Hui-chao Yan

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)

The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b0,+AT, EAAT3, y+LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b0,+AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y+LAT2 had positive correlations with body weight (0.71<correlation coefficient (CC)<0.82, P<0.0001), while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight (−0.86<CC<−0.64, P<0.0001). The gene expressions of b0,+AT, EAAT3, LAT4, PepT1, NHE2, NHE3, and y+LAT2 showed positive correlations with intestinal weight (0.80<CC<0.91, P<0.0001),while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight (−0.84<CC<−0.67, P<0.0001). It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter mRNA in the small intestine during embryonic growth of pigeons.概要目的探索家鸽胚胎期器官生长发育及其小肠营养素转运载体基因表达规律, 为家鸽胚胎期的营养调控提供理论基础。创新点第一次较全面地研究了家鸽胚胎期小肠氨基酸转运载体基因表达规律。方法利用实时荧光定量逆转录聚合酶链反应 (real-timeRT-PCR) 技术, 同时结合了多内标校正方法对小肠营养素转运载体基因表达模式进行分析。结论在家鸽胚胎期发育的过程中, 不同的器官生长模式不完全一致。营养素转运载体b0,+AT、EAAT3、LAT4、PepT1、NHE2、NHE3 和y+LAT2 基因的表达与小肠重量呈正相关关系, CAT1、CAT2 和EAAT2 基因的表达与小肠重量呈负相关关系。

Heat stress inhibits proliferation, promotes growth, and induces apoptosis in cultured Lantang swine skeletal muscle satellite cells

Proliferation suppression and apoptosis are the prominent characteristics induced by heat stress (HS) in cells, whereas the effects of HS on cell growth (mass accumulation) are unknown. In this study, Lantang swine (an indigenous breed of China) skeletal muscle satellite cells (SCs) were pre-cultured at 37 °C for 24 h. The HS group was subjected to HS at 41 °C, while the control group was maintained at 37 °C. Heat shock protein 70 (HSP70) expression and SC size are significantly increased (P<0.05) by HS, but cell proliferation is suppressed (P<0.05) and apoptosis is induced (P<0.05). HS led to a lower percentage of SCs in the G0/G1 phase (P<0.05) together with a higher percentage of SCs in the S phase (P<0.05). However, the percentage of SCs in the G2/M phase was decreased (P<0.05) at 48 h but then increased (P<0.05) at 72 h with HS. In addition, the phosphorylation ratios of protein kinase b (Akt), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 were increased (P<0.05) by HS. Nevertheless, the phosphorylation ratios of the 4E binding protein 1 and the eukaryotic initiation factor-4E were indistinguishable (P>0.05) from those of the control group. The phosphorylation ratio of the mammalian target of rapamycin (mTOR) (Ser2448) increased(P<0.05) within 48 h, and apparent differences were abrogated at 72 h (P>0.05). Moreover, cleaved caspase-3 expression was increased at 72 h (P<0.05). These findings indicate that HS induces apoptosis and disrupts cell cycle distribution to decrease the number of cells. Additionally, HS can promote SC growth via an activated Akt/mTOR/S6K signaling pathway.概要目的探究热应激对蓝塘猪骨骼肌卫星细胞增殖与生长的影响, 分析其作用机制。创新点通过体外细胞模型研究了热应激对猪骨骼肌卫星细胞增殖与生长的影响,发现热应激通过 Akt/mTOR/S6K 途径调控细胞的生长。方法本试验选用1 日龄蓝塘猪背最长肌的骨骼肌卫星细胞, 对照组为37 °C 正常培养, 热应激组培养温度为41 °C, 其它培养条件相同。采用细胞计数、MTT 法、流式细胞术等手段分析比较热应激对蓝塘猪骨骼肌卫星细胞增殖与生长的影响; 利用实时逆转录聚合酶链式反应 (RT-PCR) 和蛋白质印迹法 (Western blot) 等方法, 检测了热休克蛋白HSP70、半胱氨酸蛋白酶-3 (caspase-3) 及哺乳动物雷帕霉素靶蛋白 (mTOR) 信号通路关键基因的表达。结论(1) 41 °C 热处理可引起蓝塘猪骨骼肌卫星细胞产生热应激反应; (2) 热应激72 h 会通过改变细胞周期、抑制细胞增殖并诱导细胞凋亡等共同作用减少细胞数目; (3) 热应激通过Akt/mTOR/S6K途径调控细胞的生长。

