必需氨基酸通过哺乳动物雷帕霉素靶蛋白信号通路调控乳蛋白合成的研究进展

最近的研究表明,必需氨基酸不仅可以作为合成乳蛋白的底物,还可以作为信号分子调控乳蛋白的合成.不同的必需氨基酸通过哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控乳蛋白合成的作用也不相同,既包括正调控作用,也包括负调控作用.研究不同必需氨基酸调控乳蛋白合成的分子机制将为提高乳蛋白合成率提供理论基础.本文综述了mTOR信号通路及必需氨基酸通过mTOR信号通路调控乳蛋白的合成的研究进展,为揭示必需氨基酸作为蛋白质合成底物及细胞信号分子在乳蛋白合成过程中的作用机制提供研究方向.     

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。     

亮氨酸对κ-酪蛋白合成的影响及相关信号通路的研究

本研究旨在探讨其他氨基酸缺乏条件下补充亮氨酸对奶牛乳腺上皮细胞κ-酪蛋白基因表达和蛋白合成以及细胞增殖的影响,并从mRNA水平探究mTOR信号通路介导的蛋白质合成的重要性。处理组1在对照组(稀释100倍的培养基)的基础上补充亮氨酸,处理组2在处理组1的基础上添加mTOR信号通路上游抑制剂,分别采用RT-qPCR技术和ELISA法测定基因的相对表达量和蛋白合成量,CCK-8法测定细胞增殖。结果表明:亮氨酸显著促进了κ-酪蛋白基因表达和蛋白合成(P<0.05),而添加抑制剂极显著降低了这种作用(P<0.01);各处理对蛋白质合成信号通路相关基因mTOR、S6K1、4EBP1、eIF4E、eEF2相对表达量均有一定影响;抑制剂能显著抑制细胞增殖(P<0.05)。结果提示,补充亮氨酸能显著促进κ-酪蛋白的合成,这可能与mTOR信号通路介导蛋白质合成有关,此外,mTOR信号通路也可调控奶牛乳腺上皮细胞的增殖。     

mTOR上游信号对乳蛋白合成作用机制研究进展

牛乳中的蛋白质是反映牛奶质量的重要指标之一,其含量高低决定了牛奶的营养特性和经济价值。乳蛋白含有人体生长发育的一切必需氨基酸和其他氨基酸,其消化率远比植物蛋白质高,而且牛乳中的酪蛋白具有较强的抗变异能力,能减少癌变。我国奶牛单产差异逐步缩小,但是与世界奶牛养殖发达国家相比,牛奶中蛋白质含量差距仍然较大。因此,提高乳中蛋白质含量成为我国奶业面临的重要挑战。蛋白质的合成与哺乳动物雷帕霉素靶蛋白(mTOR)信号通路密切相关,特别是激素、氨基酸、能量等因子作为mTOR信号通路的上游信号参与乳蛋白合成,因此文章分别对这三种因子影响mTOR信号通路进而调控蛋白质合成过程进行了详细综述,揭示了这三种因子对牛乳中乳蛋白的影响,以期在细胞领域对奶牛有关蛋白质的合成研究提供一些理论参考。     

氨基酸介导mTOR上游信号通路作用机制的研究进展

氨基酸作为蛋白质营养功能的执行者,其调控细胞功能的作用已经超过其在新陈代谢中的基本作用;而细胞生理功能的调控是通过调整氨基酸转运体基因表达和信号转导途径实现的。虽然氨基酸调控基因表达的研究已经成熟,但人们对真核细胞如何感应氨基酸的营养信号尚未了解透彻。对雷帕霉素靶蛋白(mTOR)信号通路及氨基酸介导通路上游作用机制的最新研究进展进行综述,为调控蛋白质合成达到最大化提供依据。     

小G蛋白对mTOR信号通路的调控

哺乳动物雷帕霉素靶蛋白(mTOR)是一种重要的信号调控分子,通过磷酸化作用调控细胞内mRNA的翻译,参与膜蛋白转运、蛋白质降解、蛋白激酶c信号转导和核糖体合成等。不同家族的小G蛋白,如Rheb、Rag、RalA、Rac1和Rab5,在调控mTOR信号通路中发挥着重要作用。Rheb结合并直接激活mTOR,Rag和Rac1介导mTOR的定位,RalA通过磷脂酸的产生间接激活mTOR,Rab5调控mTORC1的活化和定位。本文将围绕近年来发现的多种小G蛋白家族成员参与调控mTOR信号通路的研究进展进行综述。     

