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Am J Physiol Endocrinol Metab 293: E188-E196, 2007. First published March 27, 2007; doi:10.1152/ajpendo.00037.2007
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Rapamycin blunts nutrient stimulation of eIF4G, but not PKC{varepsilon} phosphorylation, in skeletal muscle

Thomas C. Vary, Joshua C. Anthony,* Leonard S. Jefferson, Scot R. Kimball, and Christopher J. Lynch

Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania

Submitted 15 January 2007 ; accepted in final form 21 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phosphorylation of eukaryotic initiation factor 4G (eIF4G) is hypothesized to be an important contributor to the stimulation of protein synthesis in skeletal muscle following meal feeding. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in mediating the meal feeding-induced elevations in phosphorylation of eIF4G. Gastrocnemius from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled prior to and following 3 h of having the meal provided in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Pretreatment with rapamycin prevented the feeding-induced phosphorylation of mTOR, eIF4G, and S6K1 but only partially attenuated the shift in 4E-BP1 into the {gamma}-form. In contrast, the feeding-induced increase in phosphorylation of PKC{varepsilon} was not reduced by rapamycin. Rapamycin also prevented the augmented association of eIF4G with eIF4E and the decreased association of eIF4E with 4E-BP1. Similar findings were observed in gastrocnemius from animals after oral administration of leucine. Perfusion of gastrocnemius with medium containing rapamycin partially prevented the leucine-induced increase in phosphorylation of eIF4G. Thus, rapamycin attenuated a feeding- or leucine-induced phosphorylation of eIF4G in skeletal muscle both in vivo and in situ. The latter observation implies that the effects observed with rapamycin were the result of modulation of skeletal muscle signaling mechanisms responsible for eIF4G phosphorylation.

translation initiation; eukaryotic initiation factor 4G; protein kinase C{varepsilon}; mammalian target of rapamycin; ribosomal protein S6 kinase 1; leucine; hindlimb perfusion

ACCRETION OF MUSCLE PROTEIN following meal feeding occurs in part through a stimulation of protein synthesis at the level of mRNA translation initiation. One regulatory step responsible for accelerating mRNA translation initiation involves the recognition, unwinding, and binding of mRNA to the 43S preinitiation complex (4, 49, 54). These processes are catalyzed by a multisubunit complex of eukaryotic factors referred to as eukaryotic initiation factor (eIF)4F and composed of eIF4A (a RNA helicase that unwinds secondary structure in 5' untranslated region of mRNA), eIF4E (a protein that binds directly to the m7GTP cap structure present at the 5' end of most eukaryotic mRNAs), and eIF4G (a protein that functions as a scaffold for eIF4E, eIF4A, and the mRNA and the ribosome) (35, 36, 39). The association of eIF4G with eIF4E and eIF4A means that mRNA destined for translation can be unwound and rendered accessible to a 43S preinitiation complex to facilitate ribosomal binding to mRNA in order for translation to proceed. eIF4G links the mRNA cap structure, the poly(A)+ tail, and ribosomal subunit because it has binding sites not only for eIF4E but also for eIF4A and eIF3 (18). We (43) were the first to report a positive linear relationship between rates of protein synthesis and amount of eIF4G associated with eIF4E in skeletal muscle in vivo.

Assembly of active eIF4G·eIF4E complex is dependent upon both the availability of eIF4E and the extent of eIF4G phosphorylation. Availability of eIF4E to bind with eIF4G and form active eIF4F complex is regulated by a family of low-molecular-weight acid- and heat-stable proteins termed eIF4E-binding proteins (4E-BPs) (29, 31). The activity of 4E-BPs is controlled through covalent modification, whereby phosphorylation reduces the binding affinity for eIF4E, allowing eIF4E to bind to eIF4G and promote mRNA translation (10, 24). The signaling pathway leading to changes in phosphorylation of 4E-BP1 following meal feeding occurs in part through the mammalian target of rapamycin (mTOR). The activity of this serine/threonine kinase is regulated by nutrients when it is part of a complex called mTOR complex 1 (TORC1), which includes not only mTOR but also rapamycin-associated TOR protein (RAPTOR) and GbetaL (12, 38). However, the regulation of 4E-BP1·eIF4E appears more complex, as we have reported (2, 5, 23, 49) that leucine stimulates protein synthesis in perfused hindlimb preparations through a mechanism that appears to be independent of 4E-BP1 phosphorylation.

