Reevaluation of amino acid stimulation of protein synthesis in murine- and human-derived skeletal muscle cells assessed by independent techniques

Britt-Marie Iresjö, Elisabeth Svanberg, and Kent Lundholm

Department of Surgery, Surgical Metabolic Research Laboratory at Lunderberg Laboratory for Cancer Research, Sahlgrenska University Hospital, Goteborg, Sweden

Submitted 12 July 2004 ; accepted in final form 9 December 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 µM/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. "Flooding" tracee experiments did not overcome this problem.

amino acids; insulin; initiation of translation; protein synthesis; muscle cells


AROUND 45% OF THE BODY WEIGHT in adult humans is skeletal muscles, an important nitrogen reserve in different stress conditions such as trauma, infection, and starvation with balanced regulation of muscle protein synthesis and degradation to maintain muscle mass at functional levels (51). Feeding stimulates synthesis, whereas acute and chronic starvation will increase and decrease degradation, respectively (35). Mechanisms behind controlled protein balance in skeletal muscles over time have been extensively described in humans and animals based on studies in a variety of models from subcellular to cellular and tissue to organ levels (51). Yet, integrated signals behind protein balance in skeletal muscles are not fully understood, although several studies suggest amino acids in combination with hormones (insulin, IGF-I, and growth hormone) as key factors (1) communicated by intracellular phosphoproteins (22, 25, 38).

Our own investigations have indicated rapid activation of protein synthesis in skeletal muscles during refeeding, a phenomenon that was independent of both muscle innervation and circulating insulin (47). In contrast, intravenous infusion by stepwise increased loads of amino acids to unselected patients and healthy volunteers indicated that amino acids themselves are involved in the process of initiation of translation (29), whereas carbohydrates and fat entirely lacked such effects (45). Also, there was no indication that glutamine alone had stimulatory effects in vivo in human muscles (46), while we observed that branched-chain amino acids stimulated human muscle protein synthesis, whereas insulin lacked this effect at physiological concentrations (32). However, effects of amino acids to stimulate global protein synthesis in muscle cells and tissue have been reported in numerous publications (14, 16, 40, 52) and in addition to more recent studies on relationships between amino acid kinetics and activation of phosphoproteins for initiation of translation of protein synthesis in response to amino acids and insulin (19, 26). Therefore, it may seem needless to add more knowledge to this body of information, but a number of these reports were rather designed to show a requirement of omitted individual amino acids to support global protein synthesis than reactivation of translation (23). Besides, we and others have reported that experiments with traditional labeling of proteins in assessment of global protein synthesis at steady-state conditions may give incorrect quantitative estimates, even in the presence of flooding tracee conditions (10, 33). Therefore, the aim of the present study was to reevaluate semiquantitative effects by amino acids to activate protein synthesis by comparing results from entirely independent methods for assessment of net global protein synthesis in murine- and human-derived muscle cells, where tracer distribution is not limited.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Animal Refeeding Experiments

Adult weight-stable female C5Bl mice (B & K Universal, Stockholm, Sweden) were used for in vivo experiments. All animals were kept on a diurnal 12-h light cycle and were provided with tap water and standard rodent chow ad libitum (B & K Universal) 2 wk before experimentation. Protein synthesis was assessed in mixed hindlimb muscles from a group of freely fed mice and compared with mice subjected to an overnight fast (18-h-starved group) as described (47). Additional mice were subjected to overnight starvation and a subsequent period of 3-h refeeding (refed groups), after which initiation of muscle protein synthesis was measured. Refeeding was performed with standard rodent chow and pellets. Food intake was recorded by weighing the animals before and after refeeding and inspection and weighing of food content within the stomach after the animals had been killed as described (47).

Confluent Cell Cultures

L6 cells from rat skeletal muscles were used in all experiments. It is a myoblast cell line originally isolated by Yaffe (53), from primary cultures of rat quadriceps muscle. The L6 cell exhibits many skeletal muscle characteristics seen in vivo, like the ability to differentiate into myotubes, spontaneous contractility, resting membrane potential around –70 mV, active lactate transport, synthesis of actinomyosin and creatine phosphokinase. The cells may also display high intracellular glucose levels, which is in contrast to adult skeletal muscles (39).

