Fate of undigested proteins in the pig large intestine: What impact on the colon epithelium?

Apart from its obvious agronomic interest in feeding billions of people worldwide, the porcine species represents an irreplaceable experimental model for intestinal physiologists and nutritionists. In this review, we give an overview on the fate of proteins that are not fully digested in the pig small intestine, and thus are transferred into the large intestine. In the large intestine, dietary and endogenous proteins are converted to peptides and amino acids (AA) by the action of bacterial proteases and peptidases. AA, which cannot, except in the neonatal period, be absorbed to any significant level by the colonocytes, are used by the intestinal microbes for protein synthesis and for the production of numerous metabolites. Of note, the production of the AA-derived metabolites greatly depends on the amount of undigested polysaccharides in the pig's diet. The effects of these AA-derived bacterial metabolites on the pig colonic epithelium have not yet been largely studied. However, the available data, performed on colonic mucosa, isolated colonic crypts and colonocytes, indicate that some of them, like ammonia, butyrate, acetate, hydrogen sulfide (H2S), and p-cresol are active either directly or indirectly on energy metabolism in colonic epithelial cells. Further studies in that area will certainly gain from the utilization of the pig colonic organoid model, which allows for disposal of functional epithelial unities. Such studies will contribute to a better understanding of the potential causal links between diet-induced changes in the luminal concentrations of these AA-derived bacterial metabolites and effects on the colon epithelial barrier function and water/electrolyte absorption.

Delayed effects of autophagy on T-2 toxin-induced apoptosis in mouse primary Leydig cells.

T-2 toxin is a type-A trichothecene produced by Fusarium found in several food commodities worldwide. T-2 toxin causes reproductive disorders, genotoxicity, and testicular toxicity in animals. Our previous research has reported that T-2 toxin can induce apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells. However, little is known about the functions of autophagy and the cross talk between autophagy and apoptosis after exposure to T-2 toxin in Leydig cells. This study investigated these problems in mouse primary Leydig cells. Results showed that T-2 toxin treatment upregulated LC3-II and Beclin-1 expression, suggesting that T-2 toxin induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to T-2 toxin-treated group. Autophagy delayed apoptosis in the T-2 toxin-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing T-2 toxin-induced cytotoxicity.

Colonic luminal microbiota and bacterial metabolite composition in pregnant Huanjiang mini-pigs: effects of food composition at different times of pregnancy.

The gut harbours diverse and complex microbiota, which influence body health including nutrient metabolism, immune development, and protection from pathogens. Pregnancy is associated with immune and metabolic changes that might be related to microbiota compositional dynamics. We therefore investigated the colonic luminal bacteria community in Huanjiang mini-pigs fed diets with different nutrient levels from the first to third trimester of pregnancy. The concentrations of intestinal metabolites including short-chain fat acids, NH3-N, indole, skatole, and bioamines were also determined. We found that the colonic bacteria species richness estimators (Chao1 and ACE) decreased with increased gestational age. The dominant phyla identified were Firmicutes and Bacteroidetes; the dominant genera were Lactobacillus, Treponema, Ruminococcus, Clostridium, and Prevotella. In addition, microbiota displayed spatial and temporal heterogeneity in composition, diversity, and species abundance in different colonic segments from the first to third trimester of pregnancy. Furthermore, the bacterial metabolites also changed according to the diet used and the pregnancy stage. These findings suggest that colonic bacteria richness decreased as gestational age increased, and that the higher nutrient level diet increased the production of metabolites related to nitrogen metabolism. However, although the higher nutrient diet was associated with pregnancy syndrome, causal links remain to be determined.

EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells.

Excitatory amino acid transporter 3 (EAAT3, encoded by SLC1A1) is an epithelial type high-affinity anionic amino acid transporter, and glutamate is the major oxidative fuel for intestinal epithelial cells. This study investigated the effects of EAAT3 on amino acid transport and cell proliferation through activation of the mammalian target of the rapamycin (mTOR) pathway in porcine jejunal epithelial cells (IPEC-J2). Anionic amino acid and cystine (Cys) transport were increased (P<0.05) by EAAT3 overexpression and decreased (P<0.05) by EAAT3 knockdown rather than other amino acids. MTT and cell counting assays suggested that IPEC-J2 cell proliferation increased (P<0.05) with EAAT3 overexpression. Phosphorylation of mTOR (Ser2448), ribosomal protein S6 kinase-1 (S6K1, Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1, Thr70) was increased by EAAT3 overexpression and decreased by EAAT3 knockdown (P<0.05), as were levels of activating transcription factor 4 (ATF4) and cystine/glutamate antiporter (xCT) (P<0.05). Our results demonstrate for the first time that EAAT3 facilitates anionic amino acid transport and activates the mTOR pathway, promoting Cys transport and IPEC-J2 cell proliferation.

Effects of T-2 toxin on the regulation of steroidogenesis in mouse Leydig cells.

T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of testosterone secretion in primary Leydig cells derived from mouse testis. The previous study demonstrated T-2 toxin decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis directly. In this study, we further examined the direct biological effects of T-2 toxin on the process of steroidogenesis, primarily in Leydig cells of mice. Leydig cells of mature mouse were purified by Percoll gradient centrifugation and the cell purity was determined by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) staining. To examine the decrease in T-2 toxin-induced testosterone secretion, we measured the transcription level of three key steroidogenic enzymes including 3ß-HSD-1, cytochrome P450 side-chain cleavage (P450scc) enzyme, and steroidogenic acute regulatory (StAR) protein in T-2 toxin/human chorionic gonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7), 10(-8), and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with a decrease in the level of transcription of 3ß-HSD-1, P450scc, and StAR (p < 0.05).

