BACKGROUND:Improving n-3 polyunsaturated fatty acids (PUFAs) dietary intake could modify the characteristics of breast milk. The "Bleu-Blanc-Cœurr" (BBC) food products display increased α-linolenic acid (ALA) content compared with standard food items. OBJECTIVES:This study aims to evaluate whether improving ALA dietary intake by animal and vegetal BBC products during pregnancy and lactation increases ALA content of breast milk on day (d) 21 postpartum (PP) (primary aim), modifies the fatty acids (FAs), human milk oligosaccharides (HMOs), hormone and immune composition of breast milk, as well as the intestinal microbiota of neonates (secondary aims). METHODS:In a single-center, interventional, randomized, open-label trial, adult females, ≤28 wk of amenorrhea, substituted (BBC group) or not [control group (CTRL)] their usual food with BBC animal and vegetal products containing ALA from month 7 of pregnancy until d45-PP. Food intake, breast milk FAs, lipidome, insulin, leptin, adiponectin, interleukin (IL)-6, lactoferrin, immunoglobulin (Ig) A and G, HMOs, and the fecal microbiota composition of neonates were analyzed. RESULTS:Of the 66 included and randomly assigned females, 58 were analyzed. In breast milk, ALA and n-3 PUFA levels were, respectively, 75% and 52% higher at d21-PP, and 38% and 22% higher at d45-PP, in the BBC (n = 28) than in the CTRL (n = 30) groups. At d21-PP, the breast milk lipidome, insulin, IL-6, IgG (P < 0.05), and HMO levels (P < 0.01) differed. The alpha-diversity of the fecal microbiota of neonates at d21-PP was greater (P < 0.05) in the BBC than in the CTRL groups. CONCLUSIONS:Improvement in alpha-linolenic acid dietary intake modifies nutritional and non-nutritional characteristics of breast milk, as well as the fecal microbiota of neonates. Whether this could lead to potential health benefits deserves further investigation. This trial was registered at clinicaltrials.gov as NCT03805997.

Les complications métaboliques de l’obésité sont associées à une perte d’homéostasie intestinale et à une inflammation chronique de bas grade. Centrales énergétiques des entérocytes, les mitochondries jouent un rôle clé dans le maintien de cette homéostasie. Nous avons évalué si leur altération pouvait contribuer à l’hyperperméabilité intestinale observée dans l’obésité. Chez la souris, un excès de lipides alimentaires provoque une accumulation de gouttelettes lipidiques dans les entérocytes, entraînant une baisse du nombre et de l’activité mitochondriale, associée à une perte du phénotype mature et de la fonction de barrière. Dans les organoïdes jéjunaux murins, la réduction de l’activité mitochondriale limite la différenciation des progéniteurs en entérocytes matures, pouvant favoriser l’hyperperméabilité. Dans le côlon, le régime hyperlipidique induit aussi une dysfonction mitochondriale et une hyperperméabilité liée à une dysbiose marquée par les Desulfovibrionaceae et les sulfures, inhibiteurs du complexe IV mitochondrial. Ces travaux soulignent le rôle clé des mitochondries dans les déséquilibres intestinaux liés à l’obésité et ouvrent de nouvelles pistes thérapeutiques.