Cells were seeded for outgrowth in standard Dulbecco's modified Eagle’s medium (DMEM) with 4.5 g/l glucose supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and L-glutamine, and other amino acids, as shown in Table 1. Medium was changed to standard DMEM with 2% FCS for maintenance and concentrations of all amino acids at 9 mM. L6 cultures became confluent after 3–4 days in standard DMEM, and cells appeared to differentiate spontaneously into myotubes after confluence. Cells were always kept in an incubator with an environment of 95% air-5% CO2 during all experiments. RD cells (human rhabdomyosarcoma; American Type Culture Collection, LGC Promochem) were cultured and treated in the same way as murine L6 cells in all experiments, which were primarily carried out on L6 cells and in part confirmed in RD cells.


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Table 1. Amino acid concentrations in DMEM (essential AA) and nonessential AA medium

 
Starvation-Refeeding Experiments on Cell Cultures

Confluent cells were transferred for maintenance growth in "starvation medium," which was standard DMEM with all amino acids at low final concentration (0.14 mM) without FCS. Later (20 h), culture medium was changed to "refeeding" medium consisting of DMEM with all amino acids at either low (0.28 mM) or normal (9 mM) concentrations, as specified in the legends for Figs. 112. In some refeeding experiments, medium contained either low or normal amounts of amino acids with additions of insulin at physiological (100 µU/ml) or pharmacological (3 mU/ml) final concentrations specified in the legends for Figs. 112.



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Fig. 1. Incorporation rate of tyrosine (A) and phenylalanine (B) into cellular proteins. L6 cells were "starved" for 24 h in medium with low amino acid concentration (142 µM) and "refed" for 16 h in medium with either essential (Ess) or nonessential (Noness) amino acids (AA) at increasing concentrations. The essential amino acids were present at "starvation" concentrations (142 µM) in the "nonessential medium." Final concentrations of amino acid in the medium are indicated (µM). Nos. in parentheses are concentrations of the tracee (Tyr, Phe). The specific radioactivity was 6.2 µCi/nmol in all experiments.

 


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Fig. 12. Western blots of initiation factors. Confluent L6 cells were starved in medium with low amino acid concentration (0.14 mM) for 24 h and refed for 16 h in medium with low (0.28 mM) or normal (9 mM) amino acid concentration. The results confirm incorporation results in Fig. 11.

 
Estimates of Protein Synthesis

Incorporation of radioactive amino acids. Cells were seeded in 96-well plates (5 x 104 cells/well), grown to confluence, and "starved" as described above with a subsequent switch to refeeding medium containing either L-[U-14C]tyrosine, L-[U-14C]phenylalanine, or L-[U-14C]arginine at specific radioactivities of 160 µCi/nmol. Amino acids in the medium were either increased in multiples to create a concentration gradient among different wells or kept constant. The specific radioactivity was always kept constant among vials. "Essential and nonessential" amino acids refer to cell culture conditions as specified in Table 1. All other components in the medium were kept constant, corresponding to levels in standard DMEM. After incubation, the medium was removed, and the cells were loosened with trypsin-EDTA solution and harvested on glass-fiber filters in a cell harvester (Skatron). Filters with cells were rinsed free of soluble radioactivity and dried at 60°C for 30 min. Radioactivity was determined in a {beta}-scintillation counter (Wallac 1409). Preincubation of cell cultures in the presence of puromycin or cycloheximide completely inhibited (>97%) the appearance of labeled amino acids in proteins and cell components. Control experiments comparing the preparation of labeled cells either bound to filter membranes or homogenization of cells followed by lipid extraction and solubilization of denatured proteins (TCA) for scintillation counting of radioactivity gave the same results of incorporation rate. These control experiments validate our biochemical methods for quantification of amino acid incorporation into global proteins isolated by the filter technique. Incorporation rate (R) of amino acids (Phe, Tyr, and Arg) into protein was calculated as

where dpm/µg is the specific radioactivity of the labeled amino acid in proteins at the end of incorporation, and dpm/nmol is the constant specific radioactivity of the labeled amino acid in the incubation medium throughout incubations, as checked by isolation of the medium amino acids by HPLC chromatography with determination of specific radioactivity of the pure amino acids (Phe, Tyr, and Arg).

