Insulin rapidly induces the biosynthesis of elongation factor 2.

Insulin increases the rate of overall protein synthesis in many cells and tissues, while inducing the preferential expression of individual proteins. To identify and characterize such proteins, NIH 3T3 cells stably expressing more than 10(6) human insulin receptors per cell (HIR 3.5; Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5237-5241) were treated with insulin in the presence of [35S]methionine, and labeled proteins were separated using ultra-high resolution ("giant") two-dimensional gel electrophoresis. Overall protein synthesis was enhanced as much as 3-fold by insulin treatment; the synthesis of approximately 1% of the 2,500 proteins visible on the gel autoradiographs was further selectively increased. By using immunoblotting, immunoprecipitation and comigration assays, we identified one of the rapidly induced proteins (Mr 96,000; pI 6.8) as eukaryotic elongation factor 2 (EF-2), a major component of the protein translation apparatus. Insulin induced the synthesis of EF-2 within 20 min of treatment, with a half-maximal dose of 10(-11) M. It was synthesized as a precursor form that was processed to a more basic mature species within 30 min. Long term treatment with insulin led to accumulation of EF-2 within the cell and prevented the substantial decrease in EF-2 concentration that occurred during serum deprivation. Finally, we found that insulin induction of EF-2 occurred normally in the presence of the RNA-transcription inhibitor, actinomycin D. Thus, insulin rapidly induced the synthesis of EF-2 predominantly or exclusively at the level of mRNA translation.

Insulin increases the rate of overall protein synthesis in many cells and tissues, while inducing the preferential expression of individual proteins. To identify and characterize such proteins, NIH 3T3 cells stably expressing more than 10' human insulin receptors per cell (HIR 3.5; Whittaker, J., Okamoto, A. K., Thys, R., Bell, G . I., Steiner, D. F., and Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5237-5241) were treated with insulin in the presence of [s6S]methionine, and labeled proteins were separated using ultra-high resolution ("giant") two-dimensional gel electrophoresis. Overall protein synthesis was enhanced as much as %fold by insulin treatment; the synthesis of approximately 1% of the 2,500 proteins visible on the gel autoradiographs was further selectively increased. By using immunoblotting, immunoprecipitation and comigration assays, we identified one of the rapidly induced proteins (M, 96,000; PI 6.8) as eukaryotic elongation factor 2 (EF-2), a major component of the protein translation apparatus. Insulin induced the synthesis of EF-2 within 20 min of treatment, with a halfmaximal dose of 10-l' M. It was synthesized as a precursor form that was processed to a more basic mature species within 3 0 min. Long term treatment with insulin led to accumulation of EF-2 within the cell and prevented the substantial decrease in EF-2 concentration that occurred during serum deprivation. Finally, we found that insulin induction of EF-2 occurred normally in the presence of the RNA-transcription inhibitor, actinomycin D. Thus, insulin rapidly induced the synthesis of EF-2 predominantly or exclusively at the level of mRNA translation.
Insulin is an anabolic hormone that can increase the overall rate of protein synthesis in many cells and tissues. General protein synthesis begins to increase within minutes following insulin exposure and typically rises to a level 2-to %fold greater than basal levels within a few hours of treatment (Hansson and Ingelman-Sundberg, 1985;Towle et al., 1984;Okabayashi et ul., 1987). The increase in overall protein synthesis that occurs within the first few hours of insulin treatment can take place in the absence of ongoing mRNA transcription and correlates with the recruitment of previ-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section  ously synthesized messenger RNA into synthetically active polyribosomes (Wool et al., 1972;Lyons et al., 1980). However, a later, sustained increase in general protein synthesis appears to be dependent on mRNA synthesis (Thomas et al., 1981).
In addition to stimulating overall protein synthesis, insulin has been shown to regulate the preferential expression of specific proteins. A number of these specific effects are dependent upon continued mRNA synthesis (reviewed in Davis et al., 1988), whereas others can still take place without change in mRNA levels (Okabayashi et al., 1987).' Thus, both the stimulation of overall protein synthesis and specific effects on at least some proteins appear to be regulated at the level of translation.