BACKGROUND:Alcohol use disorder (AUD) is a complex condition affecting several body systems. Gut microbiota alterations, intestinal-barrier disruption, and the consequent translocation of metabolites foster chronic inflammation, lower short-chain fatty acid (SCFA) output, and depleted beneficial bacteria may contribute to transcriptional, epigenetic, and metabolic changes that influence ethanol preference. METHODS:Two experimental phases were used. T1 (8 weeks): mice received either the American Institute of Nutrition standard diet (AING) or a high-sugar-butter (HSB) diet. T2 (4 weeks): HSB animals switched to AING (SWITCH), while AING mice maintained the same diet. Each diet arm was split into ethanol (EtOH; free access to 10% ethanol) or H2O, generating four groups (AING + H2O, AING + EtOH, SWITCH + H2O, and SWITCH + EtOH). Sample processing involved colonic-content collection, 16S rRNA sequencing, microbiome profiling, functional inference, metabolic-network analysis, and SCFA/amino acid quantification. RESULTS:SWITCH + EtOH mice displayed high ethanol consumption and preference, whereas AING + EtOH mice showed ethanol aversion. Their colonic microbiota differed markedly; amino acid metabolism fell, secondary bile acid synthesis rose, and SCFA production dropped in SWITCH + EtOH animals. Direct measurements confirmed significant reductions in butyrate, acetate, propionate, and selected amino acids. Network analysis revealed enrichment of bacterial metabolism, oxidative stress, and dopamine pathway genes. CONCLUSIONS:Diet-induced dysbiosis, reflected in shifts in microbiota-derived metabolites, was associated with excessive alcohol intake; the metabolites identified can represent potential therapeutic targets for AUD.

OBJECTIVE:Obesity and overweight are associated with low-grade inflammation induced by adipose tissue expansion and perpetuated by altered intestinal homeostasis, including increased epithelial permeability. Intestinal epithelium functions are supported by intestinal epithelial cells (IEC) mitochondria function. However, diet-induced obesity (DIO) may impair mitochondrial activity of IEC and consequently, intestinal homeostasis. The aim of the project was to determine whether DIO alters the mitochondrial function of IEC, and what are the consequences on intestinal homeostasis. METHODS:C57Bl/6J mice were fed a control diet for 22 weeks or a high fat diet (58 kcal% fat). Bioenergetic adaptations of IEC were evaluated on isolated crypts and villi from mouse jejunum. To determine the link between mitochondrial function and alterations of intestinal homeostasis in response to lipid overload, we used the jejunal epithelial cell line IPEC-J2 in vitro and mouse jejunum organoids. RESULTS:Here, we report that DIO in mice induced lipid metabolism adaptations favoring lipid storage in IEC together with reduced number, altered dynamics and diminished oxidative phosphorylation activity of IEC mitochondria. Using the IPEC-J2 cell line, we showed that IEC lipid metabolism and oxidative stress machinery adaptations preceded mitochondrial bioenergetic ones. Moreover, we unraveled the intricate link between IEC energetic status and proliferation / differentiation balance since enhancing mitochondrial function with the AMPK activator AICAR in jejunal organoids reduced proliferation and initiated IEC differentiation and conversely. We confirmed that the reduced IEC mitochondrial function observed in DIO mice was associated with increased proliferation and reduced differentiation, promoting expression of the permissive Cldn2 in the jejunal epithelium of DIO mice. CONCLUSIONS:Our study provides new insights into metabolic adaptations of IEC in obesity by revealing that excess lipid intake diminishes mitochondrial number in IEC, reducing IEC differentiation that contribute to increased epithelial permeability.

BACKGROUND & AIMS:Alternating periods of excessive and restrained eating results in weight cycling ('yo-yo' effect), a suspected risk factor for eating behavior dysregulation such as binge eating. The hypothesis that recurrent diet alternation alters hedonic feeding regulation by changing either or both intestinal microbiota and brain neuronal and glial regulation in mouse is tested. METHODS:C57BL/6 mice undergo 3 cycles of 1 week of western diet (WD) separated by 2 weeks of chow diet (CYCL group) or remain under chow diet (CTRL group). RESULTS:CYCL mice exhibit weight cycling, with enhanced weight gain upon each WD feeding phase and increased energy intake specifically during the first hours following WD re-introduction, reminiscent of binge-eating episodes. Expression of reward-related genes in the striatum and thickness of the astro-glial barrier in the brain stem is enhanced in CYCL mice. Diet alternation induces caecal dysbiosis in CYCL mice. Gut microbiota transfer from CYCL mice to naive recipient mice recapitulates the altered eating behavior upon WD exposure. CONCLUSIONS:Alternation between high-energy and standard diets is established to durably remodel the gut microbiota and the brain toward a profile associated with an increase in hedonic appetite and that this microbiota signature affects hedonic feeding regulation.