Another potential mechanism modulating the assembly of eIF4G·eIF4E complex involves phosphorylation of eIF4G (5). eIF4G possesses three phosphorylation sites located in the COOH-terminal one-third of the protein corresponding to serine residues 1108, 1148, and 1192 (34). Numerous reports have linked phosphorylation of eIF4G with corresponding changes in protein synthesis. Increased phosphorylation of eIF4G on Ser1108 is associated with enhanced assembly of eIF4E·eIF4G complex following exposure of cells in culture to serum (34) or gonadotrophin-releasing hormone (26). In vivo, IGF-I-induced stimulation of phosphorylation of eIF4G correlates with acceleration of protein synthesis in muscle (19, 46). Likewise, enhanced phosphorylation of eIF4G correlated with stimulation of protein synthesis and assembly of the eIF4G·eIF4E complex following perfusion of hindlimb muscle with buffer supplemented with leucine (5, 42). On the other hand, thermal injury reduces the phosphorylation of eIF4G and assembly of eIF4E·eIF4G complex in heart (20). Hence, phosphorylation of eIF4G is a potentially important mechanism controlling protein synthesis through assembly of the eIF4G·eIF4E complex in striated muscle.

There is some evidence (9, 34, 46) in cells in culture that the signaling pathway through rapamycin-sensitive mTOR complex is responsible for phosphorylation of eIF4G, but other reports (5, 40) have implicated different signaling pathways. Thus the potential signaling pathways responsible for modulating the phosphorylation state of eIF4G in vivo remain unresolved. We tested the hypothesis that mTOR plays an important role in regulating the phosphorylation of eIF4G in skeletal muscle by inhibiting mTOR with rapamycin. In this study we show that the effects of meal feeding and leucine to increase phosphorylation of eIF4G are mediated in part through a rapamycin-sensitive pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Meal feeding. For the meal-feeding experiments, male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were adapted to a reverse light cycle (the dark cycle began at 7 AM and the light cycle began at 7 PM) as described previously (41, 47, 48). Animals were caged in pairs rather than singly to reduce anxiety-induced changes in food intake and trained over a period of ≥12 days to consume a meal when presented. Food (Teklad Rodent Diet no. 8604) was provided in two metal food cups for 3 h beginning 30 min after the beginning of the dark cycle (41, 47, 48). The concentration of leucine in Teklad Diet no. 8604 is 2.04%, and overall protein content is 24.48%. Water was provided ad libitum. The Institutional Animal Care and Use Committee at the Penn State University College of Medicine approved the animal protocols.

After the training, rats were divided into three groups: nonfed, fed, and fed plus rapamycin. Animals received an intraperitoneal injection of either vehicle (nonfed and fed groups: 0.75 ml/kg body wt) or rapamycin (fed plus rapamycin: 1 mg/ml mixed in ethanol, 0.75 ml/kg body wt) at the beginning of the dark cycle 15 min before the usual presentation of the meal. The nonfed group received no food in the morning prior to sampling the gastrocnemius. In pilot studies, acute rapamycin injection caused a decreased (15 g/day) food intake. Therefore, the fed group animals were given the same amount of rat chow as animals in the fed plus rapamycin group to match food intake between the two groups. The gastrocnemius was sampled 3 h after the presentation of the meal and immediately frozen between clamps precooled to the temperature of liquid nitrogen.

