of incretins.

5.

DIETS AND NUTRACEUTICALS

There is an intricate relationship between diets and the GM. Ingested foods influence the gut’s microbial ecology, allowing the GM to then alter the biochemical nature of a variety of nutrients.

5.1 Amino acids Amino acids are essential for protein synthesis, but they can be transformed by the GM into bioactive metabolites. For instance, intermittent leucine deprivation was shown to influence the GM by increasing Bacteriodes, Alloprevotella and Rikenellaceae species while decreasing Lachnospiraceae. These effects prevented the onset of type 2 diabetes in db/db mice[51]. Similarly, tryptophan restriction was shown to decrease Enterocateriaceae, Lactobacillus, Bacteroides and Clostridium coccoides (Blautia coccoides) numbers and increase Roseburia sp. in fecal samples of obesity-prone rats[52]. These microbial changes increased thermogenesis and reduced food intake, weight gain and energy expenditure. These effects were also associated with reduced fasting glucose, insulin and leptin levels as well as increased GLP-1, glucagon and pancreatic polypeptide levels in the circulation. On the other hand, lysine restriction increased Actinobacteria, Saccharibacteria and Synergistetes, but decreased CCK concentrations in pigs[53].

5.2 Lipids Conjugated linoleic acid increased B. coccoides, Clostridium leptum (both of the phylum Firmicutes) and Bacteriodetes abundance, but decreased the number of Bifidobacterium species in mice[54]. The same treatment also decreased food intake while increasing leptin, ghrelin, ghrelin o-acylating ghrelin o-acytransferase (GOAT, ghrelin’s acylating enzyme), resistin, GPR39, glucagon and somatostatin expression. 11

5.3 Prebiotics Prebiotics are defined as “microbial food supplements that beneficially affect the host by improving its intestinal microbial balance”[55]. Prebiotics were initially associated with the consumption of fermentable fibers, but other classes of nutraceuticals such as polyphenols were also shown to exert positive outcomes on the GM and its host. In obesity-prone mice, prebiotic carbohydrates (oligofructose) increased GLP-2 levels and reduced gut permeability and inflammation[56]. In Zucker Diabetic Fatty rats, resistant starch increased the Bacteroidetes to Firmicutes ratio as well as GLP-1 plasma levels[57]. This outcome was also associated with an increase in insulin sensitivity.

Inulin is a fructooligosaccharide modulating GM ecology and improving metabolic functions. The latter prebiotic is reported to decrease caloric intake and respiratory quotient and improve glucose control in rats submitted to obesogenic conditions[58]. In addition, inulin treatment promoted the production of CCK, PYY, CCK, GLP-1 and butyryl-CoA: acetate CoA-transferase while reducing GIP levels in the caecum and colon[58,59]. These effects were also associated with increases in Bacteroidetes and Bifidobacterium and decreases in Clostridium clusters I and IV species. In patients with metabolic syndrome, a combined treatment with seven strains of probiotic bacteria and fructooligosaccharides improved BMI and glucose control and increased GLP-1 and PYY levels in the circulation[60]. In Apo E-deficient mice submitted to an omega-3depleted diet for 12 weeks, supplementation with inulin-type fructans reversed endothelial dysfunctions in mesenteric and carotid arteries, activated NO synthase (NOS), increased NO-producing bacteria and Akkermansia muciniphila and decreased bacterial species involved in secondary bile acid synthesis[61]. The treatment was also associated with increased GLP-1 production and bile acid turnover.

Other fermentable carbohydrates have shown beneficial effects on metabolic functions. Galactooligosaccharide supplementation reduced energy intake and fat pads while increasing caecal weight, GLP-1 and PYY secretion as well as B. bifidum and B. longum counts in rats[62,63]. However, galactooligosaccharide supplementation did not improve glucose control in T2D patients[64]. Our group has previously published that ghrelin secretion 12

profiles were positively associated with fiber intake in overweight and obese post-menopausal women[65]. Although the distinct type of fiber was not characterized, this data outlined the existence of an association between GI peptides secretion and the consumption prebiotic foods in humans. In turn, this suggests how the interplay between food selection, GI peptide secretion and gut microbial ecology influences the regulation of metabolic functions such as glucose control.

Several polyphenols have been shown to exert beneficial effects on the GM and organs and tissues with a key role in the regulation of metabolic functions. Resveratrol was initially isolated form grape skin and is reported to modify GM ecology while reducing the inflammatory status in mice[66]. These effects were also associated with increases in GLP-1 secretion and in the expression of the proglucagon gene, but required the presence of the active form of the GLP-1R. Epigallocathechin-3-gallate is a polyphenol derived from green tea that increased bacterial numbers of A. muciniphila in the intestine of mice[67]. This effect was also associated with the activation of TGR5 and increases in GLP-1 and PYY expression from L cells. These results support data indicating that GLP-1 and PYY production is regulated through direct and/or indirect mechanisms involving bile acids.

