Role of milk carbohydrates in intestinal health of nursery pigs: a review

Intestinal health is essential for the resistance to enteric diseases and for nutrient digestion and absorption to support growth. The intestine of nursery pigs are immature and vulnerable to external challenges, which cause negative impacts on the structure and function of the intestine. Among nutritional interventions, the benefits of milk are significant for the intestinal health of pigs. Milk coproducts have traditionally been used in starter feeds to improve the growth of nursery pigs, but their use is somewhat limited due to the high costs and potential risks of excessive lactose on the intestine. Thus, understanding a proper feeding level of milk carbohydrates is an important start of the feeding strategy. For nursery pigs, lactose is considered a highly digestible energy source compared with plant-based starch, whereas milk oligosaccharides are considered bioactive compounds modulating intestinal immunity and microbiota. Therefore, milk carbohydrates, mainly composed of lactose and oligosaccharides, have essential roles in the intestinal development and functions of nursery pigs. The proper feeding levels of lactose in starter feeds could be variable by weaning age, body weight, or genetic lines. Effects of lactose and milk oligosaccharides have been broadly studied in human health and animal production. Therefore, this review focuses on the mechanisms of lactose and milk oligosaccharides affecting intestinal maturation and functions through modulation of enterocyte proliferation, intestinal immunity, and intestinal microbiota of nursery pigs.

Nutritional and functional values of lysed Corynebacterium glutamicum cell mass for intestinal health and growth of nursery pigs

The objective was to determine the nutritional and functional values of lysed Corynebacterium glutamicum cell mass (CGCM) as a protein supplement and a source of cell wall fragments supporting the growth and intestinal health of nursery pigs. Thirty-two pigs (21 d of age) were allotted to four treatments (n = 8) based on the randomized block design with sex and initial body weight (BW) as blocks. The main effect was the dietary supplementation of lysed CGCM (0, 0.7, 1.4, and 2.1%) replacing blood plasma and fed in two phases (10 and 11 d, respectively). Feed intake and BW were measured at the end of each phase. Pigs were euthanized on day 21 to collect jejunal tissue and mucosa to evaluate intestinal health. Ileal digesta were collected to measure the apparent ileal digestibility of nutrients in diets. Data were analyzed using Proc Mixed and Reg of SAS. Increasing daily intake of CGCM increased (linear; P < 0.05) ADG of pigs. Increasing CGCM supplementation affected (quadratic; P < 0.05) the relative abundance of Lactobacillaceae (minimum: 26.4% at 1.2% CGCM), Helicobacteraceae (maximum: 29.3% at 1.2% CGCM), and Campylobacteraceae (maximum: 9.0% at 1.0% CGCM). Increasing CGCM supplementation affected (quadratic; P < 0.05) the concentrations of immunoglobulin G (maximum: 4.94 µg/mg of protein at 1.0% CGCM) and protein carbonyl (PC; maximum: 6.12 nmol/mg of protein at 1.1% CGCM), whereas linearly decreased (P < 0.05) malondialdehyde (MDA) in the proximal jejunal mucosa. Increasing CGCM supplemention affected (quadratic; P < 0.05) intestinal enterocyte proliferation rate (maximum: 13.3% at 1.0% CGCM), whereas it did not affect intestinal morphology and the nutrient digestibility. In conclusion, supplementing 1.0% to 1.2%, reducing blood plasma supplementation by 0.7% to 0.9%, respectively, increased potential pathogenic microbiota associated in the jejunal mucosa resulting in increased immune response, enterocyte proliferation, and PC concentration. However, supplementing diets with 2.1% CGCM, replacing 1.5% blood plasma, improved growth performance, and reduced MDA without affecting nutrient digestibility, intestinal morphology, and microbiota in the jejunal mucosa. In this study, based on the polynomial contrast, supplementing 1.0% to 1.2% CGCM suppressed the benefits from blood plasma, whereas supplementing 2.1% CGCM showed functional benefits of CGCM with similar effects from blood plasma supplementation.

Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells

Intestinal NaCl, HCO3- and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical NHE isoforms, NHE2, NHE3 and NHE8. We addressed the role of NHE8 (SLC9A8) and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis, and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated vs. undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state pHi, an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi-control.