The mechanism by which insulin alters translational activity is not completely understood. Translation of mRNA into cognate polypeptide chains is considered to occur in three stages: initiation, elongation, and termination (reviewed in Moldave, 1985). Insulin has been reported to increase protein synthesis exclusively by enhancing initiation (Monier and Le Marchand-Brustel, 1982). This conclusion is consistent with observations that insulin stimulates at least two reactions that may promote initiation, namely the phosphorylation of ribosomal protein S6 (Smith et al., 1980;Thomas et ul., 1982) and the dephosphorylation of eukaryotic initiation factor 2a (e1F-2~~)' (Towle et al., 1984). Phosphorylation of S6 may enhance recruitment of ribosomes containing the modified protein into translationally active polysomes (Thomas et al., 1982), whereas dephosphorylation of phospho-eIF-2a is necessary to allow it to recycle and form another initiation complex (Panniers and Henshaw, 1983). These events may contribute to insulin's effect on overall protein synthesis, and there are indications that they may also differentially affect the rate of translation of specific mRNAs (Palen and Traugh, 1987;Kaufman et al., 1989). Although insulin has not been shown to affect stages of protein synthesis other than initiation, a number of stimuli can increase the rate of protein synthesis apparently through increases in the elongation rate of nascent peptide chains. These stimuli include heat shock (Theodorakis et Ballinger and Pardue, 1983) or treatment with cyclic AMP , estrogen and progesterone (Palmiter, 1972;Gehrke et al., 1981), and serum (Nielsen and Mc-Conkey, 1980). In this paper, we demonstrate that insulin rapidly stimulates the biosynthesis of a crucial factor in this peptide elongation reaction, elongation factor 2 (EF-2). This effect, which appears to occur almost entirely at the transla-R. M. Levenson and P. J. Blackshear, manuscript in preparation. The abbreviations used are: eIF, eukaryotic initiation factor; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; 2-D, two-dimensional; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EGTA, [ethylenebis (oxyethylenenitrilo)]tetraacetic acid; EF, elongation factor. tional level, represents a novel action of insulin that could conceivably affect overall rates of peptide chain elongation in response to insulin.
Treatment and Labeling-Cells were grown to confluence in 35mm dishes. Approximately 18 h before labeling, the cells were washed twice in methionine-free DMEM and transferred to low-methionine (5% (w/v) of normal) DMEM (GIBCO)/l% bovine serum albumin (BSA crystallized and lyophilized, Sigma). At various times, the cells were pulsed with [%]methionine (250 pCi/ml; ICN Biomedicals, Inc., Irvine, CA) for 20 min to 1 h, after which the dishes were placed on ice and washed three times with ice-cold DMEM. The cell monolayers then were lysed by the addition of 150-200 p1 of lysis buffer (9.5 M urea (ultra-pure, Schwartz/Mann Biotech), 2% Nonidet P-40 (Sigma), 5% P-mercaptoethanol, 1.6% (w/v) ampholytes, pH 5-8, and 0.4% ampholytes, pH 3.5-10 (Pharmacia LKB Biotechnology Inc.) O'Farrell, 1975). This procedure effectively solubilizes most cellular proteins (Klose and Zeindl, 1984). Alternatively, cytoplasmic and ''nuclear" proteins were prepared for two-dimensional (2-D) gel electrophoresis with a double detergent extraction (Thomas et al., 1981), in which the cells were rinsed with a hypotonic buffer (1.5 mM KCl, 2.5 mM MgClZ, and 5 mM Tris-HC1, pH 7.4), and then extracted with 1% (w/v) sodium deoxycholate and 1% (w/v) Triton X-100 (Sigma) in the same buffer. After treatment with 50 pg of ribonuclease A (Boehringer Mannheim) for 5 min at 37 "C, the samples were precipitated with 5 volumes of -20 "C acetone and the resulting pellets were dissolved in lysis buffer. Protein concentrations were determined using the Bradford colorimetric protein assay (Bradford, 1976) with reagent supplied by Bio-Rad. Trichloroacetic acid-precipitable radioactivity of the lysate was determined by spotting 4 pl of the cell lysates onto a filter paper grid which was then soaked in 10% (w/v) trichloroacetic acid, 0.1% (w/v) methionine for 30 min at 4 "C, followed by three 5-min washes in the same solution, and two 1-min washes in 95% ethanol. Regions containing the samples were cut from the dried grid and subjected to liquid scintillation counting in Biofluor (Du Pont-New England Nuclear). In order to investigate the effect of inhibition of mRNA synthesis, cells were preincubated with actinomycin D (4 pglml, Sigma) for 15 min before addition of other agents. This concentration inhibited incorporation of ['4C]uridine by more than 90% (data not shown).