Leucine gavage. Food and water were provided ad libitum. Rats were maintained on a 12:12-h light-dark cycle, with the light cycle beginning at 7 AM and the dark cycle beginning at 7 PM. On the day before the experiment, animals were deprived of food for 16 h. At time 0, the animals were randomly divided into two groups and administered vehicle (0.75 ml/kg body wt, 155 mM NaCl + 2% vol/vol ethanol) or rapamycin (1 mg/ml, 0.75 ml/kg body wt) via tail vein. Two hours later, one-half of the animals in the control and rapamycin groups were given one of the following solutions by oral gavage (2.5 ml/100 g body wt) according to their designated treatment group: control (0.155 mol NaCl/l) or leucine (54.0 g/l L-leucine) as described previously (3, 41, 48). The dose of leucine gavaged is equivalent to the amount of leucine consumed by rats of this age and strain during 24 h of free access to a commercial rodent diet (1, 3, 23). Thirty minutes later, animals were anesthetized (Nembutal 100 mg/kg body wt) and the gastrocnemius excised and immediately frozen between clamps precooled to the temperature of liquid nitrogen. At the time of tissue sampling, the plasma leucine concentrations were the following: control 127 ± 15 and leucine 2,273 ± 99 mM (23).

Hindlimb perfusions. In some experiments, the rat hindlimb was perfused with buffer supplemented with leucine according to previously described methods (13, 14, 41–43). Food was not withdrawn prior to the perfusion experiments, and perfusions were begun by 10 AM. Animals were anesthetized (Nembutal 100 mg/kg) and catheters placed in the descending aorta and vena cava below the level of the renal artery and vein. Perfusion was initiated immediately, and the first 50 ml of perfusate were discarded. The perfusate was then recirculated. Following an initial period of 15 min, perfusion was continued for an additional 60 min (41, 42). The perfusate (250 ml) consisted of a modified Krebs-Henseleit bicarbonate buffer containing 30% (vol/vol) washed bovine erythrocytes, 4.5% (wt/vol) bovine serum albumin (BSA fraction V), 11 mM glucose, and amino acids present at plasma concentrations, except leucine was added at either 1x or 10x the plasma concentration (41, 42). A concentration of 10x leucine was selected because rates of protein synthesis in perfused skeletal muscle are stimulated maximally at this concentration (21, 41, 42). Rapamycin (250 nM) was included in the perfusate and present throughout the perfusion period. At the conclusion of the perfusion, the gastrocnemius was quickly removed and frozen between clamps precooled to the temperature of liquid nitrogen.

Preparation of muscle lysates. Muscles were prepared for analysis of the phosphorylation state of eIF4G, mTOR, PKC{varepsilon}, ribosomal protein S6 kinase 1 (S6K1), PKB, and 4E-BP1, the association of eIF4G with eIF4E, and the association of 4E-BP1 with eIF4E. For this purpose, powdered muscle (0.2 g) was homogenized in 7 volumes of buffer A [20 mM HEPES (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM DTT, 50 mM NaF, 50 mM beta-glycerolphosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µmol/l microcystin LR] using a Polytron PT 10 homogenizer set at 60% of maximum power. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. An aliquot of the 10,000 g supernatant was mixed with an equal volume of 2x Laemmli-SDS sample buffer (65°C) and then subjected to protein immunoblot analysis. Another aliquot was used to measure the protein concentration by the Biuret method with crystalline BSA serving as a standard. A third aliquot was used for immunoprecipitation to determine association of eIF4E with 4E-BP1 and association of eIF4G with eIF4E following immunoprecipitation of eIF4E.

Determination of extent of eIF4G or mTOR phosphorylation. To measure the relative extent of phosphorylation of eIF4G or mTOR, proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE). Following electrophoresis the proteins transferred to polyvinylidene difluoride (PVDF; Biotrace; PALL, Pensacola, FL). The membranes were incubated overnight at 4°C with antibodies specific for phosphorylated forms of eIF4G(Ser1108), mTOR(Ser2448), or mTOR(Ser2481) (Cell Signaling Technology, Beverly, MA). The autoradiographs were scanned and analyzed. The membranes were subsequently immunoblotted with the antibodies that recognize eIF4G or mTOR independently of their phosphorylation states (Bethyl Laboratories, Montgomery, TX) and analyzed. The phosphorylated eIF4G or mTOR signal densities were normalized to the respective total eIF4G signal to reflect the relative ratio of phosphorylated eIF4G or mTOR to total eIF4G or mTOR, respectively.