Dietary supplementation with soybean oligosaccharides increases short-chain fatty acids but decreases protein-derived catabolites in the intestinal luminal content of weaned Huanjiang mini-piglets.

The improvement of gut health and function with prebiotic supplements after weaning is an active area of research in pig nutrition. The present study was conducted to test the working hypothesis that medium-term dietary supplementation with soybean oligosaccharides (SBOS) can affect the gut ecosystem in terms of microbiota composition, luminal bacterial short-chain fatty acid and ammonia concentrations, and intestinal expression of genes related to intestinal immunity and barrier function. Ten Huanjiang mini-piglets, weaned at 21 days of age, were randomly assigned to 2 groups. Each group received a standard diet containing either dietary supplementation with 0.5% corn starch (control group) or 0.5% SBOS (experimental group). The results showed that dietary supplementation with SBOS increased the diversity of intestinal microflora and elevated (P < .05) the numbers of some presumably beneficial intestinal bacteria (e.g., Bifidobacterium sp, Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Roseburia). Soybean oligosaccharide supplementation also increased the concentration of short-chain fatty acid in the intestinal lumen, and it reduced (P < .05) the numbers of bacteria with pathogenic potential (e.g., Escherichia coli, Clostridium, and Streptococcus) and the concentration of several protein-derived catabolites (e.g., isobutyrate, isovalerate, and ammonia). In addition, SBOS supplementation increased (P < .05) expression of zonula occludens 1 messenger RNA, and it decreased (P < .05) expression of tumor necrosis factor α, interleukin 1ß, and interleukin 8 messenger RNA in the ileum and colon. These findings suggest that SBOS supplementation modifies the intestinal ecosystem in weaned Huanjiang mini-piglets and has potentially beneficial effects on the gut.

Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets.

This work is aimed at investigating the effects of recombinant bovine lactoferrampin-lactoferricin (LFA-LFC) instead of chlortetracycline on intestinal microflora in weaned piglets. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, recombinant LFA-LFC was produced via fed-batch fermentation in recombinant strain Pichia pastoris (KM71) XS10. Uniform design U6(6(4)) was used to optimize the fermentation conditions. The target peptide purified via cation-exchange and size-exclusion chromatography was added into the dietary of weaned piglets. After 21 days, the Lactobacilli, Bifidobacteria, and Enterobacteria in the chyme of the gut were quantified using real-time polymerase chain reaction. The results showed that approximately 82 mg of LFA-LFC was secreted into 1 L of medium under optimized conditions. Moreover, purified peptide showed strong antimicrobial activities against all the tested microorganisms. Compared with the control group, the LFA-LFC group increased the amount of Lactobacilli and Bifidobacteria (P<0.05) in the chyme of the stomach, duodenum, jejunum, ileum, colon, and caecum. These results show that dietary supplementation with LFA-LFC can affect intestinal microflora in weaned piglets.

Toxic effects of T-2 toxin on reproductive system in male mice.

The present study was conducted to evaluate the effects of T-2 toxin on semen quality, fertility and serum testosterone concentration in mice. Adult male mice were mated with sexually mature untreated female mice after being exposed to intraperitoneal injection of T-2 toxin at 0, 5, 10 or 15 mg/kg body weight daily for 7 successive days. Semen quality, serum testosterone concentration and fertility of treated mice were assessed. The results showed that the number of abnormal spermatozoa increased significantly and a significant decrease in spermatozoa with integrated acrosome was observed in males treated with T-2 toxin at all doses, As well, the amount of live spermatozoa decreased significantly in mice treated with 10 and 15 mg/kg body weight T-2 toxin. Low pregnancy rate and high fetal resorption rate were observed when females were mated with T-2 toxin-exposed males. Testicular and cauda epididymal sperm counts, efficiency of sperm production and serum testosterone concentration were significantly reduced in mice treated with T-2 toxin at all doses in a dose-dependent manner. In conclusion, these findings indicated that T-2 toxin presented toxic effects on reproductive system of adult male mice.

Selection of component drug in activating blood flow and removing blood stasis of Chinese herbal medicinal formula for dairy cow mastitis by hemorheological method.

OBJECTIVE: In order to select the component drug in promoting blood circulation and removing blood stasis of Chinese herbal medicinal formula for dairy cow mastitis. METHODS: 25 healthy rabbits were allocated randomly into five equal groups. The rabbits in four experimental groups were administered with decoctions of giant knotweed rhizome (GKR, rhizoma polygoni cuspidati), safflower (SF, flos carthami), red sage root (RSR, radix salviae miltiorrhizae) and chuanxiong rhizome (CXR, rhizoma Chuanxiong) by gastrogavage, respectively, in control group, physiological saline, once a day for seven successive days. After the last administration, all rabbits were intravenously injected with 10% macromolecular dextran to induce blood stasis. The blood samples of all rabbits were collected before the first administration, at 2h after the last administration and 1h after injection of dextran, respectively for determination of hemorheologic parameters by MVIS-2035 hemorheology auto-analyzing system. RESULTS: The results showed that all of four kinds of herbs presented different degree of activating blood flow and removing blood stasis. CONCLUSION: Red sage root was the best especially in resisting blood stasis induced by dextran, and would be selected as main component drug of the prescription for dairy cow mastitis.

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro.

A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.