Two-dimensional Gel Electrophoresis-Details of procedures for giant two-dimensional gel electrophoresis have been described in detail (Young et al., 19831, and the advantages over conventional 2-D gel systems were discussed (Young, 1984). Briefly, cell lysates containing 5-20 X lo6 cpm of trichloroacetic acid-precipitable radioactivity were first separated by isoelectric focusing under reducing conditions in 30-cm rod gels containing 2% (w/v) ampholytes in the ratio of 20% (v/v) pH 3.5-10 and 80% (v/v) pH 5-8 for 22 h (42,000 Vh; maximum voltage, 2000 V). Rod gels then were extruded and equilibrated in 1% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.625 M Tris-HC1, pH 6.8, for 30-60 min at room temperature with gentle shaking. The equilibration solution was replaced, and the gels were then frozen in a -70 'C ethanol bath and kept at -70 "C until SDSpolyacrylamide gel electrophoresis (PAGE) was performed. The isoelectric focusing gels were placed on top of 32 X 36 X 0.075 cm, 10-16% (w/v) exponential gradient SDS-polyacrylamide slab gels. Electrophoresis was carried out for 12 h at 30 mA/gel and, subsequently, the gels were fixed with 50% (v/v) methanol 12% (v/v) acetic acid (which causes them to shrink), reswollen in 14% methanol, 7.5% acetic acid, dried, and exposed to Kodak X-AR film for 40 X lo6 cpm/ days (2-8 days). Within an experiment, equal amounts of trichloroacetic acid-precipitable radioactivity from each experimental dish were applied to the first-dimension gels. "C-Labeled molecular weight standards were obtained from Bethesda Research Laboratories.
Gel Analysis and Data Management-Autoradiographs and immunoblots blots from one-dimensional gels were quantitated using a laser densitometer (Biomed Instruments, Fullerton, CA). Two-dimen-sional autoradiographs were analyzed visually and quantified by densitometry using a microcomputer-based 2-D gel densitometer described by Levenson et al. (1986). Results are expressed in arbitrary but consistent units that are related to the film's response to a '*clabeled autoradiographic step-wedge.
Immunoprecipitation-After experimental treatments, confluent cultures of HIR 3.5 cells in 6-or 24-well culture plates were lysed in O'Farrell's lysis buffer. Samples containing equal amounts of trichloroacetic acid-precipitable radioactivity and approximately 60 pg of protein (in approximately 30 pl) were diluted 1:lO with NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HC1, pH 7.5 containing 1.5% (w/v) Nonidet P-40 and 2.5 mg/ml bovine serum albumin). After mixing, 2 pl of undiluted rabbit anti-EF-2 antiserum (Nairn et al., 1985;Nairn and Palfrey, 1987) was added and the mixture incubated for 2 h at 4 "C. 30 p1 of a 10% (v/v) suspension of formalinfixed Staphylococcus aureus, Cowen strain (Pansorbin, Calbiochem), were added and the samples incubated for a further 30 min at 4 "C with rotation. The samples were then centrifuged at approximately 12,000 X g for 2 min at 4 "C, resuspended twice with brief sonication in NET buffer containing 0.1% Nonidet P-40, and pelleted as above.
The final pellet was resuspended in 80 pl of SDS sample buffer (Laemmli, 1970) and heated to 100 "C for 3 min; 60 pl were then applied to 7.2% SDS-PAGE gels. For 2-D gels, the immunoprecipitate bound to the S. aureus beads was resuspended in 100 pl of O'Farrell's lysis buffer and incubated with shaking at room temperature for 1 h before separation by isoelectric focusing.
