Patrycja Puiman

The regulation of intestinal mucin MUC2 expression by short-chain fatty acids: implications for epithelial protection.

SCFAs (short-chain fatty acids), fermentation products of bacteria, influence epithelial-specific gene expression. We hypothesize that SCFAs affect goblet-cell-specific mucin MUC2 expression and thereby alter epithelial protection. In the present study, our aim was to investigate the mechanisms that regulate butyrate-mediated effects on MUC2 synthesis. Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability, and MUC2 protein expression were analysed. SCFA-responsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel-shift assays. The effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. Butyrate (at 1 mM), as well as propionate, induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM propionate, MUC2 mRNA levels remained increased. Promoter-regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 10-15 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. In conclusion, 1 mM butyrate and 1-15 mM propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP-1 and acetylation/methylation of histones at the MUC2 promoter.

Animal models to study neonatal nutrition in humans.

Purpose of reviewThe impact of neonatal nutrition on the health status of the newborn and incidence of disease in later life is a topic of intense interest. Animal models are an invaluable tool to identify mechanisms that mediate the effect of nutrition on neonatal development and metabolic function. This review highlights recently developed animal models that are being used to study neonatal human nutrition. Recent findingsIn recent years, mice, rats, and pigs have become the most frequently used animal models to study human neonatal nutrition. Techniques for rearing newborn mice, preterm rats, and preterm pigs have been developed. Neonatal mice have great potential for mechanistic and genomic research in postnatal nutrition and related diseases. The neonatal pig model is valuable to study acute and chronic effects of parenteral and enteral nutrition on whole-body metabolism as well as specific tissues. To date, a wealth of information from studies with neonatal pigs has been applied to humans. SummaryFurther development of neonatal animal models related to nutrition is required for the advancement of research in early postnatal nutrition. Improvement of nutritional support during this critical period of development will enhance immediate clinical outcomes and possibly prevent diseases later in life.

Continuous Parenteral and Enteral Nutrition Induces Metabolic Dysfunction in Neonatal Pigs

BACKGROUND We previously showed that parenteral nutrition (PN) compared with formula feeding results in hepatic insulin resistance and steatosis in neonatal pigs. The current aim was to test whether the route of feeding (intravenous [IV] vs enteral) rather than other feeding modalities (diet, pattern) had contributed to the outcome. METHODS Neonatal pigs were fed enterally or parenterally for 14 days with 1 of 4 feeding modalities as follows: (1) enteral polymeric formula intermittently (FORM), (2) enteral elemental diet (ED) intermittently (IEN), (3) enteral ED continuously (CEN), and (4) parenteral ED continuously (PN). Subgroups of pigs underwent IV glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP). Following CLAMP, pigs were euthanized and tissues collected for further analysis. RESULTS Insulin secretion during IVGTT was significantly higher and glucose infusion rates during CLAMP were lower in CEN and PN than in FORM and IEN. Endogenous glucose production rate was suppressed to zero in all groups during CLAMP. In the fed state, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide (GLP)-1, and GLP-2 were different between feeding modalities. Insulin receptor phosphorylation in liver and muscle was decreased in IEN, CEN, and PN compared with FORM. Liver weight was highest in PN. Steatosis and myeloperoxidase (MPO) activity tended to be highest in PN and CEN. Enterally fed groups had higher plasma GLP-2 and jejunum weight compared with PN. CONCLUSIONS PN and enteral nutrition (EN) when given continuously as an elemental diet reduces insulin sensitivity and the secretion of key gut incretins. The intermittent vs continuous pattern of EN produced the optimal effect on metabolic function.

Paneth cell hyperplasia and metaplasia in necrotizing enterocolitis.

Paneth cell dysfunction has been suggested in necrotizing enterocolitis (NEC). The aim of this study was to i) study Paneth cell presence, protein expression, and developmental changes in preterm infants with NEC and ii) determine Paneth cell products and antimicrobial capacity in ileostomy outflow fluid. Intestinal tissue from NEC patients (n = 55), preterm control infants (n = 22), and term controls (n = 7) was obtained during surgical resection and at stoma closure after recovery. Paneth cell abundance and protein expression were analyzed by immunohistochemistry. RNA levels of Paneth cell proteins were determined by real-time quantitative RT-PCR. In ileostomy outflow fluid, Paneth cell products were quantified, and antimicrobial activity was measured in vitro. In acute NEC, Paneth cell abundance in small intestinal tissue was not significantly different from preterm controls. After recovery from NEC, Paneth cell hyperplasia was observed in the small intestine concomitant with elevated human alpha-defensin 5 mRNA levels. In the colon, metaplastic Paneth cells were observed. Ileostomy fluid contained Paneth cell proteins and inhibited bacterial growth. In conjunction, these data suggest an important role of Paneth cells and their products in various phases of NEC.

Intestinal Threonine Utilization for Protein and Mucin Synthesis Is Decreased in Formula-Fed Preterm Pigs

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 ± 2%) than in COL pigs (60 ± 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 ± 31 mg·kg(-1)·d(-1) vs. 615 ± 54 mg·kg(-1)·d(-1); P = 0.003) and MUC2 synthesis (121 ± 17%/d vs. 184 ± 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC.

Enteral Arginine Does Not Increase Superior Mesenteric Arterial Blood Flow but Induces Mucosal Growth in Neonatal Pigs

Arginine is an essential amino acid in neonates synthesized by gut epithelial cells and a precursor for NO that regulates vasodilatation and blood flow. Arginine supplementation has been shown to improve intestinal integrity in ischemia-reperfusion models and low plasma levels are associated with necrotizing enterocolitis. We hypothesized that enteral arginine is a specific stimulus for neonatal intestinal blood flow and mucosal growth under conditions of total parenteral nutrition (TPN) or partial enteral nutrition (PEN). We first tested the dose dependence and specificity of acute (3 h) enteral arginine infusion on superior mesenteric artery (SMA) blood flow in pigs fed TPN or PEN. We then determined whether chronic (4 d) arginine supplementation of PEN increases mucosal growth and if this was affected by treatment with the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME). Acute enteral arginine infusion increased plasma arginine dose dependently in both TPN and PEN groups, but the plasma response was markedly higher (100-250%) in the PEN group than in the TPN group at the 2 highest arginine doses. Baseline SMA blood flow was 90% higher in the PEN (2.37 ± 0.32 L⋅kg(-1)⋅h(-1)) pigs than in the TPN pigs (1.23 ± 0.17 L⋅kg(-1)⋅h(-1)), but was not affected by acute infusion individually of arginine, citrulline, or other major gut fuels. Chronic dietary arginine supplementation in PEN pigs induced mucosal growth in the intestine, but this effect was not prevented by treatment with L-NAME. Intestinal crypt cell proliferation, protein synthesis, and phosphorylation of mammalian target of rapamycin and p70S6 kinase were not affected by dietary arginine. We conclude that partial enteral feeding, but not acute enteral arginine, increases SMA blood flow in the neonatal pig. Furthermore, supplementing arginine in partial enteral feeding modestly increases intestinal mucosal growth and was NO independent.

Modulation of the gut microbiota with antibiotic treatment suppresses whole body urea production in neonatal pigs

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.