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.     

Leucine stimulates mammalian target of rapamycin signaling in C2C12 myoblasts in part through inhibition of adenosine monophosphate-activated protein kinase

Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis. Leucine treatment upregulates mTOR signaling, which enhances protein synthesis; however, the mechanisms are not well understood. Herein, treatment of C2C12 myoblast cells with leucine enhanced the phosphorylation of mTOR and ribosomal protein S6 kinase. Leucine treatment also decreased the adenosine monophosphate/ATP ratio in myoblasts by 36.4 +/- 9.1% (P < 0.05) and reduced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) alpha subunit at Thr172 (28.6 +/- 4.9% reduction, P < 0.05) and inhibited AMPK activity (43.6 +/- 3.5% reduction, P < 0.05). In addition, leucine increased the phosphorylation of mTOR at Ser2448 by 63.5 +/- 10.0% (P < 0.05) and protein synthesis by 30.6 +/- 6.1% (P < 0.05). Applying 5-aminoimidazole-4-carbox-amide 1-beta-d-ribonucleoside, an activator of AMPK, abolished the stimulation of mTOR signaling by leucine, showing that AMPK negatively controls mTOR signaling. To further show the role of AMPK in mTOR signaling, myoblasts expressing a dominant negative AMPKalpha subunit were employed. Negative myoblasts had very low AMPK activity. The activation of mTOR induced by leucine in these cells was abated, showing that AMPK contributed to mTOR activation. In conclusion, leucine stimulates mTOR signaling in part through AMPK inhibition. This study implicates AMPK as an important target for nutritional management to enhance mTOR signaling and protein synthesis in muscle cells, thereby increasing muscle growth.     

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。     

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.     

Leucine‐induced promotion of post‐absorptive EAA utilization and hepatic gluconeogenesis contributes to protein synthesis in skeletal muscle of dairy calves

The high rate of protein synthesis in skeletal muscle of dairy calves can benefit their first lactation even lifetime milk yield. Since the rate of protein synthesis is relatively low in the post‐absorptive state, the aim of this research was to determine whether leucine supplementation could increase the post‐absorptive essential amino acid (EAA) utilization and protein synthesis in the skeletal muscle. Ten male neonatal dairy calves (38 ± 3 kg) were randomly assigned to either the control (CON, no leucine supplementation, n = 5) or supplementation with 1.435 g leucine/L milk (LEU, n = 5). Results showed that leucine significantly increased the length and protein concentration in longissimus dorsi (LD) muscle, whereas it decreased creatinine concentration and glutamic‐oxalacetic transaminase (GOT) activity. Compared to the control group, leucine supplementation also reduced the glutamic‐pyruvic transaminase (GPT) activity. Supplementation of leucine improved the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E‐binding protein 1 (4EBP1) and substrates ribosomal protein S6 kinase 1 (p70S6K). Supplementation of leucine resulted in increased concentrations of glucose, methionine, threonine, histidine and EAAs and decreased concentration of arginine in serum. Liver glucose concentration was higher and pyranic acid was lower in LEU compared to CON. In conclusion, leucine supplementation can promote post‐absorptive EAA utilization and hepatic gluconeogenesis, which contributes to protein synthesis in skeletal muscle of dairy calves.     

Developmental regulation of the activation of translation initiation factors of skeletal muscle in response to feeding in horses