Zmmunoblotting-After treatment with or without insulin for 6, 12, or 14 h, confluent cells in 12-well dishes were washed in DMEM, scraped with a rubber "policeman," and pelleted in microcentrifuge tubes (2,000 X g for 2 min at 4 "C). The cell pellets were lysed in 100 ~1 of sodium glycerophosphate (0.1 M), sucrose (0.5 M), dithiothreitol (2 mM), EGTA (2 mM), EDTA (2 mM), pH 7.5, freeze-thawed, and triturated five times through a 26-gauge needle. The samples were centrifuged at 12,000 X g for 10 min at 4 "C and the supernatant collected. Equal amounts of protein (1.7 fig) from each sample were separated by 7.2% SDS-PAGE gel in a minigel apparatus. Alternatively, cell lysates were prepared as described above with O'Farrell's lysis buffer; aliquots containing 200 pg of protein were subjected to 2-D electrophoresis, and the area corresponding to EF-2 was excised (see below). Proteins were then transferred to nitrocellulose membrane (BA-85, Schleicher & Schuell) and immune complexes were detected with horseradish peroxidase essentially as described (Blackshear et al., 1986).

Effects of Insulin on Overall Protein Synthesis in HIR 3.5
Cells-Cells were pulsed with [35S]methionine for 20 min at the end of 1-5 h of insulin treatment (70 nM). Insulin exerted a prompt (within 1 h), sustained, and substantial increase in the rate of incorporation of [35S]methionine into trichloroacetic acid-precipitable radioactivity ( Fig. l A ) , which reached a level 2.4-fold higher than that observed in control dishes 5 h after insulin addition. Moreover, long term steady-state labeling and colorimetric determinations of total protein in cell lysates indicated that there was an increase in protein mass 12 or 24 h after incubation of cells in DMEM, 1% BSA supplemented with 70 nM insulin (Fig. l B ) , which was due largely to continued cell division and an increase in cell number occurring only in the insulin-treated dishes (Hofmann et al., 1989).

Insulin-treated and untreated cells were labeled with [Y3]
methionine at various times and total cell proteins were solubilized and separated on giant 2-D gels as described under "Experimental Procedures." Characteristic autoradiographs of 2-D gels of total cell lysates are shown in Fig. 2. Approximately 2,500 35S-labeled proteins with M , between 14,000 and 200,000 and PI between 4.2 and 7.2 were visible on the original autoradiographs. At least 25 proteins were preferentially synthesized after addition of insulin,' most of these responding within the 1st h of treatment. One of the most prominent insulin-induced proteins detected in these gels was repre- . The cells were harvested as described under "Experimental Procedures" and trichloroacetic acid-precipitable counts determined. For short labeling periods, total counts incorporated reflect the rate of ongoing protein synthesis. Results shown are the means (*ranges) from duplicate cultures. B, effect of insulin on total protein mass. Quiescent HIR 3.5 cells in a 12-well plate were washed four times in DMEM and then incubated in DMEM, 1% BSA in the presence or absence of insulin (70 nM) for up to 24 h. Cells were scraped and pelleted and then resuspended in sodium glycerophosphate (0.1 M), sucrose (0.5 M), dithiothreitol (2 mM), EGTA (2 mM), EDTA (2 mM), pH 7.5, freeze-thawed, and triturated through a 26-gauge needle. After pelleting insoluble debris, protein concentrations were measured by the Bradford method. Results shown are the means (k ranges) from duplicate cultures. The increase in total protein reflects continued cell division in the insulin-treated cultures.
sented by a series of spots of M , 96,000 and PI approximately 6.8 (enclosed within the box in Fig. 2). These characteristics are shared by EF-2 (Palfrey et al., 1987;Nairn and Palfrey, 1987).
To confirm that the insulin-inducible 96-kDa protein was EF-2, we immunoprecipitated EF-2 from cellular lysates of control and insulin-treated HIR 3.5 cells using a rabbit antiserum directed against rat EF-2 (Nairn et al., 1985). Onedimensional separation of immunoprecipitates with immune and preimmune sera demonstrated that the anti-EF-2 serum A Mr PI3 7. p ( x~0 3 + The cells were washed twice in cold DMEM and lysed immediately in 2-D gel lysis buffer and frozen at -70 "C prior to isoelectric focusing. After electrophoresis in the second dimension, the gels were dried and exposed to Kodak X-AR film for approximately 30-million cpm/days. Although there are a number of insulin-sensitive changes in protein synthesis visible on the autoradiographs, only the location of a 96-kDa protein with an isoelectric point of 6.8 whose synthesis is stimulated by insulin is indicated (box).

