RESUMEN
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.
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Microbiota , Animales , Porcinos , Receptor Toll-Like 4 , Colistina , Lipopolisacáridos , Células Madre , Succinatos , Ácido Succínico , Colon , HomeostasisRESUMEN
Obesity is characterized by systemic low-grade inflammation associated with disturbances of intestinal homeostasis and microbiota dysbiosis. Mitochondrial metabolism sustains epithelial homeostasis by providing energy to colonic epithelial cells (CEC) but can be altered by dietary modulations of the luminal environment. Our study aimed at evaluating whether the consumption of an obesogenic diet alters the mitochondrial function of CEC in mice. Mice were fed for 22 weeks with a 58% kcal fat diet (diet-induced obesity [DIO] group) or a 10% kcal fat diet (control diet, CTRL). Colonic crypts were isolated to assess mitochondrial function while colonic content was collected to characterize microbiota and metabolites. DIO mice developed obesity, intestinal hyperpermeability, and increased endotoxemia. Analysis of isolated colonic crypt bioenergetics revealed a mitochondrial dysfunction marked by decreased basal and maximal respirations and lower respiration linked to ATP production in DIO mice. Yet, CEC gene expression of mitochondrial respiration chain complexes and mitochondrial dynamics were not altered in DIO mice. In parallel, DIO mice displayed increased colonic bile acid concentrations, associated with higher abundance of Desulfovibrionaceae. Sulfide concentration was markedly increased in the colon content of DIO mice. Hence, chronic treatment of CTRL mouse colon organoids with sodium sulfide provoked mitochondrial dysfunction similar to that observed in vivo in DIO mice while acute exposure of isolated mitochondria from CEC of CTRL mice to sodium sulfide diminished complex IV activity. Our study provides new insights into colon mitochondrial dysfunction in obesity by revealing that increased sulfide production by DIO-induced dysbiosis impairs complex IV activity in mouse CEC.
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Dieta Alta en Grasa , Disbiosis , Ratones , Animales , Dieta Alta en Grasa/efectos adversos , Disbiosis/metabolismo , Obesidad/metabolismo , Sulfuros/metabolismo , Mitocondrias/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Early hyperphagia and hypothalamic inflammation encountered after Western diet (WD) are linked to rodent propensity to obesity. Inflammation in several brain structures has been associated with gut dysbiosis. Since gut microbiota is highly sensitive to dietary changes, we hypothesised that immediate gut microbiota adaptation to WD in rats is involved in inflammation-related hypothalamic modifications. METHODS: We evaluated short-term impact of WD consumption (2 h, 1, 2 and 4 days) on hypothalamic metabolome and caecal microbiota composition and metabolome. Data integration analyses were performed to uncover potential relationships among these three datasets. Finally, changes in hypothalamic gene expression in absence of gut microbiota were evaluated in germ-free rats fed WD for 2 days. RESULTS: WD quickly and profoundly affected the levels of several hypothalamic metabolites, especially oxidative stress markers. In parallel, WD consumption reduced caecal microbiota diversity, modified its composition towards pro-inflammatory profile and changed caecal metabolome. Data integration identified strong correlations between gut microbiota sub-networks, unidentified caecal metabolites and hypothalamic oxidative stress metabolites. Germ-free rats displayed reduced energy intake and no changes in redox homoeostasis machinery expression or pro-inflammatory cytokines after 2 days of WD, in contrast to conventional rats, which exhibited increased SOD2, GLRX and IL-6 mRNA levels. CONCLUSION: A potentially pro-inflammatory gut microbiota and an early hypothalamic oxidative stress appear shortly after WD introduction. Tripartite data integration highlighted putative links between gut microbiota sub-networks and hypothalamic oxidative stress. Together with the absence of hypothalamic modifications in germ-free rats, this strongly suggests the involvement of the microbiota-hypothalamus axis in rat adaptation to WD introduction and in energy homoeostasis regulation.
