Protein Ingredient Quality within Infant Formulas Impacts Plasma Amino Acid Concentrations in Neonatal Minipiglets.

BACKGROUND: Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein digestion and subsequent amino acid (AA) absorption. OBJECTIVES: The objective was to understand the impact of the protein ingredient quality within IFs on postprandial plasma AA profiles. METHODS: Four isonitrogenous and isocaloric IFs were produced at a semi-industrial scale using whey proteins from different origins (cheese compared with ideal whey) and denaturation levels (IF-A, -B, -C), and caseins with different supramolecular organizations (IF-C, -D). Ten Yucatan minipiglets (12- to 27-d-old) were used as a human infant model and received each IF for 3 d according to a Williams Latin square followed by a 2-d wash-out period. Jugular plasma was regularly sampled from 10 min preprandial to 4 h postprandial on the third day to measure free AAs, urea, insulin, and glucose concentrations. Data were statistically analyzed using a mixed linear model with diet (IFs), time, and sex as fixed factors and piglet as random factor. RESULTS: IFs made with cheese whey (IF-A and -B) elicited significantly higher plasma total and essential AA concentrations than IFs made with ideal whey (IF-C and -D), regardless of the pre- and postprandial times. Most of the differences observed postprandially were explained by AA homeostasis modifications. IFs based on cheese whey induced an increased plasma concentration of Thr due to both a higher Thr content in these IFs and a Thr-limiting degrading capability in piglets. The use of a nonmicellar casein ingredient led to reduced plasma content of AA catabolism markers (IF-D compared with IF-C). CONCLUSIONS: Overall, our results highlight the importance of the protein ingredient quality (composition and structure) within IFs on neonatal plasma AA profiles, which may further impact infant protein metabolism.

Ileal Digestibility of Nitrogen and Amino Acids in Human Milk and an Infant Formula as Determined in Neonatal Minipiglets.

BACKGROUND: Infant formula (IF) has to provide at least the same amount of amino acids (AAs) as human milk (HM). AA digestibility in HM and IF was not studied extensively, with no data available for tryptophan digestibility. OBJECTIVES: The present study aimed to measure the true ileal digestibility (TID) of total nitrogen and AAs in HM and IF to estimate AA bioavailability using Yucatan mini-piglets as an infant model. METHODS: Twenty-four 19-day-old piglets (males and females) received either HM or IF for 6 days or a protein-free diet for 3 days, with cobalt-EDTA as an indigestible marker. Diets were fed hourly over 6 h before euthanasia and digesta collection. Total N, AA, and marker contents in diets and digesta were measured to determine the TID. Unidimensional statistical analyses were conducted. RESULTS: Dietary N content was not different between HM and IF, while true protein was lower in HM (-4 g/L) due to a 7-fold higher non-protein N content in HM. The TID of total N was lower (P < 0.001) for HM (91.3 ± 1.24%) than for IF (98.0 ± 0.810%), while the TID of amino acid nitrogen (AAN) was not different (average of 97.4 ± 0.655%, P = 0.272). HM and IF had similar (P > 0.05) TID for most of the AAs including tryptophan (96.7 ± 0.950%, P = 0.079), except for some AAs (lysine, phenylalanine, threonine, valine, alanine, proline, and serine), with small significant difference (P < 0.05). The first limiting AA was the aromatic AAs, and the digestible indispensable AA score (DIAAS) was higher for HM (DIAASHM = 101) than for IF (DIAASIF = 83). CONCLUSION: HM, compared to IF, had a lower TID for total N only, whereas the TID of AAN and most AAs, including Trp, was high and similar. A larger proportion of non-protein N is transferred to the microbiota with HM, which is of physiological relevance, although this fraction is poorly considered for IF manufacturing.

Perinatal short-chain fructooligosaccharides program intestinal microbiota and improve enteroinsular axis function and inflammatory status in high-fat diet-fed adult pigs.

