Oral Administration of Leucine Stimulates Ribosomal Protein Mrna Translation but Not Global Rates of Protein Synthesis in the Liver of Rats1 Tracy G

Biochemical and Molecular Action of Nutrients

Oral Administration of Leucine Stimulates Ribosomal Protein mRNA Translation but Not Global Rates of Protein Synthesis in the Liver of Rats1 Tracy G. Anthony, Joshua C. Anthony, Fumiaki Yoshizawa, Scot R. Kimball and Leonard S. Jefferson2 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033 Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021

ABSTRACT The objective of the current study was to examine the role of the branched-chain amino acid (BCAA) leucine in the regulation of hepatic protein synthesis and ribosomal protein (rp) mRNA translation in vivo. Food-deprived (18 h) male rats (200 g) were orally administered saline (control) or 270 mg leucine, isoleucine or valine and killed 1 h later. Administration of any BCAA resulted in enhanced phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) compared with controls. However, leucine was the most effective at stimulating phosphorylation of 4E-BP1 as well as the 70-kDa ribosomal protein S6 kinase (S6K1). Despite these effects on components of the translation initiation process, there were no differences in total protein synthesis rates among treatment groups. The distribution of rp (S4, S8, L26) and non-rp (albumin, ␤-actin) mRNAs across sucrose density gradients showed that the preponderance of hepatic rp mRNAs in control rats was unloaded from polysomes. Of the BCAA, only leucine was the most effective in causing a shift in the distribution of rp mRNA to polysomes compared with controls. Non-rp transcripts remained mainly polysome-associated under all conditions. These results suggest that leucine is most effective among the BCAA in its ability to stimulate translation of rp mRNA in liver. Furthermore, the translation of rp mRNA is disjointed from rates of total protein synthesis in liver and related to the degree of S6K1 phosphorylation. J. Nutr. 131: 1171–1176, 2001.

KEY WORDS: ● leucine ● liver ● ribosomal protein mRNA translation ● rats

Rapid control of protein synthesis by nutritional, hormonal translation of all mRNAs in the cell, although not necessarily and other environmental stimuli is attained at the level of to the same extent. The translation of a single or subset of translation initiation, a process that culminates in the assem- mRNAs can be discordant with overall rates of protein syn- bly of a competent 80S ribosome attached to mRNA. One thesis. One class of mRNAs that demonstrate specific regula- rate-controlling step in translation initiation involves the tion includes the ribosomal proteins (rp), a family of 80 binding of mRNA to the 40S ribosome [reviewed in (1)]. This members that associate in equimolar amounts with the ribo- step requires a multisubunit complex, referred to as eukaryotic somal RNAs to form a mature 80S ribosome. Mammalian rp initiation factor (eIF)3 4F. Assembly of the eIF4F complex is mRNAs are characterized structurally by the presence of a regulated by the phosphorylation state of the translational Terminal OligoPyrimidine tract (TOP) at the 5Ј-end of the repressor, eIF4E-binding protein 1 (4E-BP1). 4E-BP1 prevents transcript, consisting of a C residue adjacent to the m7GTP formation of the eIF4F complex by sequestering the mRNA cap site, followed by an uninterrupted sequence of 4–20 pyri- cap binding protein, eIF4E, into an inactive complex under midines (2). Genes that contain a TOP tract in their 5Ј conditions of hypophosphorylation. Hyperphosphorylation of untranslated region code for proteins involved in the produc- 4E-BP1 allows elF4E to bind elF4G, a large protein that serves tion and function of the translational apparatus (3). as a scaffold for elF4F complex assembly. Apportionment of rp mRNAs across polysome profiles sug- Alterations in the rate of protein synthesis reflect the gests two discrete populations, i.e., translationally active (and thus, polysome associated) under growth conditions and translationally inactive (and thus, resident in subpolysomal fractions) under conditions of growth inhibition (4). Recruitment 1 Supported by research grants DK13499 (L.S.J.), GM39277 (T.C. Vary) and GM08619 (J.C.A.) from the National Institutes of Health. T.G.A. is supported by an of TOP mRNAs into polysomes is associated with hyperphos- American Diabetes Association Postdoctoral Fellowship (L.S.J.). phorylation of the 70-kDa ribosomal protein S6 kinase 2 To whom correspondence should be addressed. (S6K1). Phosphorylation of rp S6 by S6K1 is thought to E-mail: [email protected]. 