Maternal Serum Albumin Redox State Is Associated with Infant Birth Weight in Japanese Pregnant Women.

BACKGROUND: Plasma albumin (ALB) reflects protein nutritional status in rats, but it is not clear whether it is associated with dietary protein insufficiency in pregnant women and/or their risk of low birth weight delivery. This study aimed to investigate whether maternal serum ALB redox state reflects maternal protein nutritional status and/or is associated with infant birth weights. METHODS: The relationship between the serum reduced ALB ratio and infant birth weight was examined in an observational study of 229 Japanese pregnant women. A rat model simulating fetal growth restriction, induced by protein-energy restriction, was used to elucidate the relationship between maternal nutritional status, maternal serum ALB redox state, and birth weight of the offspring. RESULTS: In the human study, serum reduced ALB ratio in the third trimester was significantly and positively correlated with infant birth weight. In the rat study, serum reduced ALB ratio and birth weight in the litter decreased as the degree of protein-energy restriction intensified, and a significant and positive correlation was observed between them in late pregnancy. CONCLUSIONS: Maternal serum reduced ALB ratio in the third trimester is positively associated with infant birth weight in Japanese pregnant women, which would be mediated by maternal protein nutritional status.

Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk.

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.

Dynamic Associations of Milk Components With the Infant Gut Microbiome and Fecal Metabolites in a Mother-Infant Model by Microbiome, NMR Metabolomic, and Time-Series Clustering Analyses.

Background: The gut microbiome and fecal metabolites of breastfed infants changes during lactation, and are influenced by breast milk components. This study aimed to investigate dynamic associations of milk components with the infant gut microbiome and fecal metabolites throughout the lactation period in a mother-infant model. Methods: One month after delivery, breast milk and subsequent infant feces were collected in a pair for 5 months from a mother and an exclusively breastfed infant. Composition of the fecal microbiome was determined with 16S rRNA sequencing. Low-molecular-weight metabolites, including human milk oligosaccharides (HMOs), and antibacterial proteins were measured in feces and milk using 1H NMR metabolomics and enzyme-linked immunosorbent assays. The association of milk bioactive components with the infant gut microbiome and fecal metabolites was determined with Python clustering and correlation analyses. Results: The HMOs in milk did not fluctuate throughout the lactation period. However, they began to disappear in infant feces at the beginning of month 4. Notably, at this time-point, a bifidobacterium species switching (from B. breve to B. longum subsp. infantis) occurred, accompanied by fluctuations in several metabolites including acetate and butyrate in infant feces. Conclusions: Milk bioactive components, such as HMOs, might play different roles in the exclusively breastfed infants depending on the lactation period.

1H NMR metabolomic and transcriptomic analyses reveal urinary metabolites as biomarker candidates in response to protein undernutrition in adult rats.

Protein undernutrition contributes to the development of various diseases in broad generations. Urinary metabolites may serve as non-invasive biomarkers of protein undernutrition; however, this requires further investigation. We aimed to identify novel urinary metabolites as biomarker candidates responsive to protein undernutrition. Adult rats were fed control (CT; 14 % casein) or isoenergetic low-protein (LP; 5 % casein) diets for 4 weeks. 1H NMR metabolomics was applied to urine, plasma and liver samples to identify metabolites responsive to protein undernutrition. Liver samples were subjected to mRNA microarray and quantitative PCR analyses to elucidate the mechanisms causing fluctuations in identified metabolites. Urinary taurine levels were significantly lower in the LP group than in the CT group at week 1 and remained constant until week 4. Hepatic taurine level and gene expression level of cysteine dioxygenase type 1 were also significantly lower in the LP group than in the CT group. Urinary trimethylamine N-oxide (TMAO) levels were significantly higher in the LP group than in the CT group at week 2 and remained constant until week 4. Hepatic TMAO level and gene expression levels of flavin-containing mono-oxygenase 1 and 5 were also significantly higher in the LP group than in the CT group. In conclusion, urinary taurine and TMAO levels substantially responded to protein undernutrition. Furthermore, changes in hepatic levels of these metabolites and gene expressions associated with their metabolic pathways were also reflected in their fluctuating urinary levels. Thus, taurine and TMAO could act as non-invasive urinary biomarker candidates to detect protein undernutrition.