Perinatal nutrition programs physiologic and metabolic functions, with consequences on the susceptibility to develop metabolic diseases in adulthood. The microbiota represents a key factor of such programming. We investigated whether perinatal prebiotic [short-chain fructooligosaccharides (scFOS)] supplementation improved adult metabolic health in association with microbiota changes in pigs used as human model. Sows were supplemented with scFOS or not during the end of gestation and the entire lactation, and offspring received scFOS accordingly during 1 mo after weaning. Pigs were then fed a standard diet for 5 mo, followed by a high-fat diet for 3 mo once adults. Perinatal scFOS supplementation induced a persistent modulation of the composition of the fecal microbiota in adulthood, notably by increasing the Prevotella genus. Meanwhile, scFOS animals displayed improved capacity to secrete glucagon-like peptide-1 and improved pancreas sensitivity to glucose without any changes in peripheral insulin sensitivity. Perinatal scFOS supplementation also increased ileal secretory IgA secretion and alkaline phosphatase activity and decreased TNF-α expression in adipose tissue. In conclusion, perinatal scFOS supplementation induced long-lasting modulation of intestinal microbiota and had beneficial consequences on the host physiology in adulthood. Our results highlight the key role of perinatal nutrition on later microbiota and host metabolic adaptation to an unbalanced diet.-Le Bourgot, C., Ferret-Bernard, S., Apper, E., Taminiau, B., Cahu, A., Le Normand, L., Respondek, F., Le Huërou-Luron, I., Blat, S. Perinatal short-chain fructooligosaccharides program intestinal microbiota and improve enteroinsular axis function and inflammatory status in high-fat diet-fed adult pigs.

Maternal Western diet during gestation and lactation modifies adult offspring's cognitive and hedonic brain processes, behavior, and metabolism in Yucatan minipigs.

This study explores the long-term effects of exposure to a maternal Western diet (WD) vs. standard diet (SD) in the Yucatan minipig, on the adult progeny at lean status ( n = 32), and then overweight status. We investigated eating behavior, cognitive abilities, brain basal glucose metabolism, dopamine transporter availability, microbiota activity, blood lipids, and glucose tolerance. Although both groups demonstrated similar cognitive abilities in a holeboard test, WD pigs expressed a higher stress level than did SD pigs (immobility, P < 0.05) and lower performance in an alley maze ( P = 0.06). WD pigs demonstrated lower dopamine transporter binding potential in the hippocampus and parahippocampal cortex ( P < 0.05 for both), as well as a trend in putamen ( P = 0.07), associated with lower basal brain activity in the prefrontal cortex and nucleus accumbens ( P < 0.05) compared with lean SD pigs. Lean WD pigs demonstrated a lower glucose tolerance than did SD animals (higher glucose peak, P < 0.05) and a tendency to a higher incremental area under the curve of insulin from 0 to 30 minutes after intravenous glucose injection ( P < 0.1). Both groups developed glucose intolerance with overweight, but WD animals were less impacted than SD animals. These results demonstrate that maternal diet shaped the offspring's brain functions and cognitive responses long term, even after being fed a balanced diet from weaning, but behavioral effects were only revealed in WD pigs under anxiogenic situation; however, WD animals seemed to cope better with the obesogenic diet from a metabolic standpoint.-Gautier, Y., Luneau, I., Coquery, N., Meurice, P., Malbert, C.-H., Guerin, S., Kemp, B., Bolhuis, J. E., Clouard, C., Le Huërou-Luron, I., Blat, S., Val-Laillet, D. Maternal Western diet during gestation and lactation modifies adult offspring's cognitive and hedonic brain processes, behavior, and metabolism in Yucatan minipigs.

A maternal Western diet during gestation and lactation modifies offspring's microbiota activity, blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs.