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Eje Cerebro-Intestino/fisiología , Dieta Occidental/efectos adversos , Disbiosis , Hipotálamo/metabolismo , Animales , Citocinas/metabolismo , Disbiosis/metabolismo , Disbiosis/fisiopatología , Microbioma Gastrointestinal/fisiología , Inflamación/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Gut microbiota and intestinal barrier co-develop after birth, establishing a homeostatic state whereby mucosal cells cohabit with commensal bacteria. We hypothesized that this post-natal co-development follows different timings depending on the intestinal site considered. Jejunal, ileal, and colonic luminal contents and mucosa were sampled in suckling piglets at post-natal day (PND) 0, 2, 7, 14, and 28. Jejunal, ileal, and colonic luminal microbiota (evaluated by 16S DNA sequencing followed by beta-diversity analysis) clustered at PND2 but colonic microbiota diverge afterwards (P < .05). Mucosal permeability, evaluated in Ussing chambers, increased with age in the jejunum and ileum (P < .05) but not the colon. Expression of pattern recognition receptor (PRR) exhibited different patterns (gradual or sharp increase, decrease, or no change with age, P < .05) depending on PRR and intestinal site considered. Principal component analysis of mucosa data revealed clear clustering of colonic samples, irrespective of the age and clustering of jejunal and ileal samples, with gradual changes with age. Correlation analysis highlighted three families correlating with mucosal parameters: Enterobacteriaceae in the jejunum, Peptostreptococcaceae in the ileum, and Micrococcaceae in the colon. In conclusion, small and large intestine display close microbiota composition early in life but distinct mucosal phenotype and follow very different post-natal development.
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Bacterias , Colon/microbiología , Microbioma Gastrointestinal/fisiología , Íleon/microbiología , Mucosa Intestinal/microbiología , Yeyuno/microbiología , Animales , Bacterias/clasificación , Bacterias/crecimiento & desarrollo , PorcinosRESUMEN
Oleoylethanolamide is a well-recognized anorectic compound which also has noteworthy effects on food-reward, influencing the acetylcholine (ACh)/dopamine (DA) balance in the cholinergic system. After its administration, oleoylethanolamide is quickly degraded into oleic acid and ethanolamine. The effect of oleic acid on the gut-brain axis has been extensively investigated, whereas ethanolamine has received scarce attention. However, there is scattered evidence from old and recent research that has underlined the influence of ethanolamine on the cholinergic system. In the present article, we propose a model by which the released ethanolamine contributes to the overall balance between DA and ACh after oleoylethanolamide administration.
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Acetilcolina/metabolismo , Dopamina/metabolismo , Endocannabinoides/farmacología , Etanolamina/metabolismo , Conducta Alimentaria/efectos de los fármacos , Ácidos Oléicos/farmacología , Animales , Depresores del Apetito/metabolismo , Depresores del Apetito/farmacología , Endocannabinoides/metabolismo , Humanos , Ratones , Modelos Biológicos , Ácidos Oléicos/metabolismo , RatasRESUMEN
PURPOSE: Reduced ability of cholecystokinin (CCK) to induce satiation contributes to hyperphagia and weight gain in high-fat/high-sucrose (HF/HS) diet-induced obesity, and has been linked to altered gut microbiota. Rodent models of obesity use chow or low-fat (LF) diets as control diets; the latter has been shown to alter gut microbiota and metabolome. We aimed to determine whether LF-diet consumption impacts CCK satiation in rats and if so, whether this is prevented by addition of inulin to LF diet. METHODS: Rats (n = 40) were fed, for 8 weeks, a chow diet (chow) or low-fat (10%) or high-fat/high-sucrose (45 and 17%, respectively) refined diets with either 10% cellulose (LF and HF/HS) or 10% inulin (LF-I and HF/HS-I). Caecal metabolome was assessed by 1H-NMR-based metabolomics. CCK satiation was evaluated by measuring the suppression of food intake after intraperitoneal CCK injection (1 or 3 µg/kg). RESULTS: LF-diet consumption altered the caecal metabolome, reduced caecal weight, and increased IAP activity, compared to chow. CCK-induced inhibition of food intake was abolished in LF diet-fed rats compared to chow-fed rats, while HF/HS diet-fed rats responded only to the highest CCK dose. Inulin substitution ameliorated caecal atrophy, reduced IAP activity, and modulated caecal metabolome, but did not improve CCK-induced satiety in either LF- or HF/HS-fed rats. CONCLUSIONS: CCK signaling is impaired by LF-diet consumption, highlighting that caution must be taken when using LF diet until a more suitable refined control diet is identified.