A More Oxidized Plasma Albumin Redox State and Lower Plasma HDL Particle Number Reflect Low-Protein Diet Ingestion in Adult Rats.

BACKGROUND: Plasma albumin (ALB) redox state reflects protein nutritional status, but how it differs from other protein nutrition biomarkers remains to be fully elucidated. OBJECTIVE: This study aimed to delineate the characteristics of plasma ALB redox state as a protein nutrition biomarker. METHODS: Adult male Wistar rats were maintained on an AIN-93 M [14% casein, control (CT)] diet or an AIN-93 M-based 5% casein [low protein (LP)] diet ad libitum for 4 wk. Plasma samples were repeatedly obtained from the same rats at weeks 0-4, ALB redox state was determined by HPLC, and the concentrations of conventional protein nutrition biomarkers, ALB and transthyretin (TTR), were compared between the groups by Student t test. Body mass, relative muscle masses, plasma proteome, and plasma lipids at week 4 were also compared. RESULTS: Plasma ALB redox state shifted to a more oxidized state in the LP diet group compared with the CT diet group at weeks 1-4. The LP diet group also showed significantly lower plasma ALB concentrations at weeks 1 and 2 (13% and 11% lower, respectively) and significantly lower TTR concentration at week 1 (21% lower) compared with the CT diet group, but these concentrations did not differ significantly at weeks 3 and 4. After 4 wk, body mass and relative soleus and gastrocnemius muscle masses did not differ, but the relative plantaris muscle mass tended to be 4% lower (1.75 compared with 1.68 g/kg body mass) in the LP diet group compared with the CT group (P = 0.06). The LP diet group also had a significantly lower HDL particle number than the CT group (30% lower). CONCLUSIONS: A more oxidized plasma ALB redox state and lower plasma HDL particle number reflect LP diet ingestion in adult rats, which did not exhibit changes of plasma ALB and TTR concentrations.

Plasma Albumin Redox State Is Responsive to the Amino Acid Balance of Dietary Proteins in Rats Fed a Low Protein Diet.

We recently reported that plasma albumin redox state, which correlates with albumin synthesis rate, could be associated with the quality of dietary protein. Aiming to elucidate the association between them, plasma albumin redox state was investigated in rats fed various kinds of AIN-93G-based low protein diets. Plasma albumin redox state was shifted to a more oxidized state in rats fed 3% casein (CN) diet than those fed 3% whey protein or 3% wheat gluten diet, while supplementing 3% CN diet with cystine reversed it to a more reduced state, indicating that cystine would complement the shortage of cysteine in CN, thereby increasing albumin synthesis rate. Supplementation with glutathione, a cysteine-containing antioxidative tripeptide, normalized hepatic glutathione redox state modulated by ingestion of 3% CN diet, but it only reversed the oxidized shift of plasma albumin redox state to an extent similar to cystine alone or the constituting amino acid mixture of glutathione (i.e., glutamic acid, cystine, and glycine), indicating that glutathione would primarily serve as a source of cysteine rather than exert its antioxidative activity. Plasma albumin would thus be influenced by amino acid balance in dietary proteins, and it could be useful as a biomarker that contributes to prevention of protein under-nutriton, caused by not only insufficient protein intake but also ingestion of poor-quality protein.

The Combination of Bifidobacterium breve and Three Prebiotic Oligosaccharides Modifies Gut Immune and Endocrine Functions in Neonatal Mice.

