Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway

Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine’s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg -1 ·h -1 ) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2a and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.     

Triennial Growth Symposium: leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs

The postprandial increases in AA and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to AA. We have shown that the postprandial increase in leucine, but not isoleucine or valine, acutely stimulates muscle protein synthesis in piglets. Leucine increases muscle protein synthesis by modulating the activation of mammalian target of rapamycin (mTOR) complex 1 and signaling components of translation initiation. Leucine increases the phosphorylation of mTOR, 70-kDa ribosomal protein S6 kinase-1, eukaryotic initiation factor (eIF) 4E-binding protein-1, and eIF4G; decreases eIF2α phosphorylation; and increases the association of eIF4E with eIF4G. However, leucine does not affect the upstream activators of mTOR, that is, protein kinase B, adenosine monophosphate-activated protein kinase, and tuberous sclerosis complex 1/2, or the activation of translation elongation regulator, eukaryotic elongation factor 2. The action of leucine can be replicated by α-ketoisocaproate but not by norleucine. Interference by rapamycin with the raptor-mTOR interaction blocks leucine-induced muscle protein synthesis. The acute leucine-induced stimulation of muscle protein synthesis is not maintained for prolonged periods, despite continued activation of mTOR signaling, because circulating AA fall as they are utilized for protein synthesis. However, when circulating AA concentrations are maintained, the leucine-induced stimulation of muscle protein synthesis is maintained for prolonged periods. Thus, leucine acts as a nutrient signal to stimulate translation initiation, but whether this translates into a prolonged increase in protein synthesis depends on the sustained availability of all AA.     

Regulation of mTORC1 by amino acids in mammalian cells: A general picture of recent advances

The mechanistic target of rapamycin complex 1 (mTORC1) integrates various types of signal inputs, such as energy, growth factors, and amino acids to regulate cell growth and proliferation mainly through the 2 direct downstream targets, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase 1 (S6K1). Most of the signal arms upstream of mTORC1 including energy status, stress signals, and growth factors converge on the tuberous sclerosis complex (TSC) − Ras homologue enriched in brain (Rheb) axis. Amino acids, however, are distinct from other signals and modulate mTORC1 using a unique pathway. In recent years, the transmission mechanism of amino acid signals upstream of mTORC1 has been gradually elucidated, and some sensors or signal transmission pathways for individual amino acids have also been discovered. With the help of these findings, we propose a general picture of recent advances, which demonstrates that various amino acids from lysosomes, cytoplasm, and Golgi are sensed by their respective sensors. These signals converge on mTORC1 and form a huge and complicated signal network with multiple synergies, antagonisms, and feedback mechanisms.     

Truncation of the mRNA cap-binding protein eIF4E is specific for the non-invasive implantation in pigs

The mechanism of implantation is species specific (pig: epitheliochorial, bovine: synepitheliochorial, mouse: hemochorial). Recently, we have shown that proteolytical cleavage of the prototypical 25 kDa mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E) produces a stable variant with a molecular mass of approximately 23 kDa in porcine endometrium at the time of implantation. Here, we investigate if an eIF4E truncation also takes place in the endometrium of species with other implantation forms. Thus, eIF4E and its repressor protein 4E-BP1 were investigated in porcine, murine and bovine endometrium during the time of implantation. Our results show that eIF4E truncation is specific for the porcine implantation. In bovine and mouse uterine tissue, no cleavage of eIF4E was observed. Whereas no difference of bovine 4E-BP1 was found, in murine samples, increased phosphorylation during implantation was observed. However, porcine samples exhibit an opposite behaviour, the abundance and mainly the phosphorylation of 4E-BP1 decrease. We propose that the translation initiation in the endometrium is differently regulated by the two eIF4E forms with regard to different 4E-BP1 abundance and phosphorylation as well as different eIF4E/4E-BP1 binding dynamic depending on the type of implantation.     