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Colecistoquinina/metabolismo , Dieta con Restricción de Grasas/métodos , Saciedad/fisiología , Animales , Masculino , Modelos Animales , Ratas , Ratas WistarRESUMEN
BACKGROUND: Maturity of intestinal functions is critical for neonatal health and survival, but comprehensive description of mechanisms underlying intestinal maturation that occur during late gestation still remain poorly characterized. The aim of this study was to investigate biological processes specifically involved in intestinal maturation by comparing fetal jejunal transcriptomes of two representative porcine breeds (Large White, LW; Meishan, MS) with contrasting neonatal vitality and maturity, at two key time points during late gestation (gestational days 90 and 110). MS and LW sows inseminated with mixed semen (from breed LW and MS) gave birth to both purebred and crossbred fetuses. We hypothesized that part of the differences in neonatal maturity between the two breeds results from distinct developmental profiles of the fetal intestine during late gestation. Reciprocal crossed fetuses were used to analyze the effect of parental genome. Transcriptomic data and 23 phenotypic variables known to be associated with maturity trait were integrated using multivariate analysis with expectation of identifying relevant genes-phenotypic variable relationships involved in intestinal maturation. RESULTS: A moderate maternal genotype effect, but no paternal genotype effect, was observed on offspring intestinal maturation. Four hundred and four differentially expressed probes, corresponding to 274 differentially expressed genes (DEGs), more specifically involved in the maturation process were further studied. In day 110-MS fetuses, Ingenuity® functional enrichment analysis revealed that 46% of DEGs were involved in glucose and lipid metabolism, cell proliferation, vasculogenesis and hormone synthesis compared to day 90-MS fetuses. Expression of genes involved in immune pathways including phagocytosis, inflammation and defense processes was changed in day 110-LW compared to day 90-LW fetuses (corresponding to 13% of DEGs). The transcriptional regulator PPARGC1A was predicted to be an important regulator of differentially expressed genes in MS. Fetal blood fructose level, intestinal lactase activity and villous height were the best predicted phenotypic variables with probes mostly involved in lipid metabolism, carbohydrate metabolism and cellular movement biological pathways. CONCLUSIONS: Collectively, our findings indicate that the neonatal maturity of pig intestine may rely on functional development of glucose and lipid metabolisms, immune phagocyte differentiation and inflammatory pathways. This process may partially be governed by PPARGC1A.
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Desarrollo Fetal/genética , Perfilación de la Expresión Génica , Glucosa/metabolismo , Intestinos/embriología , Intestinos/inmunología , Metabolismo de los Lípidos/genética , Animales , Inmunidad/genética , Mucosa Intestinal/metabolismo , Fenotipo , PorcinosRESUMEN
Obesity and its related disorders have been associated with the presence in the blood of gut bacteria-derived lipopolysaccharides (LPS). However, the factors underlying this low-grade elevation in plasma LPS, so-called metabolic endotoxemia, are not fully elucidated. We aimed to investigate the effects of Western diet (WD) feeding on intestinal and hepatic LPS handling mechanisms in a rat model of diet-induced obesity (DIO). Rats were fed either a standard chow diet (C) or a Western Diet (WD, 45% fat) for 6 wk. They were either fed ad libitum or pair-fed to match the caloric intake of C rats for the first week, then fed ad libitum for the remaining 5 wk. Six-week WD feeding led to a mild obese phenotype with increased adiposity and elevated serum LPS-binding protein (LBP) levels relative to C rats, irrespective of initial energy intake. Serum LPS was not different between dietary groups but exhibited strong variability. Disrupted ileal mucus secretion and decreased ileal Reg3-γ and -ß gene expression along with high ileal permeability to LPS were observed in WD compared with C-fed rats. Ileal and cecal intestinal alkaline phosphatase (IAP) activity as well as Verrucomicrobia and Bifidobacterium cecal levels were increased in WD-fed rats compared with C-fed rats. WD consumption did not impact mRNA levels of LPS-handling hepatic enzymes. Correlation analysis revealed that ileal passage of LPS, IAP activity, Proteobacteria levels and hepatic aoah gene expression correlated with serum LPS and LBP, suggesting that ileal mucosal defense impairment induced by WD feeding contribute to metabolic endotoxemia.