Excessive consumption of high-fat, high-sugar diets promotes obesity, metabolic syndrome, and chronic inflammation through increased fat accumulation. While adopting a balanced diet promotes weight loss and improvements in various physiological parameters, persistent alterations in microbiota composition and function, metabolic imbalances, and behavioral changes may increase vulnerability to ethanol consumption and preference. In this context, the present study aims to investigate how switching from a high-sugar, high-saturated fat diet (HSB) to a standard diet (AIN93G) affects: (I) cecal microbiota composition and function, (II) colonic homeostasis, and (III) vulnerability to voluntary ethanol consumption in mice. To conduct the study, six animals were maintained on the standard diet, while 12 were given the HSB diet for 8 weeks. After this period, the HSB mice were switched to AIN93G for 4 weeks, with one subgroup given access exclusively to water (SWITCH) and another to water or a 10% ethanol solution (SWITCH+EtOH). The evaluation included measurements of body weight, adiposity index, cecal microbial composition, metabolomic profile, gut and hepatic morphology, transcriptional regulation of genes involved in colonic homeostasis and striatal dopaminergic neurotransmission, as well as ethanol consumption and preference. The results indicate that switching to the standard diet does not completely reverse obesity-induced alterations. Persistent cecal dysbiosis, metabolic imbalances, and dopaminergic sensitization increase the predisposition to compulsive alcohol consumption and perpetuate epithelial and hepatic dysfunction. Therefore, post-obesity interventions should combine weight management with strategies to restore microbiota and intestinal barrier function, along with measures to reduce vulnerability to reinforcement-seeking behaviors.

An impaired gut barrier has emerged as a potential driver of the low-grade inflammation that accompanies obesity and its complications. Among these, hyperglycemia per se has been proposed to sustain such increased intestinal permeability and subsequent translocation of bacterial endotoxins in the systemic circulation. Because reduced insulin signaling in the gut epithelium has also been reported upon obese conditions, we hypothesized that, beyond hyperglycemia, defective intestinal insulin signaling could directly compromise epithelial integrity. To mimic this diabesity feature, we induced deletion of the insulin receptor (IR) in the adult gut epithelium of IRΔGUT mice. Remarkably, gut IR loss persistently maintained normal body weight and glucose homeostasis, thereby allowing the specific role of insulin action to be investigated. While IRΔGUT mice exhibited increased intestinal paracellular permeability, mechanistic characterization of this gut leakiness revealed that IRΔGUT mice displayed a rapid and drastic decline in Paneth cells anti-microbial defenses. This paralleled the onset of a cecal dysbiosis, as characterized by increased abundance of Pseudomonadota , and enhanced microbiota encroachment. Of note, IRΔGUT mice exhibited intestinal stem cell (ISC) defects, as evidenced by reduced expression of ISC markers and ISC-mediated growth of intestinal organoids. Although expression of niche factors such as Wnt3a was diminished in Paneth cells isolated from IRΔGUT mice, pharmacological activation of the canonical Wnt pathway failed to rescue the growth defects of IR-deleted gut organoids. The direct contribution of IR-downstream signaling to ISCs homeostasis was confirmed by the transcriptional reprogramming of FACS-sorted ISCs from IRΔISC mice. Finally, while gut IR loss did not worsen endotoxemia or impaired glycemic control upon HFD-feeding, IRΔGUT displayed a higher susceptibility to chemically induced colitis and enteric infections ( S. typhimurium , C. rodentium ), underscoring intestinal insulin signaling as a key determinant of barrier integrity and epithelial homeostasis. ### Competing Interest Statement The authors have declared no competing interest. European Foundation for the Study of Diabetes, EFSD/Novo Nordisk Programme 2014 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE14-0044-01, GutBarrIR Société Francophone du Diabète, https://ror.org/05gdhxj91, SFD/Roche Diabetes Care 2018 Université Paris Cité, https://ror.org/05f82e368, IdEx Emergence 2020, InidEx DiabetEx 2025, DHU AUTHORS 2016 Fondation pour la Recherche Médicale