3 Abbreviations used: BCAA, branched-chain amino acids; Con, food-de- facilitate the initiation process by enhancing the affinity of the prived rats; 4E-BP1, eIF4E-binding protein 1; eIF, eukaryotic initiation factor; Ile, ribosome for binding TOP mRNAs (5). In mitogen-stimulated food-deprived rats orally administered 270 mg isoleucine; Leu, food-deprived rats cells, the efficiency of translation of TOP mRNAs is mediated orally administered 270 mg leucine; rp, ribosomal protein; S6K1, 70-kDa ribosomal protein S6 kinase; TOP, terminal oligopyrimidine tract; Val, food-deprived by the activity of S6K1 (6). This effect is specific for TOP rats orally administered 270 mg valine. mRNAs because treatment of cells with rapamycin, an inhib-

1171 1172 ANTHONY ET AL. itor of S6K1 phosphorylation, selectively suppresses transla- 254 nm was recorded continuously and 5-mL fractions were collected tion of TOP mRNAs (7). for subsequent extraction of total RNA. Previous work in our laboratory demonstrated that oral Total RNA extraction from whole liver and sucrose gradients. administration of the branched-chain amino acid (BCAA) Total RNA from frozen liver samples was isolated using Tri Reagent leucine alone stimulates the synthesis of total mixed proteins (13) (Molecular Research Center, Cincinnati, OH). Sucrose fractions were acid phenol/chloroform extracted twice (1:1, v/v, sucrose/ in muscle concomitant with increased eIF4F assembly and acid phenol, pH 4.5, Ambion, Austin, TX) and ethanol precipitated S6K1 phosphorylation (8,9). However, it is not known overnight. The RNA concentration and purity of all samples were whether feeding leucine stimulates translation initiation and determined by measuring the UV absorbance at 260 nm and the ratio rates of protein synthesis in tissues other than skeletal muscle. of optical densitites 260 nm/280 nm, respectively. The purpose of this study was to investigate the role of leucine Preparation of RNA probes. The full-length cDNAs for ribo- in regulating total vs. speciﬁc protein synthesis in the liver. somal proteins S4, S8 and L26 were kindly provided by Dr. Ira Wool Particular emphasis was placed on the regulation of rp mRNA (Department of Biochemistry and Molecular Biology, University of translation and the role of S6K1 in mediating enhanced trans- Chicago, Chicago, IL). The plasmid pUC8 was digested with PstI to produce 250-, 212- and 185-bp fragments for L26, S4 and S8, respec-

lation of TOP-containing mRNAs in vivo. Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021 tively. The cDNA for albumin was in the pBR322 vector and the plasmid pAlb576 (14) was digested with EcoRV followed by AccI to produce a 280-bp albumin fragment. The four fragments were sub- MATERIALS AND METHODS cloned individually into the pBluescript II SK ϩ vector (Stratagene, La Jolla, CA) and transformed into bacteria (TOP10 One-Shot cells, Animals and experimental design. The animal facilities and Invitrogen, Carlsbad, CA). Colonies were isolated and DNA was protocol were reviewed and approved by the Institutional Animal purified (Wizard Plus Mini and Maxi-Preps DNA Purification System, Care and Use Committee of The Pennsylvania State University Promega, Madison, WI). Plasmid stocks of pBluescript-L26, pBlue- College of Medicine. Male Sprague-Dawley rats (ϳ200 g) were main- script-S4, pBluescript-S8 and pBluescript-Alb were then linearized tained on a 12-h light:dark cycle with food (Harlan-Teklad Rodent with EcoRI, BamHI, HindIII and AccI, respectively, to produce DNA Chow, Madison, WI) and water provided freely. Rats were deprived of food for 18 h and then randomly assigned to continue as food- template stocks of each probe for in vitro transcription. A linearized deprived (Con) or to receive one of three dietary treatments by oral ␤-actin DNA template (126 bp) was purchased from Ambion. One microgram of each DNA template was mixed with 25 ␮mol/L gavage as follows: L-leucine (Leu), L-isoleucine (Ile) or L-valine (Val). 