Autoradiogram. Cells were seeded in 25-cm2 flasks, grown to confluence, and treated with "starvation" medium as described. L-[35S]methionine (10 µCi/ml) was added to the refeeding medium. The specific radioactivity of methionine was kept constant among various vials for 16 h. Cells were detached by trypsination at the end of the incubation, diluted in medium, and centrifuged 1,200 g for 10 min. Pelleted cells were dissolved in 300 µl of 10 mM Tris·HCl buffer, pH 7.4, with 10 µl 10% Triton X-100. The samples were kept on ice for 30 min and centrifuged for 2 min at 10,000 g. Protein content from lysed cells was determined in the supernatants (Coomassie Plus; Pierce). An equal amount of protein was subjected to electrophoresis on a precast Nupage minigel with the 2-(N-morpholino)ethanesulfonic acid buffer system (Invitrogen) and was transferred to a 0.2-µm polyvinylidene difluoride (PVDF) membrane (Bio-Rad), which was exposed against film (Hyperfilm MP; Amersham).

Ribosome profiles. Cells were seeded in 75-cm2 cell culture flasks, grown to confluence, and then treated with starvation/refeeding medium with unlabeled amino acids as described above. Cells were loosened by trypsination and diluted in medium, and cell concentrations were adjusted to 1 x 107 cells /400 µl. Triton X-100 (10%) was added (10 µl/300 µl cell suspension), and the cells were left on ice for 10 min and then centrifuged at 8,500 g for 10 min. Supernatant (400 µl) was layered on top of a sucrose gradient made as described elsewhere (37). Gradients were centrifuged in a Beckman sw-41 rotor for 195 min at 40,000 g. The test tubes were punctured in the bottom with a Beckman Fraction recovery system, the gradients were pumped trough a flow cell detector at 254 nm (Bio-Rad), and the absorbance was registered. The detector was connected to a computer with a program for area integration (Millenium 32 Waters). The area under the absorbance curve was registered. The relative distribution of polysomes to monosomes and subunits was calculated as the area under the polysome peak divided by the total area for RNA absorbance.

Initiation factors of protein synthesis.
ANALYSIS OF EIF4E·4E-BP1 COMPLEX. Cells were grown to confluence on 10-cm petri dishes and treated with "starvation/refeeding" medium as described above to initiate protein synthesis. Culture medium was removed, and cells were scraped for homogenization into ice-cold buffer A (20 mM HEPES, pH 7.4, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 50 mM {beta}-glycerophosphate, 0.1 mM AEBSF, 1 mM benzamidine, 0.5 mM sodium vanadate, 2.5% Triton X-100, and 0.25% deoxycholate). The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and aliquots of the supernatant were immunoprecipitated using a monoclonal 4E antibody (kindly provided by Dr. S. R. Kimball, Pennsylvania State University, College of Medicine, Hershey, PA). The antibody-antigen complex was collected by incubation for 1 h with goat anti-mouse Biomag IgG beads (Perseptive Diagnostics). Before use, the beads were washed in 1% nonfat dry milk in buffer B (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% {beta}-mercaptoethanol, and 1% Triton X-100). The beads were captured using a magnetic stand and washed two times in buffer B and one time in buffer C (50 mM Tris·HCl, 500 mM NaCl, 5 mM EDTA, 0.04% {beta}-mercaptoethanol, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS). Protein bound to the beads was eluted by resuspending the beads in SDS sample buffer and boiling for 5 min. The beads were collected by centrifugation, and the supernatants were subjected to electrophoresis on a 15% polyacrylamide gel. Proteins were transferred to a PVDF membrane, and the membranes were incubated with a rabbit anti-rat 4E-BP1 antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were then developed using an enhanced chemiluminescence Western Blotting Kit according to the manufacturers description (Amersham Pharmacia).


