Meimei Geng

Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d

Lactoferrin has antimicrobial activity associated with peptide fragments lactoferricin (LFC) and lactoferrampin (LFA) released on digestion. These two fragments have been expressed in Photorhabdus luminescens as a fusion peptide linked to protein cipB. The construct cipB-LFC-LFA was tested as an alternative to antimicrobial growth promoters in pig production. Sixty piglets with an average live body weight of 5.42 (sem 0.59) kg were challenged with enterotoxigenic Escherichia coli and randomly assigned to four treatment groups fed a maize-soyabean meal diet containing either no addition (C), cipB at 100 mg/kg (C+B), cipB-LFC-LFA at 100 mg/kg (C+L) or colistin sulfate at 100 mg/kg (C+CS) for 3 weeks. Compared with C, dietary supplementation with C+L for 3 weeks increased daily weight gain by 21 %, increased recovery from diarrhoea, enhanced serum glutathione peroxidase (GPx), peroxidase (POD) and total antioxidant content (T-AOC), liver GPx, POD, superoxide dismutase and T-AOC, Fe, total Fe-binding capacity, IgA, IgG and IgM levels (P < 0.05), decreased the concentration of E. coli in the ileum, caecum and colon (P < 0.05), increased the concentration of lactobacilli and bifidobacteria in the ileum, caecum and colon (P < 0.05), and promoted development of the villus-crypt architecture of the small intestine. Growth performance was similar between C+L- and C+CS-supplemented pigs. The present results indicate that LFC-LFA is an effective alternative to the feed antibiotic CS for enhancing growth performance in piglets weaned at age 21 d.

Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates

The objective of this study was to determine developmental changes in mRNA and protein levels for N-acetylglutamate synthase (NAGS; a key enzyme in synthesis of citrulline and arginine from glutamine/glutamate and proline) in the small intestine of suckling piglets. The porcine NAGS gene was cloned using the real-time polymerase-chain reaction (RT-PCR) method. The porcine NAGS gene encoded 368 amino acid residues and had a high degree of sequence similarity to the “conserved domain” of human and mouse NAGS genes. The porcine NAGS gene was expressed in E. coli BL21 and a polyclonal antibody against the porcine NAGS protein was developed. Real-time RT-PCR and western-blot analyses were performed to quantify NAGS mRNA and protein, respectively, in the jejunum and ileum of 1- to 28-day-old pigs. Results indicated that intestinal NAGS mRNA levels were lower in 7- to 28-day-old than in 1-day-old pigs. Immunochemical analysis revealed that NAGS protein was localized in enterocytes of the gut. Notably, intestinal NAGS protein abundance declined progressively during the 28-day suckling period. The postnatal decrease in NAGS protein levels was consistent with the previous report of reduced NAGS enzymatic activity as well as reduced synthesis of citrulline and arginine in the small intestine of 7- to 28-day-old pigs. Collectively, these results suggest that intestinal NAGS expression is regulated primarily at the post-transcriptional level. The findings also provide a new molecular basis to explain that endogenous synthesis of arginine is impaired in sow-reared piglets and arginine is a nutritionally essential amino acid for the neonates.

Molecular cloning, tissue distribution and ontogenetic expression of the amino acid transporter b0,+ cDNA in the small intestine of Tibetan suckling piglets