18
specifically precipitated a protein whose synthesis was markedly stimulated by insulin (Fig. 3). This protein could also be metabolically labeled with [32P]orthophosphate (Fig. 3), mostly on threonine residues (data not shown), both characteristics of EF-2 (Nairn et aZ., 1987). Whole cell lysates and immunoprecipitates were separated in parallel by giant 2-D gel electrophoresis. Only one radiolabeled protein was visible (Fig. 4C) that was not precipitated by nonimmune serum (  (1987). Right lunes, for [32P]orthophosphate-labeling, the cells were incubated in DMEM containing 1% BSA for 48 h before being transferred to phosphate-free DMEM containing 1% BSA. The cells were prelabeled for 2 h with 1 mCi/ml [32P]orthophosphate and insulin (70 nM) was then added for 10 min. The cells were lysed and the lysate immunoprecipitated with anti-EF-2 antiserum as above. Both sets of immunoprecipitates were electrophoresed on the same 7.2% SDS-polyacrylamide gel and visualized by autoradiography on Kodak X-AR film.  ). This immunoprecipitated protein co-migrated with the 96-kDa protein detected in whole cell lysates (Fig. 4A). In addition, [35S]methionine-labeled proteins in the crude cell lysates were separated on giant 2-D gels, and the region surrounding the 96-kDa protein was cut out and the proteins transferred to nitrocellulose. The 96-kDa protein (localized by autoradiography of the blot) was also visualized by immunoblotting with anti-EF-2 antiserum, peroxidase-coupled second antibody, and color reagent (Fig. 5, A and B ) .
In briefly labeled cells, radioactive EF-2 was resolved by 2-D gel electrophoresis into 4 isoforms (identified as numbers 1 through 4 in Fig. 5A, from basic to acidic). However, only 2 of these isoforms (numbers 1 and 3) were detected in the immunoblot. This is not due to failure of the antibody to recognize isoforms 2 and 4, since all 4 forms could be immunoprecipitated from lysates of briefly labeled cells (see Fig.  4C). Instead, forms 1 and 3 are detected in the immunoblot because they comprise the bulk of cellular EF-2; they, but not isoforms 2 and 4, are visible by Coomassie Blue staining (data not shown). Isoform 3 (more acidic) was determined to be the phosphorylated form of isoform 1 (and isoform 4 the phosphorylated form of isoform 2) by inspection of stained and autoradiographed 2-D gels of 32P-phosphorylated EF-2 (data not shown).
The less abundant species (numbers 2 and 4) appeared to be transient post-translational processing intermediates, since they were prominent in cells pulsed briefly with [35S] methionine (Fig. 5C), but relatively depleted in cells labeled continuously for 24 h (Fig. 5 0 ) . Pulse-chase experiments (not shown) indicated that these newly synthesized forms were processed to the mature EF-2 species within 30 min, with form 2 being converted to form 1 and form 4 to form 3. Since forms 1 and 3 represent the unphosphorylated and phosphorylated forms of the mature EF-2 molecule, respectively, and since EF-2 appears to be phosphorylated with a stoichiometry of -1 mol phosphate/mol protein (Nairn and Palfrey, 1987), .
-, , "..,.>..  the distance between these two forms is due to the presence of a single phosphate group and its 2 negative charges on form 3. Since form 2 lies approximately equidistant from forms 1 and 3, it evidently differs from its mature form by a single (negative) charge. Thus, maturation of form 2 to form 1 involves the loss of one negative charge (see "Discussion").
Synthesis of EF-2-EF-2 is an essential component of the cellular protein synthetic machinery (Moldave, 1985). To investigate whether insulin increased the amount of EF-2 relative to total cellular protein, cells were incubated in DMEM supplemented only with 1% BSA in the presence or absence of insulin for 6, 12, and 24 h. Immunoblots of equal amounts of proteins from these cells were probed with anti-EF-2 antiserum and analyzed by densitometry (Fig. 6). In the absence of insulin, the amount of EF-2 in the cell declined by over 50% after 12 h in serum-free medium, whereas the amount of EF-2 increased in cells incubated in serum-free medium containing 70 nM insulin and reached a level by 12 h that was 40% higher than that seen in control cells at the 6h time point and 260% higher than in the corresponding 12h controls.
