Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C18-C27 ◽  
Author(s):  
W. V. Everson ◽  
K. E. Flaim ◽  
D. M. Susco ◽  
S. R. Kimball ◽  
L. S. Jefferson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Synthetic Activity ◽  
Perfused Liver ◽  
Amino Acid Deprivation ◽  
Isolated Rat Hepatocytes ◽  
Reticulocyte Lysate

Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals A Novel Mechanism for the Control of Translation Initiation by Amino Acids, Mediated by Phosphorylation of Eukaryotic Initiation Factor 2B

2007 ◽  
Vol 28 (5) ◽  
pp. 1429-1442 ◽  
Author(s):  
Xuemin Wang ◽  
Christopher G. Proud
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Translation Initiation ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Amino Acid Deprivation ◽  
Signaling Mechanism

ABSTRACT Eukaryotic initiation factor 2B (eIF2B) plays a key role in controlling the initiation of mRNA translation. eIF2B is heteropentamer whose catalytic (ε) subunit promotes GDP/GTP exchange on eIF2. We show here that depriving human cells of amino acids rapidly results in the inhibition of eIF2B, independently of changes in eIF2 phosphorylation. Although amino acid deprivation also inhibits signaling through the mammalian target of rapamycin complex 1 (mTORC1), the inhibition of eIF2B activity by amino acid starvation is independent of mTORC1. Instead, amino acids repress the phosphorylation of a novel site in eIF2Bε. We identify this site as Ser525, located adjacent to the known phosphoregulatory region in eIF2Bε. Mutation of Ser525 to Ala abolishes the regulation of eIF2B and protein synthesis by amino acids. This indicates that phosphorylation of this site is crucial for the control of eIF2B and protein synthesis by amino acids. These findings identify a new way in which amino acids regulate a key step in translation initiation and indicate that this involves a novel amino acid-sensitive signaling mechanism.

AMINO ACID CONTROL OF PROTEIN SYNTHESIS AND DEGRADATION IN ISOLATED RAT HEPATOCYTES*

1980 ◽  
Vol 349 (1) ◽  
pp. 1-17 ◽  
Author(s):  
Per O. Seglen ◽  
Anne E. Solheim ◽  
Bjørn Grinde ◽  
Paul B. Gordon ◽  
Per E. Schwarze ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Acid Control ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation ◽  
Isolated Rat

scholarly journals Microcystin-LR induced an inhibition of protein synthesis in isolated rat hepatocytes

1995 ◽  
Vol 306 (3) ◽  
pp. 693-696 ◽  
Author(s):  
S Claeyssens ◽  
A Francois ◽  
A Chedeville ◽  
A Lavoinne
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Maximum Effect ◽  
Isolated Rat Hepatocytes ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Phosphatase Activation ◽  
Isolated Rat

The effect of microcystin-LR, an inhibitor of protein phosphatases PP1 and PP2A, was studied on protein synthesis by measuring the incorporation of labelled amino acid into protein in isolated rat hepatocytes. Microcystin-LR inhibited protein synthesis in the first minutes of the incubation period, and half-maximum effect was obtained at about 60 nM. Such an inhibition was also observed in the presence of different protein phosphatase inhibitors, i.e. okadaic acid, calyculin A and microcystin-RR. This effect was observed in whole hepatocytes, in the supernatant of the post-mitochondrial fraction and in the microsomal fraction. It was independent of a substrate supply and of the labelled amino acid used. Furthermore, this inhibition preceded the previously reported glucose-6-phosphatase activation induced by microcystin-LR [Claeyssens, Chédeville and Lavoinne (1993) FEBS Lett. 315, 7-10].

scholarly journals Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos

1991 ◽  
Vol 276 (3) ◽  
pp. 809-816 ◽  
Author(s):  
A Moreno ◽  
R Mendez ◽  
C de Haro
Keyword(s):  
Protein Synthesis ◽  
Developmental Stages ◽  
Wide Spectrum ◽  
Initiation Factor ◽  
Synthetic Activity ◽  
Globin Mrna ◽  
Free System ◽  
Early Developmental Stages ◽  
Reticulocyte Lysate ◽  
Early Developmental

We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5′-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity in the undeveloped embryo lysate and made it capable of directing the synthesis of both endogenous mRNAs and exogenous (globin) mRNA.

Invited Review: Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise

2002 ◽  
Vol 93 (3) ◽  
pp. 1168-1180 ◽  
Author(s):  
Scot R. Kimball ◽  
Peter A. Farrell ◽  
Leonard S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Amino Acid Availability

Protein synthesis in skeletal muscle is modulated in response to a variety of stimuli. Two stimuli receiving a great deal of recent attention are increased amino acid availability and exercise. Both of these effectors stimulate protein synthesis in part through activation of translation initiation. However, the full response of translation initiation and protein synthesis to either effector is not observed in the absence of a minimal concentration of insulin. The combination of insulin and either increased amino acid availability or endurance exercise stimulates translation initiation and protein synthesis in part through activation of the ribosomal protein S6 protein kinase S6K1 as well as through enhanced association of eukaryotic initiation factor eIF4G with eIF4E, an event that promotes binding of mRNA to the ribosome. In contrast, insulin in combination with resistance exercise stimulates translation initiation and protein synthesis through enhanced activity of a guanine nucleotide exchange protein referred to as eIF2B. In both cases, the amount of insulin required for the effects is low, and a concentration of the hormone that approximates that observed in fasting animals is sufficient for maximal stimulation. This review summarizes the results of a number of recent studies that have helped to establish our present understanding of the interactions of insulin, amino acids, and exercise in the regulation of protein synthesis in skeletal muscle.

scholarly journals Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

2008 ◽  
Vol 295 (4) ◽  
pp. E876-E883 ◽  
Author(s):  
Fiona A. Wilson ◽  
Agus Suryawan ◽  
Renán A. Orellana ◽  
Hanh V. Nguyen ◽  
Asumthia S. Jeyapalan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Translation Initiation ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Growing Pigs ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Skeletal Muscle Protein

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) ( P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level ( P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation ( P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

scholarly journals Mechanisms involved in the nutritional regulation of mRNA translation: features of the avian model

2006 ◽  
Vol 19 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Sophie Tesseraud ◽  
Mourad Abbas ◽  
Sophie Duchene ◽  
Karine Bigot ◽  
Pascal Vaudin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Muscle Growth ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Nutritional Regulation ◽  
Translational Repressor

Abstract:Insulin and amino acids are key factors in regulating protein synthesis. The mechanisms of their action have been widely studied for several years. The insulin signal is mediated by the activation of intracellular kinases such as phosphatidylinositol–3'kinase and the mammalian target of rapamycin (mTOR), affecting the phosphorylation of some major effectors involved in the regulation of translation initiation, i.e. p70 S6 kinase (p70S6K) and the translational repressor eukaryotic initiation factor 4E binding protein (4E-BP1). The amino acid–induced signalling cascade also originates from mTOR and promotes p70S6K and 4E–BP1 activation. However, the mechanisms of regulation are complex and little understood, especially in vivo. Elucidating these mechanisms is important for both fundamental physiology and nutritional applications, i.e. better control of the use of nutrients and optimisation of dietary amino acid supplies in various physiological and physiopathological situations. In comparative physiology, the chicken is an interesting model to gain better understanding of the nutritional regulation of mRNA translation because of the very high rates of muscle growth and protein synthesis, and the unusual features compared with mammals. In the present review we provide an overview of the roles of insulin and amino acids as regulators of protein synthesis in both mammals and avian species.

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