Determination of extent of 4E-BP1 phosphorylation. The various phosphorylated forms of 4E-BP1 (designated {alpha}, beta, and {gamma}) in gastrocenemius homogenates were separated by SDS-PAGE electrophoresis and quantitated by protein immunoblot analysis as described previously (43–45, 50, 51).

Determination of extent of S6K1, PKB, PKC{alpha}, PKC{zeta}/{lambda}, and PKC{varepsilon} phosphorylation. Homogenates from gastrocnemius muscle were mixed with 2x Laemmli-SDS sample buffer and subjected to electrophoresis on 12.5% SDS-PAGE Criterion gels (Bio-Rad, Hercules, CA) (22, 23, 43, 50). A separate gel was electrophoresed for each individual protein assayed. Following transfer to PVDF membranes, the blots were probed with phosphospecific antibodies that recognize phospho-S6K1(Thr389), phospho-PKB(Thr308), phospho-PKB(Ser473), phospho-PKB{alpha}(Ser657), phospho-PKC{zeta}/{lambda}(Ser473) (Cell Signaling Technology, Beverly, MA), or phospho-PKC{varepsilon}(Ser729) (Upstate Cell Signaling, Lake Placid, NY) and quantified as described above for eIF4G phosphorylation. The blots were then probed with antibodies that recognize total S6K1, PKB, PKC{alpha}, PKC{zeta}, or PKC{varepsilon} (i.e., both phosphorylated and unphosphorylated forms of the proteins), respectively, and analyzed. Results are presented as the ratio of the densitometric analysis of blot for phosphorylated form of the protein divided by sum of both the phosphorylated and unphosphorylated forms of the proteins performed on the same gel.

Statistics. Results are presented as means ± SE of multiple densitometric analyses for each group. Results were compared using a two-tailed, two-sample Student's t-test to assess differences between two treatment groups. When comparisons between more than two means were required, ANOVA statistical analysis was performed. When ANOVA indicated a significant difference among means, post hoc tests for statistical analysis were performed with a Sidak post hoc test. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Meal feeding. mTOR is reported to be a common intermediate in growth factor- and nutrient-induced stimulation of mRNA translation. mTOR exists in two complexes; one of these contains the adaptor protein RAPTOR and is sensitive to rapamycin (TORC1), whereas the other complex contains the adaptor protein RICTOR (a.k.a. pianissimo) (TORC2) and is rapamycin insensitive (11, 12, 38). Phosphorylation of mTOR on residues Ser2448 and Ser2481 often correlates with changes in mTOR activity (30). Therefore, we examined the phosphorylation of mTOR using phosphospecific antibodies as an index of mTOR activity following pretreatment with rapamycin prior to meal feeding. As illustrated in Fig. 1, the extent of phosphorylation of mTOR at both Ser2441 and Ser2448 was significantly increased in response to meal feeding. To this end, the extent of phosphorylation of mTOR at Ser2441 and Ser2488 were both significantly reduced in animals that were pretreated with rapamycin prior to meal feeding. Rapamycin, administered prior to meal feeding, maintained mTOR phosphorylation on both sites at values equivalent to those observed in nonfed control animals.