DETERMINATION OF 4E-BP1 PHOSPHORYLATION STATE. Aliquots of the supernatant were combined with an equal amount of SDS sample buffer and then subjected to electrophoresis on a 15% polyacrylamide gel. The proteins were transferred to a PVDF membrane and treated as described above.


DETERMINATION OF P70S6K PHOSPHORYLATION STATE. Aliquots of the supernatant were combined with an equal amount of SDS sample buffer and then subjected to electrophoresis on a 7.5% polyacrylamide gel. The proteins were transferred to a PVDF membrane and treated as described above using a p70S6K antibody (Santa Cruz Biotechnology).

Statistics

Results are presented as means ± SE. Statistical comparisons among two and several groups were performed by ANOVA. P < 0.05 was considered statistically significant in two-tailed tests. Presented results are based on five to seven individual experiments for statistical evaluations.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Culture Experiments

Effects of amino acids on incorporation of labeled amino acids. Essential amino acids stimulated the incorporation rate of phenylalanine and tyrosine into cellular proteins to the same extent, whereas nonessential amino acids in the incubation medium lacked stimulation (Fig. 1A). Increased incorporation to cellular protein was also seen when the medium concentration of either tyrosine (6–405 µM) or phenylalanine (6–405 µM) was increased only in combination with constant specific radioactivity of L-[14C]tyrosine or L-[14C]phenylalanine, respectively (data not shown). However, increased concentration of tyrosine did not stimulate the incorporation rate of phenylalanine or vice versa. Also, a lack of stimulation of either tyrosine or phenylalanine incorporation rate by the essential amino acids was evident when tyrosine or phenylalanine was present at high medium concentration (405 µM) in a flooding-dose experiment (Fig. 1B). Similar results were seen when groups of amino acids (aromatics, branched-chain amino acids, sulfur-containing amino acids, Arg, Gln, plus others) were used to evaluate potential stimulation of tyrosine and arginine incorporation (Fig. 2, A and B). Thus stimulation of tyrosine, phenylalanine, and arginine incorporation rates was demonstrated by groups of amino acids that contained the tracer at increasing tracee concentration, i.e., the aromatics stimulated the incorporation of tyrosine but not of arginine and vice versa. Similar results were obtained when amino acid incorporation was evaluated among different proteins separated by gel electrophoresis. Confluent cells were cultured in standard DMEM in the presence of low (0.28 mM) and normal amino acids concentrations (9 mM) with [35S]methionine at constant specific radioactivity (50 µCi/µmol) to evaluate stimulation of protein synthesis by amino acids among different protein fractions. Autoradiograms of labeled cell proteins after separation in SDS gel gradient electrophoresis revealed that a large number of proteins were stimulated up to fivefold by normal amino acid concentrations (Fig. 3). However, when the same experiment was repeated and both specific radioactivity of [35S]methionine and tracee concentration was kept constant, it was observed that only some protein fractions indicated a truly increased incorporation, i.e., the tracer was incorporated more extensively in the presence of high tracee concentrations compared with low tracee concentrations in the medium (Fig. 4). These results indicate that incorporation of amino acids to mixed proteins was not from one homogenous precursor pool or that amino acids initiated various mRNAs differently. FCS (5%) additionally stimulated a number of proteins in the presence of normal concentrations (9 mM) of all amino acids, although the synthesis of a large number of proteins was unaffected (Fig. 5).



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Fig. 2. Incorporation rate of tyrosine (A) and arginine (B) into cellular proteins. L6 cells were starved for 24 h in medium with low amino acid concentration (142 µM) and refed for 16 h in medium with groups of amino acids at increasing concentrations, whereas the concentrations of the other amino acids remained at starvation concentration. Both experiments are identical except for using different 14C-labeled amino acids (L-[U-14C]tyrosine or L-[U-14C]arginine) for measurement of incorporation rates. Final concentrations of amino acids in the incubation medium are indicated (µM). Nos. in parentheses are concentrations of the tracee. BCAA, branched-chain amino acid; ao.AA, and other AA.