32 The amount of each amino acid administered was 135 mg/100 g body, [ P]UTP (800 Ci/mmol, 30 TBq/mmol; Amersham) and limiting prepared as 54.0 g/L in distilled water. Food-deprived rats (Con) (0.1 mmol/L) unlabeled UTP in a 20-␮L transcription reaction received an equal volume of saline (0.155 mol/L) by oral gavage. according to the manufacturer’s instructions (MAXIscript In Vitro Sample collection. Exactly 1 h after oral gavage, rats were killed Transcription Kit, Ambion) to produce single-stranded RNA probes. by decapitation. Trunk blood was collected and centrifuged at 1,800 The resulting reactions were treated with DNase I, heat denatured ϫ g for 10 min to obtain serum. The whole liver was excised, blotted, and gel purified by loading onto a 5% acrylamide-8 mol/L urea weighed and divided into three parts. One portion of liver was mini-slab gel. After electrophoresis, the full-length probes were lo- weighed and homogenized in 3 volumes of Buffer A, consisting of (in cated by short-term (20 s) exposure of the gel to X-ray film. The mmol/L) 40 N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid full-length RNA probes were subsequently excised from the gel with (pH 7.5), 100 potassium chloride and 5 magnesium chloride. The a clean blade and incubated overnight in probe elution buffer (0.5 Buffer A homogenate was centrifuged at 3000 ϫ g for 15 min at 4°C mol/L ammonium acetate, 1 mmol/L EDTA, 0.2% SDS; RPA III, for subsequent polysome profile analysis, as described below. A second Ambion). The gel was briefly reexposed to X-ray film after excision to portion of liver was homogenized in 7 volumes of Buffer B, consisting ensure that the correct bands were isolated. of (in mmol/L) 20 N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic Ribonuclease protection assay. Total RNA (5 ␮g) isolated from acid (pH 7.4), 100 potassium chloride, 0.2 EDTA, 2 ethylene glycol- either whole liver or sucrose density gradient fractions was coprecipi- bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid, 1 dithiothrei- tated with L26, S4, S8 and ␤-actin RNA probes in a single tube. tol, 50 sodium fluoride, 50 ␤-glycerophosphate, 0.1 phenylmethylsul- Albumin mRNA expression was detected in a separate ribonuclease fonyl fluoride, 1 benzamidine and 0.5 sodium vanadate. An aliquot of protection assay. Samples were heat denatured and allowed to hy- the Buffer B homogenate was used for the measurement of protein bridize to the RNA probes overnight in a 42°C water bath in synthesis as described below. The remaining Buffer B homogenate hybridization buffer (RPA III, Ambion). Samples were digested with was immediately centrifuged at 10,000 ϫ g for 10 min at 4°C. The RNaseA/TI the next day, and the protected, double-stranded mRNA resulting supernatant was used to examine the phosphorylation of fragments were ethanol precipitated, heat denatured and loaded onto 4E-BP1, S6K1 and rpS6 as described below. The remaining portion of 5% acrylamide-8 mol/L urea slab gels. After electrophoresis, gels were liver was quickly frozen in liquid nitrogen for the subsequent isolation wrapped in plastic and exposed to X-ray film at Ϫ70°C for up to 4 h. of total RNA. All serum and tissue samples were stored at Ϫ70°C. Examination of 4E-BP1 phosphorylation state. An aliquot of Measurement of liver protein synthesis. A bolus (1.0 mL/100 g the 10,000 ϫ g supernatant (Buffer B) was boiled for 10 min and then 3 body) of L-[2,3,4,5,6- H] phenylalanine (150 mmol/L containing 3.70 centrifuged at 10,000 ϫ g for 30 min at 4°C. The resulting superna- GBq/L, Amersham, Piscataway, NJ) was injected via the tail vein 50 tant was mixed with an equal volume of 2X sample buffer and then min after oral gavage for the measurement of hepatic protein synthe- subjected to protein immunoblot analysis as described previously sis (10). The elapsed time from injection of the metabolic tracer until (15). homogenization of liver was recorded as the actual time for incorpo- Quantitation of eIF4G ⅐ eIF4E complexes. eIF4E was immuno- ration of radiolabeled amino