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.     

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.     

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-²H₃]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.     

Antagonistic effects of leucine and glutamine on the mTOR pathway in myogenic C<Subscript>2</Subscript>C<Subscript>12</Subscript> cells

This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C(2)C(12) cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70(S6k), S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70(S6k) and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70(S6k) pathway.     

Hypoxia inhibits protein synthesis through a 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer cells

Hypoxia is a state of low oxygen availability that limits tumor growth. The mechanism of protein synthesis inhibition by hypoxia and its circumvention by transformation are not well understood. Hypoxic breast epithelial cells are shown to downregulate protein synthesis by inhibition of the kinase mTOR, which suppresses mRNA translation through a novel mechanism mitigated in transformed cells: disruption of proteasome-targeted degradation of eukaryotic elongation factor 2 (eEF2) kinase and activation of the regulatory protein 4E-BP1. In transformed breast epithelial cells under hypoxia, the mTOR and S6 kinases are constitutively activated and the mTOR negative regulator tuberous sclerosis complex 2 (TSC2) protein fails to function. Gene silencing of 4E-BP1 and eEF2 kinase or TSC2 confers resistance to hypoxia inhibition of protein synthesis in immortalized breast epithelial cells. Breast cancer cells therefore acquire resistance to hypoxia by uncoupling oxygen-responsive signaling pathways from mTOR function, eliminating inhibition of protein synthesis mediated by 4E-BP1 and eEF2.     

Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.     

Effects of simian virus 40 large and small tumor antigens on mammalian target of rapamycin signaling: small tumor antigen mediates hypophosphorylation of eIF4E-binding protein 1 late in infection

We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.     

L-arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells

Impairment of placental growth is a major factor contributing to intrauterine growth retardation (IUGR) in both human pregnancy and animal production. Results of recent studies indicate that administration of L-arginine (Arg) to gestating pigs or sheep with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that Arg stimulates the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis in porcine conceptus trophectoderm (pTr2) cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's Ham medium containing 10, 50, 100, 200, 350 or 500 μM Arg. Cell numbers, protein synthesis and degradation, as well as total and phosphorylated levels of mTOR, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. The pTr2 cells exhibited time (0-6 days)- and Arg concentration (10-350 μM)-dependent increases in proliferation. Addition of 100 and 350 μM Arg to culture medium dose-dependently increased (a) protein synthesis and decreased protein degradation and (b) the abundance of total and phosphorylated mTOR, p70S6K and 4EBP1 proteins. Effects of 350 μM Arg on intracellular protein turnover were only modestly affected when nitric oxide synthesis was inhibited. Collectively, these results indicate a novel and important role for Arg in promoting growth of porcine placental cells largely via a nitric-oxide-independent pathway. Additionally, these findings help to explain beneficial effects of Arg supplementation on improving survival and growth of embryos/fetuses in mammals.     

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

BACKGROUND: The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. RESULTS: Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. CONCLUSIONS: Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.     

Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation

Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.     

Predominant nuclear localization of mammalian target of rapamycin in normal and malignant cells in culture

Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells.     

The effect of ketone bodies on protein turnover in isolated skeletal muscle from the fed and fasted chick

1. The addition of 4 mM acetoacetate or DL-beta-hydroxybutyrate to the incubation medium decreased the rate of protein synthesis without influencing the rate of protein degradation in extensor digitorum communis (EDC) muscles from fed chicks and decreased the rates of protein synthesis and degradation in muscles from fasted chicks. 2. Ketone bodies markedly decreased intracellular concentrations of glutamine in EDC muscles from fed chicks by increasing glutamine oxidation. 3. The addition of 0.5 mM glutamine to incubation media containing 1.0 mM glutamine reversed the ketone body-induced decrease in intracellular glutamine concentration to the control value and blocked the inhibiting effect of ketone bodies on protein synthesis in skeletal muscles from fed chicks. 4. The addition of 5 mM pyruvate blocked the ability of ketone bodies to increase glutamine oxidation and prevented the associated decrease in intracellular glutamine concentration and the rate of protein synthesis in EDC muscles from fed chicks. 5. These results suggest that ketone bodies can act directly on skeletal muscle to inhibit the rate of protein synthesis in muscles from fed chicks by decreasing intracellular glutamine concentration by increasing its oxidation.     

Effects of glutamine,methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by cortocisteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175–179). The transfer of cells from high (1–5 mM) to low (0.2–0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0–1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.     

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.     

The rapid activation of protein synthesis by growth hormone requires signaling through mTOR

An important function of growth hormone (GH) is to promote cell and tissue growth, and a key component of these effects is the stimulation of protein synthesis. In this study, we demonstrate that, in H4IIE hepatoma cells, GH acutely activated protein synthesis through signaling via the mammalian target of rapamycin (mTOR) and specifically through the rapamycin-sensitive mTOR complex 1 (mTORC1). GH treatment enhanced the phosphorylation of two targets of mTOR signaling, 4E-BP1 and ribosomal protein S6. Phosphorylation of S6 and 4E-BP1 was maximal at 30-45 min and 10-20 min after GH stimulation, respectively. Both proteins modulate components of the translational machinery. The GH-induced phosphorylation of 4E-BP1 led to its dissociation from eIF4E and increased binding of eIF4E to eIF4G to form (active) eIF4F complexes. The ability of GH to stimulate the phosphorylation of S6 and 4E-BP1 was blocked by rapamycin. GH also led to the dephosphorylation of a third translational component linked to mTORC1, the elongation factor eEF2. Its regulation followed complex biphasic kinetics, both phases of which required mTOR signaling. GH rapidly activated both the MAP kinase (ERK) and PI 3-kinase pathways. Signaling through PI 3-kinase alone was, however, sufficient to activate the downstream mTORC1 pathway. Consistent with this, GH increased the phosphorylation of TSC2, an upstream regulator of mTORC1, at sites that are targets for Akt/PKB. Finally, the activation of overall protein synthesis by GH in H4IIE cells was essentially completely inhibited by wortmannin or rapamycin. These results demonstrate for the first time that mTORC1 plays a major role in the rapid activation of protein synthesis by GH.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.