Growth curves and age-related changes in carcass characteristics, organs, serum parameters, and intestinal transporter gene expression in domestic pigeon (Columba livia)

Two experiments were conducted to fit growth curves, and determine age-related changes in carcass characteristics, organs, serum biochemical parameters, and gene expression of intestinal nutrient transporters in domestic pigeon (Columba livia). In experiment 1, body weight (BW) of 30 pigeons was respectively determined at 1, 3, 7, 14, 21, 28, and 35 days old to fit growth curves and to describe the growth of pigeons. In experiment 2, eighty-four 1-day-old squabs were grouped by weight into 7 groups. On d 1, 3, 7, 14, 21, 28, and 35, twelve birds from each group were randomly selected for slaughter and post-slaughter analysis. The results showed that BW of pigeons increased rapidly from d 1 to d 28 (a 25.7-fold increase), and then had little change until d 35. The Logistic, Gompertz, and Von Bertalanffy functions can all be well fitted with the growth curve of domestic pigeons (R2>0.90) and the Gompertz model showed the highest R2value among the models (R2=0.9997). The equation of Gompertz model was Y=507.72×e-(3.76exp(-0.17t))(Y=BW of pigeon (g); t=time (day)). In addition, breast meat yield (%) increased with age throughout the experiment, whereas the leg meat yield (%) reached to the peak on d 14. Serum total protein, albumin, globulin, and glucose concentration were increased with age, whereas serum uric acid concentration was decreased (P<0.05). Furthermore, the gene expressions of nutrient transporters (y+LAT2, LAT1, B0AT1, PepT1, and NHE2) in jejunum of pigeon were increased with age. The results of correlation analysis showed the gene expressions of B0AT1, PepT1, and NHE2 had positive correlations with BW (0.73<correlation coefficient<0.80,P<0.0001). It can be concluded that, 28-day-old should be the latest market age for meat type pigeon. And the various physiological and functional properties of organs, serum profiles, and gene expression of nutrient transporters in small intestine might cause the differences in their development patterns.

L-Glutamate deficiency can trigger proliferation inhibition via down regulation of the mTOR/S6K1 pathway in pig intestinal epithelial cells

The objective of this study was to investigate the effects of L-glutamate (Glu) deficiency or L-trans pyrrolidine-2,4-dicarboxylic acid (PDC) supplementation on the proliferation of pig intestinal epithelial cells (IPEC-1). First, IPEC-1 cells were cultured in normal growing medium supplemented with 0 (Control), 50, 100, or 200 µmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 µmol/L Glu (Glu deficiency), 50 µmol/L Glu (Control), or 50 µmol/L Glu plus 100 µmol/L PDC (PDC supplementation). Cell proliferation ( = 24), cell cycle distribution ( = 6), cell apoptosis ( = 6), and expression levels of proteins of interest ( = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited ( < 0.05) by 50, 100, and 200 µmol/L PDC supplementation at 24 and 48 h after treatment. Variance analysis was performed using the GLM procedure, and the results demonstrated that Glu deficiency or PDC supplementation led to the inhibition ( < 0.05) of cell proliferation, a greater ( < 0.05) percentage of cells in the G1 phase, and a lower ( < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced ( < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphor-mammalian target of rapamycin (p-mTOR; Ser2448), p-ribosomal protein S6 kinase 1 (S6K1; Thr389), and p-S6 (Ser235/236). This study demonstrates that Glu deficiency or PDC supplementation inhibits proliferation of IPEC-1 cells via downregulation of the mTOR/S6K1 pathway and EAAT3 expression indicating that Glu deficiency may lead to the disturbances of intestinal epithelial renewal in pigs, particularly in neonates.

CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/β-Catenin Signaling Pathways

Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H –thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways.

Focal adhesion kinase and paxillin promote migration and adhesion to fibronectin by swine skeletal muscle satellite cells