Objective-To determine whether feeding-induced activation of translation initiation factors, specifically protein kinase B, ribosomal protein S6 kinase (S6K1), ribosomal protein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1, in horses is affected by age. Animals-6 yearlings, six 2-year-old horses, and 6 mature horses. Procedures-After an 18-hour period of feed withholding, horses consumed a high-protein meal (2 g/kg) at time 0 and 30 minutes (postprandial state) or continued to have feed withheld (postabsorptive state). Blood samples were collected for the duration of the experimental procedures and used to determine plasma concentrations of glucose, insulin, and amino acids. At 90 minutes, biopsy specimens were collected from a gluteal muscle and used to measure phosphorylation of translation initiation factors. Results-Plasma glucose, insulin, and amino acid concentrations were elevated for the postprandial state, compared with results for the postabsorptive state, regardless of age. Phosphorylation of protein kinase B, S6K1, rpS6, and eukaryotic initation factor 4E binding protein 1 was increased for the postprandial state. There was an effect of age with increased phosphorylation of S6K1 at Thr(389) and rpS6 at Ser(235/236) in the yearlings and mature horses, compared with results for the 2-year-old horses. Conclusions and Clinical Relevance-Food consumption resulted in an increase in the activation of translation initiation factors, with the highest degree of responsiveness in the yearlings. This indicated that increased muscle accretion seen during growth could be a result of increased rates of muscle protein synthesis in response to a meal stimulus.     

雷帕霉素靶蛋白（mTOR）结构功能及其对骨骼肌蛋白质合成影响的研究进展

雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）是丝氨酸/苏氨酸蛋白激酶,能感受细胞中的氨基酸、生长因子、能量及环境压力等信号,通过下游效应蛋白调控翻译起始因子,调控蛋白质的合成。作者对近年来mTOR研究的新进展及其在骨骼肌蛋白质合成中的作用进行了综述。     

奶牛乳腺中4F2hc基因的表达模式及亮氨酸对其表达的调控研究

为了研究4F2hc在奶牛乳腺中的表达模式及调控方式，进一步明确氨基酸在奶牛乳腺上皮细胞中的跨膜转运过程，本研究采用Western blotting和实时荧光定量PCR技术检测了4F2hc在泌乳期和干奶期奶牛乳腺组织中的表达变化；在体外培养的泌乳期奶牛乳腺上皮细胞中添加亮氨酸，采用Western blotting和实时荧光定量PCR技术检测其对奶牛乳腺上皮细胞中4F2hc表达的影响；采用雷帕霉素抑制剂抑制mTOR信号通路，使用Western blotting方法检测mTOR信号抑制后奶牛乳腺上皮细胞中4F2hc表达以及乳蛋白合成的变化。结果显示，在泌乳期的奶牛乳腺组织中4F2hc的mRNA和蛋白表达水平均显著或极显著高于干奶期（P<0.05，P<0.01）；在体外培养的奶牛乳腺上皮细胞中添加亮氨酸可以极显著提高乳腺上皮细胞中4F2hc的mRNA和蛋白质表达水平（P<0.01）；亮氨酸刺激可以激活细胞内的mTOR信号通路（P<0.05），而雷帕霉素处理则可以显著抑制mTOR信号分子的磷酸化并极显著抑制亮氨酸诱导的4F2hc的表达（P<0.05，P<0.01），进而极显著抑制β-Casein的合成（P<0.01）。以上研究结果表明，4F2hc基因的表达与奶牛乳腺的泌乳活性之间呈正相关，亮氨酸可以通过激活mTOR信号通路来调节4F2hc基因的表达，进而影响乳蛋白的合成。     

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-euglycemiohyperaminoacidemic, 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., unc51-1ike kinase 1 （UKL1）, microtubule-associated protein light chain 3 （LC3）, and lysosomal-associated membrane protein 2 （Lamp-2）. For comparison, we measured ribosomal protein 56 （rpS6） and eukaryotic initiation factor 4E （elF4E） 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 LC3-11/LC3-1 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 elF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of elF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the response     

