Protocol for the preclinical evaluation of gut barrier function and immune interaction in an HT-29/PBMC co-culture model.

Here, we present a protocol for the pre-clinical evaluation of gut barrier function and immune interaction in an HT-29/PBMC co-culture model. We describe steps for culturing HT-29 cells and peripheral blood mononuclear cells (PBMCs), assembling the co-culture model, and testing cell viability. We then detail procedures for performing efficacy tests through stress challenges and barrier permeability assays. For complete details on the use and execution of this protocol, please refer to Abot et al.1.

Role of the intestinal microbiota in contributing to weight disorders and associated comorbidities.

SUMMARYThe gut microbiota is a major factor contributing to the regulation of energy homeostasis and has been linked to both excessive body weight and accumulation of fat mass (i.e., overweight, obesity) or body weight loss, weakness, muscle atrophy, and fat depletion (i.e., cachexia). These syndromes are characterized by multiple metabolic dysfunctions including abnormal regulation of food reward and intake, energy storage, and low-grade inflammation. Given the increasing worldwide prevalence of obesity, cachexia, and associated metabolic disorders, novel therapeutic strategies are needed. Among the different mechanisms explaining how the gut microbiota is capable of influencing host metabolism and energy balance, numerous studies have investigated the complex interactions existing between nutrition, gut microbes, and their metabolites. In this review, we discuss how gut microbes and different microbiota-derived metabolites regulate host metabolism. We describe the role of the gut barrier function in the onset of inflammation in this context. We explore the importance of the gut-to-brain axis in the regulation of energy homeostasis and glucose metabolism but also the key role played by the liver. Finally, we present specific key examples of how using targeted approaches such as prebiotics and probiotics might affect specific metabolites, their signaling pathways, and their interactions with the host and reflect on the challenges to move from bench to bedside.

Effect of the dietary supplement PERMEAPROTECT+ TOLERANCE© on gut permeability in a human co-culture epithelial and immune cells model.

Background and objective: The leaky gut syndrome is characterized by an intestinal hyperpermeability observed in multiple chronic disorders. Alterations of the gut barrier are associated with translocation of bacterial components increasing inflammation, oxidative stress and eventually dysfunctions of cellular interactions at the origin pathologies. Therapeutic and/or preventive approaches have to focus on the identification of novel targets to improve gut homeostasis. In this context, this study aims to identify the role of PERMEAPROTECT + TOLERANE©, known as PERMEA, a food complement composed of a combination of factors (including l-Glutamine) known to improve gut physiology. Methods: We tested the effects of PERMEA or l-Glutamine alone (as reference) on gut permeability (FITC dextran method, expression of tight junctions) and its inflammatory/oxidative consequences (cytokines and redox assays, RT-qPCR) in a co-culture of human cells (peripheral blood mononuclear cells and intestinal epithelial cells) challenged with TNFα. Results: PERMEA prevented intestinal hyperpermeability induced by inflammation. This was linked with its antioxidant and immunomodulatory properties showing a better efficacity than l-Glutamine alone on several parameters including permeability, global antioxidant charge and production of cytokines. Conclusion: PERMEA is more efficient to restore intestinal physiology, reinforcing the concept that combination of food constituents could be used to prevent the development of numerous diseases.

Limosilactobacillus reuteri BIO7251 administration improves metabolic phenotype in obese mice fed a high fat diet: an inter-organ crosstalk between gut, adipose tissue and nervous system.

Gut microbiota is implicated in the control of host physiology by releasing bioactive actors that could exert a direct or indirect effect on tissue. A dysfunction of the gut microbiota to tissue axis could participate in the development of pathological states such as obesity and diabetes. The aim of this study was to identify the metabolic effect of Limosilactobacillus reuteri (known as Lactobacillus reuteri) BIO7251 (L. reuteri BIO7251) isolated from Corsican clementine orange. Body weight gain, adiposity, glucose tolerance, glucose absorption and food intake were measured in mice fed a high-fat diet in response to a preventive oral administration of L. reuteri BIO7251. This strain of bacteria exerts a beneficial effect on body weight gain by decreasing the subcutaneous adipose tissue mass. The treatment with L. reuteri BIO7251 decreases glucose absorption and food intake in obese/diabetic mice. L. reuteri BIO7251 could be tested as new probiotic strain that could manage body weight during obesity.

Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Pasteurized Akkermansia muciniphila improves glucose metabolism is linked with increased hypothalamic nitric oxide release.