A suboptimal early nutritional environment (i.e., excess of energy, sugar, and fat intake) can increase susceptibility to diseases and neurocognitive disorders. The purpose of this study was to investigate in nonobese Yucatan minipigs (Sus scrofa) the impact of maternal diet [standard diet (SD) vs. Western diet (WD)] during gestation and 25 d of lactation on milk composition, blood metabolism, and microbiota activity of sows (n = 17) and their piglets (n = 65), and on spatial cognition (n = 51), hippocampal plasticity (n = 17), and food preferences/motivation (n = 51) in the progeny. Milk dry matter and lipid content, as well as plasma total cholesterol and free fatty acid (FFA) concentrations (P < 0.05) were higher in WD than in SD sows. Microbiota activity decreased in both WD sows and 100-d-old piglets (P < 0.05 or P < 0.10, depending on short-chain FAs [SCFAs]). At weaning [postnatal day (PND) 25], WD piglets had increased blood triglyceride and FFA levels (P < 0.01). Both SD and WD piglets consumed more of a known SD than an unknown high-fat and -sucrose (HFS) diet (P < 0.0001), but were quicker to obtain HFS rewards compared with SD rewards (P < 0.01). WD piglets had higher working memory (P = 0.015) and reference memory (P < 0.001) scores, which may reflect better cognitive abilities in the task context and a higher motivation for the food rewards. WD piglets had a smaller hippocampal granular cell layer (P = 0.03) and decreased neurogenesis (P < 0.005), but increased cell proliferation (P < 0.001). A maternal WD during gestation and lactation, even in the absence of obesity, has significant consequences for piglets' blood lipid levels, microbiota activity, gut-brain axis, and neurocognitive abilities after weaning.-Val-Laillet, D., Besson, M., Guérin, S., Coquery, N., Randuineau, G., Kanzari, A., Quesnel, H., Bonhomme, N., Bolhuis, J. E., Kemp, B., Blat, S., Le Huërou-Luron, I., Clouard, C. A maternal Western diet during gestation and lactation modifies offspring's microbiota activity, blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs.

A mixture of milk and vegetable lipids in infant formula changes gut digestion, mucosal immunity and microbiota composition in neonatal piglets.

PURPOSE: Although composition of infant formula has been significantly improved during the last decade, major differences with the composition and structure of breast milk still remain and might affect nutrient digestion and gut biology. We hypothesized that the incorporation of dairy fat in infant formulas could modify their physiological impacts by making their composition closer to that of human milk. The effect of milk fat and milk fat globule membrane (MFGM) fragments in infant formulas on gut digestion, mucosal immunity and microbiota composition was evaluated. METHODS: Three formulas containing either (1) vegetable lipids stabilized only by proteins (V-P), (2) vegetable lipids stabilized by a mixture of proteins and MFGM fragments (V-M) and (3) a mixture of milk and vegetable lipids stabilized by a mixture of proteins and MFGM fragments (M-M) were automatically distributed to 42 newborn piglets until slaughter at postnatal day (PND) 7 or 28, and compared to a fourth group of sow's suckling piglets (SM) used as a breast-fed reference. RESULTS: At both PND, casein and ß-lactoglobulin digestion was reduced in M-M proximal jejunum and ileum contents compared to V-P and V-M ones leading to more numerous ß-Cn peptides in M-M contents. The IFNγ cytokine secretion of ConA-stimulated MLN cells from M-M piglets tended to be higher than in V-P ones at PND 7 and PND 28 and was closer to that of SM piglets. No dietary treatment effect was observed on IL-10 MLN cell secretion. Changes in faecal microbiota in M-M piglets resulted in an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes phyla compared to V-P ones. M-M piglets showed higher abundances of Parabacteroides, Escherichia/Shigella and Klebsiella genus. CONCLUSIONS: The incorporation of both milk fat and MFGM fragments in infant formula modifies protein digestion, the dynamic of the immune system maturation and the faecal microbiota composition.

Comparing the intestinal transcriptome of Meishan and Large White piglets during late fetal development reveals genes involved in glucose and lipid metabolism and immunity as valuable clues of intestinal maturity.

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.

Maternal short-chain fructo-oligosaccharide supplementation increases intestinal cytokine secretion, goblet cell number, butyrate concentration and Lawsonia intracellularis humoral vaccine response in weaned pigs.