We also examined the dose dependence and time course of insulin induction of EF-2. To determine the dose-response curve, we incubated HIR 3.5 cells in concentrations of insulin ranging from to lo" M for 1 h, pulsing the cells with [35S]methionine for the last 20 min. EF-2 was immunoprecipitated from lysates containing equal numbers of trichloroacetic acid-precipitable counts and the EF-2 synthesis rates quantitated by densitometry. The concentration of insulin necessary for a half-maximal response was approximately lo-" M, and a maximal effect was observed at insulin concentrations as low as lo-'' M (0.1 nM). This dose response parallels that observed for activation of glycogen synthesis by insulin in these cells (Whittaker et aZ., 1987) and suggests that the effect is mediated through insulin receptors and not through insulin-like growth factor I receptors, which are approximately two orders of magnitude less responsive to insulin (Jacobs et aZ., 1983). The synthesis of EF-2 was also stimulated by 5 and 10% fetal bovine serum (data not shown).
To determine the time course of insulin's effects, HIR 3.5 cells were exposed to insulin (70 nM) for various times over a 3-h period. The amount of immunoprecipitated, metabolically labeled EF-2 was determined densitometrically (Fig. 7). An  6. Abundance of immunoreactive EF-2 in HIR 3.5 cells incubated in serum-free medium in the absence or presence of insulin. HIR 3.5 cells were grown to confluence in a 12-well plate in DMEM, 10% calf serum and washed four times in DMEM before incubation in DMEM, 1% BSA. At 6, 12, and 24 h, duplicate wells were washed and cytoplasmic proteins extracted as described under "Experimental Procedures." Aliquots containing equal amounts of protein (1.7 pg) were separated on 7.2% SDS-PAGE using a minigel apparatus. The proteins were transferred to nitrocellulose and the blots probed with anti-EF-2 (1:200). The immunoblot was quantitated densitometrically. EF-2 mass decreased in cells incubated in serumfree medium alone. Addition of insulin (70 nM) blocked this decrease and caused an accumulation of EF-2 to levels 40% greater than control levels (6 h) by 12 h of treatment. . Time course of insulin-stimulated EF-2 synthesis. Confluent, quiescent HIR 3.5 cells in 6-well plates were incubated in DMEM, 1% BSA overnight and transferred to methionine-free DMEM, 1% BSA. At appropriate intervals, insulin was added to individual wells. 10 min before the end of the incubation, ["SI methionine (300 pCi/ml) was added to each well. After the 10-min labeling period, the plates were placed on ice and washed rapidly with ice-cold DMEM in the same order as the insulin had been added. The cells were then lysed, and aliquots containing equal amounts of trichloroacetic acid-precipitable radioactivity (to allow for the increase in general protein synthesis) were immunoprecipitated with anti-EF-2 antiserum. The immunoprecipitates were separated on 7.2% SDS-PAGE, autoradiographed, and quantitated densitometrically. The time course of total protein synthesis (trichloroacetic acidprecipitable cpm) and of the immunoprecipitated EF-2 are shown. increase in EF-2 labeling was detected between 10 and 20 min after insulin addition. The rate of EF-2 biosynthesis reached a maximum within 1 h and remained elevated for at least 3 h. In other experiments, increased EF-2 biosynthetic rates were sustained for at least 5 h (data not shown). Increases in both total protein synthesis and EF-2 synthesis in particular began at approximately the same time after insulin addition (Fig. 7); however, the rate of overall protein synthesis, which reached a plateau between 90 and 150 min after treatment, increased again at 3 h, while the rate of EF-2 synthesis did not. The timing of this second wave of protein synthesis varied from experiment to experiment. In the experiment shown in Fig. 1, for example, the second phase was not seen until after the 4th h of insulin treatment, but once again, EF-2 synthesis did not increase relative to total protein synthesis at that time (data not shown).