Recent Advances in Swine Amino Acid Nutrition

Recent advances in swine protein nutrition are characterized by the development of functional amino acids (AA) in regulating fetal and postnatal survival, growth and development. These AA include arginine, glutamine, glutamate, proline, leucine, cysteine and tryptophan. Due to limited knowledge on AA nutrition, pork producers have traditionally paid little attention to supplementing the arginine family of AA to swine diets. Results of recent studies indicate that functional AA serve important regulatory functions in nutrient metabolism, protein turnover, and immune function, therefore enhancing efficiency of feed utilization by pigs. The underlying mechanisms include activation of nitric oxide, mammalian target of rapamycin, gaseous signaling, and AMP-activated protein kinase pathways, as well as anti-oxidative function. Dietary supplementation with arginine, glutamine, proline or leucine to weanling piglets enhances their growth performance. Arginine or glutamine is also effective in increasing milk production by lactating sows. Furthermore, supplementing arginine to the diet of pregnant gilts between days 30 and 114 of gestation increases the number of live-born piglets and litter birth-weight. Availability of feed-grade functional AA holds great promise for improving animal health and nutrient utilization in pig production worldwide. Additionally, feedstuffs of animal origin [e.g., blood meal (ring dried), feather meal (hydrolyzed), meat and bone meal, porcine protein meal, and poultry by-product meal (both feed- and petfood-grades)] are excellent and cost-effective sources of both essential and functional AA for formulating balanced swine diets. New knowledge on AA nutrition provides a much needed scientific basis for revising the next edition of swine nutrient requirements.     

Recent Advances in Swine Amino Acid Nutrition

Recent advances in swine protein nutrition are characterized by the development of functional amino acids(AA)in regulating fetal and postnatal survival,growth and development. These AA include arginine,glutamine,glutamate,proline,leucine, cysteine and tryptophan. Due to limited knowledge on AA nutrition,pork producers have traditionally paid little attention to supplementing the arginine family of AA to swine diets. Results of recent studies indicate that functional AA serve important regulatory functions in nutrient metabolism,protein turnover, and immune function,therefore enhancing efficiency of feed utilization by pigs. The underlying mechanisms include activation of nitric oxide,mammalian target of rapamycin,gaseous signaling,and AMP-activated protein kinase pathways, as well as anti-oxidative function. Dietary supplementation with arginine,glutamine, proline or leucine to weanling piglets enhances their growth performance. Arginine or glutamine is also effective in increasing milk production by lactating sows. Furthermore,supplementing arginine to the diet of pregnant gilts between days 30 and 114 of gestation increases the number of live-born piglets and litter birth-weight. Availability of feed-grade functional AA holds great promise for improving animal health and nutrient utilization in pig production worldwide. Additionally, feedstuffs of animal origin[e. g. , blood meal (ring dried),feather meal(hydrolyzed),meat and bone meal, porcine protein meal,and poultry by-product meal(both feed- and petfood-grades )]are excellent and cost-effective sources of both essential and functional AA for formulating balanced swine diets.New knowledge on AA nutrition provides a much needed scientific basis for revising the next edition of swine nutrient requirements.     

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.     

mTOR对信号通路调控的研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)信号通路是最近新出现的细胞内重要信号途径,该途径在进化上高度保守,主要通过PI3K/Akt/mTOR信号通路磷酸化激活来调控细胞分裂、促进转录、信号翻译等,从而控制蛋白合成来调节细胞生长。mTOR作为一种重要的调节基因通过调节细胞周期、蛋白质合成、细胞能量代谢等多种途径发挥重要的生理功能,在细胞增殖、生长、分化过程中起着中心调控点的作用。     

Effects of methionine on muscle protein synthesis and degradation pathways in broilers

