l-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells

This study tested the hypothesis that l-arginine (Arg) may stimulate cell proliferation and prevent lipopolysaccharide (LPS)-induced death of intestinal cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 4 days in Arg-free Dulbecco’s modified Eagle’s-F12 Ham medium (DMEM-F12) containing 10, 100 or 350 μM Arg and 0 or 20 ng/ml LPS. Cell numbers, protein concentrations, protein synthesis and degradation, as well as mammalian target of rapamycin (mTOR) and Toll-like receptor 4 (TLR4) signaling pathways were determined. Without LPS, IPEC-1 cells exhibited time- and Arg-dependent growth curves. LPS treatment increased cell death and reduced protein concentrations in IPEC-1 cells. Addition of 100 and 350 μM Arg to culture medium dose-dependently attenuated LPS-induced cell death and reduction of protein concentrations, in comparison with the basal medium containing 10 μM Arg. Furthermore, supplementation of 100 and 350 μM Arg increased protein synthesis and reduced protein degradation in both control and LPS-treated IPEC-1 cells. Consistent with the data on cell growth and protein turnover, addition of 100 or 350 μM Arg to culture medium increased relative protein levels for phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase-1, while reducing the relative levels of TLR4 and phosphorylated levels of nuclear factor-κB in LPS-treated IPEC-1 cells. These results demonstrate a protective effect of Arg against LPS-induced enterocyte damage through mechanisms involving mTOR and TLR4 signaling pathways, as well as intracellular protein turnover.     

Reactive oxygen species mediated placental oxidative stress,mitochondrial content,and cell cycle progression through mitogen-activated protein kinases in intrauterine growth restricted pigs

Intrauterine growth restriction (IUGR) remains a significant obstacle in pig production; however, information regarding the relationship between reactive oxygen species (ROS)-induced placental dysfunction and IUGR is still unknown. This study aimed to explore the placental redox status, mitochondrial content, cellular progression, and mitogen-activated protein kinase (MAPK) pathways in IUGR. Placental tissues were collected from normal intrauterine gestation (NIUG) and IUGR fetuses at delivery. Compared with the NIUG, placental ROS production, lipid peroxidation, and DNA damage were increased in IUGR. Placental mitochondrial DNA (mtDNA) content and mtDNA-encoded gene expression decreased in IUGR. Moreover, p21 phosphorylation increased, cyclin E expression decreased in IUGR cases, which showed senescence characteristics. Analysis of signaling pathways showed that the ERK1/2 phosphorylation increased whereas the p38 and JNK phosphorylation decreased in IUGR. In cultured porcine trophectoderm (pTr) cells, exogenous H₂O₂ increased intracellular ROS production, decreased cell viability in a dose-dependent manner. Cell cycle distribution was found to arrest in S and G2/M phases. Our findings suggested that IUGR was associated with greater placental ROS and oxidative injury, which might be a factor that resulted in lower mitochondrial content, microvilli loss and senescence, and activation of MAPK pathways.     

Weekly intra-amniotic IGF-1 treatment increases growth of growth-restricted ovine fetuses and up-regulates placental amino acid transporters

Frequent treatment of the growth-restricted (IUGR) ovine fetus with intra-amniotic IGF-1 increases fetal growth. We aimed to determine whether increased growth was maintained with an extended dosing interval and to examine possible mechanisms. Pregnant ewes were allocated to three groups: Control, and two IUGR groups (induced by placental embolization) treated with weekly intra-amniotic injections of either saline (IUGR) or 360 μg IGF-1 (IGF1). IUGR fetuses were hypoxic, hyperuremic, hypoglycemic, and grew more slowly than controls. Placental glucose uptake and SLC2A1 (GLUT2) mRNA levels decreased in IUGR fetuses, but SLC2A3 (GLUT3) and SLC2A4 (GLUT4) levels were unaffected. IGF-1 treatment increased fetal growth rate, did not alter uterine blood flow or placental glucose uptake, and increased placental SLC2A1 and SLC2A4 (but not SLC2A3) mRNA levels compared with saline-treated IUGR animals. Following IGF-1 treatment, placental mRNA levels of isoforms of the system A, y(+), and L amino acid transporters increased 1.3 to 5.0 fold, while the ratio of phosphorylated-mTOR to total mTOR also tended to increase. Weekly intra-amniotic IGF-1 treatment provides a promising avenue for intra-uterine treatment of IUGR babies, and may act via increased fetal substrate supply, up-regulating placental transporters for neutral, cationic, and branched-chain amino acids, possibly via increased activation of the mTOR pathway.     