Figure 1
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  		Fig. 1. Effect of rapamycin on the extent of phosphorylation of mammalian target of rapamycin (mTOR) following meal feeding. Animals were fasted overnight. On the morning of the experiment, gastrocnemius muscle was sampled 0.5 h before feeding (nonfed) and then 3 h after the feeding regimen was initiated. Animals receiving the meal were injected with either vehicle (0.75 ml ethanol/kg body wt) or rapamycin (1 mg/ml mixed in ethanol, 0.75 ml/kg body wt). Equal amounts of protein in homogenates from gastrocnemius of animals were immunoblotted with antibodies specific for the phosphorylated form of mTOR(Ser2448) (top) and mTOR(Ser2481) (bottom). The blots were stripped and immunoblotted with an antibody that recognizes both the phosphorylated and nonphosphorylated forms of mTOR. The bar graph shows the means of individual densitometric analysis of several immunoblots of mTOR phosphorylation on Ser2448 and Ser2481 in homogenates corrected for the total amount of mTOR as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 8 in each group. Phospho-mTOR(Ser2448): ANOVA P < 0.001, F = 15.29; phospho-mTOR(Ser2481): ANOVA P < 0.001, F = 6.08 *P < 0.05 vs. nonfed or meal feeding + rapamycin.

 
Both S6K1 and 4E-BP1 are activated by multisite phosphorylation that results in phosphorylated forms exhibiting retarded electrophoretic mobility when subjected to SDS-PAGE (7, 17, 29, 53). S6K1 (7) and 4E-BP1 (for review, see Refs. 32 and 33) are believed to be downstream substrates for phosphorylation by the TORC1 complex under certain circumstances. Indeed, phosphorylation of S6K1 on residue Thr389 is associated with an increased activity of the kinase (52, 53). As shown in Fig. 2, top, meal feeding induced a robust S6K1 phosphorylation (~11-fold higher) over nonfed rats. Pretreatment of animals with rapamycin completely prevented the meal feeding-induced phosphorylation of S6K1 at residue Thr389 and reduced the extent of phosphorylation to values observed in nonfed controls.


Figure 2
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  		Fig. 2. Effect of rapamycin on the extent of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF)-binding protein 1 (4E-BP1) phosphorylation following meal feeding. Homogenates of gastrocnemius obtained from animals in Fig. 1 were analyzed for phosphorylated S6K1. Equal amounts of protein in homogenates from gastrocnemius were immunoblotted with antibodies specific for the phosphorylated form of S6K1(Thr389) (top). The blots were stripped and immunoblotted with an antibody that recognizes both the phosphorylated and nonphosphorylated forms of S6K1. The figure shows a bar graph of the means of individual densitometric analysis of several immunoblots where S6K1 phosphorylation on Thr389 was measured and corrected for the total amount of S6K1 as described in MATERIALS AND METHODS. The phosphorylation state of 4E-BP1 (bottom) was measured in homogenates of gastrocnemius obtained from animals in Fig. 1 as described in MATERIALS AND METHODS. 4E-BP1 in the {gamma}-phosphorylated form is as described in MATERIALS AND METHODS. The bar graph shows the means of individual densitometric analysis of several immunoblots of 4E-BP1 in the {gamma}-form divided by the total amount of 4E-BP1 as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 8 in each group. S6K1: ANOVA P < 0.05, F = 5.01; *P < 0.05 vs. nonfed or meal feeding + rapamycin. 4E-BP1: ANOVA P < 0.0001, F = 30.21; §P < 0.001 vs. nonfed. {dagger}P < 0.01 vs. all other groups.

 
4E-BP1, when phosphorylated, resolves into distinct electrophoretic forms ({alpha}, beta, and {gamma}), with the {gamma}-form representing the highest phosphorylated form (29). The results reveal that meal feeding promoted a significant, 3.5-fold increase in the proportion of protein in the {gamma}-form (Fig. 2, bottom). The extent of phosphorylation of 4E-BP1 was significantly reduced 30% following pretreatment with rapamycin prior to feeding and remained elevated relative to nonfed controls.

The changes in phosphorylation of 4E-BP1 would be expected to modify the association of eIF4E with 4E-BP1. As shown in Fig. 3, meal feeding decreased the association of eIF4E with 4E-BP1 to 25% of the value observed in nonfed controls. Pretreating with rapamycin prior to meal feeding prevented the meal feeding-induced reduction of eIF4E association with 4E-BP1.