Background and objective: Pasteurized Akkermansia muciniphila cells have shown anti-diabetic effects in rodents and human. Although, its primary site of action consists in maintaining the gut barrier function, there are no study exploring if A. muciniphila controls glycemia via a gut to brain axis. Targeting the gut motility represents an alternative pathway to treat hyperglycemia. Here, we tested the impact of pasteurized A. muciniphila on gut motility, gut-brain axis and glucose metabolism. Methods: We used mice fed a 45% high-fat (HFD) treated or not with pasteurized A. muciniphila MucT during 12 weeks. We measured the effects of the treatment on body weight gain, glucose metabolism (insulin, glycemia, glucose tolerance), gut contraction and enteric neurotransmitter release, and hypothalamic nitric oxide (NO) release. Results: We show that pasteurized A. muciniphila exerts positive effects on different metabolic parameters such as body weight, fat mass, insulin, glycemia and glucose tolerance. This could be explained by the ability of pasteurized A. muciniphila supplementation to decrease duodenal contraction and to increase hypothalamic NO release in HFD mice. Conclusion: We demonstrate a novel mode of action of pasteurized A. muciniphila explaining its beneficial impact on the control of glycemia in a preclinical model of type 2 diabetes via gut-brain axis signaling.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

How does apelin affect LH levels? An investigation at the level of GnRH and KNDy neurons.

Hypothalamic control of reproduction relies on GnRH and kisspeptin (KP) secretions. KP neurons are sensitive to sex steroids and metabolic status and their distribution overlaps with neurons producing apelin, a metabolic hormone known to decrease LH secretion in rats. Here, we observed neuroanatomical contacts between apelin fibers and both KP and GnRH neurons in the hypothalamus of male rodents. Intracerebroventricular apelin infusion for 2 weeks in male mice did not decrease LH levels nor did it affect gene expression for KP, neurokinin B and dynorphin. Finally, increasing apelin concentrations did not modulate Ca2+ levels of cultured GnRH neurons, while 10 µM apelin infusion on forskolin pretreated GnRH neurons revoked a rhythmic activity in 18% of GnRH neurons. These results suggest that acute apelin effect on LH secretion does not involve modulation of gene expression in KP neurons but may affect the secretory activity of GnRH neurons.

Glucose Stimulates Gut Motility in Fasted and Fed Conditions: Potential Involvement of a Nitric Oxide Pathway.

(1) Background: Type 2 diabetes (T2D) is associated with a duodenal hypermotility in postprandial conditions that favors hyperglycemia and insulin resistance via the gut-brain axis. Enterosynes, molecules produced within the gut with effects on the enteric nervous system, have been recently discovered and pointed to as potential key modulators of the glycemia. Indeed, targeting the enteric nervous system that controls gut motility is now considered as an innovative therapeutic way in T2D to limit intestinal glucose absorption and restore the gut-brain axis to improve insulin sensitivity. So far, little is known about the role of glucose on duodenal contraction in fasted and fed states in normal and diabetic conditions. The aim of the present study was thus to investigate these effects in adult mice. (2) Methods: Gene-expression level of glucose transporters (SGLT-1 and GLUT2) were quantified in the duodenum and jejunum of normal and diabetic mice fed with an HFD. The effect of glucose at different concentrations on duodenal and jejunal motility was studied ex vivo using an isotonic sensor in fasted and fed conditions in both normal chow and HFD mice. (3) Results: Both SGLT1 and GLUT2 expressions were increased in the duodenum (47 and 300%, respectively) and jejunum (75% for GLUT2) of T2D mice. We observed that glucose stimulates intestinal motility in fasted (200%) and fed (400%) control mice via GLUT2 by decreasing enteric nitric oxide release (by 600%), a neurotransmitter that inhibits gut contractions. This effect was not observed in diabetic mice, suggesting that glucose sensing and mechanosensing are altered during T2D. (4) Conclusions: Glucose acts as an enterosyne to control intestinal motility and glucose absorption through the enteric nervous system. Our data demonstrate that GLUT2 and a reduction of NO production could both be involved in this stimulatory contracting effect.

Camu-Camu Reduces Obesity and Improves Diabetic Profiles of Obese and Diabetic Mice: A Dose-Ranging Study.