mTOR及其底物在HeLa细胞的细胞周期不同时相中的表达

为探讨细胞生长的机制 ,用RT PCR、Western印迹及蛋白激酶活性测定等方法对同步化的HeLa细胞的细胞周期不同时相中mTOR(mammaliantargetofrapamycin) ,p70S6激酶 (p70S6K)的α1 、α2 、β1 、β2 不同亚型及起始因子 4E结合蛋白 1 (4EBP1 )的表达进行了检测 .RT PCR的结果表明 :在G1 、S1 、G2 、M1 、M2 几个细胞周期时相中 ,mTOR的mRNA表达无明显变化 .mTOR的底物P70S6K的亚型α1 、α2 、β1 、β2 在M期表达均有明显增加 .4EBP1的表达在M期明显减少 .免疫印迹的结果与RT PCR的一致 ,即M期p70S6K的α1 、α2 、均有增加 ,4EBP1在M期减少 .活性测定表明 ,G2 期、M期mTOR较其它期有明显增加 ,4EBP1在M期活性有所下降 .研究结果表明 :mTOR、p70S6K、4EBP1很可能在HeLa细胞的生长中起重要的调节作用     

Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells

α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.     

Angiotensin IV enhances phosphorylation of 4EBP1 by multiple signaling events in lung endothelial cells

Angiotensin IV (Ang IV)-stimulated cell proliferation is regulated through activation of multiple signaling modules in lung endothelial cells (EC). Because eukaryotic intitiation factor 4E (eIF4E) binding protein 1 (4EBP1) plays a critical role in the RNA translation and the regulation of cell growth, we examined whether Ang IV modulates expression and/or phosphorylation of eIF4E and 4EBP1 as well as the role of multiple signaling events associated with 4EBP1 phosphorylation in EC. Ang IV stimulation increased phosphorylation but not expression of eIF4E and 4EBP1 proteins. Ang IV stimulation selectively phosphorylated Thr46 > Thr70 > Ser65 but not Thr37 residues in 4EBP1. Pretreatment of cells with PD-98059 and rapamycin, inhibitors of mitogen-activated protein kinase (ERK1/2) and mammalian target for rapamycin (mTOR), respectively, partially blocked Ang IV-mediated phosphorylation of 4EBP1. In contrast, overexpression of p70 ribosomal S6 kinase (p70S6K) and protein kinase B (Akt) enhanced phosphorylation of 4EBP1 and eIF4E binding affinity to the cap region of mRNA. These results support critical roles of multiple signaling and phosphorylation of 4EBP1 by Ang IV in translation process and protein synthesis.     

Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

Background

The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70?kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles.

Results

In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2?C10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2?C16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3?C13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2?C18 fold in atrophic denervated muscle.

Conclusions

The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.     

Insulin-like growth factor-1 (IGF-1) and leucine activate pig myogenic satellite cells through mammalian target of rapamycin (mTOR) pathway

Myogenic satellite cells are adult stem cells and have important roles in skeletal muscle growth, repair, and regeneration. Both insulin-like growth factor-1 (IGF-1) and leucine stimulate skeletal muscle growth, which link to the activation and proliferation of myogenic satellite cells in skeletal muscle. Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis and cell proliferation. Thus, IGF-1 and leucine may stimulate activation of myogenic satellite cells through mTOR signaling. In this study, myogenic satellite cells were isolated from 6-month-old pigs and subjected to IGF-1 and leucine treatments. Both IGF-1 and leucine upregulated mTOR signaling in myogenic satellite cells. The phosphorylation of mTOR at Ser(2448) increased 83.8 +/- 7.7% by IGF-1 (P < 0.05) and 83.4 +/- 5.7% by leucine (P < 0.05). The downstream targets of mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) were also phosphorylated due to IGF-1 and leucine treatments. Treatment with IGF-1 and leucine induced the phosphorylation of tuburin (TSC2), a key mediator upstream of mTOR signaling, by 272.8 +/- 26.4% and 94.2 +/- 28.7%, respectively. Treatment of cells with both IGF-1 and leucine did not show synergistic effect on mTOR signaling. Inhibition of mTOR by rapamycin abolished the protein synthesis and cell proliferation stimulated by both IGF-1 and leucine. In summary, our data showed that in preliminary cultured myogenic satellite cells mTOR signaling was activated due to IGF-1 and leucine treatments, and this mTOR activation is necessary for the activation of myogenic satellite cells.     

Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.     

Regulation of protein turnover by L-glutamine in porcine intestinal epithelial cells

L-Glutamine (Gln) plays an important role in sustaining the intestinal mucosal mass of humans and animals. However, the underlying mechanisms are largely unknown. This study tested the hypothesis that Gln regulates protein turnover in intestinal epithelial cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 3 h (short-term study) or 96 h (long-term study) in Gln-free Dulbecco's modified Eagle-F12 Ham medium containing 0, 0.5 or 2.0 mM Gln. To determine effects of ammonia (a metabolite of Gln, i.e., 0.18 mM ammonia produced from 2 mM Gln in 3 h) on protein turnover, additional experiments were conducted in which medium contained 0.5 mM Gln and 0, 0.2, 0.5 or 2.0 mM NH(4)Cl. Variables of analysis included cell growth, protein synthesis, proteolysis and mammalian target of rapamycin (mTOR) signaling. IPEC-1 cell growth increased with extracellular Gln concentrations. Compared with 0 mM Gln, the addition of 0.5 and 2 mM Gln to medium stimulated protein synthesis and inhibited protein degradation in those cells in both the short- and long-term studies. Ammonia (0.05 to 2.0 mM) did not affect protein synthesis, although higher levels of ammonia (0.5 and 2.0 mM) reduced protein degradation in IPEC-1 cells. Consistent with the data on protein turnover, 0.5 and 2 mM Gln increased abundance of phosphorylated eIF4E-binding protein-1 and phosphorylated S6 kinase-1 proteins. Collectively, these results demonstrate that physiological levels of Gln regulate protein turnover independent of ammonia production in intestinal cells through the mTOR signaling pathway.     

D-glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K.     

Amino acids stimulate glycyl-tRNA synthetase nuclear localization for mammalian target of rapamycin expression in bovine mammary epithelial cells

Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.     

AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2alpha

Prostaglandin F2alpha (PGF2alpha) is an important mediator of corpus luteum (CL) regression, although the cellular signaling events that mediate this process have not been clearly identified. It is established that PGF2alpha binds to a G-proteincoupled receptor (GPCR) to stimulate protein kinase C (PKC) and Raf-MEK-Erk signaling in luteal cells. The present experiments were performed to determine whether PGF2alpha stimulates the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway in steroidogenic luteal cells. We demonstrate that PGF2alpha treatment results in a timeand concentration-dependent stimulation of the phosphorylation and activation of S6K1. The stimulation of S6K1 in response to PGF2alpha treatment was abolished by the mTOR inhibitor rapamycin. Treatment with PGF2alpha did not increase AKT phosphorylation but increased the phosphorylation of Erk and the tumor suppressor protein tuberous sclerosis complex 2 (TSC2), an upstream regulator of mTOR. The effects of PGF2alpha were mimicked by the PKC activator PMA and inhibited by U0126, a MEK1 inhibitor. The activation of mTOR/S6K1 and putative down stream processes involving the translational apparatus (i.e. 4EBP1 phosphorylation, release of 4EBP1 binding in m(7)G cap binding assays, and the phosphorylation and synthesis of S6) were completely sensitive to treatment with rapamycin, implicating mTOR in the actions of PGF2alpha. Taken together, our data suggest that GPCR activation in response to PGF2alpha stimulates the mTOR pathway which increases the translational machinery in luteal cells. The translation of proteins under the control of mTOR may have implications for luteal development and regression and offer new strategies for therapeutic intervention in PGF2alpha-target tissues.     

Regulation of placental amino acid transporter activity by mammalian target of rapamycin

The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of l-type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.     

Arachidonic acid activation of translation initiation signaling in vascular smooth muscle cells

To understand the role of arachidonic acid (AA) in regulating vascular smooth muscle cell (VSMC) growth, its effects on phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E were studied. Arachidonic acid stimulated phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E in a time-dependent manner in VSMC. Arachidonic acid stimulation of phosphorylation of the above signaling molecules is specific, as these events were not affected by other unsaturated or saturated fatty acids. Metabolic conversion of AA via the LOX/MOX and/or COX pathways, to some extent, was required for its effects on the phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E. In addition, AA increased PI3K activity in a time-dependent manner in VSMC. LY294002, an inhibitor of PI3K, completely blocked AA-induced phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E, suggesting a role for PI3K in these effects. Consistent with its effects on translation initiation signaling events, AA induced global protein synthesis in VSMC and this response was dependent, to some extent, on its metabolism via the LOX/MOX and/or COX pathways, and mediated by the PI3K/Akt/mTOR pathway. Thus, the above observations provide the first biochemical evidence for the role of AA in the activation of translation initiation signaling in VSMC.     

