Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts.

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at -80 degrees C. Cell lysates were fractionated on 15-60% sucrose gradients by centrifugation at 200,000 x g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P < 0.05) in monosome peak height and a increase (P < 0.05) in polysome percentage (P < 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P < 0.05). None of the growth factors had a significant effect (P > 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.     

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes.

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.     

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum.

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium. 2. Insulin (100 microU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum. 3. Dexamethasone inhibited the effects of 100 microU insulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 microU and 1 mU/ml respectively.     

Regulation of translation rate during morphogenesis in the fungus Mucor

The present study was undertaken in order to elucidate the molecular mechanisms responsible for regulating changes in the specific rate of protein synthesis during the yeast-to-hyphae morphogenesis in the fungus, Mucor racemosus. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by means of pulse-labeling and density gradient fractionation techniques. The percentage of ribosomes active in protein synthesis was observed to decrease throughout the morphological transition. The rate of amino acid addition to nascent polypeptide chains was calculated and the transit time of messenger RNA translation was measured. The results showed a significant increase in the velocity of ribosome movement along the message which was continuously adjusted throughout hyphal development.     

Morphological changes in Krebs II ascites tumour cells induced by insulin are associated with differences in protein composition and altered amounts of free,cytoskeletal-bound and membrane-bound polysomes

A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183–193,1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture- Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more then 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins. (Mol Cell Biochem 118: 131–140, 1992)     

Simultaneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E after mitogenic stimulation of Swiss 3T3 cells

Ribosomal protein L32 mRNA moved from messenger ribonucleoprotein particles into polysomes following serum activation of quiescent Swiss 3T3 cells. This redistribution of the mRNA into a translationally active state began by 1 h and was complete by 3 h after activation. In contrast, actin mRNA showed no translational control, being found predominantly in polysomes in both quiescent and activated cultures. The phosphorylation state of eukaryotic initiation factor (eIF) 4E, which binds mRNA caps, was examined in parallel. eIF-4E phosphorylation was elevated by 1 h following serum activation and reached a peak by 3-5 h. Treatment of resting cells with phorbol ester also simultaneously stimulated eIF-4E phosphorylation and the movement of L32 mRNA into polysomes. These results are consistent with a model in which mitogen-induced phosphorylation increases the pool of active eIF-4E molecules, which in turn cause the recruitment of translationally controlled mRNAs to actively synthesizing ribosomes.     

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium.2. Insulin (100 μU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum.3. Dexamethasone inhibited the effects of 100 μU ulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 μU and 1 mU/ml respectively.     

Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.     

Protein synthesis in resting and growth-stimulated human peripheral lymphocytes : Evidence for regulation by a non-messenger RNA

Stimulation of lymphocyte growth is accompanied by an early increase in the rate of protein synthesis. This increase is dependent upon the flow of inactive free ribosomes into polysomes, which is limited by a rate-controlling step at initiation [2]. Addition of actinomycin D (actD) to lymphocytes caused a gradual reduction in protein synthesis in resting cells, but rapidly inhibited both the elevation of protein synthesis and the activation of free ribosomes which normally follow exposure to mitogens. Since actD does not affect protein synthesis in enucleated lymphocytes [4], the effect in intact cells must be mediated by a nuclear event, which available data indicate is RNA synthesis. ActD prevented the accumulation of 80S initiation complexes which normally occurs in resting lymphocytes treated with pactamycin and cycloheximide, showing that its locus of action was at some point in initiation. The decline in rate of protein synthesis began without detectable lag when resting lymphocytes were treated with actD. However, after growth stimulation, a delay of ca 50 min occurred before the protein synthetic rate declined in response to actD. These observations agree with the hypothesis that the concentration of some moderately short-lived RNA is rate-limiting for protein synthesis in resting lymphocytes, and that an early event in growth stimulation is a rise in the amount of this component to levels which are no longer rate-limiting. This permits an increased flow of ribosomes into polysomes and a consequent rise in protein synthesis. Available evidence indicates that the regulatory RNA is neither mRNA nor rRNA, but may either be one of the small cytoplasmic RNAs whose function is unknown, or tRNA_i^met.     

Polysomes, Ribonucleic Acid, and Protein Synthesis During Germination of Neurospora crassa Conidia

The level of polysomes in ungerminated conidia of Neurospora crassa depends on the method used to collect spores. Spores harvested and exposed to hydration contain 30% of their ribosomes as polysomes, whereas those not exposed to hydration contain only 3% of their ribosomes as polysomes. During the germination process, the percentage of the ribosomes which sediment as polysomes increases rapidly to a level of approximately 75% during the first 15 to 30 min of germination. This rapid increase has been shown to require a carbon source. During the first 30 min of germination, spores synthesize ribosomal ribonucleic acid (RNA) and heterogeneously sedimenting RNA, i.e., presumptive messenger RNA.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Modulation by dexamethasone of the pyruvate dehydrogenase-complex activity in 3T3-L1 adipocytes.