 


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Fig. 3. Autoradiogram of [35S]methionine-labeled proteins from L6 cells grown in medium with low (0.28 mM) and normal (9 mM) amino acid concentration with the same specific radioactivity but with different radioactive dose of [35S]methionine. Proteins were separated in a 4–12% Nupage minigel with an 2-(N-morpholino)ethanesulfonic acid (MES) buffer system in the presence of SDS. Equal amounts of protein for both samples were applied on the gel. The results indicate that amino acids stimulated incorporation of both tracer and tracee despite the same medium specific radioactivity in the presence of different doses of radioactivity.

 


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Fig. 4. Autoradiograms of [35S]methionine-labeled proteins from L6 cells grown in medium with low (0.28 mM) and normal (9 mM) amino acid concentration with the same specific radioactivity and dose of [35S]methionine. Proteins were separated in a 4–12% Nupage minigel with an MES buffer system in the presence of SDS. Equal amounts of protein for both samples were applied on the gel. The results demonstrate that only some protein fractions had significantly increased incorporation, which is in contrast to results in Fig. 3.

 


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Fig. 5. Incorporation of labeled [35S]methionine into proteins when L6 cells were grown and prepared as described in Figs. 3 and 4 in medium with normal (9 mM) amino acid concentration, plus FCS (5%). The results demonstrate that FCS can stimulate synthesis of some protein fractions independently of alterations in medium amino acid concentrations as used in Fig. 4.

 
Ribosome profiles. Amino acids at normal concentrations stimulated protein synthesis, as indicated by significantly more polysomes in such cells assessed by fractionation of RNA in sucrose gradients. This effect was related to the essential amino acids (Fig. 6), whereas nonessential amino acids did not promote formation of polyribosomes to the same extent (P < 0.01).



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Fig. 6. Distribution of cellular RNA in sucrose gradient, which was centrifuged for 195 min at 40,000 g. Confluent L6 cells were starved for 24 h in a medium with low amino acid concentration (0.14 mM). Thereafter cells were refed for 16 h in medium with increased amino acid concentrations of either essential or nonessential amino acids (9 or 7 mM, respectively). Essential amino acids were present at starvation concentration in the nonessential amino acid medium. Data are representative of one of several similar experiments.

 
Effects of amino acids on initiation of translation. Effects of amino acids on protein synthesis were also evaluated by techniques independent of labeling, such as phosphorylation/dephosphorylation of initiation factors. Phosphorylated/dephosphorylated forms of 4E-BP1 protein exhibited altered mobility when separated in an SDS-PAGE electrophoresis gel, where phosphorylation showed retarded mobility. The {alpha}- and {beta}-forms of the protein bind to eIF4E, whereas the most phosphorylated form ({gamma}) is free. Much of the 4E-BP1 was found in complex with eIF4E in cells treated with low amino acid concentrations (0.28 mM). In contrast, cells exposed to normal amino acid concentration (9 mM) had almost no 4E-BP1 bound to eIF4E. Western blot analysis of 4E-BP1 detects both the free and the bound 4E-BP1, showing that cells provided with normal amounts of amino acids (9 mM) had almost all 4E-BP1 in the {gamma} form, whereas cells incubated in the presence of low amino acid concentration had no 4E-BP1 present in the highest phosphorylated {gamma} form. An increased amount of eIF4G·eIF4E in complex was also seen in cells provided with normal amino acid concentrations. This confirms that global protein synthesis was more active in the presence of 9 mM amino acids in the medium than in 0.28 mM (Fig. 7).



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Fig. 7. Western blots of initiation factors of protein synthesis in skeletal muscle tissue from mice and cells in starvation/refeeding experiments. Mice were fasted overnight (18 h) and refed complete chow for 3 h. Cells were cultured in medium with low amino acid concentration (0.14 mM) for 24 h and refed for 16 h in medium with either low (0.28 mM; S) or normal (9 mM; RF) amino acid concentration.