不同蛋白源对生长大鼠骨骼肌蛋白质代谢相关基因表达的影响

本试验旨在研究酪蛋白(CAS)、大豆分离蛋白(SPI)和玉米醇溶蛋白(ZEIN)对大鼠骨骼肌蛋白质代谢基因表达的影响。试验选用60只生长大鼠进行饲养试验,按照体重一致原则随机分为3个处理,每个处理20只大鼠,饲喂等能等氮半纯合日粮。结果表明:玉米醇溶蛋白(ZEIN)日粮使试鼠腓肠肌具有较高的mTOR、RhebmR NA水平及磷酸化mTOR蛋白量(P<0.01)和较高的Atrogin1(MAFbx)、MuR F1、pUb和FoxO mR NA表达量(P<0.05或P<0.01)。本研究揭示出蛋白源对蛋白质代谢基因表达产生的差异,可能主要由饲料蛋白质氨基酸组成模式不同所体现,尤其表现为亮氨酸(Leu)对组织蛋白质代谢进行调控。可见,蛋白质合成代谢调控与mTOR途径有关,蛋白质分解代谢与泛素-蛋白酶蛋白降解途径(UPP)有关。     

氨基酸对奶牛乳腺蛋白质合成调控及其分子机制的研究进展

乳蛋白中含有大量人体所需的必需氨基酸,其组成平衡、含量丰富,是一种具有极高营养价值的蛋白质,而乳中 90%以上的蛋白质是乳腺利用氨基酸从头合成的,因此氨基酸对奶牛乳蛋白合成发挥着重要的作用。此外,氨基酸不仅是合成乳蛋白不可或缺的前体物质,而且还是重要的信号调控因子,通过哺乳动物雷帕霉素靶蛋白（mTOR） 信号通路调控乳蛋白的合成。基于此,作者就影响奶牛乳腺氨基酸供应、摄取、利用的因素及氨基酸的信号传导作用的研究进展进行综述,以期为提高乳蛋白的合成提供一定的理论基础。     

营养素和激素对乳蛋白合成过程中哺乳动物雷帕霉素靶蛋白信号通路调节作用的研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)是一种非典型丝氨酸/苏氨酸蛋白激酶,是mTOR信号通路的重要分子。mTOR可整合氨基酸、能量和激素等多种细胞外信号,参与基因转录、蛋白质翻译等生物过程。本文总结了mTOR信号通路特点及信号途径,重点介绍了营养素(氨基酸、能量底物)和激素(主要是胰岛素)在乳蛋白合成过程中对mTOR信号通路的调节作用。     

奶牛乳蛋白产量的营养调节效应与乳腺真核起始因子-2（eIF2）和核糖体s6（S6）激酶-1（S6K1）的磷酸化有关（摘要）

日粮蛋白质和能量同时影响泌乳动物乳蛋白的产量,必需氨基酸单独不能完全解释营养素对乳蛋白产量的影响。生长动物组织中,营养素对蛋白质合成受哺乳动物雷帕霉素靶点（mTOR）和整合应激网络（ISR）的调控。本试验旨在探讨乳腺内营养素信号是否也通过mTOR和ISR网络调控乳蛋白的合成。泌乳奶牛饥饿22 h后,静脉内分别灌注必需氨基酸（EAA）和葡萄糖混合物,葡萄糖、l-Met＋l-Lys、l-His或l-Leu 9 h。结果表明,EAA和葡萄糖混合物或葡萄糖能分别提高乳蛋白产量的33%和27%。     

用甲基组氨酸估计实验动物的蛋白质周转

用测定3 -甲基组氨酸排泄量的方法估计了实验动物的骨骼肌蛋白质合成和降解速度。结果表明 :用纯合饲粮饲喂时 ,3周龄肉鸡的蛋白质降解速度为3.63 %/天 ,蛋白质合成速度为11.71 %/天。60日龄成年小白鼠的平均蛋白质降解速度为3.66 %/天 ,平均蛋白质合成速度为6.86 %/天。     

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.     