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Dieta Occidental , Ingestión de Alimentos/fisiología , Endotoxemia/metabolismo , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Animales , Células Cultivadas , Endotoxemia/patología , Conducta Alimentaria , Íleon/patología , Mucosa Intestinal/patología , Masculino , Técnicas de Cultivo de Órganos , Permeabilidad , Ratas , Ratas WistarRESUMEN
Obesity is characterized by altered gut homeostasis, including dysbiosis and increased gut permeability closely linked to the development of metabolic disorders. Milk oligosaccharides are complex sugars that selectively enhance the growth of specific beneficial bacteria in the gastrointestinal tract and could be used as prebiotics. The aim of the study was to demonstrate the effects of bovine milk oligosaccharides (BMO) and Bifidobacterium longum ssp. infantis (B. infantis) on restoring diet-induced obesity intestinal microbiota and barrier function defects in mice. Male C57/BL6 mice were fed a Western diet (WD, 40% fat/kcal) or normal chow (C, 14% fat/kcal) for 7 wk. During the final 2 wk of the study, the diet of a subgroup of WD-fed mice was supplemented with BMO (7% wt/wt). Weekly gavage of B. infantis was performed in all mice starting at wk 3, yet B. infantis could not be detected in any luminal contents when mice were killed. Supplementation of the WD with BMO normalized the cecal and colonic microbiota with increased abundance of Lactobacillus compared with both WD and C mice and restoration of Allobaculum and Ruminococcus levels to that of C mice. The BMO supplementation reduced WD-induced increase in paracellular and transcellular flux in the large intestine as well as mRNA levels of the inflammatory marker tumor necrosis factor α. In conclusion, BMO are promising prebiotics to modulate gut microbiota and intestinal barrier function for enhanced health.
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Disbiosis , Leche/metabolismo , Animales , Bovinos , Dieta , Inflamación , Ratones , Ratones Obesos , Oligosacáridos/metabolismo , PermeabilidadRESUMEN
The human microbiota consists of 100 trillion microorganisms that provide important metabolic and biological functions benefiting the host. However, the presence in host plasma of a gut-derived bacteria component, the lipopolysaccharide (LPS), has been identified as a causal or complicating factor in multiple serious diseases such as sepsis and septic shock and, more recently, obesity-associated metabolic disorders. Understanding the precise mechanisms by which gut-derived LPS is transported from the gut lumen to the systemic circulation is crucial to advance our knowledge of LPS-associated diseases and elaborate targeted strategies for their prevention. The aim of this review is to synthetize current knowledge on the host mechanisms limiting the entry and dissemination of LPS into the systemic circulation. To prevent bacterial colonization and penetration, the intestinal epithelium harbors multiple defense mechanisms including the secretion of antimicrobial peptides and mucins as well as detoxification enzymes. Despite this first line of defense, LPS can reach the apical site of intestinal epithelial cells (IECs) and, because of its large size, likely crosses IECs via transcellular transport, either lipid raft- or clathrin-mediated endocytosis or goblet cell-associated passage. However, the precise pathway remains poorly described. Finally, if LPS crosses the gut mucosa, it is directed via the portal vein to the liver, where major detoxification processes occur by deacetylation and excretion through the bile. If this disposal process is not sufficient, LPS enters the systemic circulation, where it is handled by numerous transport proteins that clear it back to the liver for further excretion.