The small intestine is the main absorption place of peptides and free amino acids in mammals. The amino acid transporter system b(0,+) mediates apical uptake of basic amino acids, especially lysine, arginine and cysteine. The aim of the current study was to clone Tibetan porcine amino acid transporter b(0,+)AT (SLC7A9) for comparing the sequences of Tibetan and common (Sus scrofa) pigs, and investigating the tissue distribution and ontogenetic expression in the small intestine of Tibetan suckling piglets. The Tibetan porcine SLC7A9 gene was first cloned from the porcine small intestine and found to encode the amino acid transporter b(0,+)AT. The entire open reading frame (ORF) of the SLC7A9 is 1464 bp and codes for 487 amino acid residues, with a higher degree of sequence similarity with common pig (99.59%) and horse counterparts (91.2%) than with monkey (89.5%) or human (88.7%). The deduced protein has 12 putative transmembrane domains. In this study, SLC7A9 mRNA was detected in brain, kidney, duodenum, jejunum, ileum, heart, liver, lung and muscle from Tibetan pigs at 7 and 21 days by PCR. We also investigated the age-dependent expression of b(0,+)AT in Tibetan suckling piglets in duodenum, anterior jejunum, posterior jejunum, ileum and kidney from day 1 to 35. The abundance of SLC7A9 mRNA in duodenum and jejunum was highest and lowest, respectively. Expression patterns were similar in duodenum and anterior jejunum, where the mRNA level was decreased before the suckling period and increased until day 35. Posterior jejunum expression was increasing steadily with age, except on day 7. The ileum has the highest expression at day 14 and became steady after day 28. The mRNA abundance in the kidney is opposite to duodenum, increasing until day 14 and reducing thereafter. Our results showed the pattern of b(0,+)AT expressed in small intestine of Tibetan pig and lay the foundation for in depth investigations of the regulation of b(0,+)AT in vivo.

Chemerin regulates proliferation and differentiation of myoblast cells via ERK1/2 and mTOR signaling pathways.

Obesity in human is an alarming major public health crisis worldwide and insulin resistance is a hallmark of it. The negative cross-talk between skeletal muscle and adipose tissue through adipokines is now accepted as one of the leading cause of insulin resistance. Chemerin is a novel adipokine previously reported to induce insulin resistance in primary human skeletal muscle cells. To investigate the role of chemerin in myogenesis, C2C12 cells were used and treated with chemerin in proliferation and differentiation stages. Our results showed that chemerin promoted proliferation and suppressed differentiation of C2C12 cells through extracellular-signal regulated kinase-1/2 (ERK1/2) and mammalian target of rapamycin (mTOR) signaling pathways, and these two pathways were interacted with each other in C2C12 cells treated with chemerin. It is concluded from this in vitro study that chemerin which expression is increased during myoblast differentiation appears to be able, likely in an autocrine/paracrine manner, to increase myoblast proliferation and decrease myoblast differentiation.

Soy isoflavones modulate adipokines and myokines to regulate lipid metabolism in adipose tissue, skeletal muscle and liver of male Huanjiang mini-pigs

Although a growing body of evidence suggests that soy isoflavones help regulate lipid metabolism, the underlying mechanism has not yet been thoroughly clarified. The present study was undertaken to determine the effects of soy isoflavones on the expression of genes involved in lipid metabolism in different adipose tissue depots, skeletal muscle and liver of male Huanjiang mini-pigs, as well as the expression of adipokines and myokines. A total of 36 male Huanjiang mini-pigs were fed basal diet (control, Con), low-dose soy isoflavones (LSI) and high-dose soy isoflavones (HSI). The results showed that LSI and HSI regulated the expression of genes involved in the anabolism and catabolism of fatty acids in dorsal subcutaneous (DSA), abdominal subcutaneous (ASA) and perirenal (PRA) adipose tissue depots, as well as longissimus dorsi muscle (LDM) and liver. LSI and HSI also regulated the expression of adipokines in DSA, ASA and PRA, and the expression of myokines in LDM in male Huanjiang mini-pigs. In addition, soy isoflavones regulated plasma glucose, leptin and adiponectin contents after treatment for two months. Our results indicate that soy isoflavones, by regulating the expression of adipokines and myokines, may regulate the metabolism of lipids and could have potential therapeutic applications in lipid abnormalities.