BACKGROUND: Several types of oligosaccharides are used in infant formula to improve the gut microbiota of formula-fed infants. We previously reported that a combination of 3 oligosaccharides (lactulose, raffinose, and galacto-oligosaccharides; LRG) and Bifidobacterium breve effectively increased B. breve numbers, acetate, and the expression of several immune- and gut hormone-related mRNAs in neonatal mice gut. OBJECTIVE: We investigated whether changes in neonatal gut microbiota alter gut immune and endocrine development. METHODS: We first compared postnatal day (PD) 14 with PD21 in C57BL/6J male mouse pups to identify the physiologic immune and endocrine changes during development. In a separate study, we administered phosphate-buffered saline (control group; CON), B. breve M-16V (M-16V), or M-16V + LRG to male mouse pups from PD6 to PD13, and analyzed the gut microbiota and immune and endocrine parameters on PD14 to evaluate whether M-16V + LRG accelerates gut immune and endocrine development. RESULTS: The proportion of regulatory T (Treg) cells in the CD4+ cells of large intestinal lamina propria lymphocytes (LPLs) was significantly increased (63% higher) at PD21 compared with PD14. The serum glucagon-like peptide (GLP)-1 tended to be lower (P = 0.0515) and that of GLP-2 was significantly lower (58% lower) at PD21 than at PD14. M-16V + LRG significantly increased the Treg proportion in large intestinal LPL CD4+ cells (20% and 29% higher compared with CON and M-16V, respectively) at PD14. M-16V + LRG also caused significant changes in expression of large intestinal mRNAs that are consistent with developmental progression, and increased serum concentrations of GLP-1 (207% and 311% higher compared with CON and M-16V, respectively) and GLP-2 (57% and 97% higher compared with CON and M-16V, respectively) at PD14. CONCLUSIONS: Neonatal administration of M-16V + LRG alters the gut microbiota and enhances gut immune and endocrine development in suckling mice.

Casein materials show different digestion patterns using an in vitro gastrointestinal model and different release of glucagon-like peptide-1 by enteroendocrine GLUTag cells.

Physicochemical properties of casein (CN) materials manufactured using different processes are well studied; however, data on their bioaccessibility or bioactivity are limited. We compared the digestion patterns and glucagon-like peptide-1 (GLP-1)-releasing activities of micellar CN concentrate (MCC) and sodium caseinate (SCN). MCC and SCN mixed with whey protein isolate (SCN + WPI) were subjected to in vitro gastrointestinal digestion; the digestibility of MCC was higher than that of SCN + WPI, and both CN materials showed different patterns of peptides released after in vitro digestion. A comparison of GLP-1-releasing activities showed that MCC induced GLP-1 secretion to a greater extent than SCN + WPI. Candidate peptides identified from CN digesta were chemically synthesized to test their GLP-1-releasing activity. GPVRGPFPIIV identified only in the MCC digesta, could stimulate GLP-1 release. In conclusion, the digestion patterns and GLP-1-releasing activity of CN materials depend on the production process.

Increased Ratio of Non-mercaptalbumin-1 Among Total Plasma Albumin Demonstrates Potential Protein Undernutrition in Adult Rats.

The redox state of plasma albumin shifts in response to dietary protein intake in growing rats, and the shift is more sensitive than that of plasma albumin level, a classical marker of protein nutritional status. While it has been suggested that plasma albumin redox state could be useful as a novel marker of protein nutritional status, the above animal model is highly sensitive to dietary protein intake and the observation may not be extrapolated widely to humans. This study aimed to investigate whether albumin redox state also reflects protein nutritional status in adult rats, which have a lower dietary protein requirement and are less responsive to protein intake. Male adult rats were placed on AIN-93M diet (14% casein), or AIN-93M-based low protein diets (10 or 5% casein) ad libitum for 24 weeks. Whereas there was no significant difference in body weight between the groups at the end of the experimental period, the 5% casein diet group had the smallest gastrocnemius muscle weight among the groups, which was significantly lower than that of the 10% casein diet group. Plasma albumin level was also lower in the 5% casein diet group compared with the other groups, but the differences were limited and inconsistent during the experimental period. Among the albumin redox isoforms such as mercaptalbumin, non-mercaptalbumin-1, and non-mercaptalbumin-2, the ratio of non-mercaptalbumin-1 among total albumin was significantly higher in the 5% casein diet group, and the increase remained constant throughout the experimental period. Increased non-mercaptalbumin-1 ratio would thus demonstrate the presence of potential protein undernutrition in adult rats, as manifested only by a decreased gain in a specific type of skeletal muscle; non-mercaptalbumin-1 among total albumin ratio could be useful as a robust marker of protein nutritional status, contributing to prevention of protein undernutrition-related diseases such as frailty and sarcopenia.