Chronic exposure of 3T3-L1 pre-adipocytes to dexamethasone plus 3-isobutyl-1-methylxanthine (IBMX) with or without insulin caused a significant increase in the specific activity of 'total' pyruvate dehydrogenase complex (PDC) and in the percentage of the 'active' form of the complex compared with cells exposed to a chronic insulin treatment or an acute treatment (2 days) with dexamethasone plus IBMX. In acute-drug-switch-over experiments, dexamethasone also caused an increase in the percentage of 'active' PDC in 3T3-L1 adipocytes. The results show that, in 3T3-L1 adipocytes, dexamethasone, even in the absence of insulin, increases the proportion of PDC in its 'active' form. The mechanism of the dexamethasone effect remains to be investigated.     

CONCOMITANT EFFECTS OF INSULIN ON SURFACE MEMBRANE CONFORMATION AND POLYSOME PROFILES OF SERUM-STARVED BALB/C 3T3 FIBROBLASTS

By scanning and transmission electron microscopy we have shown that insulin rapidly reversed changes in surface membrane conformation and polysome profile induced by the transfer of actively growing Balb/c 3T3 fibroblasts from a serum-containing to a serum-free medium. Morphometric analysis of polysome profiles revealed a 94% aggregation of total f ribosomes during logarithmic growth. This figure fell to 78% after 18 h of serum starvation. The number of f ribosomes per unit area of cytoplasm also fell. 1 h of insulin treatment restored aggregation to 92% and increased the number of f ribosomes per unit area of cytoplasm by 22%. Scanning electron microscopy of logarithmically growing cells revealed an abundance of surface microvilli, whereas serum starvation promoted a smooth surface with few microvilli. After 1 h of insulin treatment, microvilli reappeared with a distribution and subcellular organization characteristic of exponential growth. This study shows the combined and rapid effect of insulin on the regulation of polysome formation and the promotion of a specific surface membrane conformation in cultured cells. The observations are consistent with the knowledge that insulin, acting on the surface membrane, can influence such parameters as membrane transport, and the rates of protein and RNA synthesis.     

Differential kinetics of changes in the state of phosphorylation of ribosomal protein S6 and in the rate of protein synthesis in MPC 11 cells during tonicity shifts

Mouse myeloma (MPC 11) cells respond rapidly to hypertonic conditions by shutting down protein synthesis at the level of polypeptide chain initiation. Translational activity recovers equally quickly upon a return to isotonicity. Disaggregation and reformation of polysomes occur in parallel to the changes in protein synthesis. Ribosomal protein S6 becomes dephosphorylated under hypertonic conditions and rephosphorylated when isotonic conditions are restored. The kinetics with which these changes occur are, however, too slow to account for the changes in protein synthesis. Treatment of the cells with a low concentration of cycloheximide allows reformation of polysomes under hypertonic conditions; conversely, puromycin prevents the restoration of polysomes which otherwise occurs on return to isotonicity. Neither inhibitor prevents the changes in S6 phosphorylation resulting from the tonicity shifts. We conclude that the overall extent of phosphorylation of S6 neither regulates nor is determined by the rate of protein synthesis and is not obligatorily related to the proportion of ribosomes in polysomes.     

Free ribosomes and growth stimulation in human peripheral lymphocytes: Activation of free ribosomes as an essential event in growth induction

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

Temperature-sensitive Chinese hamster fibroblast mutant with a defect in RNA metabolism

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5 degrees C, the rates of DNA, RNA, and protein synthesis declined rapidly (to < or = 50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5 degrees C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5 degrees C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.     

Content and Aggregation of Ribosomes during Formation, Dormancy and Sprouting of Tubers of Helianthus tuberosus

The ribosomes and their qualitative (monosomes-polysomes) and quantitative variations over a whole vegetative period of the tuber of Helianthus tuberosus L. (cv. OB 1) were examined. Tubers in different phases of growth, dormancy and sprouting or slices of dormant tubers activated with 2 x 10^-6M indol-3-acetic acid were used. The ribosomes were analyzed by a linear sucrose gradient. During flowering, polysomes of tuber disappeared almost completely and rRNA decreased in comparison with the level present at the beginning of tuber formation. After flowering, there was a new synthesis of monosomes and polysomes until the onset of dormancy; this last period was characterized by a marked increase in polysomes and a proportional increase in monosomes. The level remained almost constant till the break of dormancy. When the tubers sprouted, ribosomes, present almost exclusively as monosomes, decreased considerably; on the contrary the non-photosynthetic sprouts contained many monosomes and polysomes. The first phases of activation (3 h) of tuber slices were characterized by a RNA synthesis, which occurred during one hour, in the subunit region of the gradient. Successively (10 h of activation) the ³²P incorporation was seen also in the polysome region and increased with time. Some possible interpretations of these last results are discussed.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.