The focal adhesion kinase (FAK) signaling pathway contributes to the cell migration and adhesion that is critical for wound healing and regeneration of damaged muscle, but its function in skeletal muscle satellite cells (SCs) is less clear. We compared the migration and adhesion of SCs derived from two species of pig (Lantang and Landrace) in vitro, and explored how FAK signaling modulates the two processes. The results showed that Lantang SCs had greater ability to migrate and adhere to fibronection (P < 0.05) than Landrace SCs. Compared to Landrace SCs, Lantang SCs expressed many more focal adhesion (FA) sites, which were indicated by the presence of p-paxillin (Tyr118), and exhibited less F-actin reorganization 24 h after seeding onto fibronectin. Levels of p-FAK (Tyr397) and p-paxillin (Tyr118) were greater (P < 0.05) in Lantang SCs than Landrace SCs after migration for 24 h. Similarly, Lantang SCs showed much higher levels of p-FAK (Tyr397), p-paxillin (Tyr118) and p-Akt (Ser473) than Landrace SCs 2 h after adhesion. Treatment with the FAK inhibitor PF-573228 (5 or 10 μmol/L) inhibited Lantang SC migration and adhesion to fibronectin (P < 0.05), decreased levels of p-paxillin (Tyr118) and p-Akt (Ser473) (P < 0.05), and suppressed the formation of FA sites on migrating SCs. Thus FAK appears to play a key role in the regulation of SC migration and adhesion necessary for muscle regeneration.

CDX2 increases SLC7A7 expression and proliferation of pig intestinal epithelial cells

Nutrient absorption mediated by nutrient transporters expressed in the intestinal epithelium supplies substrates to support intestinal processes, including epithelial cell proliferation. We evaluated the role of Caudal type homeobox 2 (CDX2), an intestine-specific transcription factor, in the proliferation of pig intestinal epithelial cells (IPEC-1) and searched for novel intestinal nutrient transporter genes activated by CDX2. Our cloned pig CDX2 cDNA contains a “homeobox” DNA binding motif, suggesting it is a transcriptional activator. CDX2 overexpression in IPEC-1 cells increased cell proliferation, the percentage of cells in S/G2 phase, and the abundance of transcripts of the cell cycle-related genes Cyclin A2; Cyclin B; Cyclin D2; proliferating cell nuclear antigen; and cell cycle cyclin-dependent kinases 1, 2 and 4, as well as the predicted CDX2 target genes SLC1A1, SLC5A1 and SLC7A7. In addition, luciferase reporter and chromatin immunoprecipitation assays revealed that CDX2 binds directly to the SLC7A7 promoter. This is the first report of CDX2 function in pig intestinal epithelial cells and identifies SLC7A7 as a novel CDX2 target gene. Our findings show that nutrient transporters are activated during CDX2-induced proliferation of normal intestinal epithelial cells.

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.

Dietary Supplementation with Pioglitazone Hydrochloride and Chromium Methionine Improves Growth Performance, Meat Quality, and Antioxidant Ability in Finishing Pigs

This work was designed to investigate the synergistic effects of pioglitazone hydrochloride (PGZ) and chromium methionine (CrMet) on meat quality, muscle fatty acid profile, and antioxidant ability of pigs. Pigs in four groups were fed a basic diet or basic diet supplemented with 15 mg/kg of PGZ, 200 μg/kg of CrMet, or 15 mg/kg of PGZ + 200 μg/kg of CrMet. In comparison to the control group, the average daily feed intake, feed/gain ratio, and serum high-density lipoprotein level decreased in the PGZ + CrMet group. Dietary PGZ + CrMet supplementation increased carcass dressing percentage, intramuscular fat, and marbling score. The percentages of C18:1ω-9c, C18:2ω-6c, C18:3ω-3, and polyunsaturated fatty acid (PUFA) in the longissimus thoracis muscle were increased in the PGZ + CrMet group. Greater superoxide dismutase and glutathione peroxidase activities were observed in the PGZ + CrMet group compared to the control group. Collectively, these findings suggested that feed with PGZ and CrMet improved the growth performance and meat quality, especially for PUFA proportions and antioxidant ability.

LGR5 and BMI1 Increase Pig Intestinal Epithelial Cell Proliferation by Stimulating WNT/β-Catenin Signaling

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) are markers of fast-cycling and quiescent intestinal stem cells, respectively. To determine the functions of these proteins in large animals, we investigated their effects on the proliferation of intestinal epithelial cells from pigs. Our results indicated that LGR5 and BMI1 are highly conserved proteins and that the pig proteins have greater homology with the human proteins than do mouse proteins. Overexpression of either LGR5 or BMI1 promoted cell proliferation and WNT/β-catenin signaling in pig intestinal epithelial cells (IPEC-J2). Moreover, the activation of WNT/β-catenin signaling by recombinant human WNT3A protein increased cell proliferation and LGR5 and BMI1 protein levels. Conversely, inhibition of WNT/β-catenin signaling using XAV939 reduced cell proliferation and LGR5 and BMI1 protein levels. This is the first report that LGR5 and BMI1 can increase proliferation of pig intestinal epithelial cells by activating WNT/β-catenin signaling.