Influence of insulin and substrate concentration on protein synthetic rate in fetal tissues

The influence of fetal plasma substrate and insulin concentrations on the fractional synthesis rate or turnover of the mixed proteins in the organs of fetal lambs was calculated from measurements of the rate of uptake of L-14C lysine by the protein in the steady state in utero. The control values for protein synthesis rate in the fetal organs of Welsh mountain ewes, 130 to 140 days' pregnant, were similar to those previously observed in the fetuses of Border Leicester ewes: the mean turnover rates were 38, 72, 40 and 63 per cent per day for the brain, liver, cardiac muscle and the placenta respectively; that for skeletal muscles was 6.8 per cent per day; the corresponding half lives were 2.15, 1.27, 1.8 and 1.42 days respectively and 11.3 days for skeletal muscle. A fivefold increase in the plasma glucose levels had no influence on protein turnover rate over a six hour period. A fourfold increase in the concentration of some amino acids, following a mixed infusion, decreased turnover rate in brain, heart and liver. The half life was increased by 30 per cent, but a small reduction in half life was observed in skeletal muscle. Insulin caused a fall in intracellular lysine concentration in skeletal muscle and in plasma, which suggests that the hormone can reduce muscle protein catabolism in the fetal lamb in the last trimester of gestation. Enhancement of synthesis rate occurred in the presence of the amino acid infusions only; the half life of the proteins was reduced by a factor of three. Insulin also increased placental protein synthesis rate by 50 per cent but had no influence on the other tissues studied.     

Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression

Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.     

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.     

肌肉生长抑制素对骨骼肌作用研究进展

肌肉生长抑制素(MSTN)是转化生长因子β超家族的成员之一,又称生长分化因子8。MSTN主要在骨骼肌中广泛表达,并可在心肌、脂肪、乳腺等多个组织中表达,其作用主要体现在抑制骨骼肌生长发育、诱发肌萎缩等方面。MSTN可以通过多种途径协同作用于骨骼肌,即通过激活TGF-β、p38MAPK、ERK1/2、JNK等信号途径以及抑制IGF-AKT、Wnt信号途径来抑制肌细胞增殖分化;通过调控AKT途径、泛素-蛋白水解酶系统、自噬溶酶体系统来影响骨骼肌蛋白的合成与分解;MSTN还参与了与骨骼肌生成相关的脂肪代谢及骨形成等生理活动。论文重点阐述MSTN在肌细胞增殖分化、肌蛋白合成与分解、脂肪代谢、骨骼发育等方面的作用机制,并对其应用前景进行展望,为相关科学研究提供参考。     

Differential ammonia metabolism and toxicity between avian and mammalian species,and effect of ammonia on skeletal muscle: A comparative review

Comparative aspects of ammonia toxicity, specific to liver and skeletal muscle and skeletal muscle metabolism between avian and mammalian species are discussed in the context of models for liver disease and subsequent skeletal muscle wasting. The purpose of this review is to present species differences in ammonia metabolism and to specifically highlight observed differences in skeletal muscle response to excess ammonia in avian species. Ammonia, which is produced during protein catabolism and is an essential component of nucleic acid and protein biosynthesis, is detoxified mainly in the liver. While the liver is consistent as the main organ responsible for ammonia detoxification, there are evolutionary differences in ammonia metabolism and nitrogen excretory products between avian and mammalian species. In patients with liver disease and all mammalian models, inadequate ammonia detoxification and successive increased circulating ammonia concentration, termed hyperammonemia, leads to severe skeletal muscle atrophy, increased apoptosis and reduced protein synthesis, altogether having deleterious effects on muscle size and strength. Previously, an avian embryonic model, designed to determine the effects of increased circulating ammonia on muscle development, revealed that ammonia elicits a positive myogenic response. Specifically, induced hyperammonemia in avian embryos resulted in a reduction in myostatin, a well‐known inhibitor of muscle growth, expression, whereas myostatin expression is significantly increased in mammalian models of hyperammonemia. These interesting findings imply that species differences in ammonia metabolism allow avians to utilize ammonia for growth. Understanding the intrinsic physiological mechanisms that allow for ammonia to be utilized for growth has potential to reveal novel approaches to muscle growth in avian species and will provide new targets for preventing muscle degeneration in mammalian species.     

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.