Overweight, obesity, and their comorbidities are currently considered a major public health concern. Today considerable efforts are still needed to develop efficient strategies able to attenuate the burden of these diseases. Nutritional interventions, some with plant extracts, present promising health benefits. In this study, we evaluated the action of Camu-Camu (Myrciaria dubia), an Amazonian fruit rich in polyphenols and vitamin C, on the prevention of obesity and associated disorders in mice and the abundance of Akkermansia muciniphila in both cecum and feces. Methods: We investigated the dose-response effects of Camu-Camu extract (CCE) in the context of high-fat-diet (HFD)-induced obesity. After 5 weeks of supplementation, we demonstrated that the two doses of CCE differently improved glucose and lipid homeostasis. The lowest CCE dose (62.5 mg/kg) preferentially decreased non-HDL cholesterol and free fatty acids (FFA) and increased the abundance of A. muciniphila without affecting liver metabolism, while only the highest dose of CCE (200 mg/kg) prevented excessive body weight gain, fat mass gain, and hepatic steatosis. Both doses decreased fasting hyperglycemia induced by HFD. In conclusion, the use of plant extracts, and particularly CCE, may represent an additional option in the support of weight management strategies and glucose homeostasis alteration by mechanisms likely independent from the modulation of A. muciniphila abundance.

Reactive Oxygen Species/Reactive Nitrogen Species as Messengers in the Gut: Impact on Physiology and Metabolic Disorders.

Significance: The role of reactive oxygen/nitrogen species as "friend" or "foe" messengers in the whole body is well characterized. Depending on the concentration in the tissue considered, these molecular actors exert beneficial or deleterious impacts leading to a pathological state, as observed in metabolic disorders such as type 2 diabetes and obesity. Recent Advances: Among the tissues impacted by oxidation and inflammation in this pathological state, the intestine is a site of dysfunction that can establish diabetic symptoms, such as alterations in the intestinal barrier, gut motility, microbiota composition, and gut/brain axis communication. In the intestine, reactive oxygen/nitrogen species (from the host and/or microbiota) are key factors that modulate the transition from physiological to pathological signaling. Critical Issues: Controlling the levels of intestinal reactive oxygen/nitrogen species is a complicated balance between positive and negative impacts that is in constant equilibrium. Here, we describe the synthesis and degradation of intestinal reactive oxygen/nitrogen species and their interactions with the host. The development of novel redox-based therapeutics that alter these processes could restore intestinal health in patients with metabolic disorders. Future Directions: Deciphering the mode of action of reactive oxygen/nitrogen species in the gut of obese/diabetic patients could result in a future therapeutic strategy that combines nutritional and pharmacological approaches. Consequently, preventive and curative treatments must take into account one of the first sites of oxidative and inflammatory dysfunctions in the body, that is, the intestine. Antioxid. Redox Signal. 37, 394-415.

Identification of new enterosynes using prebiotics: roles of bioactive lipids and mu-opioid receptor signalling in humans and mice.

OBJECTIVE: The enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes. DESIGN: We measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human. RESULTS: We found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic. CONCLUSIONS: Using a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

Targeting the Enteric Nervous System to Treat Metabolic Disorders? "Enterosynes" as Therapeutic Gut Factors.

The gut-brain axis is of crucial importance for controlling glucose homeostasis. Alteration of this axis promotes the type 2 diabetes (T2D) phenotype (hyperglycaemia, insulin resistance). Recently, a new concept has emerged to demonstrate the crucial role of the enteric nervous system in the control of glycaemia via the hypothalamus. In diabetic patients and mice, modification of enteric neurons activity in the proximal part of the intestine generates a duodenal hyper-contractility that generates an aberrant message from the gut to the brain. In turn, the hypothalamus sends an aberrant efferent message that provokes a state of insulin resistance, which is characteristic of a T2D state. Targeting the enteric nervous system of the duodenum is now recognized as an innovative strategy for treatment of diabetes. By acting in the intestine, bioactive gut molecules that we called "enterosynes" can modulate the function of a specific type of neurons of the enteric nervous system to decrease the contraction of intestinal smooth muscle cells. Here, we focus on the origins of enterosynes (hormones, neurotransmitters, nutrients, microbiota, and immune factors), which could be considered therapeutic factors, and we describe their modes of action on enteric neurons. This unsuspected action of enterosynes is proposed for the treatment of T2D, but it could be applied for other therapeutic solutions that implicate communication between the gut and brain.

Pharmacological inhibition of the F1 -ATPase/P2Y1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation.

AIM: The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. METHOD: Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. RESULTS: Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. CONCLUSIONS: Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

Central Effects of Beta-Blockers May Be Due to Nitric Oxide and Hydrogen Peroxide Release Independently of Their Ability to Cross the Blood-Brain Barrier.