Dietary protein reduces energy intake in following meals by signaling directly or indirectly to the brain. We recently observed differences in plasma amino acid kinetics and intra-gastric behavior between micellar casein (MC) and sodium caseinate (SC) in pigs, two factors that impact food intake. Our objective was to clarify whether the supramolecular structure of casein, given as a preload to pigs, impacts on subsequent food intake. Overnight fasted pigs were allowed to consume casein drinks differing in casein macromolecular structure (SC vs MC) within 5 min in a cross-over study. Ad libitum intake of their regular feed was assessed during 1 h, either 1 or 4 h after casein drink ingestion. To evaluate the potential mechanisms at play, gastric emptying of the casein drinks radiolabeled with 99Tc-colloïd was followed using gamma-scintigraphy while plasma kinetics of ghrelin, GLP-1, insulin and free amino acids were evaluated. The amount of feed consumed 1, but not 4 h, after SC ingestion was lower than the amount of feed consumed after MC ingestion (P = 0.03). Gastric emptying parameters, plasma ghrelin, GLP-1 and insulin kinetics after both types of casein ingestion were not significantly different (P > 0.05). However, plasma free amino acid concentrations, known to reduce food intake, increased after both SC and MC ingestion but was greater after SC than MC ingestion from 60 to 120 min (P = 0.009). In conclusion, casein supramolecular structure in a preload drink impacts differently subsequent energy intake, likely due to difference in amino acid bioavailability. Micellar casein exhibits less anorectic effect than sodium caseinate, a property that could benefit population with high protein need but low appetite such as elderly.

Background & Aims: Alternating periods of excessive and restrained eating results in weight cycling, a known risk factor for eating behavior dysregulation such as binge eating. Diet alternation also induces changes in intestinal microbiota composition. We tested the hypothesis that recurrent diet alternation alters hedonic feeding regulation by changing either or both intestinal microbiota and brain homeostasis in mouse. Methods: C57BL/6 mice underwent 3 cycles of 1 week of western diet (WD, 45% kcal from fat) separated by 2 weeks of chow diet (CYCL group) or staid under chow diet (CTRL group). Food intake was monitored after each dietary change. Striatum, hypothalamus, brainstem and caecal content were collected before the third WD introduction in CYCL mice and in CTRL mice. Microbiota transfer from CYCL or CTRL mice into naive recipient mice was performed to investigate whether gut microbiota per se could explain differences in eating behavior during weight cycling. Results: Diet alternation in CYCL mice resulted in weight cycling, with enhanced weight gain upon each WD feeding phase. CYCL mice increased their energy intake specifically during the first hours following WD re-introduction, reminiscent of binge-eating episodes. Expression of reward-related genes in the striatum and thickness of the astro-glial barrier in the brain stem were enhanced in CYCL compared to CTRL mice. Diet alternation also induced caecal dysbiosis in CYCL mice. Gut microbiota transfer from CYCL mice to naive recipient mice recapitulated the altered eating behavior upon WD exposure. Conclusions: Alternation between high-energy and standard diet durably remodels the gut microbiota and the brain towards a profile associated with an increase in hedonic appetite. Using gut microbiota transfer, we established that this microbiota signature affects hedonic feeding regulation. These results open the ways to microbiota-targeted strategies to prevent development of eating disorders in weight cycling patients. ### Competing Interest Statement The authors have declared no competing interest.

Dietary fibers (DF) are important components of human and animal diets. However, they can decrease protein digestibility and absorption and thus the nutritional value of a food. The aim of this study was to investigate how the form of delivery of pea DF impacted the integrity of the intestinal barrier and, thereby, the potential absorption of molecules. To this end, two pea flours, with either intact or ruptured cell walls, and two controls, pea fibers and pea protein, were digested in vitro and the digesta obtained applied onto a jejunum porcine cell line (IPEC-J2 cells). Cell viability and integrity were evaluated by transepithelial electrical resistance measurement, colorimetric assay (MTS), and immunohistochemistry for tight junction proteins. Additionally, the diffusion of FITC-dextran (FD4) and lucifer yellow (LY) through the epithelial cell monolayers was monitored. The digested pea samples did not alter the IPEC-J2 viability and permeability. For instance, no difference in the diffusion of molecules either FD4 or LY across the monolayers was observed between the different digesta and the control. Similarly, no effect was observed in ZO-1 labeling intensity compared to the control. This study demonstrated that intestinal integrity was maintained whether pea cell walls were intact or ruptured.

Introduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described.Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3 days with 250 µM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids.Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0.

Background: Intestinal organoids are promising tools in the context of animal experiment reduction but a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig. Methods: Organoids were derived from jejunal and colonic ISC obtained from 1-, 7-, 28-, 36- and 180-day-old pigs and passaged three times. Results: We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (p < 0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, p < 0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (p < 0.05), spheroids were larger during the primary culture but smaller after two passages (p < 0.05) and organoids were smaller after one passage (p > 0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) was observed between organoids originating from 7- or 180-day-old pigs, but differences between intestinal site origins were noticed. Conclusion: In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence the phenotype.

The gut microbiota plays a key role in the postnatal development of the intestinal epithelium. However, the bacterial members of the primocolonizing microbiota driving these effects are not fully identified and the mechanisms underlying their long -term influence on epithelial homeostasis remain poorly described. Here, we used a model of newborn piglets treated during the first week of life with the antibiotic colistin in order to deplete specific gram-negative bacteria that are transiently dominant in the neonatal gut microbiota. Colistin depleted Proteobacteria and Fusobacteriota from the neonatal colon microbiota, reduced the bacterial predicted capacity to synthetize lipopolysaccharide (LPS), and increased the concentration of succinate in the colon. The colistin-induced disruption of the primocolonizing microbiota was associated with altered gene expression in the colon epithelium including a reduction of toll -like receptor 4 (TLR4) and lysozyme (LYZ). Our data obtained in porcine colonic organoid cell monolayers suggested that these effects were not driven by the variation of succinate or LPS levels nor by a direct effect of colistin on epithelial cells. The disruption of the primocolonizing microbiota imprinted colon epithelial stem cells since the expression of TLR4 and LYZ remained lower in organoids derived from colistin-treated piglet colonic crypts after several passages when compared to control piglets. Finally, the stable imprinting of LYZ in colon organoids was independent of the H3K4me3 level in its transcription start site. Altogether, our results show that disruption of the primocolonizing gut microbiota alters epithelial innate immunity in the colon and imprints stem cells, which could have long -term consequences for gut health.

We aimed to assess if casein structure affects its digestion and its subsequent amino acid delivery kinetic. Higher nitrogen levels were recovered in dialysates after in vitro digestions of sodium caseinate (SC, formed of small aggregates) compared to micellar casein (MC, native form of casein) and calcium caseinate (CC, intermediate structure). Likewise, plasma indispensable amino-acid concentration peak was higher after SC compared to MC or CC ingestion in healthy volunteers in a randomized, double blind, cross-over study. In pigs, gamma-scintigraphy using labelled meals revealed that SC was mainly localized in the proximal part of the stomach whereas MC was distributed in the whole gastric cavity. Caseins were found in both solid and liquid phases and partly hydrolyzed casein in the solid phase shortly after SC drink ingestion. These data support the concept of slow (MC) and rapid (SC) casein depending of casein structure, likely due to their intra-gastric clotting properties.

Aim The current study investigated the performance of 4 widely used DNA extraction kits using different types of high (stool) and low biomass samples (chyme, broncho alveolar lavage and sputum). Methods Qiagen Powerfecal Pro DNA kit, Macherey Nucleospin Soil kit, Macherey Nucleospin Tissue Kit and MagnaPure LC DNA isolation kit III were evaluated in terms of DNA quantity, quality, diversity and composition profiles. Results The quantity and quality of DNA varied among the four kits. The microbiota of the stool samples showed similar diversity and compositional profiles for the 4 kits. Conclusion Despite differences in DNA quality and quantity, the 4 kits yielded similar results for stool samples, while all kits were not sensitive enough for low biomass samples.