Actinomycin Sensitivity-Many early protein synthetic responses to growth factors are controlled at the post-transcriptional level (Thomas et aZ., 1981;Thomas and Thomas, 1986;Okabayashi et aZ., 1987). Accordingly, we attempted to determine whether any of the increases in insulin-induced protein synthesis detected in HIR 3.5 cells were dependent on de mvo mRNA synthesis. We incubated HIR 3.5 cells in 4 pg/ml actinomycin D (an inhibitor of mRNA synthesis; Goldberg and Reich, 1964) for 15 min before the addition of 70 nM insulin. At this concentration of actinomycin D, incorporation of [I4C]uridine into RNA was inhibited by at least 90% (data not shown), and approximately 25% of the insulin inductions detected on giant gels was prevented.' Actinomycin D treatment did not inhibit either the basal or insulin-stimulated synthesis of EF-2. In fact, actinomycin D alone caused a small but detectable stimulation of EF-2 biosynthesis (Fig. 8); interestingly, insulin and actinomycin D together enhanced EF-2 synthesis by 400% over control levels.
EF-2 within the cell, while incubation in serum-and insulinfree medium resulted in a marked decline in its intracellular concentration. Induction of EF-2 was not unique to insulin; both 5 and 10% fetal bovine serum, as well as platelet-derived

DISCUSSION
We have shown in this paper that insulin treatment of HIR 3.5 cells resulted in a 2-to %fold increase in the rate of overall cellular protein synthesis. When metabolically labeled individual proteins were separated on giant 2-D gels, more than 25 of the 2500 proteins visible were synthesized at a rate substantially above the rate of total cellular protein synthesis. ' We identified one of these proteins as EF-2, an essential component of the cellular protein synthesis machinery (Moldave, 1985). The earliest increase in EF-2 synthesis coincided with the increase in overall protein synthesis that occurred approximately 20 min after the addition of insulin to these cells.
Long term treatment with insulin led to elevated levels of growth factor and phorbol 12-myristate 13-acetate (data not shown), also induced it. Thus, EF-2 induction appears to be part of a common response seen in cells treated with peptide mitogens and tumor promoters, all agents that promote increased protein synthesis in these fibroblastic cells. Induction of EF-2 was apparently effected at a post-transcriptional step, since actinomycin D, an inhibitor of mRNA synthesis, did not block insulin's ability to increase EF-2 synthesis. EF-2 synthesis did not decline appreciably even after 4 h of treatment with actinomycin D suggesting that EF-2 mRNA is quite stable under these conditions. Effectiveness of the inhibitory action of actinomycin D on mRNA synthesis was assessed both directly, by measuring [14C]uridine incorporation (greater than 90% inhibition), and indirectly, by qualitatively observing its ability to abrogate induction of other insulin-sensitive proteins (data not shown). Actinomycin D has frequently been used to establish the dependence of protein inductions on mRNA synthesis, despite side effects which include inhibition of mRNA transport (Dolecki et al., 1979) and stabilization of message (Lodish, 1976). The validity of this approach was tested directly by Thomas and Thomas (1986). Of the 20 serum-stimulated proteins detected on 2-D gels, they found that 10 were completely inhibited by actinomycin D, while expression of the remaining 10 was unaffected. Translational regulation of this latter group was confirmed by in vitro translation of mRNA isolated from treated and untreated cells. They also found that all of the translationally regulated proteins had been correctly identified by actinomycin D treatment. Therefore, it appears that the enhanced synthesis of EF-2 induced by insulin apparently occurred through the increased translation of stable, previously synthesized EF-2 mRNA.
Translational regulation of EF-2 is not a unique response of these cells to insulin treatment; on the contrary, we found that approximately 75% of the early insulin-induced increases in the synthetic rates of specific proteins were resistant to inhibition by actinomycin D.' That insulin can act though stimulation of mRNA translation has been suggested by previous studies. For example, insulin was shown to induce the synthesis of several major proteins in pancreatic acini exclusively through translational regulation without affecting levels of the corresponding mRNAs (Okabayashi et aL, 1987). Nevertheless, it should be appreciated that only a small fraction of previously synthesized mRNA is susceptible to insulinsensitive specific translational regulation, because less than 1% (approximately 20 out of 2500) of the proteins visible on giant 2-D gels was specifically induced in an actinomycin Dresistant manner after insulin treatment. The mechanisms responsible for preferential translation of specific mRNAs are still obscure and cannot be readily accounted for by alterations in the phosphorylation status of ribosomal protein S6 or of eIF-2a; it is likely that these involve specific structural features of the responsive mRNAs. To our knowledge, such regions have not been characterized for the mRNA encoding EF-2, but recent studies on iron-regulated translation of ferritin heavy chain mRNA (Leibold and Munro, 1988;Rouault et al., 1988) have begun to shed some light in this area.