acid into protein. Fractional rates of precipitated from 10,000 ϫ g (Buffer B) supernatants of liver homog- protein synthesis were estimated from the rate of incorporation of enates using a monoclonal antibody to eIF4E (15). Next, samples radioactive phenylalanine into liver protein, using the specific radio- were subjected to immunoblot analysis using a polyclonal antibody to activity of serum phenylalanine as an indication of the precursor pool eIF4G to determine the association of eIF4G with eIF4E (15). Results (11). The use of serum phenylalanine as the precursor pool was were normalized to the amount of eIF4E in the immunoprecipitates. validated (12). Phosphorylation of S6K1 and ribosomal protein S6. Phosphor- Polysome profiles. One volume of detergent (10% Triton X-100, ylation of S6K1 and rpS6 was determined in 10,000 ϫ g (Buffer B) 10% sodium deoxycholate) was mixed with nine volumes of post- supernatants by protein immunoblot analysis as previously described nuclear supernatant (Buffer A) and layered over 10–70% linear (16). Phosphorylation of S6K1 at Thr389, a site whose phosphory- sucrose density gradients. The gradients were centrifuged at 90,000 lation is associated with maximal activation of the kinase, was de- ϫ g for 210 min at 4°C in a Beckman SW28 rotor (Palo Alto, CA). termined using an anti-phospho-S6K1 (Thr389) antibody (Cell Sig- After centrifugation, the gradients were fractionated on an Isco naling Technology, Beverly, MA). The anti-rpS6 antibody was raised density gradient fractionator (Lincoln, NE). The UV absorption at against a phosphopeptide that included five phosphorylation sites in LEUCINE STIMULATES RIBOSOMAL PROTEIN mRNA TRANSLATION 1173

ined. In contrast to our previously published work in skeletal muscle (8,9,17), oral administration of leucine did not stimulate fractional rates of protein synthesis in the liver of food- deprived rats (Fig. 1). Isoleucine and valine were additionally without effect. Although BCAA administration did not stimulate fractional rates of protein synthesis in the liver, alterations in 4E-BP1 phosphorylation and eIF4F assembly were noted. 4E- BP1 resolves into three bands on SDS polyacrylamide gels, with the top band (␥-form) corresponding to the most highly phosphorylated species. Hyperphosphorylation of 4E-BP1 results in the release of the mRNA cap binding protein, eIF4E, FIGURE 1 Fractional rate of liver protein synthesis in rats deprived allowing it to participate in eIF4F assembly. All three BCAA of food for 18 h and then orally administered saline (Con) or 270 mg increased the phosphorylation of 4E-BP1 relative to food- Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021 leucine (Leu), isoleucine (Ile) or valine (Val). Measurements were made deprived controls, but the effect was graded, with leucine 1 h after oral gavage. Values are means Ϯ SEM, n ϭ 6. There were no Ͼ valine and isoleucine intermediary (Fig. 2A).To support the differences among treatment groups. concept that increases in 4E-BP1 phosphorylation promote eIF4F assembly, the association of eIF4G with eIF4E was examined. Oral leucine administration significantly enhanced the C-terminus of the protein (generously provided by Dr. M. Birn- the binding of eIF4G to eIF4F, whereas oral administration of baum, University of Pennsylvania). isoleucine and valine were not effective (Fig. 2B). Statistical analysis. All data were analyzed by the Statistica Similar to 4E-BP1, administration of any BCAA promoted statistical software package for the Macintosh, volume II (StatSoft, phosphorylation of S6K1 (Fig. 3A). S6K1 resolves on SDS Tulsa, OK). Data were analyzed using one-way ANOVA to assess polyacrylamide gels into multiple electrophoretic forms, with main effects, with treatment group as the independent variable. more slowly migrating species corresponding to more highly When a significant overall effect was detected, differences among phosphorylated forms of the protein. To determine whether individual means were assessed with Duncan’s Multiple Range post- the observed phosphorylation patterns corresponded to a po- hoc test. The level of significance was set at P Ͻ 0.05 for all statistical tests. tential change in activity, we examined phosphorylation of S6K1 at Thr389, a site that has been shown to be associated RESULTS with maximal activity of the protein when phosphorylated (18). Similar to 4E-BP1, phosphorylation at this site was In this investigation, the ability of the BCAA to stimulate graded, with leucine Ͼ isoleucine Ͼ valine compared with translation initiation and protein synthesis in liver was exam- controls (Fig. 3B). Finally, we examined the phosphorylation