The Embryonic Pregnancy Signal Oestradiol Influences Gene Expression at the Level of Translational Initiation in Porcine Endometrial Cells

In the pig, conceptus-derived oestrogens (days 11 and 12 of pregnancy) seem to be a critical component of the signalling mechanism for maternal recognition of pregnancy. Embryonic oestrogens can mediate effects on endometrial function by interactions with epithelial and stromal oestrogen receptors (ER). Recent data demonstrate that cell membrane ER interacts with the phosphatidylinositol 3-kinase/Akt pathway in several types of cells. The protein kinase Akt is involved in the control of cell growth, survival and proliferation. One distinct function of the Akt signalling cascade is its ability to phosphorylate the eukaryotic initiation factor-4E (eIF-4E)-binding protein 1 (4E-BP1). This phosphorylation suppresses the inhibitory effect of 4E-BP1 on the translation initiation factor eIF4E and in such a way potentially stimulates gene expression at the level of translational initiation. The aim of the present study was to examine if embryonic oestradiol (E(2)) transmits its effect by such a mechanism. Endometrial cells of cyclic gilts (day 13 of the oestrous cycle, n = 4) were cultured and supplemented with vehicle (control), E(2) (50 and 100 pm/l) or with the selective ER modulator raloxifen (10 and 1000 nm/l), and incubated for 24 h. The cell viability was detected by MTT assay, the abundance and phosphorylation of Akt, 4E-BP1 and ERalpha was analysed by Western blotting. Incubation with E(2) or raloxifen did not alter endometrial cell viability. The phosphorylation of Akt at Ser(473) seems to be increased by E(2) (p < 0.05) and decreased by raloxifen (p > 0.05). Raloxifen (1000 nm/l) induced a band shift in 4E-BP1 to the highest electrophoretic mobility which reflects a decrease in phosphorylation (p < 0.05), whereas an influence of E(2) on 4E-BP1 phosphorylation could not be detected. The decrease (p < 0.05) of the abundance of the 80 kDa ERalpha form both by E(2) and raloxifen indicates that the E(2)-stimulated Akt phosphorylation and the inhibition of 4E-BP1 phosphorylation by raloxifen is an E(2) ER-transmitted process. Therefore, embryonic oestrogens can potentially transmit their effect by influencing signalling cascades which modulate gene expression at the level of translational initiation.     

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10(-8)M, 36h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10(-8)M, 30min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10(-8)M, 10min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1mM, 36h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.     

Comparison of the molecular effects of the mycotoxins beta-zearalenol and deoxynivalenol in porcine endometrial cells--a review

The mycotoxins beta-zearalenol (beta-ZOL) and deoxynivalenol (DON) produce toxic effects that result in diseases in humans and animals. The molecular mechanisms that control the mycotoxin-mediated effects are far from being completely understood. Various results show that these mycotoxins could inhibit cell proliferation. In the present short communication, the influence of beta-ZOL and DON on the abundance and phosphorylation state of kinases that are included in regulation of the initiation of mRNA translation (which is correlated with cell proliferation) was compared in porcine endometrial cells (PEC). Our results indicate that these mycotoxins modulate the expression and phosphorylation of these factors in a different manner. Whereas beta-ZOL mainly had an impact on the biological activity of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E (eIF4E) and its repressor 4E binding protein 1 (4E-BP1), DON reduced the abundance of p38 MAPk, Akt and specific 4E-BP1 bands. In summary, these results indicate that beta-ZOL influences molecular events that are included in the initiation of mRNA translation in the porcine endometrium but DON does not alter such processes clearly.     

Inhibition of host protein synthesis in B95a cells infected with the HL strain of measles virus

The shut-off of host protein synthesis in virus-infected cells is one of the important mechanisms for viral replication. In this report, we showed that the HL strain of measles virus (MeV-HL) as well as other field isolates, which were isolated from human blood lymphocytes using B95a cells, induce the shut-off in B95a cells. Since the Edmonston strain of MeV failed to induce the shut-off in B95a cells, the ability to induce the shut-off was considered to be dependent on virus strains. Although, the modification of eukaryotic translation initiation factors (eIF) including eIF4G, eIF4E, and 4E-BP1 was reported for shut-off by various viruses, the involvement of these eIFs was not observed in MeV-HL-infected B95a cells. Instead, the accumulation of phosphorylated eIF2alpha was found to coincide to the decrease of host protein synthesis, suggesting the involvement of phosphorylation of eIF2alpha in inhibition of translation as one of the mechanisms of the shut-off.     

槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制。猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2%DMSO处理。采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)法测定氨基酸和小肽转运载体以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的mRNA相对表达量;采用Western blot法测定mTOR信号通路相关基因的蛋白表达。结果表明:与对照组相比,1)0.4和0.8 mg/L槲皮素均极显著增加猪肠上皮细胞中蛋白质的含量(P<0.01)。2)1.6 mg/L槲皮素极显著提高猪肠上皮细胞中兴奋性氨基酸转运载体1(EAAC1)、谷氨酰胺载体2(ASCT2)、氨基酸转运载体A2(ATA2)、L型氨基酸转运载体2(LAT2)、阳离子氨基酸转运载体1(CAT1)、b 0,+系统氨基酸转运载体(rBAT)、y+L系统氨基酸转运载体1(y+LAT1)、y+L系统氨基酸转运载体2(y+LAT2)和寡肽转运载体1(PepT1)mRNA相对表达量(P<0.01)。3)0.4 mg/L槲皮素极显著降低猪肠上皮细胞中结节性硬化复合物1(TSC1)mRNA相对表达量(P<0.01);0.8 mg/L槲皮素极显著增加mTOR和核糖体蛋白S6(RPS6)mRNA相对表达量并极显著降低TSC1 mRNA相对表达量(P<0.01);1.6 mg/L槲皮素极显著增加mTOR、真核起始因子4E结合蛋白1(4E-BP1)、真核细胞翻译起始因子4E(eIF4E)、真核细胞翻译起始因子4B(eIF4B)、真核细胞翻译起始因子4A(eIF4A)和RPS6 mRNA相对表达量(P<0.01)。4)0.1和1.6 mg/L槲皮素极显著提高猪肠上皮细胞中mTOR、eIF4E和eIF4A蛋白表达量并极显著降低4E-BP1蛋白表达量(P<0.01)。由此可见,槲皮素可通过调控氨基酸转运载体、小肽转运载体及mTOR信号通路相关基因的表达来促进猪肠上皮细胞对蛋白质的利用。     

Study on Effect of IGF-1 on Proliferation and Apoptosis of Bovine Mammary Epithelial Cells by Adding Inhibitor

The test was aimed to study the effect of insulin-like growth factor 1(IGF-1) on the proliferation of bovine mammary epithelial cells (BMECs),and provide a theoretical basis for the subsequent regulating mammary gland development using IGF-1. The optimal IGF-1 concentration for inhibiting BMECs apoptosis was obtained by measuring the apoptosis rate at 12, 24, 48 and 72 h after the addition of IGF-1 at four groups (0, 10, 50 and 100 μg/mL). Then divided into six groups:BMECs group, BMECs+IGF-1 group, BMECs+LY294002 group, BMECs+IGF-1+LY294002 group, BMECs+RAPA group and BMECs+IGF-1+RAPA group,and the apoptosis rates were detected by flow cytometry. The results showed that the heterologous IGF-1 had an inhibitory effect on the apoptosis of BMECs with an optimal concentration of 50 μg/mL. The apoptosis rates of BMECs+IGF-1+LY294002 and BMECs+IGF-1+RAPA groups were extremely significantly higher than BMECs+IGF-1 group (P<0.01). This study suggested that IGF-1 could activate the PI3K/Akt/mTOR signaling pathway and thereby inhibit the apoptosis of BMECs. Moreover, IGF-1 might also promote the "repair" mechanism for the inhibited PI3K/Akt/mTOR signaling pathway, and therefore make it reparticipate in BMECs life process.     