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Bacterias/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Lipopolisacáridos/sangre , Hígado/metabolismo , Sepsis/sangre , Animales , Bacterias/inmunología , Traslocación Bacteriana , Interacciones Huésped-Patógeno , Humanos , Intestinos/inmunología , Intestinos/microbiología , Lipopolisacáridos/química , Hígado/inmunología , Hígado/microbiología , Permeabilidad , Sepsis/inmunología , Sepsis/microbiologíaRESUMEN
A causal relationship between the pathophysiological changes in the gut epithelium and altered gut microbiota with the onset of obesity have been suggested but not defined. The aim of this study was to determine the temporal relationship between impaired intestinal barrier function and microbial dysbiosis in the small and large intestine in rodent high-fat (HF) diet-induced obesity. Rats were fed HF diet (45% fat) or normal chow (C, 10% fat) for 1, 3, or 6 wk; food intake, body weight, and adiposity were measured. Barrier function ex vivo using FITC-labeled dextran (4,000 Da, FD-4) and horseradish peroxidase (HRP) probes in Ussing chambers, gene expression, and gut microbial communities was assessed. After 1 wk, there was an immediate but reversible increase in paracellular permeability, decrease in IL-10 expression, and decrease in abundance of genera within the class Clostridia in the ileum. In the large intestine, HRP flux and abundance of genera within the order Bacteroidales increased with time on the HF diet and correlated with the onset of increased body weight and adiposity. The data show immediate insults in the ileum in response to ingestion of a HF diet, which were rapidly restored and preceded increased passage of large molecules across the large intestinal epithelium. This study provides an understanding of microbiota dysbiosis and gut pathophysiology in diet-induced obesity and has identified IL-10 and Oscillospira in the ileum and transcellular flux in the large intestine as potential early impairments in the gut that might lead to obesity and metabolic disorders.
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Grasas de la Dieta/metabolismo , Absorción Intestinal , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiopatología , Microbiota/fisiología , Obesidad/microbiología , Obesidad/fisiopatología , Animales , Dieta Alta en Grasa , Masculino , Ratas , Ratas WistarRESUMEN
Dietary peptides are absorbed along the intestine through peptide transporter 1 (PepT-1) which is highly responsive to dietary protein level. PepT-1 is also involved in gut homeostasis, both initiating and resolving inflammation. Low-birth-weight (LBW) neonates are routinely fed a high-protein (HP) formula to enhance growth. However, the influence of this nutritional practice on PepT-1 activity is unknown. Intestinal PepT-1 activity was compared in normal-birth-weight (NBW) and LBW piglets. The effect of HP v. normal-protein (NP) formula feeding on PepT-1 activity and gut homeostasis in LBW piglets was evaluated, during the neonatal period and in adulthood. Flux of cephalexin (CFX) across the tissue mounted in Ussing chambers was used as an indicator of PepT-1 activity. CFX flux was greater in the ileum, but not jejunum or colon, of LBW than NBW piglets during the neonatal period. When LBW piglets were formula-fed, the HP formula increased colonic CFX during the 1st week of life. Later in life, intestinal CFX fluxes and barrier function were similar whether LBW pigs had been fed NP or HP formula. However, colonic permeability of HP- but not NP-fed pigs increased when luminal pH was brought to 6·0. The formyl peptide N-formyl methionyl-leucyl-phenylalanine conferred colonic barrier protection in HP-fed piglets. Heat shock protein 27 levels in the colonic mucosa of HP-fed LBW pigs correlated with the magnitude of response to the acidic challenge. In conclusion, feeding a HP formula enhanced colonic PepT-1 activity in LBW pig neonates and increased sensitivity of the colon to luminal stress in adulthood.