Supplemental Insulin-Like Growth Factor-1 and Necrotizing Enterocolitis in Preterm Pigs

Background: Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP-3) is currently tested as a therapy in preterm infants but possible effects on the gut, including necrotizing enterocolitis (NEC), have not been tested. The aim of this study was to evaluate if rhIGF-1/BP-3 supplementation in the first days after birth negatively affects clinical variables like growth, physical activity, blood chemistry and hematology and gut maturation (e.g., intestinal permeability, morphology, enzyme activities, cytokine levels, enterocyte proliferation, NEC lesions), using NEC-sensitive preterm pigs as a model for preterm infants.Methods: Preterm pigs were given twice daily subcutaneous injections of rhIGF-1/BP-3 or vehicle. Blood was collected for IGF-1 measurements and gut tissue for NEC evaluation and biochemical analyses on day 5.Results: Baseline circulating IGF-1 levels were low in preterm pigs compared with near-term pigs reared by their mother (<20 vs. 70 ng/ml). Injection with rhIGF-1/BP-3 resulted in increased plasma IGF-1 levels for up to 6 h after injection (>40 ng/mL). rhIGF-1/BP-3 treatment reduced the incidence of severe NEC lesions (7/24 vs.16/24, p = 0.01) and overall NEC severity (1.8 ± 0.2 vs. 2.6 ± 0.3, p < 0.05, with most lesions occurring in colon). In the small intestine, villi length (405 ± 25 vs. 345 ± 33 μm) and activities of the brush border peptidases aminopeptidase N and dipeptidylpeptidase IV were increased in rhIGF-1/BP-3 treated pigs, relative to control pigs (+31–44%, both p < 0.05). The treatment had no effects on body weight, blood chemistry or hematology, except for an increase in blood leucocyte and neutrophil counts (p < 0.05, i.e., reduced neonatal neutropenia). Likewise, rhIGF-1/BP-3 treatment did not affect intestinal tissue cytokine levels (IL-1β, IL-6, IL-8, TNFα,), enterocyte proliferation, goblet cell density, permeability or bacterial translocation to the bone marrow.Conclusion: Supplemental rhIGF-1/BP-3 did not negatively affect any of the measured variables of clinical status or gut maturation in preterm pigs. Longer-term safety and efficacy of exogenous rhIGF-1/BP-3 to support maturation of the gut and other critical organs in preterm newborns remain to be investigated in both pigs and infants.

Loss of Slfn3 induces a gender-dependent repair vulnerability after 50% bowel resection

Bowel resection accelerates enterocyte proliferation in the remaining gut that may have suboptimal absorptive and digestive capacity because of proliferation-associated decrease in functional differentiation markers. We hypothesized that although Slfn3 is an important regulator of murine enterocytic differentiation, Slfn3 would have less impact on the bowel resection adaptation where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers of mucosa from resected and postoperative bowel of wild type (WT) and Slfn3 knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and was even longer in Slfn3KO mice, while female Slfn3KO mice displayed even deeper crypts than both WT sexes after resection. Mitotic marker, Phh3+, and proliferation marker expression of Lgr5, FoxL1, and PDGFRα were increased after resection in male WT but this effect was blunted in male Slfn3KO mice. Cell shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, while Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice but differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased. This suggests that enterocytic differentiation effort is incompatible with a rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Slfn3 may therefore be an important regulator not only of "normal" enterocytic differentiation but also the response to bowel resection.

Dietary Dihydroartemisinin Supplementation Alleviates Intestinal Inflammatory Injury Through TLR4/NODs/NF-κB Signaling Pathway in Weaned Piglets with Intrauterine Growth Retardation