This study investigated the hypothesis that supplementation of methionine (Met) to broiler diets increases muscle growth due to regulation of molecular pathways related to protein synthesis and degradation depending on the Met source. Day‐old male Cobb‐500 broilers (n = 240) were phase‐fed three different wheat–soya bean meal‐based basal diets during days 1–10, 11–21 and 22–35. Basal diets (Met‐ group, Met + Cys concentration 15% below NRC recommendations) were supplemented with 0.10% or 0.40% Met either as DL‐Met (DLM) or DL‐2‐hydroxy‐4‐(methylthio) butanoic acid (DL‐HMTBA) (equimolar comparison). Breast muscle weights were lower in the Met‐ group compared to all Met‐supplemented groups and were lower in broilers supplemented with 0.10% of DL‐HMTBA compared to the other groups fed Met‐supplemented diets. However, the expression of genes or relative phosphorylation and thus activation state of proteins involved in the somatotropic axis, the mammalian target of rapamycin (mTOR) pathway of protein synthesis, the ubiquitin–proteasome pathway (UPP) and autophagy–lysosomal pathway of protein degradation, the GCN2/eIF2a pathway involved in the inhibition of protein synthesis and in the myostatin–Smad2/3 pathway involved in myogenesis were not affected by Met source. Feeding diets with suboptimum Met + Cys concentrations, however, decreased expression of GHR and IGF1 in liver and muscle and increased that of MURF1 involved in the UPP in the broiler's muscle at day 10 and 21, while that of FOXO and atrogin‐1 and FOXO phosphorylation remained unaffected. Additionally, suboptimum dietary Met concentrations increased expression of the autophagy‐related genes ATG5 and BECN1 at day 35. Met supplementation neither affected gene expression nor phosphorylation of proteins involved in the GNC2/eIF2a and mTOR pathways. These data indicate that protein synthesis was not affected on the molecular level, while protein degradation was marginally affected by dietary Met dosage.     

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.     

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.     

牛乳腺上皮细胞SNAT2对氨基酸调节乳合成的影响

试验旨在研究氨基酸转运体钠离子依赖的中性氨基酸转运蛋白（the sodium-dependent neutral amino acid transporter 2,SNAT2）在牛乳腺上皮细胞（bovine mammary epithelial cell,BMEC）中对氨基酸调节乳合成的影响。利用组织块法成功培养原代BMEC,添加不同氨基酸（蛋氨酸（Met）、赖氨酸（Lys）和亮氨酸（Leu）刺激BMEC后,通过实时荧光定量PCR、Western blotting技术和甘油三酯试剂盒检测SNAT2、酪蛋白（β-casein）基因的表达量和BMEC培养液上清甘油三酯的分泌量;将N-flag-SNAT2真核表达载体及SNAT2 siRNA分别转入细胞中进行SNAT2基因的过表达和敲低试验;通过Western blotting和甘油三酯试剂盒分别检测SNAT2、哺乳动物雷帕霉素靶蛋白（the mammalian target of rapamycin,mTOR）、β-casein蛋白表达量和BMEC培养液上清的甘油三酯含量。结果显示,3种氨基酸（Met、Lys、Leu）均能显著促进BMEC分泌乳蛋白和乳脂,并激活mTOR信号途径,其中Met、Lys还能够显著上调SNAT2基因表达;SNAT2能够正向调节BMEC乳蛋白和乳脂肪的合成,并激活mTOR信号通路,说明氨基酸激活mTOR信号通路是通过SNAT2基因介导完成的,进而调节了BMEC乳蛋白和乳脂肪合成。     

口蹄疫病毒前导蛋白结构及功能研究进展

口蹄疫病毒前导蛋白为一种木瓜蛋白半胱氨酸酶,用一个半胱氨酸区作为亲和试剂.由于结构上的特殊性,使其能够进行自身切割,从正合成的多聚蛋白中游离自己,并通过切割宿主蛋白eIF的两种相似因子4GⅠ和4GⅡ减弱宿主细胞转录自身mRNA的能力,尤其使α/β干扰素的翻译受到抑制,从而为病毒的复制及感染创造有利条件.文章从结构特点、功能特性方面对其进行阐述.     