 
Additional experiments were performed where groups of interrelated amino acids were checked for stimulation of initiation of translation of global proteins. In these experiments, it was confirmed that essential amino acids activated initiation of translation, whereas nonessential amino acids had no such effect (Fig. 8). Branched-chain amino acids (Leu, Ile, and Val) stimulated initiation of translation, whereas the aromatics (Trp, Phe, and Tyr) and sulfatic amino acids (Met and Cys) had no stimulation effect (Fig. 9). A group of amino acids with glutamine, histidine, threonine, arginine, and lysine showed increased initiation of translation. Further exploration of this effect suggested that the presence of all three branched amino acids in combination were significantly more potent than any of the individual branched-chain amino acids. Also, glutamine in combination with histidine, arginine, threonine, and lysine were significantly more potent than glutamine alone (Fig. 10).



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Fig. 8. Western blots of initiation factors. Confluent L6 cells were starved for 24 h in medium with low amino acid concentration (0.14 mM) and refed for 16 h in medium with normal concentrations of either essential (9 mM) or nonessential (7 mM) amino acids. Essential amino acids were present at starvation (0.14 mM) concentration in the nonessential medium.

 


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Fig. 9. Western blots of initiation factors. Confluent L6 cells were starved for 24 h in medium with low amino acid concentration (0.14 mM) and refed for 16 h with medium with groups of amino acids at increased concentration. The remaining amino acids were kept at starvation concentration (0.14 mM).

 


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Fig. 10. Western blots of initiation factors. Confluent L6 cells were starved for 24 h in medium with low amino acid concentration (0.14 mM) and refed for 16 h with medium with either several or individual amino acids at increasing concentrations. The remaining amino acids were kept at starvation concentration (0.14 mM).

 
Effects of insulin. The net effect on global protein synthesis by insulin was evaluated at two different concentrations of amino acids (0.28 and 9 mM) after culture in starvation medium (0.14 mM). The addition of insulin at physiological concentration (100 µU/ml) had no significant effect on amino acid incorporation at either low or normal concentrations of amino acids, whereas pharmacological insulin concentrations (3 mU/ml) stimulated phenylalanine incorporation at low amino acid concentration (+23 ± 5%) but significantly more at normal amino acid concentration (9 mM; +38 ± 4%, P < 0.05; Fig. 11). Similar results were obtained by Western blot analyses on initiation factors (Fig. 12).



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Fig. 11. Effects of insulin at physiological (100 µU/ml) or pharmacological (3 mU/ml) concentrations in incubation medium with low (0.28 mM) or normal (9 mM) concentrations of amino acids, which had a permissive effect on insulin (Ins, 3 mU/ml) to activate protein synthesis evaluated in L6 cells by incorporation of L-[14C-U]phenylalanine, which was stimulated by 23 vs. 38%, respectively (P < 0.05).

 
In Vivo Experiments

These experiments were performed to confirm that results from refeeding of cultured cells by amino acids agreed with in vivo refeeding experiments (complete food). Analyses of eIF4G·eIF4E complex, 4E-BPI·eIF4E complex, eIF4E, 4E-BPI phosphorylation state, and p70S6K by immunoprecipitation and subsequent electrophoretic separation confirmed that initiation of translation of protein synthesis was increased compared with muscles from starved mice (Fig. 7). Starvation/refeeding of normal mice (C57 black) reduced and then stimulated initiation of global muscle protein synthesis by ~40% (P < 0.01).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Understanding the normal control of protein balance in skeletal muscles of living animals and humans is important in veterinary and clinical medicine. During decades, several models have been developed to understand the tuned balance of contractile function and working capacity in muscle groups over time, which is important for survival. Thus it is necessary that protein content in muscles is kept constant despite variations in food availability. Accordingly, skeletal muscle mass is balanced by a control of both breakdown and resynthesis. This is particularly evident in stressed conditions where breakdown accelerates the loss of contractile proteins at the expense of functional capacity, whereas protein synthesis is depressed because of hormonal alterations to deviate substrates to more immediate needs as synthesis of acute phase reactants (11). Clinically, resumed resynthesis of muscle proteins does not occur until oral feeding is normalized during convalescence (20, 50), although it was initially believed that artificial nutrition was able to refeed individuals to conditions of normal muscle mass and function. Such observations emphasize the important role feeding has to support muscle function (15). However, despite extensive research, it is still unclear how substrate and hormones integrate resynthesis of muscle proteins in response to normal feeding (9). Our own investigations have demonstrated that this feeding response is independent of muscle innervation and circulating levels of insulin (44, 47). Also, stepwise provision of carbohydrate, fat, and amino acids to healthy volunteers indicated that nonprotein substrates had no stimulatory effect on protein balance in skeletal muscles (45), while increasing loads of amino acids switched outflow of muscle amino acids to net uptake across peripheral tissues without a corresponding change in serum insulin or IGF-I (29). Such observations indicate that amino acids themselves have stimulatory effects within the amino acid transporting capacity beyond their role of being protein components. However, artificial nutritional support to weight-losing patients has clearly indicated that provision of amino acids and nonprotein calories are not enough to elicit a normal refeeding response in skeletal muscles, evident in steady-state measurements of amino acid balance across muscle beds in resting individuals (5, 6, 36), based on constant infusion of labeled amino acids (5, 6, 20, 36, 50). However, similar studies, usually based on isotope infusions, have indicated comparatively pronounced activation of muscle protein synthesis in response to both artificial and normal feeding (41, 42). Long-term artificial nutrition to weight-losing cancer patients has also indicated improved muscle RNA content and increased amino acid incorporation in vitro to muscle proteins (30). Thus discrepant results on the role feeding may have, particularly in provision of amino acids with and without insulin in activation of muscle protein synthesis, may in part be explained by method and investigative differences.