添加通路阻断剂对IGF-1影响奶牛乳腺上皮细胞增殖与凋亡的研究

试验旨在研究胰岛素样生长因子-1（insulin-like growth factor 1,IGF-1）对奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）增殖的影响,为后期利用IGF-1调控乳腺发育奠定理论基础。以奶牛乳腺上皮细胞为材料,首先分4组分别外源添加0（对照组）、10、50、100 μg/mL IGF-1且分别培养12、24、48、72 h,测定抑制BMECs凋亡率的最佳浓度;然后分6组：单纯BMECs组、BMECs+IGF-1组、BMECs+LY294002组、BMECs+IGF-1+LY294002组、BMECs+RAPA组和BMECs+IGF-1+RAPA组,采用流式细胞术测定各组细胞凋亡率。结果表明,外源性添加IGF-1对BMECs凋亡率具有抑制作用,最佳浓度为50 μg/mL;BMECs+IGF-1+LY294002与BMECs+IGF-1+RAPA组细胞凋亡率均极显著高于BMECs+IGF-1组（P<0.01）。推断IGF-1能够活化PI3K/Akt/mTOR信号通路,参与BMECs凋亡的调控作用,进而抑制BMECs细胞凋亡;IGF-1可能会对被抑制PI3K/Akt/mTOR信号通路产生"修复"机制,使其能够重新参与BMECs的生命进程。     

Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

Background

The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation.

Results

Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development.

Conclusions

The rapid growth of neonatal muscle is in part due to the positive balance between the activation of protein synthesis and degradation signaling. Insulin, amino acids, and, particularly, leucine, act as signals to modulate muscle protein synthesis and degradation in neonates.     

A local increase in the mammary IGF-1: IGFBP-3 ratio mediates the mammogenic effects of estrogen and growth hormone

A single epithelium-free mammary fat pad was surgically prepared in each of twenty-five one-month-old, Friesian heifers. At 18 mo of age, heifers were randomly assigned to one of four treatment groups. Treatments were: control (C), growth hormone (GH), estrogen (E) or growth hormone + estrogen (GE). Hormones were administered for 40 hr before the animals were sacrificed to provide mammary samples of parenchyma (PAR), intact fat pad (MFP), and epithelium-free or "cleared" fat pad (CFP). IGF-1 and IGF binding protein-3 (IGFBP-3) mRNA was highest in CFP and MFP whereas the protein products were highest in PAR. IGFBP-2, a 28-kDa IGFBP and a 24-kDa IGFBP were more abundant in CFP and MFP. E and GH increased incorporation of [(3)H]thymidine into DNA of PAR. Incorporation of [(3)H]thymidine into the DNA of MFP or CFP was minimal. Coincident with the changes observed in mammary epithelial proliferation, E increased IGF-1 protein in MFP and PAR, and to a lesser extent in CFP. E tended to increase IGF-1 mRNA levels in MFP, but not CFP implying that the regulation of IGF-1 expression is modulated by adjacent epithelium. GH and E reduced IGFBP-3 protein in PAR and increased the 24-kDa IGFBP in CFP and MFP. Increased proliferation of mammary parenchymal cells was associated with increased IGF-1 and reduced IGFBP-3 protein in mammary tissue. An increase in the ratio of mammary IGF-1: IGFBP-3 likely increases the proportion of the mammary IGF-1 available to stimulate proliferation. These data also indicate that stromal: epithelial interactions regulate the IGF-1 axis in mammary tissue.     

氨基酸缺乏诱导细胞自噬的哺乳动物雷帕霉素靶蛋白复合体C1信号通路机制研究进展

自噬是机体维持自身稳态的一种重要生理活动,当体内氨基酸或葡萄糖等营养缺乏时,细胞会启动自噬。自噬受到多种信号通路的调节,哺乳动物雷帕霉素靶蛋白复合体C1(m TORC1)信号通路是其中重要的一条,它可以使自噬相关基因13(Atg13)磷酸化,抑制自噬起始。本文将围绕近年来报道的氨基酸缺乏诱导细胞自噬的m TORC1信号通路,包括小G蛋白、腺苷酸活化蛋白激酶(AMPK)、微小RNA(miRNA)、氨基酰-tRNA合成酶在其中的作用等研究进展进行综述。     

乳腺对氨基酸的摄取及其调控机制研究进展

乳蛋白是乳中重要的营养成分之一,超过90％的乳蛋白是乳腺利用从血液中摄取的氨基酸从头合成,因此在保证氨基酸充足供给的前提下,乳腺对氨基酸摄取率的高低是影响乳蛋白产量的关键因素.血液中的氨基酸不能自由扩散进出乳腺,需要由乳腺上皮细胞膜上特异的氨基酸转运载体(AAT)协助完成.而乳腺AAT活性受到营养物质和激素水平的调节,...     