Ingestion of partially hydrolyzed whey protein suppresses epicutaneous sensitization to ß-lactoglobulin in mice.

BACKGROUND: Epicutaneous sensitization to food allergens can occur through defective skin barriers. However, the relationship between oral tolerance and epicutaneous sensitization remains to be elucidated. We aimed to determine whether prior oral exposure to whey proteins or their hydrolysates prevents epicutaneous sensitization and subsequent food-allergic reaction to the whey protein, ß-lactoglobulin (ß-LG), and investigated the underlying mechanisms. METHODS: BALB/c mice were given whey protein concentrate (WPC), two kinds of partial whey protein hydrolysate (PWH1 or PWH2), or extensive whey protein hydrolysate (EWH) in drinking water for 21 days. The mice were then epicutaneously sensitized with ß-LG on tape-stripped skin. Sensitization was assessed by basophil activation tests and by measuring the level of serum ß-LG-specific antibodies and cytokines secreted from ß-LG-restimulated spleen and mesenteric lymph node (MLN) cells. Development of an allergic reaction was assessed by monitoring body temperature and by measuring mast cell protease-1 level in plasma after the ß-LG oral challenge. Activated T-cell population among ß-LG-restimulated MLN cells was also analyzed. RESULTS: In mice fed with WPC, PWH1, or PWH2, sensitization and the development of an allergic reaction were totally reduced. The acceleration of cytokine release from the spleen and MLN cells or T-cell activation was not evident after ß-LG restimulation. In EWH-fed mice, a suppressive effect, though milder than that in WPC-, PWH1-, or PWH2-fed mice, was observed during the development of the allergic reaction. CONCLUSIONS: Prior oral exposure to partially hydrolyzed whey protein prevents epicutaneous sensitization and subsequent allergic response to ß-LG in mice.

Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

Bifidobacterium breve alters immune function and ameliorates DSS-induced inflammation in weanling rats.

BACKGROUND: Bifidobacterium breve M-16V (M16V) is a probiotic bacterial strain with a long tradition of use in neonatal intensive care units in some countries. Previous study showed that the effects of M16V administration on gene expression were greater during the weaning period than in the neonatal period and were greater in the colon than in the small intestine and spleen, suggesting that M16V has anti-inflammatory effects. In this study, we evaluated the effects of inflammation during the weaning period and the effects of M16V on normal and inflammatory conditions. METHODS: From postnatal day (PD) 21 to 34, weanling rats were administered of 2.5 × 10(9) of M16V daily, and colitis was induced by administration of 2% dextran sulfate sodium from PD28 to 35. Colitis severity, immune function, and microbiota were investigated. RESULTS: Colitis caused a reduction in body weight gain, colon shortening, poor nutritional status, anemia, changes in blood and spleen lymphocyte populations, spleen T-cell malfunctions, and alterations in colon microbiota. M16V administration improved some but not all of the changes induced by colitis. CONCLUSION: M16V could suppress inflammation and, therefore, can be considered a safe strain to use not only during the neonatal period but also the weaning period.

Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages.