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Animales Recién Nacidos/metabolismo , Dieta/veterinaria , Proteínas en la Dieta/administración & dosificación , Intestinos/crecimiento & desarrollo , Proteínas de Transporte de Membrana/metabolismo , Sus scrofa/fisiología , Animales , Peso al Nacer , Colon/metabolismo , Proteínas en la Dieta/efectos adversos , Femenino , Íleon/metabolismo , Recién Nacido de Bajo Peso , Absorción Intestinal , Intestinos/química , Intestinos/fisiología , Masculino , Proteínas de Transporte de Membrana/genética , Transportador de Péptidos 1 , ARN Mensajero/análisis , SimportadoresRESUMEN
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 ß-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.
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BACKGROUND: Propionibacterium freudenreichii is a food grade bacterium consumed both in cheeses and in probiotic preparations. Its promising probiotic potential, relying largely on the active release of beneficial metabolites within the gut as well as the expression of key surface proteins involved in immunomodulation, deserves to be explored more deeply. Adaptation to the colon environment is requisite for the active release of propionibacterial beneficial metabolites and constitutes a bottleneck for metabolic activity in vivo. Mechanisms allowing P. freudenreichii to adapt to digestive stresses have been only studied in vitro so far. Our aim was therefore to study P. freudenreichii metabolic adaptation to intra-colonic conditions in situ. RESULTS: We maintained a pure culture of the type strain P. freudenreichii CIRM BIA 1, contained in a dialysis bag, within the colon of vigilant piglets during 24 hours. A transcriptomic analysis compared gene expression to identify the metabolic pathways induced by this environment, versus control cultures maintained in spent culture medium.We observed drastic changes in the catabolism of sugars and amino-acids. Glycolysis, the Wood-Werkman cycle and the oxidative phosphorylation pathways were down-regulated but induction of specific carbohydrate catabolisms and alternative pathways were induced to produce NADH, NADPH, ATP and precursors (utilizing of propanediol, gluconate, lactate, purine and pyrimidine and amino-acids). Genes involved in stress response were down-regulated and genes specifically expressed during cell division were induced, suggesting that P. freudenreichii adapted its metabolism to the conditions encountered in the colon. CONCLUSIONS: This study constitutes the first molecular demonstration of P. freudenreichii activity and physiological adaptation in vivo within the colon. Our data are likely specific to our pig microbiota composition but opens an avenue towards understanding probiotic action within the gut in further studies comparing bacterial adaptation to different microbiota.
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Adaptación Fisiológica , Colon/microbiología , Probióticos , Propionibacterium/metabolismo , Transcriptoma , Animales , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas , Propionibacterium/genética , PorcinosRESUMEN
The interplay between the colonic microbiota and gut epithelial and immune cells during the neonatal period, which establishes the structure of the microbiota and programs mucosal immunity, is affected by the diet. We hypothesized that protein-enriched milk formula would disturb this interplay through greater flux of protein entering the colon, with consequences later in life. Piglets were fed from postnatal day (PND) 2 to 28 either a normal-protein formula (NP; 51 g protein/L) or high-protein formula (HP; 77 g protein/L) and weaned at PND28, when they received standard diets until PND160. HP feeding transiently increased the quantity of protein entering the colon (PND7) but did not change the microbiota composition at PND28, except for a higher production of branched-chain fatty acids (BCFAs) in an in vitro fermentation test (P < 0.05). HP piglets had greater colonic mucosa densities of cluster of differentiation (CD) 3(+) and CD172(+) cells and lower Il-1ß and Tnfα mRNA levels at PND28 (P < 0.05). Later in life (PND160), HP females, but not males, had a higher increase in colonic permeability after ex vivo oxidative stress and higher cytokine secretion in response to lipopolysaccharide in colonic explant cultures than NP females (P < 0.05). HP females also had lower colonic amounts of F. prausnitzii and BCFAs (P < 0.05). BCFAs displayed a dose-dependent protection against inflammation-induced alteration of barrier function in Caco-2 cells (P < 0.05). In conclusion, protein-enriched formula had little impact on colonic microbiota, but it modified colonic immune cell development and had a long-term effect on adult colonic mucosa sensitivity to inflammatory insults, probably through microbiotal and hormonal factors.