Prebiotics of different biochemical nature such as non-digestible carbohydrates and polyphenols have shown promising effects on glucose control in animal and human studies; however, the mechanisms through which these effects are mediated remain to be fully elucidated.

5.4 Probiotics Probiotics are live micro-organisms that induce health benefits when ingested by a host. Lactobacillus reuteri supplementation was associated with increased glucose-stimulated insulin secretion and the upregulation of GLP-1 and GLP-2 release in healthy humans[68]. In mice, Bacteriodes acidifaciens supplementation reduced weight gain and fat mass through increases in GLP-1 serum levels and in the expression of peroxisomeproliferator-activated receptor α (PPARα) in adipose tissue[69]. In rats treated with streptozotocin and submitted to a high fat diet, Lactobacillus rhamnosus NCDC treatment increased GLP-1 secretion while improving glucose 13

control and reducing circulating levels of markers of oxidative stress, free fatty acids, triglycerides and LDL cholesterol[70]. This intervention also increased Bifidobacteria and Lactobacillus counts in the caecum and adiponectin levels while decreasing TNF-α and IL-6 expression in epididymal fat tissues. Both the expression of GLP-1 or its analogue exendin-4 in a Lactoccocus lactis strain increased insulin secretion and glucose tolerance while reducing apoptosis in vitro in INS-1 cells or mice primary islets and in vivo in mice[71,72]. These results were proposed to be mediated through GLP-1R signaling by the activation of the PI3-K/AKT pathway.

6.

GASTROINTESTINAL PEPTIDES AND THE GUT MICROBIOTA

It was recently suggested that distinct GI peptides influence nutrient absorption/processing and inflammation through their effects on microbial ecology in the intestine (see Figure 2 and Table 1) [73,74]. This observation is highly pertinent since ghrelin-, PYY- and GLP-1-secreting cells are present in the lamina priora (where most fermenting bacteria are located) in the colon, so hormones are released towards the lumen[73,74]. For instance, ghrelin and PYY could exert their effects on the GM through their cationic antimicrobial properties against Gram-negative bacteria[75,76]. Accordingly, ghrelin levels were negatively correlated with the enrichment in Bifidobacterium, Lactobacillus and the Blautia coccoides-Eubacterium rectale group, but were positively correlated with Prevotella and Bacteroides numbers[77].

Other GI peptides were also shown to display antimicrobial effects with potential influence on the GM. Intestinal permeability is a key function which influences GM and its capacity to translocate and trigger systemic subchronic inflammation. In piglets submitted to E. coli K88 infection, treatment with VIP reduced gut permeability (upregulation of occludin in the ileal mucosa) and diarrhea, and promoted growth performance[78]. This treatment inhibited the production or expression of several pro-inflammatory factors such as IL-2, IL-6, IL12p40, IFN-γ, TNF-α, TLR2, TLR4, MyD88 and NF-κB, and increased the levels of anti-inflammatory markers such as IL-4, IL-10, TGF-β and S-IgA from the serum or ileal mucosal tissue. In addition, VIP reduced the phosphorylation of IκB-α, p-ERK, p-JNK and p-p38 in the ileum. These results indicate that VIP acts as a regulator of the GM, of the integrity of intestinal epithelial cells and of GI inflammation. It is noteworthy that 14

ghrelin was also shown to promote intestinal epithelial cell proliferation via a GHSR-1-dependent mechanism in vitro[79]. This indicates that some GI peptides influence intestinal permeability through the regulation of tight Recently, VIP and secretin were shown to display antibacterial activity against E. coli. Interestingly, another duodenal hormone sharing a high degree of homology with VIP and secretin, pituitary adenylate cyclaseactivating peptide (PACAP) was reported to exert antimicrobial effects against Gram-negative (E. coli, P. aeruginosa) and Gram-positive (S. aureus) bacteria[80]. In addition, VIP was also shown to exert antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, enterococcus faecalis, E. coli P. aeruginosa and Candida albicans[81]. This suggests the importance of further investigating the roles of secretin/VIP/PACAP in the regulation of the GM.

Glucagon-like peptide 1 agonists are potent anti-diabetic molecules used by clinicians. In mice, treatment with a GLP-1 agonist (Liraglutide) changed the overall structure of the GM and the relative abundance of its phylotypes[82]. Interestingly, this effect was not observed with saxagliptin, a DPP-IV inhibitor. This indicates that this GLP-1 agonist modulates GM composition, but questions remain regarding whether these effects are exerted directly after GLP-1 is transported towards the lumen, or indirectly mediated through yet uncharacterized neuroendocrine functions. In Wistar and Goto-Kakizaki rats, Liraglutide reduced body weight and improved glucose and lipid metabolism but, unexpectedly decreased overall GM abundance and diversity[83]. In addition, GLP-1 receptor agonists increased Bacteroides vulgatus, Alistipes spp., Faecalibacterium prausnitzii, A. muciniphila and Peptostreptococccus anaerobius growth/presence in T2D patients[84]. On the other hand, GLP-2 is another bioactive gene product of the glucagon gene with important bioactivity in the intestine. In animal models, GLP-2 reduced endotoxemia, bacterial translocation and subchronic inflammation. Interestingly, GLP-2 did not influence the GM of young rats, but reduced the number of pathogenic bacteria in the gut of older animals[85].