We found that actinomycin D acted synergistically with insulin to elevate EF-2 synthesis to levels more than 400% higher than that found in control cells. This stimulatory effect of actinomycin D was also noted for a number of other insulinstimulated proteins (data not shown). How actinomycin D increases translation of previously synthesized messages is not clear, although previous examples of this "superinduction" phenomenon have been described by others (Panuska et a[. Unanue and Kiely, 1977). It is possible that actinomycin D blocks the synthesis of mRNA coding for one or more labile translation regulatory factors. Alternatively, an actinomycin D-sensitive factor may be an RNA species that interacts with ribosomal components or directly with mRNA to modulate translation of specific messages, as has been observed in bacterial plasmids by Gerdes et al. (1988). However, to our knowledge, no mammalian counterpart of this phenomenon has yet been described.
Even in the basal, unstimulated state, EF-2 is an abundant cellular protein, one of the 20 most abundant proteins seen on our Coomassie Blue-stained 2-D gels. Consequently, the insulin-sensitive increase in its rate of synthesis that we describe here is unlikely to affect total protein synthesis rates at times shortly after hormone stimulation. EF-2-dependent regulation of protein synthesis is more likely exerted through phosphorylation-dephosphorylation reactions, because it has been shown that only the dephosphorylated form of EF-2 can catalyze elongation in rabbit reticulocyte translation systems (Nairn et al., 1987;Sitikov et al., 1988). However, it is possible that the insulin-stimulated induction of EF-2 may be part of a long term adaptation by the cell to permit sustained increased protein synthesis levels. In keeping with this speculation, we found that insulin-treated HIR 3.5 cells incubated in serum-free medium expressed 2.6-fold more immunoreactive EF-2 than controls after 12 h. Others have shown that EF-2 accumulates in mouse skin treated for 17 h with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (Gschwendt et al., 1987). Moreover, Thomas and Thomas (1986) have shown increased synthesis of elongation factor 1 (a species too basic to be resolved on our gel system) in serumtreated fibroblasts and Duncan and Hershey (1985) have documented preferential synthesis of initiation factors eIF-3p24, eIF-3p44, and eIF-4A during recovery from serum deprivation (Duncan and Hershey, 1985). Thus, cellular accumulation of EF-2 in response to insulin may be part of a long term accommodation to an anabolic state characterized by increased protein synthesis.
It remains possible that insulin-induced synthesis of EF-2 may have functional effects other than accumulation of the protein. We have shown that post-translational processing of recently synthesized EF-2 occurs (see Fig. 5). Apparently, the protein is synthesized as two nascent isoforms that are rapidly transformed into two corresponding mature isoforms (isoforms within each pair differ from each other only with respect to their phosphorylation status). The maturation of the EF-2 molecule, which involves no change in apparent molecular weight, is accompanied by the loss of one negative charge (see "Results"). The nature of this processing step is not obvious because most post-translational modifications (e.g. phosphorylation, glycosylation, sulfation, acylation, ADP-ribosylation, methylation, proteolysis) involve change in apparent M , on gels or the addition of groups that confer additional negative (acidic) charges or both. It is known that EF-2 i s posttranslationally modified by a conversion of a histidine residue to a unique amino acid derivative, diphthamide, which becomes the site for ADP-ribosylation of this molecule (Kohno et al., 1986). This is accomplished by addition of a modifying side chain derived from methionine, followed by the addition of three methyl groups and finally the addition of an amide group (Chen and Bodley, 1988). Interestingly, this last step in the conversion of histidine to diphthamide involves the amidation of a carboxyl group on the "diphthine" intermedi-ate, resulting in the loss of a single negative ~h a r g e .~ We propose that forms 2 and 4 represent the unamidated precursors of the (phosphorylated and unphosphorylated) mature diphthamide-containing EF-2. It is conceivable that these relatively transient unamidated EF-2 precursors may have functions or substrate specificities that differ from those of the mature EF-2 molecules. It is therefore possible that insulin could alter the specificity of translationally regulated protein synthesis by increasing the proportion of these transient intermediates in the population of EF-2 molecules.