Figure 3
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  		Fig. 3. Effect of rapamycin on eIF4E associated with 4E-BP1, eIF4G phosphorylation, and eIF4G associated with eIF4E following meal feeding. Gastrocnemius from animals described in Fig. 1 were homogenized. Equal volumes of eIF4E immunoprecipitated from gastrocnemius were immunoblotted with antibodies specific for 4E-BP1 or eIF4G. The abundance of eIF4E was determined by measuring the eIF4E in the immunoprecipitates. Equal amounts of protein in homogenates from gastrocnemius were immunoblotted with antibodies specific for the phosphorylated form of eIF4G(Ser1108). The blots were stripped and immunoblotted with an antibody that recognizes both the phosphorylated and nonphosphorylated forms of eIF4G. The graph shows the means of individual densitometric analysis of several immunoblots of eIF4G phosphorylation on Ser1108 corrected for the total amount of eIF4G as described in MATERIALS AND METHODS. The graph shows the means of individual densitometric analysis of several immunoblots of 4E-BP1 or eIF4G divided by the amount of eIF4E in the immunoprecipitate as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 8 in each group (eIF4E associated with 4E-BP1 ANOVA P < 0.05, F = 5.47; eIF4G phosphorylation ANOVA P < 0.001, F = 25.16; eIF4G associated with eIF4E ANOVA P < 0.05, F = 4.49). *P < 0.05.

 
Having established that rapamycin in this feeding paradigm prevents the phosphorylation of mTOR, S6K1, and 4E-BP1, we next investigated the effect of inhibitor on eIF4G(Ser1108) phosphorylation following meal feeding (Fig. 3). Meal feeding increased eIF4G(Ser1108) phosphorylation sevenfold, an effect completely prevented by rapamycin.

The abundance of the eIF4G·eIF4E complex was determined by immunoprecipitating eIF4E with a monoclonal antibody followed by Western immunoblot analysis for eIF4E and eIF4G. Meal feeding brought about a pronounced fourfold increase in the abundance of eIF4G associated with eIF4E (Fig. 3), whereas pretreatment with the inhibitor prevented this effect. The changes in abundance of eIF4G·eIF4E complex were not the result of an increased expression of eIF4E or eIF4G protein, as there were no significant differences in the eIF4E or eIF4G content in gastrocnemius at any of time points investigated (data not shown).

Direct inhibition of TORC1 on the phosphorylation of PKC{varepsilon} in skeletal muscle has not previously been evaluated, even though meal feeding and leucine gavage cause an increase in PKC{varepsilon} phosphorylation in this tissue (41). Therefore, we assessed the possibility that rapamycin might reduce phosphorylation of PKC{varepsilon} in gastrocnemius of meal-fed rats (Fig. 4). Meal feeding resulted in a 50% stimulation in the level of PKC{varepsilon} phosphorylation. Rapamycin did not affect the meal feeding-induced stimulation of PKC{varepsilon} phosphorylation.


Figure 4
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  		Fig. 4. Effect of rapamycin on the extent of PKC{varepsilon} phosphorylation following meal feeding. Gastrocnemius from animals described in Fig. 1 were homogenized. Phosphorylation of PKC{varepsilon}(Ser729) was measured in homogenates of gastrocnemius from animals described in Fig. 1 by means of Western blot techniques, using phosphospecific antibodies. Equal amounts of protein were loaded onto each lane. The extent of phosphorylation of PKC{varepsilon}(Ser729) was determined on the basis of the immunoreactivity signals and normalized as a function of total PKC{varepsilon}. The graph shows the means of individual densitometric analysis of several immunoblots of phospho-PKC{varepsilon}(Ser729) divided by the amount of PKC{varepsilon} as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 8 in each group (ANOVA P < 0.05, F = 4.07). *P < 0.05 vs. nonfed.