FIGURE 2 Phosphorylation state of 4E-BP1 and the amount of elF4G associated with elF4E in the liver of food-deprived rats (Con) 1 h after oral administration of leucine (Leu), isoleucine (Ile), or valine (Val). (A) 4E-BP1 resolves into three bands on SDS polyacrylamide gels, with the top band (␥-band) corresponding to the most highly phosphorylated species. Hyperphosphorylation of 4E-BP1 results in the release of the mRNA cap binding protein, elF4E, allowing it to participate in elF4F assembly. Bar graph displays the amount of 4E-BP1 in the ␥-phosphorylated form, expressed as a proportion of the total 4E-BP1. Inset shows a representative immunoblot with positions of ␣-, ␤-, ␥-forms of 4E-BP1 noted to the right. (B) Amount of elF4G bound to elF4E. Inset shows a representative immunoblot with elF4G noted to the right. All data were normalized to the amount of elF4E in the immunoprecipitates. Values are means Ϯ SEM;nϭ 6. Means not sharing the same letter are different, P Ͻ 0.05. 1174 ANTHONY ET AL. of the ribosomal protein S6. Phosphorylation of rpS6 was signiﬁcantly increased only in rats administered leucine (Fig. 3C). These results suggest that among the BCAA, leucine is most effective at stimulating the activation of the S6K1 signaling pathway in liver. The translation of TOP-containing mRNAs (which in- clude genes encoding the ribosomal proteins) is demonstrated in cell culture systems to be related to the activation of the S6K1 signaling pathway. Therefore, we were interested in exploring the relationship between phosphorylation of S6K1 and rpS6 and the speciﬁc translation of rp mRNAs by BCAA administration. To address this, sucrose density gradients were fractionated into four equal parts. On the basis of the polysome

profiles obtained (Fig. 4), the first two fractions were desig- Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021 nated “subpolysomal.” These fractions contained protein, RNA, free ribosomal subunits and monosomes. The third and fourth fractions were then designated “polysomal.” These fractions contained disomes, trisomes and polysomes of increasing ribosome number bound to mRNA. The total RNA from each sucrose fraction was extracted and the content of both rp and non-rp mRNAs was determined in each fraction. The results of this analysis provide a relative measure of how actively each message was being translated under the experimental conditions. The distribution of three TOP-containing messages, rpL26, rpS4 and rpS8, was compared with that of albumin and ␤-actin (both non-TOP mRNAs) (Fig. 4). Total mRNA expression of all proteins examined was not altered 1 h after BCAA administration in whole liver (Fig. 5). In all treatment groups, the ␤-actin gene was constitutively FIGURE 4 Distribution of hepatic ribosomal protein (rp)L26, rpS4, rpS8, ␤-actin and albumin mRNAs across polysome profiles in food- deprived rats (Control) orally administered leucine (Leucine), isoleucine (Isoleucine) or valine (Valine). Left: Representative profiles obtained by sucrose density gradient fractionation. Nine volumes of liver homogenate were mixed with one volume of detergent and loaded onto linear 10–70% sucrose gradients as described in Materials and Methods. The gradients were centrifuged for 210 min at 4°C and subsequently fractionated on an Isco gradient fractionator. The UV absorption at 254 nm was continuously recorded. Four sucrose fractions of equal volume (shown as 1–4) were collected for the subsequent extraction of total RNA. Right: Representative autoradiograms showing the distribution of hepatic rpL26, rpS4, rpS8, ␤-actin and albumin transcripts across polysome profiles as determined by ribonuclease protection assay. Arrows to the right indicate each specific transcript. Data represent 3–6 rats per treatment group.

