Search

The intestine is the central organ of early weaning stress in piglets; however, the response of intestinal stem cells (ISCs) that drive epithelial renewal between the different breeds of piglets is not yet known. Therefore, this investigation was to explore the sensitivity of ISCs to early weaning stress. Taoyuan Black and Duroc piglets were slaughtered at 21 days of age (on the day of weaning) and 24 days of age (3 days after weaning) with 10 pigs, respectively. And the results showed that Taoyuan Black pigs are more tolerant to early weaning stress, while the mucosal mass of jejunum was significantly decreased in 24-day-old Duroc piglets compared with the 21-day-old Duroc piglets (P < 0.05). The weaning stress damaged the jejunal structure, decreased the expression of tight junction related proteins ZO-1, Occludin, and Claudin-1, and increased intestinal permeability in Duroc piglets (P < 0.05). The ex vivo intestinal organoids (IOs) derived from the jejunal crypt of Duroc piglets after weaning exhibited lower expansion efficiency (P < 0.05). Furthermore, the levels of proliferating cell marker PCNA, terminally differentiated cell marker KRT20, absorptive cell marker Villin, goblet cell marker MUC2, and Paneth cell marker lysozyme were significantly reduced in the jejunum, crypt, and jejunal organoids of 24-day-old Duroc (P < 0.05). ROS levels in the jejunum of Duroc piglets were increased along with the inhibition of the Nrf2 pathway compared with 21-day-old Duroc piglets (P < 0.05). Collectively, the Taoyuan Black pigs had stronger tolerance to weaning stress; however early weaning suppressed the Nrf2 pathway, decreased proliferation and differentiation activity of ISCs, and disrupted intestinal homeostasis in Duroc piglets.

Wnt/β-catenin pathways have an important role in porcine skeletal muscle growth. Nevertheless, the mechanism for how it respond to environmental stimulations, especially nutrients, has not been well-investigated. In this study, piglets and skeletal muscle satellite cells (MuSCs) were used to investigate the work process of the lysine (Lys) signal transmitted to β-catenin in governing skeletal muscle growth. Briefly, the piglets and MuSCs were divided into the control group, the Lys deficiency group, and the Lys rescue group, taken together with the specific inhibitor and target gene knockdown for further study. We found that the transmembrane frizzled7 (FZD7) receptor displayed the same changes with the Wnt/β-catenin pathway and positively correlated with Lys levels in skeletal muscle and MuSCs. Meanwhile, the molecular docking analyses showed that FZD7 and Lys form connections at Gly17, Phe18, Cys19, and Asp84. In contrast, FZD7 specific inhibitor Fz7-21-TFA suppressed Lys rescued MuSC viability (P< 0.05). The distribution of Ki67 and active β-catenin in the Lys rescue group were also decreased by FZD7 inhibitor (P< 0.05). Furthermore, the decreased FZD7 level caused Lys re-activated TCF4/LEF activity and the Wnt/β-catenin pathway suppression (P< 0.05). Additionally, FZD7 knockdown restricted C2C12 viability and proliferation ability (P< 0.05). Besides, FZD7 knockdown inhibited the Wnt/β-catenin pathway and Lys deficiency caused much more serious inhibition of the Wnt/β-catenin pathway (P< 0.05), and these restrictions fail to be rescued by re-supplemented Lys or Wnt3a (P< 0.05). Moreover, we titrated Lys solution into recombinant pig FZD7 (rpFZD7) protein solution by using isothermal titration calorimetry, and the heat release during titration demonstrated there was an interaction between Lys and rpFZD7. Collectively, these results indicated that Lys is not only a molecular block for protein synthesis, but is also a ligand that binds to FZD7, directly activating the Wnt/β-catenin pathway to stimulate MuSCs to promote skeletal muscle growth.

The type of myofiber is important for porcine meat quality. Meanwhile, the nt/Ca2+ pathway has been showed multiple roles in skeletal muscle formation; however, the distinct mechanism is still unclear. In this study, the weaned piglets and satellite cells were designed into the control group, lysine deficiency group and lysine rescue group to investigate the function of Wnt/Ca2+ pathway in governing skeletal muscle typing. After we confirm the growth of weaned piglets was controlled by lysine, the isobaric tag for relative and absolute quantification (iTRAQ) analysis of skeletal muscle detected that Wnt/Ca2+ pathway was involved in the transition of fast and slow fiber. Then, we found the ratio of type I myofiber in Semimembranous (fast muscle) was significantly increased after lysine deficiency (P < 0.05), and decreased by lysine rescue (P < 0.05). In contrast, the ratio of type I myofiber in Semitendinous muscle (slow muscle) was significantly decreased in the lysine deficiency group, and increased in the lysine rescue group (P < 0.05). Furthermore, the Wnt/Ca2+ pathway was significantly increased in Semimembranous muscle, while decreased in Semitendinous muscle with lysine deficiency, and this phenomenon was inversed after lysine rescue (P < 0.05). Meanwhile, the Wnt/Ca2+ pathway was stronger in satellite cells isolated from Semitendinous muscle (StSCs) than that of Semimembranous satellite cell (SmSCs) (P < 0.05). And we also found the StSCs enter in differentiation is more easily than SmSCs (P < 0.05). Besides, the ratio of type I myofiber originated from StSCs showed greater than StSCs (P < 0.05). In summary, we conclude that satellite cells participate in the Wnt/Ca2+ pathway controlled porcine myofiber determination.

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.