Background: Intrauterine growth retardation (IUGR) leads to impaired intestinal morphology and function. IUGR infants are at a risk for intestinal inflammatory diseases. Dihydroartemisinin (DHA) is a derivative of artemisinin, which possesses anti-inflammatory activity and immunomodulatory effect. However, little is known about the effects of DHA on IUGR piglets. Therefore, the present study was conducted to investigate whether dietary DHA supplementation could attenuate intestinal injury in IUGR weaned piglets. Methods: Piglets with normal birth weight or IUGR were fed the basal diet or basal diet supplemented with 80 mg/kg DHA from 21 d to 49 days of age. At 49 days of age, eight piglets from each group with nearly similar body weight were sacrificed. The jejunal and ileal samples were collected for further analysis.Results: IUGR impaired intestinal morphology, increased intestinal inflammatory response, raised enterocyte apoptosis and reduced enterocyte proliferation and activated TLR4/NODs/NF-κB signaling pathway. DHA supplementation improved intestinal morphology, indicated by higher villus height, villus height to crypt depth ratio, villus surface area and lower villus width of IUGR piglets (P < 0.05). DHA inclusion exhibited higher apoptosis index and the expression of caspase-3, and lower proliferation index and the expression of proliferating cell nuclear antigen in the intestine of IUGR piglets (P < 0.05). Diet supplemented with DHA could attenuate intesitnal inflammation, indicated by higher concentrations of intestinal inflammatory cytokines and lipopolysaccharides in IUGR piglets (P < 0.05). In addition, DHA down-regulated the related mRNA expressions of TLR4/NODs/NF-κB signaling pathway and up-regulated mRNA expressions of TLR4 and NODs signaling negative regulators in the intestine of IUGR piglets (P < 0.05). Dietary DHA supplementation decreased the protein expressions of toll-like receptors 4, phosphorylated NF-κB (pNF-κB) inhibitor α, nuclear pNF-κB, and increased the protein expression of cytoplasmic pNF-κB in the intestine of IUGR piglets (P < 0.05). Conclusions: Dietary DHA supplementation may have beneficial effects in improving intestinal morphology, regulating enterocyte proliferation and apoptosis, and alleviating intestinal inflammation through TLR4/NODs/NF-κB signaling pathway in IUGR weaned piglets.

Mutational inactivation of Apc in the intestinal epithelia compromises cellular organisation

The tumour suppressor adenomatous polyposis coli (Apc) regulates diverse effector pathways essential for cellular homeostasis. Truncating mutations in Apc, leading to the loss of its Wnt pathway and microtubule regulatory domains, are oncogenic in human and murine intestinal epithelia and drive malignant transformation. Whereas uncontrolled proliferation via Wnt pathway deregulation is an unequivocal consequence of oncogenic Apc mutations, it is not known whether loss of its other control systems contribute to tumorigenesis. Here we employ in vitro models of tumorigenesis to unmask the molecular barriers erected by Apc that maintain normal epithelial homeostasis in the murine intestinal epithelia. We determine that (i) enterocyte proliferation, (ii) microtubule dynamics and (iii) epithelial morphology are controlled by three independent molecular pathways, each corrupted by oncogenic Apc mutations. The key result of the study is to establish that Apc regulates three individual biological fates in the intestinal epithelia, through three distinct effector pathways, a significant advance to our understanding of normal tissue homeostasis, the molecular architecture of epithelial tissue and the aetiology of intestinal cancer.

B. Infantis EVC001 Metabolites Improve Enterocyte Proliferation in Vitro

Objectives Bacterial metabolites, including short chain fatty acids (SCFA) are essential for host cells to maintain homeostasis. Previous findings showed exclusively breastfed infants colonized with B. infantis EVC001 have significantly increased fecal organic acid concentrations, specifically lactate and acetate as compared to infants not colonized with B. infantis, we investigated the effect of fecal water (FW) from two distinct populations on enterocyte proliferation and morphology in vitro. Methods FW were derived from fecal samples from infants colonized with B. infantis EVC001 (EVC001) and infants not colonized with B. infantis (controls) were added to adult and premature enterocyte cell lines to assess growth, proliferation and cytotoxicity. Microscopic images were taken to observe morphological differences. Results Intestinal epithelial cells (IECs; Caco-2 and HIEC-6 cells) exposed to EVC001 FW showed significantly increased proliferation shown by ATP expression compared to medium alone and control FW (P &lt; 0.0001). Conversely, significantly decreased lactate dehydrogenase, an indication of decreased membrane integrity, was detected in enterocytes exposed to EVC001 FW compared to controls FW (P &lt; 0.01). Specific bacterial metabolites, lactate and acetate at median physiological concentrations found in EVC001 infant FW significantly increased Caco-2 intestinal integrity measured by transepithelial electrical resistance compared to medium alone (P &lt; 0.01, P &lt; 0.05, respectively), while control levels did not significantly increase proliferation. Furthermore, control FW altered the morphology of enterocytes compared to cells exposed to EVC001 FW or medium alone. Conclusions EVC001 FW and specific bacterial metabolites, lactate and acetate significantly increased enterocyte proliferation compared to control FW and medium alone, while control FW negatively affected cell growth, membrane integrity and cell morphology; suggesting metabolites produced by B. infantis EVC001 promote enterocyte growth and improve intestinal integrity in both adult and premature infants. Funding Sources Evolve Biosystems.