present in the polysome fractions (Fig. 4 and Table 1). The albumin gene demonstrated slight-to-moderate movement among fractions when food deprivation was compared with BCAA administration, but this difference was not significant (ANOVA, P Ͻ 0.06). The tendency for albumin mRNA to shift into polysomes with BCAA administration agrees with a previous study by Yap et al. (19) showing that feeding a single or mixture of amino acids to food-deprived rats increases the proportion of albumin mRNA resident in free and membrane- bound polysomes. In any case, the majority (61–81%) of albumin mRNA remained associated with heavy polysomes in all treatment groups (Fig. 4 and Table 1). In contrast to these FIGURE 3 Phosphorylation of the 70-kDa ribosomal protein S6 non-TOP transcripts, most (70–75%) of the mRNAs encod- kinase (S6K1) and of the ribosomal protein S6 (rpS6) in the liver of ing the ribosomal proteins were unloaded from polysomes in food-deprived rats (Con) orally administered leucine (Leu), isoleucine food-deprived controls, residing in the subpolysomal fractions (Ile) or valine (Val). (A) Arrows indicate multiple electrophoretic forms of S6K1, with the most highly phosphorylated species exhibiting the slow- (Fig. 4 and Table 1). Oral administration of isoleucine or est electrophoretic mobility. (B) Phosphorylation of S6K1 on Thr389, a valine did not significantly shift the proportion of these residue whose phosphorylation is associated with maximal activation mRNAs to the polysome fractions compared with controls of the protein. (C) Phosphorylation of rpS6. Data shown represent 6 rats (Fig. 4 and Table 1). In contrast, oral administration of leucine per treatment group. promoted significant redistribution of ribosomal protein LEUCINE STIMULATES RIBOSOMAL PROTEIN mRNA TRANSLATION 1175

(referred to as TOP) (2). The relationship between S6K1 activation and the selective translation of TOP mRNA was established using the immunosuppressant drug rapamycin, an inhibitor of S6K1 phosphorylation and activation (7,25). Treatment of serum-stimulated NIH 3T3 cells with rapamycin blocks S6K1 phosphorylation and recruitment of rp mRNA into polysomes (7). These inhibitory effects are nulliﬁed upon conversion of the principal rapamycin-sensitive phosphorylation site on S6K1 to a residue refractory to the macrolide (7). Additionally, disruption of the 5ЈTOP sequence also prevents the inhibitory effects of rapamycin on rp mRNA translation, highlighting the importance of the cis-regulatory element in the selective control of this class of genes.

In this study, we examined the relationship between Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021 BCAA-induced activation of the S6K1 signaling pathway and the selective movement of rp mRNAs into polysomes. S6K1 activation has been linked to leucine availability in hepatoma FIGURE 5 Total mRNA expression of ribosomal protein (rp)L26, cells and perfused liver (20, 26), and the connection between rpS4, rpS8, ␤-actin and albumin in the liver of food-deprived rats (Con) S6K1, rpS6 and the translation of TOP mRNAs has been orally administered leucine (Leu), isoleucine (Ile) or valine (Val). There described (4). However, there are no reports testing the effects were no differences in hepatic abundance of each transcript among of amino acid availability on rp mRNA translation in vivo. treatment groups 1 h after meal administration. Data represent 4–6 rats Herein we show for the first time the unique role of leucine in per treatment group. stimulating redistribution of rp mRNAs into polysomes, sug- gesting increased translation of these proteins. This type of regulation may provide temporal control of gene expression mRNAs, more than doubling the proportion of transcripts under conditions of altered growth rate. existing in the polysome fractions (Fig. 4 and Table 1). These In this study, leucine promoted the hyperphosphorylation data suggest that among the BCAA, leucine alone favors of 4E-BP1 and enhanced eIF4F assembly, implying increased increased translation of hepatic ribosomal proteins. This effect translation initiation in the liver. Studies in perfused rat liver appears to relate to the degree of S6K1 phosphorylation in similarly demonstrate leucine alone to enhance eIF4F assem- vivo. bly without altering the