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Colon/microbiología , Proteínas en la Dieta/administración & dosificación , Mediadores de Inflamación/metabolismo , Metagenoma , Alimentación Animal/análisis , Animales , Animales Recién Nacidos , Células CACO-2 , Colon/efectos de los fármacos , Citocinas/metabolismo , Femenino , Humanos , Inmunidad Mucosa/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Lipopolisacáridos/metabolismo , Estrés OxidativoRESUMEN
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.
DNA extraction is a major factor affecting the microbial profile of various samples. Considering that different kits are commonly used such as QIAamp PowerFecal Pro DNA kit (QPFPD, QIAGEN), Macherey Nucleospin Soil (MNS, MACHEREY-NAGEL) Macherey Nucleospin Tissue (MNT, MACHEREY-NAGEL) and MagnaPure LC DNA isolation kit III (MPLCD, ROCHE), this study aimed to assess their performance using high (feces) and low-biomass samples. The kits were equally effective for feces samples but not sensitive enough for low biomass samples (chyme, bronchoalveolar lavage fluid and sputum).
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Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.
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Mucosa Intestinal , Intestinos , Humanos , Animales , Porcinos , Mucosa Intestinal/metabolismo , Animales Domésticos , Células Epiteliales , Organoides/metabolismoRESUMEN
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.
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Aminoácidos , Caseínas , Estudios Cruzados , Digestión , Animales , Caseínas/química , Caseínas/metabolismo , Estómago/metabolismo , Porcinos , Humanos , Voluntarios SanosRESUMEN
Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L(-1) casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.
Asunto(s)
Leche/microbiología , Probióticos/metabolismo , Propionibacterium/metabolismo , Animales , Bovinos , Femenino , Fermentación , Tracto Gastrointestinal/microbiología , Humanos , Ácido Láctico/metabolismo , Masculino , Viabilidad Microbiana , Propionatos/metabolismo , Propionibacterium/citología , PorcinosRESUMEN
BACKGROUND: Systemic low-grade inflammation observed in diet-induced obesity has been associated with dysbiosis and disturbance of intestinal homeostasis. This latter relies on an efficient epithelial barrier and coordinated intestinal epithelial cell (IEC) renewal that are supported by their mitochondrial function. However, IEC mitochondrial function might be impaired by high fat diet (HFD) consumption, notably through gut-derived metabolite production and fatty acids, that may act as metabolic perturbators of IEC. SCOPE OF REVIEW: This review presents the current general knowledge on mitochondria, before focusing on IEC mitochondrial function and its role in the control of intestinal homeostasis, and featuring the known effects of nutrients and metabolites, originating from the diet or gut bacterial metabolism, on IEC mitochondrial function. It then summarizes the impact of HFD on mitochondrial function in IEC of both small intestine and colon and discusses the possible link between mitochondrial dysfunction and altered intestinal homeostasis in diet-induced obesity. MAJOR CONCLUSIONS: HFD consumption provokes a metabolic shift toward fatty acid ß-oxidation in the small intestine epithelial cells and impairs colonocyte mitochondrial function, possibly through downstream consequences of excessive fatty acid ß-oxidation and/or the presence of deleterious metabolites produced by the gut microbiota. Decreased levels of ATP and concomitant O2 leaks into the intestinal lumen could explain the alterations of intestinal epithelium dynamics, barrier disruption and dysbiosis that contribute to the loss of epithelial homeostasis in diet-induced obesity. However, the effect of HFD on IEC mitochondrial function in the small intestine remains unknown and the precise mechanisms by which HFD induces mitochondrial dysfunction in the colon have not been elucidated so far.