We reported previously that microRNA (miRNA) are present in whey fractions of human breast milk, bovine milk, and rat milk. Moreover, we also confirmed that so many mRNA species are present in rat milk whey. These RNA were resistant to acidic conditions and to RNase, but were degraded by detergent. Thus, these RNA are likely packaged in membrane vesicles such as exosomes. However, functional extracellular circulating RNA in bodily fluids, such as blood miRNA, are present in various forms. In the current study, we used bovine raw milk and total RNA purified from exosomes (prepared by ultracentrifugation) and ultracentrifuged supernatants, and analyzed them using miRNA and mRNA microarrays to clarify which miRNA and mRNA species are present in exosomes, and which species exist in other forms. Microarray analyses revealed that most mRNA in milk whey were present in exosomes, whereas miRNA in milk whey were present in supernatant as well as exosomes. The RNA in exosomes might exert functional effects because of their stability. Therefore, we also investigated whether bovine milk-derived exosomes could affect human cells using THP-1 cells. Flow cytometry and fluorescent microscopy studies revealed that bovine milk exosomes were incorporated into differentiated THP-1 cells. These results suggest that bovine milk exosomes might have effects in human cells by containing RNA.

Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.

Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats.

Purification of RNA from milk whey.

MicroRNAs (miRNAs) are small regulatory RNA molecules that modulate specific target mRNAs and play very important roles in physiological processes. They were recently detected in body fluids such as blood, urine, saliva, and milk. These body fluid miRNAs have been studied thoroughly as potential diagnostic biomarkers. However, there have been few studies of milk miRNAs, and their roles are not clearly understood. Milk is the only nutritional source for newborn infants, and bovine milk is used widely as a dairy product. Thus, it is important to study milk miRNAs. In general, body fluid RNA concentrations are extremely low and of diverse existence types. In this chapter, we compare two silica membrane column-based RNA purification kits, and also compare RNA obtained directly from whey with that isolated from whey-derived exosomes.

Effects of Bifidobacterium breve on inflammatory gene expression in neonatal and weaning rat intestine.

INTRODUCTION: To examine the immune-modulatory effects of probiotics during early infancy, Bifidobacterium breve M-16V (B. breve) was administered to rat pups during the newborn or weaning period, and the expression of inflammatory genes was investigated using a cDNA microarray and real-time PCR. RESULTS: After B. breve administration, significant increases in the numbers of Bifidobacterium in both the cecum and colon were confirmed during the newborn period. The numbers of upregulated and downregulated genes were greater during the weaning period than in the newborn period and were greatest in the colon, with fewer genes altered in the small intestine and the fewest in the spleen. The expression of inflammation-related genes, including lipoprotein lipase (Lpl), glutathione peroxidase 2 (Gpx2), and lipopolysaccharide-binding protein (Lbp), was significantly reduced in the colon during the newborn period. In weaning rat pups, the expression of CD3d, a cell surface receptor-linked signaling molecule, was significantly enhanced in the colon; however, the expression of co-stimulatory molecules was not enhanced. DISCUSSION: Our findings support a possible role for B. breve in mediating anti-inflammatory and antiallergic reactions by modulating the expression of inflammatory molecules during the newborn period and by regulating the expression of co-stimulatory molecules during the weaning period. METHODS: Gene expression in the intestine was investigated after feeding 5 × 10(8) cfu of B. breve every day to the F344/Du rat from days 1 to 14 (newborn group) and from days 21 to 34 (weaning group). mRNA was extracted from intestine, and the expression of inflammatory gene was analyzed by microarray and real-time PCR.

ω-3 fatty acids attenuate mucosal inflammation in premature rat pups.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although ω-3 fatty acids are known to have antiinflammatory effects, their effect against NEC remains unclear. METHODS: Mother rats fed a soybean-based, docosahexaenoic acid (DHA)- or eicosapentaenoic acid (EPA)-enriched diet from days 7 to 20 of gestation were examined. On day 20, the rat pups were delivered by abdominal incision, their intestines were removed, and messenger RNA was extracted. A rat NEC model was used to confirm the effects of ω-3 fatty acids on the inflamed intestine (n = 20-28). The expression of inflammatory molecules was analyzed by real-time polymerase chain reaction (n = 11-14). RESULTS: The concentrations of DHA and EPA in the intestine were significantly increased in the DHA and EPA groups (P < .01). The expression of the antiinflammatory prostaglandin E2 receptor EP3 was increased in the DHA (P < .05) and EPA groups (P < .01). In the NEC model, the reduced incidence of colitis was confirmed in the DHA and EPA groups. The expression of peroxisome proliferator-activated receptor γ was increased (P < .05), and the inhibitor of nuclear factor-κB α/ß decreased in both the DHA (P < .01) and EPA groups (P < .05). CONCLUSION: Our findings indicate that ω-3 fatty acids are beneficial for protecting the premature intestine from inflammation by regulating eicosanoid- and nuclear factor-κB-related metabolite expression.