synthesis of total mixed protein (20). This apparent paradox may be resolved by considering that the DISCUSSION translation of mRNAs with highly structured 5Ј-untranslated regions may have been selectively upregulated under the ex- Liver growth and protein mass are keenly sensitive to perimental conditions and that the proportion of total mRNA dietary amino acid supply. Increases in amino acid availability, represented by this subset of mRNAs is low. Changes in eIF4E engendered either by feeding a protein-containing meal or via availability are proposed to specifically modulate the synthesis balanced hyperaminoacidemia in a perfused liver system, give of a family of mRNAs that possess significant secondary struc- rise to elevations in hepatic protein biosynthesis (20,21). On ture in their 5Ј-untranslated regions (23,27). Although exam- the other hand, essential amino acid deprivation or imbalance causes disaggregation of polysomes and represses global rates of protein synthesis in the liver (20,22). TABLE 1 Beyond their use as substrates, the branched-chain amino Proportion of hepatic mRNA encoding rpL26, rpS4, rpS8, acids, and in particular leucine, function as signaling mole- cules to promote the initiation of mRNA translation in skel- albumin and ␤-actin that resides in polysomes in etal muscle (8,9,23). Contrary to our published findings in food-deprived rats orally administered leucine, skeletal muscle, oral administration of leucine, isoleucine or isoleucine or valine1,2 valine did not enhance the synthesis of total liver protein. Treatment Supporting these findings, perfusion of rat liver with leucine 3 alone at a concentration 4 times that reported in plasma of group rpL26 rpS4 rpS8 ␤-Actin Albumin food-deprived rats resulted in liver protein synthesis rates that % were not different from those of rats perfused with medium containing all amino acids at 1X concentrations (20). Addi- Control 25 Ϯ 5b 23 Ϯ 3b 30 Ϯ 4b 100 Ϯ 061Ϯ 6 tionally, in the only published report examining the short- Leucine 58 Ϯ 5a 50 Ϯ 4a 62 Ϯ 10a 100 Ϯ 075Ϯ 4 term effect of force-feeding a single BCAA on liver polysome Isoleucine 39 Ϯ 8ab 27 Ϯ 5b 43 Ϯ 11ab 100 Ϯ 081Ϯ 1 profiles and protein synthesis, isoleucine did not stimulate Valine 31 Ϯ 4b 25 Ϯ 2b 35 Ϯ 5ab 100 Ϯ 078Ϯ 5 polysome aggregation or liver protein synthesis in rats (24). 1 The proportion of mRNAs present in polysomes was determined Collectively, the data indicate that the BCAA by themselves by measuring the signal intensities of each mRNA band across all four are not sufficient to stimulate synthesis of total liver protein. gradient fractions and then expressing the signal present in the poly- Studies in cells and in vivo demonstrate that amino acids some fractions (fractions 3 and 4) as a percentage of the total. Data stimulate protein synthesis congruous with the hyperphospho- expressed as mean Ϯ SEM; n ϭ 3–6. Means in a column not sharing a rylation of 4E-BP1 and S6K1, two proteins important in the letter are different, P Ͻ 0.05. 2 selective control of mRNA translation (8,17,21,23). Hyper- All measurements made 1 h after meal administration. 3 Groups: Control, 18 h food-deprived rats administered a bolus of phosphorylation of S6K1 appears to augment the translation of saline by oral gavage; Leucine, food-deprived rats administered a specific class of genes characterized by the presence of an leucine; Isoleucine, food-deprived rats administered isoleucine; Valine, oligopyrimidine tract at the immediate 5Ј end of the transcript food-deprived rats administered valine. 1176 ANTHONY ET AL. ples of this class of genes (such as ornithine decarboxylase) skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J. Nutr. 130: 139–145. were not examined in the current investigation, on the basis of 9. Anthony, J. C., Yoshizawa, F., Gautsch Anthony, T., Vary, T. C., Jefferson, our eIF4F assembly results, it is likely that leucine would have L. S. & Kimball, S. R. (2000) Leucine stimulates translation initiation in skeletal affected the translation of this class of genes to some degree. muscle of post-absorptive rats via a rapamycin-sensitive pathway. J. Nutr. 130: On the other hand, TOP mRNAs do not contain highly 2413–2419. 