Amino acids regulate energy utilization through mammalian target of rapamycin complex 1 and adenosine monophosphate activated protein kinase pathway in porcine enterocytes

As major fuels for the small intestinal mucosa, dietary amino acids (AA) are catabolized in the mitochondria and serve as sources of energy production. The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes. Intestinal porcine epithelial cells (IPEC-J2) were treated with different concentrations of AA, inhibitor, or agonist of mammalian target of rapamycin complex 1 (mTORC1) and adenosine monophosphate activated protein kinase (AMPK), and mitochondrial respiration was monitored. The results showed that AA treatments resulted in enhanced mitochondrial respiration, increased intracellular content of pyruvic acid and lactic acid, and increased hormone-sensitive lipase mRNA expression. Meanwhile, decreased citrate synthase, isocitrate dehydrogenase alpha, and carnitine palmitoyltransferase 1 mRNA expression were also observed. We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated-p70 ribosomal protein S6 kinase, and phosphorylated-4E-binding protein 1. What is more, the protein levels of phosphorylated AMPK α (p-AMPKα) and nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-1 (SIRT1) were decreased by AA treatments in a time depending manner. Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORC1 or AMPK. Moreover, AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta (Ikbkβ), integrin-linked protein kinase (ILK), unconventional myosin-Ic (Myo1c), ribosomal protein S6 kinase beta-2 (RPS6Kβ2), and vascular endothelial growth factor (VEGF)-β, which are downstream effectors of mammalian target of rapamycin (mTOR). The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PIK3CD) and 5′-AMP-activated protein kinase subunit gamma-1 (PRKAG1), which are upstream regulators of mTOR, were also up-regulated by AMPK activation. On the other hand, AMPK activation also down-regulated FK506-binding protein 1A (FKBP1A), serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform, phosphatase and tensin homolog (PTEN), and unc-51 like autophagy activating kinase 1 (Ulk1), which are up-stream regulators of mTORC1. Taken together, these data indicated that AA regulated cellular energy metabolism through mTOR and AMPK pathway in porcine enterocytes. These results demonstrated interactions of AMPK and mTORC1 pathways in AA catabolism and energy metabolism in intestinal mucosa cells of piglets, and also provided reference for using AA to remedy human intestinal diseases.     

Nutrient restriction differentially modulates the mammalian target of rapamycin signaling and the ubiquitin-proteasome system in skeletal muscle of cows and their fetuses

The mammalian target of rapamycin (mTOR) signaling controls nutrient-stimulated protein synthesis in skeletal muscle, whereas ubiquitin-proteasome systems control the degradation of myofibrillar proteins. The objective of this study was to elucidate the effect of nutrient restriction on the mTOR signaling and ubiquitin-proteasome system in the skeletal muscle of cows and their fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet that provided 100% nutrient requirements or a nutrient-restricted diet at 68.1% of NE(m) and 86.7% of metabolizable protein requirement. Cows were slaughtered on 125 d of gestation, and the LM of both cows and fetuses was sampled for the measurement of mTOR, ribosomal protein S6, adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein ubiquitylation. When comparing the muscle samples from nutrient-restricted and control cows and their fetuses, no difference was observed for the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser(2448) and ribosomal protein S6 at Ser(235/336) were greater (P < 0.05) in control muscle than in muscle from nutrient-restricted animals. Because the phosphorylation of mTOR and ribosomal protein S6 upregulates translation, these results showed that nutrient restriction inhibits protein synthesis in muscle. The activity of AMPK in the muscle of nutrient-restricted cows was significantly lower (P = 0.05) than that of control cows. The protein ubiquitylation, however, was greater (P < 0.05) in the muscle from nutrient-restricted cows, showing accelerated protein degradation. No difference in the protein ubiquitylation was detected for fetal muscle. Data suggested that the decreased protein synthesis and promoted protein degradation resulted in muscle atrophy of pregnant cows, but not in fetal muscle. Results of this study show that in response to nutrient restriction, protein degradation was differentially regulated between cow and fetal muscle. The atrophy of cow muscle during nutrient deficiency may involve the enhanced degradation of muscle proteins.