microRNA as a new immune-regulatory agent in breast milk.

BACKGROUND: Breast milk is a complex liquid that provides nutrition to the infant and facilitates the maturation of the infant's immune system. Recent studies indicated that microRNA (miRNA) exists in human body fluid. Because miRNAs are known to regulate various immune systems, we hypothesized that human breast milk contains miRNAs that may be important for the development of the infant's immune system. FINDINGS: We profiled miRNA expression in human breast milk and detected high expression levels of immune-related miRNAs in the first 6 months of lactation. Furthermore, these miRNA molecules are stable even in very acidic conditions, indicating that breast milk allows dietary intake of miRNAs by infants. CONCLUSIONS: Our findings provide new insight into how breast milk can modulate the development of the infant's immune system. This study suggests the transfer of genetic material as miRNA from human to human occurs by means other than through sexual reproduction.

Overfeeding can cause NEC-like enterocolitis in premature rat pups.

BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. The mortality rate associated with NEC is quite high and in most reports ranges from 20 to 30%. Despite extensive studies, the pathogenesis of NEC remains poorly understood. OBJECTIVES: To investigate the mechanisms of NEC in terms of inflammatory signaling in the intestine. METHODS: A new enterocolitis model was established and examined the expression of inflammatory and anti-inflammatory signals in the intestines of rat pups. The premature rat pups, delivered by abdominal incision on day 20 of gestation (day 21 is considered as full term), were divided into three groups, and they were given a single administration of 0.05, 0.1, and 0.15 ml of formula milk via an orogastric catheter. After 24 h, the development of enterocolitis was evaluated by the presence of hemorrhagic enterocolitis, and the expression of signaling molecules, inhibitor of nuclear factor-kappaB (IkappaB)-alpha/beta and peroxisome proliferator-activated receptor (PPAR)-gamma mRNA was examined by reverse transcription-polymerase chain reaction from inflamed and non-inflamed intestinal samples. RESULTS: The incidence of enterocolitis increased with the volume of milk, and 50% of rat pups showed enterocolitis with a volume of 0.15 ml of milk. Expression of IkappaB-alpha/beta and PPAR-gamma mRNA increased in inflamed intestine. CONCLUSIONS: Increased expression of IkappaB-alpha/beta suggested that the inflammatory mediator nuclear factor-kappaB is deeply involved in the pathogenesis of enterocolitis that can be easily introduced by overfeeding of milk ingestion in premature rat pups which mimic those seen in NEC. Increased expression of PPAR-gamma may possibly regulate further development of enterocolitis in this system.

Administration of anti-glucagon-like peptide-2 serum suppresses epithelial cell proliferation of the distal small intestine in weanling rats.

We investigated the effects of endogenous glucagon-like peptide-2 (GLP-2) on the development of intestinal mucosa in weanling rats. Three-week-old male weanling Sprague-Dawley rats were administered either anti-GLP-2 or normal rabbit serum every other day for 2 weeks. We then measured length, weight, and bromodeoxyuridine incorporation in the intestine on day 13 following the first injection. Administration of anti-GLP-2 serum significantly inhibited both epithelial proliferation in the distal ileum and elongation of the small intestine. These results suggest that intrinsic GLP-2 contributes to the growth of the small intestine during the weanling period.