Amino acid kinetics have long since been recognized with limitations in accurate determination of the specific radioactivity in the immediate precursor pool(s) for protein synthesis (4, 7, 12, 27, 31, 43). The flooding-dose approach was therefore developed to minimize such limitations (18), which unrecognized would severely influence quantitative assessments (17). However, results in our laboratory indicated that a flooding dose of the tracee could not entirely compensate for unknown precursor pool dilutions in calculation of protein synthesis rate (33). Therefore, the current study reevaluates effects by amino acids to stimulate net global protein synthesis in cultured muscle cells assessed by different independent techniques with incubation conditions where label distribution among different cellular compartments should not be limited.

Our results confirm the difficulties there are to quantify global protein synthesis rate by using precursors of labeled amino acids. Incorporation rates of both tyrosine and phenylalanine were clearly stimulated by increasing extracellular concentrations of all amino acids or essential amino acids only in the incubation medium. However, this effect was also observed when tracee concentrations were increased alone in combination with constant specific radioactivity as also observed in vivo (10). Although it may be conceptually possible that tyrosine or phenylalanine may activate protein synthesis, it is noteworthy that tyrosine did not stimulate the incorporation of phenylalanine or vice versa. Furthermore, it was evident that provision of essential amino acids to the incubation medium did not further stimulate incorporation rate of phenylalanine when phenylalanine concentrations were already high in the incubation medium. Thus it is evident that increasing incorporation rate of amino acids in this kind of experiment was to some extent a mathematical phenomenon in calculations where the amount of tracer is multiplied by increasing concentrations of tracee despite long-term incubations at steady state where compromised equilibration of the labeled amino acid(s) among monolayer cells should not occur.

It appears that determination of protein synthesis by calculation of incorporation rate of amino acids in compartment models is not entirely accurate in a variety of investigative conditions. This observation was further emphasized by labeling experiments where incorporation of [35S]methionine into cellular proteins was evaluated by autoradiography after separation of cellular proteins, experiments in which all proteins should recruit precursor amino acids from a common homogenous pool of tRNA. In these studies, it was evident that normal amino acid concentrations (9 mM) stimulated incorporation of the tracer in the majority of protein fractions (Fig. 3). However, when methionine was held constant at high tracee levels (200 µM), it was obvious that the majority of protein fractions had the same labeling both in the presence of normal (9 mM) and low (0.48 mM) levels of all other amino acids (Fig. 4). These results indicate that reutilization of intracellular amino acids from protein breakdown must be an essential contribution of dilution of the tracer through pathways for flux of amino acids through any precursor pool(s), which is assumed to be a single homogeneous tRNA pool. However, our previous experiments have suggested that mathematical modeling of incorporation kinetics of amino acids into protein cannot entirely explain labeling of cellular proteins from a homogenous tRNA pool (33). Based on previous and current results, it is concluded that flooding of tracee or primed constant infusion of labeled precursors does not entirely overcome problems with unknown and incomplete dilution of specific radioactivity in precursor pool(s). Therefore, alternative methods must be applied in experiments where quantitative estimates of protein synthesis are critical for conclusions.