Although it is mainly produced by the adipose tissue, leptin is also co-secreted by ghrelin-producing cells in the stomach and its levels were positively correlated with the abundance of Bifidobacterium and Lactobacillus, but negatively associated with the abundance of Clostridium, Bacteriodes and Prevotella[77]. 15

Although the role of GI peptides on the regulation of metabolic functions is abundantly reported in the literature, the fact that some exert anti-microbial activities remains intriguing. Are they secreted or transported towards the lumen to control microbial ecology in the intestine? Do they play a complementary systemic role to that of the immune system? Nevertheless, GI peptides were shown to interact with the GM and influence key metabolic functions such as glucose control, but is there a stronger link between these two functions than initially expected?

7.

PERSPECTIVES AND CONCLUSION

Recent advances in gut microbial ecology have highlighted and supported the important role of the GM on the regulation of GI functions, GM metabolite production, intestinal permeability, bacterial/metabolite translocation, oxidative stress and inflammation (see Figure 1). The present review article was designed to highlight novel evidence supporting the role of an intricate crosstalk between the GM and GI peptides in the maintenance of glucose homeostasis. While it was previously shown that the GM influences the secretion of key GI peptides such as ghrelin, GLP-1 and PYY, it is newly proposed that GI peptides also modulate GM ecology and function. However, essential questions remain to be addressed regarding how these interactions take place, and their impact on vital functions such as glucose homeostasis.

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8.

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25

GP Bowel movement Inflammation

Food

intake Figure 2. Potential mechanisms through which gastrointestinal peptides (GP) could modulate the gut microbiota (GM). Gastrointestinal peptides regulate food intake and metabolic functions through neuroendocrine pathways. This affects both quality and quantity of nutrients reaching the GM. Neuronal responses to GP also modulate bowel movement and food retention within the different parts of the intestine and consequently regulate the ecological niche of the GM. The crosstalk between the host and the GM is also influenced by GPs’ regulation of inflammatory pathways. Other mechanisms through which GP potentially exert their roles on GM include the modulation of intestinal permeability and their direct antibacterial effects.

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Table 1: Effects of gastrointestinal peptides (GP) on the gut microbiota (GM) GP

Model

Procedure

Effects

Ref

Direct effects In vivo BALB/c mice sepsis model

GHRL (1 nmol/mouse, IP) 12 and 18 h after induction of sepsis

Important reduction in tissue bacterial load

In vitro Bacterial culture

Treatment of a mid-log E. coli bacterial culture with different concentrations of GHRL

Direct bacterial growth inhibition and bactericidal effect produced through membrane destabilization

PYY

In vitro Bacterial culture

Growth medium was supplemented with different concentrations of a PYY derivative (SPYY) to determine the Minimum Inhibition Concentration (MIC)

SPYY inhibited the growth of E. coli, Enterococcus faecalis, several molds, and Protista through disruption of their lipid membrane

73

PACAP

In vitro Bacterial culture

For PACAP and its derivatives MIC was determined using radial diffusion and broth dilution assays in several bacterial species

PACAP and its derivatives potently inhibited E.coli, Staphylococcus aureus, Pseudomonas aeruginosa while having a marginal effect on Bacillus aeruginosa

76

VIP

In vitro Bacterial culture

MIC was determined using a radial diffusion assay

VIP inhibited E. coli, growth in Pseudomonas aeruginosa, Candida albicans strains. Effect were limited in Streptococcus mutans

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VIP

In vivo Piglet model of diarrhea.

Liraglutide (GLP-1 receptor agonist)

In vivo Hyperglycemic mice

ApoE -/- mice received a single injection of streptozotocin. Mice were submitted Liraglutide reduced microbial diversity in normoglycemic mice but restored it in to a high fat diet (HFD) and injected daily with liraglutide (0.4 mg/kg, SC) hyperglycemic animals. After treatments, GM of hyperglycemic and lean mice was closely related

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In vivo Obese rats with or without diabetes

Wistar and Goto-Kakizaki (diabetes-prone) rats were submitted to HFD and administered dailsy with liraglutide (0.4 mg/kg, SC)

Liraglutide reduced the diversity and richness of GM independently of the glycemic status. GM of treated animals was closely related to the one of lean rats

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In vivo Aging rats

3- and 26-month old rats were injected daily with GLP-2 for 14 days

Small effect of GLP-2 on the GM. Reduced Treponema and Spirochaetae abundance and higher Anaerovibrio numbers in aged mice

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Ghrelin (GHRL)

GLP-2

Indirect or uncharacterized effects Four and six days after an oral administration of enterohemorrhagic E. coli, VIP restored diversity, abundance and composition of the gut microbiota (GM) piglets were injected with VIP (10 nmol/pig, IP)

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