10. Garlick, P. J., McNurlan, M. A. & Preedy, V. R. (1980) A rapid and structured 5Ј-untranslated regions (2), and therefore, are un- convenient technique for measuring the rate of protein synthesis in tissues by likely candidates for direct regulation via 4E-BP1 hyperphos- injection of [3H] phenylalanine. Biochem. J. 192: 719–723. phorylation. Studies using S6K1 mutants resistant to rapamy- 11. Kimball, S. R., Vary, T. C. & Jefferson, L. S. (1992) Age-dependent decrease in the amount of eukaryotic initiation factor 2 in various tissues. Bio- cin demonstrate the drug to repress 4E-BP1 phosphorylation chem. J. 286: 263–268. despite continued S6K1 activation and rp mRNA translation 12. Davis, T. A., Fiorotto, M. L., Nguyen, H. V. & Burrin, D. G. (1999) (7,28). Therefore, it is unlikely that redistribution of rp Aminoacyl-tRNA and tissue free amino acid pools are equilibrated after a flooding mRNAs in the current study is directly linked to 4E-BP1 dose of phenylalanine. Am. J. Physiol. 277: E103–E109. 13. Chomczynski, P. (1993) A reagent for the single-step simultaneous phosphorylation or eIF4F assembly. isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15: 532–537. In summary, we report the unique action of leucine to Downloaded from https://academic.oup.com/jn/article/131/4/1171/4687011 by guest on 28 September 2021 promote the movement of hepatic ribosomal protein mRNAs 14. Kimball, S. R., Horetsky, R. L. & Jefferson, L. S. (1995) Hormonal regulation of albumin gene expression in primary cultures of rat hepatocytes. into polysomes. This event is disjointed from overall rates of Am. J. Physiol. 268: E6–E14. liver protein synthesis and is associated with increased phos- 15. Kimball, S. R., Jurasinski, C. V., Lawrence, J. C. & Jefferson, L. S. (1997) phorylation of S6K1 and rpS6. These results suggest that Insulin stimulates protein synthesis in skeletal muscle by enhancing the associ- leucine is both necessary and sufficient as a signaling molecule ation of eIF4E and eIF4G. Am. J. Physiol. 272: C754–C759. 16. Gautsch, T. A., Anthony, J. C., Kimball, S. R., Paul, G. L., Layman, D. K. to promote ribosomal protein mRNA translation in the liver & Jefferson, L. S. (1998) Availability of eIF4E regulates skeletal muscle protein via the S6K1 signaling pathway. On the other hand, increases synthesis during recovery from exercise. Am. J. Physiol. 43: C406–C414. in the synthesis of total liver proteins require the activation of 17. Anthony, J. C., Gautsch Anthony, T. & Layman, D. K. (1999) Leucine supplementation enhances skeletal muscle recovery in rats following exercise. J. other signaling pathways that may be contingent on other Nutr. 129: 1102–1106. factors, such as a balanced supply of amino acids. 18. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H. & Sabatini, D. M. (1998) Raft1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. U.S.A. 95: 1432–1437. ACKNOWLEDGMENTS 19. Yap, S. H., Stair, R. K., & Shafritz, D. A. (1978) Identification of albumin mRNPs in the cytosol of fasting rat liver and influence of tryptophan or a mixture The authors are grateful to Sharon Rannels for her superior of amino acids. Biochem. Biophys. Res. Commun. 83; 427–433. technical assistance and to Tom Vary for determining the specific 20. Shah, O. J., Antonetti, D. A., Kimball, S. R. & Jefferson, L. S. (1999) radioactivity of serum phenylalanine. Leucine, glutamine, and tyrosine reciprocally modulate the translation initiation factors eIF4F and eIF2B in perfused rat liver. J. Biol. Chem. 274: 36168–36175. 21. Yoshizawa, F., Kimball, S. R., Vary, T. C. & Jefferson, L. S. (1998) LITERATURE CITED Effect of dietary protein on translation initiation in rat skeletal muscle and liver. 1. Pain, V. M. (1996) Initiation of protein synthesis in eukaryotic cells. Eur. Am. J. Physiol. 38: E814–E820. J. Biochem. 236: 747–771. 22. Pronczuk, A., Rogers, Q. & Munro, H. (1970) Liver polysome patterns 2. Amaldi, F. & Pierandrei-Amaldi, P. (1997) TOP genes: a translationally of rats fed amino acid imbalanced diets. J. Nutr. 100: 1249–1258. controlled class of genes including those coding for ribosomal proteins. Prog. 23. Kimball, S. R., Shantz, L.M., Horetsky, R. L. & Jefferson, L. S. (1999) Mol. Subcell. Biol. 18: 1–17. Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR- 3. Levy, S., Avni, D., Hariharan, N., Perry, R. 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