Propranolol is the first-line treatment for infants suffering from infantile hemangioma. Recently, some authors raised the question of potential neurologic side effects of propranolol due to its lipophilic nature and thus its ability to passively cross the blood-brain barrier (BBB) and accumulate into the brain. Hydrophilic beta-blockers, such as atenolol and nadolol, where therefore introduced in clinical practice. In addition to their classical mode of action in the brain, circulating factors may modulate the release of reactive oxygen/nitrogen species (ROS/RNS) from endothelial cells that compose the BBB without entering the brain. Due to their high capacity to diffuse across membranes, ROS/RNS can reach neurons and modify their activity. The aim of this study was to investigate other mechanisms of actions in which these molecules may display a central effect without actually crossing the BBB. We first performed an oral treatment in mice to measure the accumulation of propranolol, atenolol and nadolol in different brain regions in vivo. We then evaluated the ability of these molecules to induce the release of nitric oxide (NO) and hydrogen peroxide (H2O2) ex vivo in the hypothalamus. As expected, propranolol is able to cross the BBB and is found in brain tissue in higher amounts than atenolol and nadolol. However, all of these beta-blockers are able to induce the secretion of signaling molecules (i.e., NO and/or H2O2) in the hypothalamus, independently of their ability to cross the BBB, deciphering a new potential deleterious impact of hydrophilic beta-blockers in the brain.

Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction.

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.

Bisphenol S exposure affects gene expression related to intestinal glucose absorption and glucose metabolism in mice.

Bisphenol S, an industrial chemical, has raised concerns for both human and ecosystem health. Yet, health hazards posed by bisphenol S (BPS) exposure remain poorly studied. Compared to all tissues, the intestine and the liver are among the most affected by environmental endocrine disruptors. The aim of this study was to investigate the molecular effect of BPS on gene expression implicated in the control of glucose metabolism in the intestine (apelin and its receptor APJ, SGLT1, GLUT2) and in the liver (glycogenolysis and/or gluconeogenesis key enzymes (glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK)) and pro-inflammatory cytokine expression (TNF-α and IL-1ß)). BPS at 25, 50, and 100 µg/kg was administered to mice in water drink for 10 weeks. In the duodenum, BPS exposure reduces significantly mRNA expression of sodium glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2), apelin, and APJ mRNA. In the liver, BPS exposure increases the expression of G6Pase and PEPCK, but does not affect pro-inflammatory markers. These data suggest that alteration of apelinergic system and glucose transporters expression could contribute to a disruption of intestinal glucose absorption, and that BPS stimulates glycogenolysis and/or gluconeogenesis in the liver. Collectively, we reveal that BPS heightens the risk of metabolic syndrome.

Impact of Intestinal Peptides on the Enteric Nervous System: Novel Approaches to Control Glucose Metabolism and Food Intake.

The gut is one of the most important sources of bioactive peptides in the body. In addition to their direct actions in the brain and/or peripheral tissues, the intestinal peptides can also have an impact on enteric nervous neurons. By modifying the endogenousproduction of these peptides, one may expect modify the "local" physiology such as glucose absorption, but also could have a "global" action via the gut-brain axis. Due to the various origins of gut peptides (i.e., nutrients, intestinal wall, gut microbiota) and the heterogeneity of enteric neurons population, the potential physiological parameters control by the interaction between the two partners are multiple. In this review, we will exclusively focus on the role of enteric nervous system as a potential target of gut peptides to control glucose metabolism and food intake. Potential therapeutic strategies based on per os administration of gut peptides to treat type 2 diabetes will be described.

Effects of Bisphenol S on hypothalamic neuropeptides regulating feeding behavior and apelin/APJ system in mice.

Since 2010, Bisphenol A (BPA), an endocrine disruptor has been restricted and replaced by analogues like Bisphenol S (BPS). However, little is known about BPS effects and growing concern have suspected the "BPA-free" Label. Several recent studies suggest that BPS is associated with increased risk of diabetes and obesity. However, the underlying mechanisms remain unidentified. The current study investigates investigate BPS effects on hypothalamic neuropeptides regulating feeding behavior, either orexigenic or anorexigenic in Swiss Albino mice. We also studied the effect of BPS on the apelinergic system (apelin/apelin receptor (APJ)) as an original physiological system with pleiotropic actions. Bisphenol S at 25, 50, 100 µg/kg was administered to mice in water drink for 10 weeks started after weaning. Our results showed that BPS exposure alters orexigenic hypothalamic neuropeptide (AgRP) regulating feeding behavior but not anorexigenic neuropeptides (POMC, CART). Such orexigenic alterations may underlay appetite disorders leading to a concomitant food intake and body weight gain increase. In addition, data show that BPS affects the hypothalamic apelinergic system. We found a significant decrease in APJ mRNA but not in apelin expression. Based on hypothalamic APJ distribution, we suggested a potent specific physiological alteration of this receptor in mediating neuroendocrine responses in hypothalamus. Thus, our findings provide that BPS exposure could contribute to the development of obesity and metabolic disorders.