Prebiotic supplementation modulates immune system development and function. However, less is known about the effects of maternal prebiotic consumption on offspring intestinal defences and immune system responsiveness. We investigated the effects of maternal short-chain fructo-oligosaccharide (scFOS) supplementation on mucin-secreting cells, ileal secretory IgA and cytokine secretion of weaned offspring and their humoral response to an oral vaccine against obligate intracellular Lawsonia intracellularis. Sows were fed a control diet (CTRL) or scFOS-supplemented diet during the last third of gestation and throughout lactation. At weaning, each litter was divided into two groups receiving a post-weaning CTRL or scFOS diet for a month. Pigs from the four groups were either non-vaccinated (n 16) or vaccinated (n 117) at day 33. Biomarkers related to intestinal defences and immune parameters were analysed 3 weeks later. SCFA production was assessed over time in suckling and weaned pigs. Maternal scFOS supplementation improved ileal cytokine secretions (interferon (IFN)-γ, P<0·05; IL-4, P=0·07) and tended to increase caecal goblet cell number (P=0·06). It increased IgA vaccine response in the serum (P<0·01) and ileal mucosa (P=0·08). Higher bacterial fermentative activity was observed during lactation (total faecal SCFA, P<0·001) and after weaning (colonic butyrate, P=0·10) in pigs from scFOS-supplemented mothers. No synergistic effect between maternal and post-weaning scFOS supplementation was observed. Therefore, maternal scFOS supplementation has long-lasting consequences by strengthening gut defences and immune response to a vaccine against an intestinal obligate intracellular pathogen. Prebiotic consumption by gestating and lactating mothers is decisive in modulating offspring intestinal immunity.

Splanchnic tissues respond differently when piglets are offered a diet 30 % deficient in total sulfur amino acid for 10 days.

PURPOSE: A deficient total sulfur amino acid (TSAA) supply has been reported to differently affect the amino acid composition of tissues, but limited information is available about its effects on the morphology and metabolic properties of splanchnic tissues. METHODS: The amino acid composition, protein metabolism, glutathione concentration of the liver, proximal and distal jejunum, ileum and kidneys, and intestinal architecture were compared in 42-day-old piglets pair-fed either a diet deficient (TSAA-; 28 % deficiency) or sufficient (TSAA+) in TSAA for 10 days. RESULTS: The supply of TSAA had no effect on tissue weights, but influenced the amino acid composition in a tissue-dependent manner. Compared with animals receiving diet TSAA+, the concentrations of Met and Ser were higher in liver protein of TSAA- animals while the Cys concentration in protein was lower in the liver but higher in the distal jejunum. The TSAA supply had no effect on protein synthesis and proteolytic activities of tissues. Villus width and surface, and crypt surface were lower in the proximal jejunum of TSAA- versus TSAA+ pigs. Crypt surface in the ileum of TSAA- pigs was higher. Pigs receiving diet TSAA- had lower GSH and GSSG concentrations in the liver and proximal jejunum, but the GSH/GSSG ratio was decreased only in the liver. CONCLUSIONS: A greater nutritional priority appears to be given to splanchnic tissues so that its growth and protein metabolism can be maintained when the TSAA supply is limiting. The amino acid composition, glutathione status, and intestinal mucosa architecture are affected in a tissue-dependent manner.

Dysregulation of sirtuins and key metabolic genes in skeletal muscle of pigs with spontaneous intrauterine growth restriction is associated with alterations of circulating IGF-1.

Prenatal and early postnatal life determines future health, and intrauterine growth restriction (IUGR) - associated low birth weight predisposes to metabolic syndrome in adulthood. We hypothesize here that IUGR might induce hormonal and gene expression alterations predisposing to metabolic disease. Using a porcine model of spontaneous IUGR, we determined in utero (71, 112days post-conception) and early-postnatal (2days post-birth) IGF-1, insulin and leptin levels, and in parallel we investigated, in skeletal muscle, the developmental expression patterns of sirtuins and metabolic and signaling genes IRS1, GLUT4, HK2 and GAPDH. IUGR was associated with impaired IGF-1 plasmatic levels. Gene expression of sirtuin 1, 5, 6, 7, GLUT4 and HK2 exhibited significant correlations with gestational age or body weight. SIRT1 and HK2 expression displayed an age- and weight-dependent downregulation in controls, which was lost in IUGR pigs. Conversely, SIRT2 and GLUT4 were upregulated in IUGR pigs. Within the set of genes studied, we found a significant correlation between IGF-1 levels and gene expression in control, but not IUGR samples, indicating that lower IGF-1 may be a limiting factor in IUGR. IUGR-dependent gene alterations were partly linked to epigenetic changes on histone H3 acetylation and methylation. Overall, our data indicate that several sirtuins and metabolic genes display specific gene expression trajectories during fetal and early postnatal life. Gene expression alterations observed in IUGR are correlated to IGF-1 dysregulation. Given the importance of the genes studied in metabolic control, their perinatal alterations might contribute to the predisposition to metabolic disease of adulthood.