The market for amino acids: understanding supply and demand of substrate for more efficient milk protein synthesis

For dairy production systems, nitrogen is an expensive nutrient and potentially harmful waste product. With three quarters of fed nitrogen ending up in the manure, significant research efforts have focused on understanding and mitigating lactating dairy cows' nitrogen losses. Recent changes proposed to the Nutrient Requirement System for Dairy Cattle in the US include variable efficiencies of absorbed essential AA for milk protein production. This first separation from a purely substrate-based system, standing on the old limiting AA theory, recognizes the ability of the cow to alter the metabolism of AA. In this review we summarize a compelling amount of evidence suggesting that AA requirements for milk protein synthesis are based on a demand-driven system. Milk protein synthesis is governed at mammary level by a set of transduction pathways, including the mechanistic target of rapamycin complex 1(mTORC1), the integrated stress response(ISR), and the unfolded protein response(UPR). In tight coordination, these pathways not only control the rate of milk protein synthesis, setting the demand for AA, but also manipulate cellular AA transport and even blood flow to the mammary glands, securing the supply of those needed nutrients. These transduction pathways, specifically mTORC1, sense specific AA, as well as other physiological signals, including insulin, the canonical indicator of energy status. Insulin plays a key role on mTORC1 signaling, controlling its activation, once AA have determined mTORC1 localization to the lysosomal membrane.Based on this molecular model, AA and insulin signals need to be tightly coordinated to maximize milk protein synthesis rate. The evidence in lactating dairy cows supports this model, in which insulin and glucogenic energy potentiate the effect of AA on milk protein synthesis. Incorporating the effect of specific signaling AA and the differential role of energy sources on utilization of absorbed AA for milk protein synthesis seems like the evident following step in nutrient requirement systems to further improve N efficiency in lactating dairy cow rations.     

低蛋白质饲粮补充过瘤胃赖氨酸和过瘤胃蛋氨酸对荷斯坦公牛生长性能、屠宰性能、肉品质及氮代谢的影响

本试验旨在研究低蛋白质饲粮补充过瘤胃赖氨酸(RPLys)和过瘤胃蛋氨酸(RPMet)对荷斯坦公牛生长性能、屠宰性能、肉品质及氮代谢的影响.选取36头健康无疾病的荷斯坦公牛,随机分为3组,每组12头牛.对照组(Ⅰ组)饲喂蛋白质水平为13％的正常饲粮,2个试验组(Ⅱ、Ⅲ组)均饲喂蛋白质水平为11％的低蛋白质饲粮,同时Ⅱ组补...     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

The effect of dietary protein levels on the expression of genes coding for four selected protein translation initiation factors in muscle tissue of Wujin pig

The objective of this study was to investigate the regulatory mechanism underlying the increased muscle protein accumulation in pigs while were fed a high protein diet. The eukaryotic initiation factors (eIFs) have been reported to involve in muscle protein synthesis. We investigated the mRNA and protein expression levels of eIF2B1, 4A1, 4B and 4E in Wujin pigs fed either a high protein (HP: 18%) or a low protein (LP: 14%) diet at 30, 60 or 100 kg body weight, based on real‐time PCR and western blotting analyses. Our results indicated that the expression levels of eIF2B1 mRNA and protein were increased by HP diet at all body weight. The HP diet showed higher mRNA and protein levels of eIF4B gene at 60 and 100 kg. The protein expression of eIF4E phosphorylation was increased by HP diet only at 30 kg. These data suggested that the HP diet promoted porcine muscle protein accumulation mainly by up‐regulating eIF2B1, 4B and 4E rather than 4A1 expression along the growth stages.