Effect of pulsatile growth hormone administration to the growth-restricted fetal sheep on somatotrophic axis gene expression in fetal and placental tissues

We have previously reported (Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. J Endocrinol 177: 83-92, 2003) that a chronic pulsatile infusion of growth hormone (GH) to intrauterine growth-restricted (IUGR) ovine fetuses increased fetal circulating IGF-I levels without increasing fetal growth. We hypothesized a cortisol-induced upregulation of fetal hepatic GH receptor (GH-R) mRNA levels, secondary increases in IGF-I mRNA levels, and circulating IGF-I levels, but a downregulation of the type I IGF receptor (IGF-IR) as an explanation. We, therefore, measured mRNA levels of genes of the somatotrophic axis by real-time RT-PCR in fetal and placental tissues of fetuses with IUGR (induced by uteroplacental embolization from 110- to 116-days gestation) that received either a pulsatile infusion of GH (total dose 3.5 mg/day) or vehicle from 117-126 days and in control fetuses (n = 5 per group). Tissues were collected at 127 days (term, 145 days). Fetal cortisol concentrations were significantly increased in IUGR fetuses. However, in liver, GH-R, but not IGF-I or IGF-IR, mRNA levels were decreased in both IUGR groups. In contrast, in placenta, GH-R, IGF-I, and IGF-IR expression were increased in IUGR vehicle-infused fetuses. GH infusion further increased placental GH-R and IGF-IR, but abolished the increase in IGF-I mRNA levels. GH infusion reduced IGF-I expression in muscle and increased GH-R but decreased IGF-IR expression in kidney. IUGR increased hepatic IGF-binding protein (IGFBP)-1 and placental IGFBP-2 and -3 mRNA levels with no further effect of GH infusion. In conclusion, the modest increases in circulating cortisol concentrations in IUGR fetuses did not increase hepatic GH-R mRNA expression and, therefore, do not explain the increased circulating IGF-I levels that we found with GH infusion, which are likely due to reduced clearance rather than increased production. We demonstrate tissue-specific regulation of the somatotrophic axis in IUGR fetuses and a discontinuity between GH-R and IGF-I gene expression in GH-infused fetuses that is not explained by alterations in phosphorylated STAT5b.     

Effect of maternal nutrient restriction in sheep on the development of fetal skeletal muscle

The effect of maternal nutrient restriction on mTOR (mammalian target of rapamyosin) signaling and the ubiquitin system as well as their possible relation to growth of fetal muscle was determined. Ewes were fed to 50% (nutrient-restricted) or 100% (control-fed) of total digestible nutrients (National Research Council requirement) from Days 28 to 78 of gestation. Ewes were killed at Day 78 of gestation, and the fetal longissimus dorsi muscle was sampled for the measurement of mTOR, ribosomal protein S6, AMP-activated protein kinase (AMPK), calpastatin, and protein ubiquitylation. No difference was observed in the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser2448 and ribosomal protein S6 at Ser235/336 were reduced (P <0.05) in muscle from nutrient-restricted fetuses. Because phosphorylation of mTOR and ribosomal protein S6 up-regulates protein translation, these results show that nutrient restriction down-regulates protein synthesis in fetal muscle. No difference in AMPK activity was detected. The lack of difference in calpastatin and ubiquitylized protein content shows that nutrient restriction did not affect degradation of myofibrillar proteins in fetal muscle. Fetuses of nutrient-restricted ewes showed retarded development of muscles and skeleton. Muscle from nutrient-restricted fetuses contained fewer secondary myofibers than muscle from control fetuses, and the average area of fasciculi was smaller (P <0.05). The decreased number of secondary myofibers in nutrient-restricted fetuses may result from the decreased mTOR signaling. Lower activation of mTOR signaling in nutrient-restricted fetuses may reduce the proliferation of myoblasts and, thus, reduce the formation of secondary myofibers. This decrease in secondary myofibers in fetuses may predispose fetuses to metabolic diseases, such as diabetes and obesity, in their postnatal lives.     

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.     

Raptor,a binding partner of target of rapamycin (TOR), mediates TOR action

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.