Critical review evaluating the pig as a model for human nutritional physiology.

The present review examines the pig as a model for physiological studies in human subjects related to nutrient sensing, appetite regulation, gut barrier function, intestinal microbiota and nutritional neuroscience. The nutrient-sensing mechanisms regarding acids (sour), carbohydrates (sweet), glutamic acid (umami) and fatty acids are conserved between humans and pigs. In contrast, pigs show limited perception of high-intensity sweeteners and NaCl and sense a wider array of amino acids than humans. Differences on bitter taste may reflect the adaptation to ecosystems. In relation to appetite regulation, plasma concentrations of cholecystokinin and glucagon-like peptide-1 are similar in pigs and humans, while peptide YY in pigs is ten to twenty times higher and ghrelin two to five times lower than in humans. Pigs are an excellent model for human studies for vagal nerve function related to the hormonal regulation of food intake. Similarly, the study of gut barrier functions reveals conserved defence mechanisms between the two species particularly in functional permeability. However, human data are scant for some of the defence systems and nutritional programming. The pig model has been valuable for studying the changes in human microbiota following nutritional interventions. In particular, the use of human flora-associated pigs is a useful model for infants, but the long-term stability of the implanted human microbiota in pigs remains to be investigated. The similarity of the pig and human brain anatomy and development is paradigmatic. Brain explorations and therapies described in pig, when compared with available human data, highlight their value in nutritional neuroscience, particularly regarding functional neuroimaging techniques.

A high-protein formula increases colonic peptide transporter 1 activity during neonatal life in low-birth-weight piglets and disturbs barrier function later in life.

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.

Maternal short chain fructo-oligosaccharides supplementation during late gestation and lactation influences milk components and offspring gut metabolome: a pilot study.

Breast milk composition is influenced by maternal diet. This study aimed to evaluate if supplementation of maternal diet with a prebiotic fibre, through its potential effect on milk composition, can be a leverage to orientate the gut microbiota of infants in a way that would be beneficial for their health. Twelve sows received a diet supplemented with short chain fructo-oligosaccharides or maltodextrins during the last month of gestation and the lactation. Oligosaccharidic and lipidomic profiles of colostrum and mature milk (21 days), as well as faecal microbiota composition and metabolomic profile of 21 day-old piglets were evaluated. The total porcine milk oligosaccharide concentration tended to be lower in scFOS-supplemented sows, mainly due to the significant reduction of the neutral core oligosaccharides (in particular that of a tetrahexose). Maternal scFOS supplementation affected the concentration of 31 lipids (mainly long-chain triglycerides) in mature milk. Faecal short-chain fatty acid content and that of 16 bacterial metabolites were modified by scFOS supplementation. Interestingly, the integrative data analysis gave a novel insight into the relationships between (i) maternal milk lipids and PMOs and (ii) offspring faecal bacteria and metabolites. In conclusion, scFOS-enriched maternal diet affected the composition of mature milk, and this was associated with a change in the colonisation of the offspring intestinal microbiota.

Two human milk-like synthetic bacterial communities displayed contrasted impacts on barrier and immune responses in an intestinal quadricellular model.

The human milk (HM) microbiota, a highly diverse microbial ecosystem, is thought to contribute to the health benefits associated with breast-feeding, notably through its impact on infant gut microbiota. Our objective was to further explore the role of HM bacteria on gut homeostasis through a "disassembly/reassembly" strategy. HM strains covering the diversity of HM cultivable microbiota were first characterized individually and then assembled in synthetic bacterial communities (SynComs) using two human cellular models, peripheral blood mononuclear cells and a quadricellular model mimicking intestinal epithelium. Selected HM bacteria displayed a large range of immunomodulatory properties and had variable effects on epithelial barrier, allowing their classification in functional groups. This multispecies characterization of HM bacteria showed no clear association between taxonomy and HM bacteria impacts on epithelial immune and barrier functions, revealing the entirety and complexity of HM bacteria potential. More importantly, the assembly of HM strains into two SynComs of similar taxonomic composition but with strains exhibiting distinct individual properties, resulted in contrasting impacts on the epithelium. These impacts of SynComs partially diverged from the predicted ones based on individual bacteria. Overall, our results indicate that the functional properties of the HM bacterial community rather than the taxonomic composition itself could play a crucial role in intestinal homeostasis of infants.