On the basis of the above reasoning, we evaluated the effects of essential and nonessential amino acids on the formation of polyribosomes in cultured cells. The results demonstrate that essential amino acids alone increase the formation of significantly more polyribosomes concomitant with lowered amounts of monosomes and subunits. The results confirm that essential amino acids activate global protein synthesis, although the method may not be sensitive enough to detect small but significant alterations in translation. Therefore, we extended measurements to include Western blot analyses of initiation factors of protein synthesis in skeletal muscle tissue and cultured muscle cells in starvation/refeeding experiments. Mice were fasted overnight for 18 h and refed complete chow for 3 h, whereas cultured cells were exposed to reduced amino acid concentrations (0.14 mM) for 24 h and refed for 16 h with low (0.28 mM) or normal (9 mM) concentrations of amino acids. The results confirm that the active complex 4G·4E appeared at increased amount in refed conditions, whereas BP1·4E disappeared in the expected way, which was explained by dephosphorylation of the 4E·BP1-P complex (2, 49). In addition, the p70S6K were significantly more phosphorylated in refed conditions (28), effects confirmed to be related to the essential amino acids (Fig. 8 and Ref. 8). Additional experiments with different groups of amino acids confirm that branched-chain amino acids had a clear-cut effect on initiation of translation in both murine (21, 24, 34) and human (32) muscles, while sulfur-containing amino acids and the aromatics had no such effect, which is in contrast to in vivo findings based on labeling experiments (3). A group of amino acids including glutamine also had intermediate effects to activate initiation of translation in vitro, although assumed positive in vivo effects by glutamine are highly questionable (13, 46). In general, our present experiments revealed that several amino acids in combination exerted more pronounced stimulation than provision of isolated amino acids, a process that is independent of insulin during refeeding both in vitro and in vivo (44, 48).

The effect of insulin was also evaluated by different methods. Incorporation experiments revealed that insulin at physiological levels did not stimulate overall phenylalanine incorporation while pharmacological concentrations (3 mU/ml) increased incorporation significantly, more so at normal than at low medium concentrations of amino acids, as confirmed by Western blots of initiation factors (2). These results indicate that pathways for initiation of translation by insulin and amino acids are in part different (19, 26), suggesting that insulin and amino acids may stimulate different proteins. Insulin may also control transcription either positively or negatively. Therefore, our results with insulin refer to net overall changes in protein synthesis rate.

In conclusion, our results demonstrate that studies on protein synthesis may give incorrect results when based on labeled amino acids, even when flooding tracee doses are applied to circumvent dilution problems of the specific radioactivity in the immediate precursor pool for protein synthesis. This dilemma is probably not overcome by any known tracer technique, which all seem to be hampered by similar uncertainties, probably because of unknown compartmentalization, restricted distribution of amino acids among pathways of protein synthesis, and metabolic interconversions. Therefore, alternative methods that are independent of amino acid kinetics should be applied as well. By this approach, it was confirmed that essential amino acids and individual groups of amino acids have the full potential to activate initiation of translation independent of insulin in skeletal muscle cells. Thus present results emphasize that all kinds of quantitative measures of protein synthesis should be viewed with caution when based on labeled amino acid kinetics.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported, in part, by grants from the Swedish Cancer Society (nos. 2014 and 4261), the Swedish Research Council (nos. 08712 and 13268), Assar Gabrielsson Foundation (AB Volvo), Jubileumskliniken Foundation, IngaBritt & Arne Lundberg Research Foundation, Swedish and Göteborg Medical Societies, and the Medical Faculty, Göteborg University.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Lundholm, Dept. of Surgery, Sahlgrenska Univ. Hospital, SE 413 45 Goteborg, Sweden (E-mail: kent.lundholm{at}surgery.gu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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