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 1β, 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 protein intake affects expression of genes for lipid metabolism in porcine skeletal muscle in a genotype-dependent manner

Skeletal muscle is a major site for the oxidation of fatty acids (FA) in mammals, including humans. Using a swine model, we tested the hypothesis that dietary protein intake regulates the expression of key genes for lipid metabolism in skeletal muscle. A total of ninety-six barrows (forty-eight pure-bred Bama mini-pigs (fatty genotype) and forty-eight Landrace pigs (lean genotype)) were fed from 5 weeks of age to market weight. Pigs of fatty or lean genotype were randomly assigned to one of two dietary treatments (low- or adequate-protein diet), with twenty-four individually fed pigs per treatment. Our data showed that dietary protein levels affected the expression of genes involved in the anabolism and catabolism of lipids in the longissimus dorsi and biceps femoris muscles in a genotype-dependent manner. Specifically, Bama mini-pigs had more intramuscular fat, SFA and MUFA, as well as elevated mRNA expression levels of lipogenic genes, compared with Landrace pigs. In contrast, Bama mini-pigs had lower mRNA expression levels of lipolytic genes than Landrace pigs fed an adequate-protein diet in the growing phase. These data are consistent with higher white-fat deposition in Bama mini-pigs than in Landrace pigs. In conclusion, adequate provision of dietary protein (amino acids) plays an important role in regulating the expression of key lipogenic genes, and the growth of white adipose tissue, in a genotype- and tissue-specific manner. These findings have important implications for developing novel dietary strategies in pig production.

High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens

Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively.

Regulation of soy isoflavones on weight gain and fat percentage: evaluation in a Chinese Guangxi minipig model

This study was conducted to determine the effects of soy isoflavones on changes in body and tissue weight and on insulin-like factor I (IGF-I) and peroxisome proliferator-activated receptor-γ (PPARγ) gene and protein expression in muscle and adipose tissues in Chinese Guangxi minipig, as a model for studying human nutrition. A total of 72 male Chinese Guangxi minipigs were fed basal diet (control, Con), low dose of soy isoflavones and high dose of soy isoflavones (HSI). The results showed that HSI increased the body weight (BW) gain and fat percentage of minipigs (P < 0.05). In addition, the serum concentrations of IGF-I and interleukin-6 were increased by high levels of soy isoflavones (P < 0.05). Furthermore, a diet containing soy isoflavones enhanced IGF-I mRNA expression levels in longissimus muscle, but decreased these levels in perirenal fat. However, the mRNA and protein expression levels of PPARγ in longissimus muscle and subcutaneous adipose tissue were both increased when compared with the Con. The data indicated that soy isoflavones regulated the BW gain and fat percentage of Chinese Guangxi minipigs, which also showed changes in IGF-I system and PPARγ. However, further research is required to clarify the causative relationship.

Molecular cloning, tissue distribution and ontogenetic expression of Xiang pig Chemerin and its involvement in regulating energy metabolism through Akt and ERK1/2 signaling pathways

Chemerin, as a new member of adipokines family, is highly expressed in adipose tissue in rodent and its expression increases with obesity. Moreover, chemerin has been reported to have significant relationship with metabolic syndrome and insulin sensitivity. Here, the gene encoding chemerin from Xiang pig was cloned. The open reading frame of this cDNA encodes 163 deduced amino acid residues. The putative protein has a N-terminal signaling peptide and a nuclear localization signal profile which are highly conserved among the vertebrate orthologs. Both chemerin and chemerinR are highly expressed in lung, kidney and small intestine in adult Xiang pig. Besides these tissues, chemerin is abundant in liver and backfat, and chemerinR is abundant in spleen and skeletal muscle. We also investigated the age-dependent expression of chemerin in suckling Xiang piglets in various tissues, which showed an interaction between age and segments in abundance of chemerin and chemerinR from day 1 to day 21. For chemerinR, it was abundant in skeletal muscle of both adult and fetal Xiang pig. Further, we treated differentiated C2C12 cells with chemerin. The result showed that chemerin regulated energy metabolism partly through Akt and ERK1/2 signaling pathway. Taken together, our findings provide basic molecular information for the deeper investigation on the function of chemerin.