Dietary protein excess during neonatal life alters colonic microbiota and mucosal response to inflammatory mediators later in life in female pigs.

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.

Spontaneous intra-uterine growth restriction modulates the endocrine status and the developmental expression of genes in porcine fetal and neonatal adipose tissue.

Low birth weight is correlated with low adiposity at birth, a phenotype that influences neonatal survival and later adiposity. A better understanding of events affecting the fetal adipose tissue development and its functionality around birth is thus needed. This study was undertaken to examine the impact of spontaneous intra-uterine growth restriction (IUGR) on circulating concentrations of hormones and nutrients together with the developmental expression patterns of various genes in subcutaneous adipose tissue of pig fetus during the last third of pregnancy and just after birth. At 71 and 112 days post-conception and 2 days postnatal, pairs of same-sex piglets were chosen within litters to have either a medium (MBW) or a low (LBW) weight (n=6 pairs at each stage). The results indicate that IUGR counteracts the temporal fall of DLK1 gene expression in developing adipose tissue across gestation. It also attenuates the time-dependent increase in expression levels of many genes promoting adipocyte differentiation (PPARG, CEBPA) and lipogenesis (LPL, SREBF1, FASN, FABP4). Opposite responses to IUGR were observed for the IGF system, so that IGF1 mRNA levels were lower (P<0.001) but IGF2 mRNA levels were greater in adipose tissue of LBW piglets compared with MBW piglets. The plasma insulin concentration and the mRNA levels of insulin receptor (INSR) and insulin-responsive glucose transporter (GLUT4) in adipose tissue were also greater in LBW piglets at day 2 postnatal. The data indicate that IUGR delays the normal ontogeny of adipose tissue across gestation and affects the insulin and IGF axes around birth.

Exploration of fMRI brain responses to oral sucrose after Roux-en-Y gastric bypass in obese yucatan minipigs in relationship with microbiota and metabolomics profiles.

BACKGROUND & AIMS: In most cases, Roux-en-Y gastric bypass (RYGBP) is an efficient intervention to lose weight, change eating behavior and improve metabolic outcomes in obese patients. We hypothesized that weight loss induced by RYGBP in obese Yucatan minipigs would induce specific modifications of the gut-brain axis and neurocognitive responses to oral sucrose stimulation in relationship with food intake control. METHODS: An integrative study was performed after SHAM (n = 8) or RYGBP (n = 8) surgery to disentangle the physiological, metabolic and neurocognitive mechanisms of RYGBP. BOLD fMRI responses to sucrose stimulations at different concentrations, brain mRNA expression, cecal microbiota, and plasma metabolomics were explored 4 months after surgery and integrated with WGCNA analysis. RESULTS: We showed that weight loss induced by RYGBP or SHAM modulated differently the frontostriatal responses to oral sucrose stimulation, suggesting a different hedonic treatment and inhibitory control related to palatable food after RYGBP. The expression of brain genes involved in the serotoninergic and cannabinoid systems were impacted by RYGBP. Cecal microbiota was deeply modified and many metabolite features were differentially increased in RYGBP. Data integration with WGCNA identified interactions between key drivers of OTUs and metabolites features linked to RYGBP. CONCLUSION: This longitudinal study in the obese minipig model illustrates with a systemic and integrative analysis the mid-term consequences of RYGBP on brain mRNA expression, cecal microbiota and plasma metabolites. We confirmed the impact of RYGBP on functional brain responses related to food reward, hedonic evaluation and inhibitory control, which are key factors for the success of anti-obesity therapy and weight loss maintenance.

Protein ingredient quality of infant formulas impacts their structure and kinetics of proteolysis under in vitro dynamic digestion.

Infant formula (IF) is a complex matrix requiring numerous ingredients and processing steps. The objective was to understand how the quality of protein ingredients impacts IF structure and, in turn, their kinetics of digestion. Four powdered IFs (A/B/C/D), based on commercial whey protein (WP) ingredients, with different protein denaturation levels and composition (A/B/C), and on caseins with different supramolecular organisations (C/D), were produced at a semi-industrial level after homogenization and spray-drying. Once reconstituted in water (13 %, wt/wt), the IF microstructure was analysed with asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The rehydrated IFs were subjected to simulated infant in vitro dynamic digestion (DIDGI®). Digesta were regularly sampled to follow structural changes (confocal microscopy, laser-light scattering) and proteolysis (OPA, SDS-PAGE, LC-MS/MS, cation-exchange chromatography). Before digestion, different microstructures were observed among IFs. IF-A, characterized by more denatured WPs, presented star-shaped mixed aggregates, with protein aggregates bounded to casein micelles, themselves adsorbed at the fat droplet interface. Non-micellar caseins, brought by non-micellar casein powder (IF-D) underwent rearrangement and aggregation at the interface of flocculated fat droplets, leading to a largely different microstructure of IF emulsion, with large aggregates of lipids and proteins. During digestion, IF-A more digested (degree of proteolysis + 16 %) at 180 min of intestinal phase than IF-C/D. The modification of the supramolecular organisation of caseins implied different kinetics of peptide release derived from caseins during the gastric phase (more abundant at G80 for IF-D). Bioactive peptide release kinetics were also different during digestion with IF-C presenting a maximal abundance for a large proportion of them. Overall, the present study highlights the importance of the structure and composition of the protein ingredients (WPs and caseins) selected for IF formulation on the final IF structure and, in turn, on proteolysis. Whether it has some physiological consequences remains to be investigated.

Air-displacement plethysmography for determining body composition in neonates: validation using live piglets.

INTRODUCTION: Air-displacement plethysmography (ADP) was developed as a noninvasive tool to assess body composition, i.e., the proportion of fat mass (%FM) and lean body mass. The results of previous studies comparing ADP with labeled water dilution in infants and with chemical analysis in phantoms have validated the ADP approach indirectly. We assessed the precision and accuracy of measurements of % FM proportions in live animals, using ADP in comparison with biochemical analyses. METHODS: Three groups of 12 piglets each underwent four consecutive body composition assessments at 2, 7, and 21 d and were euthanized to determine whole-body lipid content by direct chemical analysis. RESULTS: The average body weights were 1,490, 2,210, and 5,610 g at d2, d7, and d21, respectively. The mean %FM values determined by biochemical analysis and ADP were 8.63 ± 4.08% and 8.01 ± 4.03%, respectively. Linear regression and Bland-Altman analyses indicated good agreement for %FM. The root mean square coefficient of variation (RMS-CV) for ADP was 17.9%, with a better precision in the higher fat mass range. DISCUSSION: Despite its relatively poor precision in the low range of %FM, ADP measures fat mass with reasonable precision and accuracy in the range of body weight encountered in low-birth-weight infants.

In vivo digestion of infant formula in piglets: protein digestion kinetics and release of bioactive peptides.

The first months of life correspond to a key period in human life where dramatic physiological changes (establishment of microbiota, development of the immune system, etc.) occur. In order to better control these changes it is necessary to understand the behaviour of food in the gastrointestinal tract of the newborn. Infant formula is the only food for the newborn when breast-feeding is impossible. The kinetics of digestion of milk proteins and the nature of the peptides liberated in the small intestine throughout infant formula digestion have never been extensively investigated so far and were therefore studied using the piglet as a model of the newborn child. Piglets were fed infant formula by an automatic delivery system during 28 d, and slaughtered 30, 90 and 210 min after the last meal. Contents of stomach, proximal and median jejunum and ileum were collected and characterised. The extent of ß-lactoglobulin (ß-lg), α-lactalbumin (α-la) and casein proteolysis was monitored by inhibition ELISA, SDS-PAGE, immunoblotting and MS. At 30 min after the last meal, caseins were shown to be extensively hydrolysed in the stomach. Nevertheless, peptides originating mainly from ß-caseins (from 509 to 2510 Da) were identified in the jejunum and ileum of the piglets. ß-Lg partially resisted gastric digestion but completely disappeared in the stomach after 210 min. α-La had a similar behaviour to that of ß-lg. Two large peptides (4276 and 2674 Da) generated from ß-lg were present in the ileum after 30 and 210 min and only one (2674 Da) after 90 min.