 
We also examined the response of PKB to rapamycin following meal feeding. PKB lies upstream of TORC1 and therefore should be relatively unaffected by inhibition of mTOR by rapamycin. The level of PKB phosphorylation on Thr308 was not significantly elevated by meal feeding or meal feeding plus rapamycin compared with fasted controls (Table 1). In contrast, PKB phosphorylation on Ser473 was significantly elevated by meal feeding compared with fasted controls, and rapamycin did not significantly alter this response (Table 1). We examined the potential role of plasma insulin concentrations as a possible explanation for rise in PKB phosphorylation with meal feeding. Plasma insulin concentrations were increased 65% (P < 0.05) with meal feeding (827 ± 59 pmol/l) compared with nonfed controls (500 ± 84 pmol/l). Rapamycin did not significantly affect the meal feeding-induced increase in plasma insulin (meal fed 827 ± 69 pmol/l vs. meal fed plus rapamycin 1,085 ± 141 pmol/l), suggesting similar nutrient intake in the meal-fed groups.


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  		Table 1. Effect of rapamycin on level of phosphorylation of PKB, tuberin, PKC{alpha}, and PKC{zeta}/{lambda} following meal feeding

 
Neither meal feeding nor rapamycin affected the phosphorylation of PKC{alpha}, PKC{zeta}/{lambda}, or tuberin (the GTPase activating protein regulateing mTOR; Table 1).

Leucine gavage. The meal provides both energy and protein macronutrients. To assess which components of the meal were responsible for increased eIF4G phosphorylation, we examined the effects of providing only one of the protein constituents, namely leucine via gavage-fasted animals. In these experiments, one-half of the animals were injected with rapamycin and then administered leucine via gavage. In fasted animals, injection of rapamycin lowered the extent of eIF4G phosphorylation by 66% compared with nonfed animals (Fig. 5). Oral administration of leucine raised the extent of eIF4G phosphorylation approximately twofold compared with untreated fasted animals. Pretreatment of fasted animals with rapamycin prior to leucine gavage prevented the phosphorylation of eIF4G by 84%.


Figure 5
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  		Fig. 5. Effect of rapamycin on the extent of eIF4G phosphorylation following oral leucine gavage. Rats were food deprived for 16 h and then randomly administered 0.75 mg rapamycin/kg body wt or an equal volume of vehicle (fasting, 0.155 mol/l NaCl, 2% vol/vol ethanol) via the tail vein. Two hours later, one-half of the rats in the fasting and fasting + rapamycin groups were orally administered 1.35 g L-leucine/kg body wt as described in MATERIALS AND METHODS (leucine and leucine + rapamycin, respectively). Rats not receiving leucine were gavaged with 2.5 ml saline/100 g body wt (0.155 mol/l). One hour later, gastrocnemius was harvested and homogenized. Equal amounts of protein in homogenates from gastrocnemius were immunoblotted with antibodies specific for the phosphorylated form of eIF4G(Ser1108). The blots were stripped and immunoblotted with an antibody that recognizes both the phosphorylated and nonphosphorylated forms of eIF4G. The graph shows the means of individual densitometric analysis of several immunoblots of eIF4G phosphorylation on Ser1108 corrected for the total amount of eIF4G as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 4 in each group (ANOVA P < 0.001, F = 11.154). {dagger}P < 0.05 vs. nonrapamycin-treated rats in each group. *P < 0.05 vs. fasting group.

 
Rapamycin did not significantly lower the phosphorylation of S6K1 in gastrocnemius compared with fasted animals (Fig. 6). Administration of leucine via the gavage raised the extent of phosphorylation of S6K1 11-fold compared with fasted animals gavaged with saline, a fold change similar to that observed following meal feeding. Pretreatment of fasted animals given leucine via gavage with rapamycin lowered the phosphorylation of S6K1 at residue Thr389 by 98% compared with animals given a leucine gavage (Fig. 6). There were no significant differences between leucine- and saline-gavaged animals injected with rapamycin.


Figure 6
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  		Fig. 6. Effect of rapamycin on the extent of S6K1 and 4E-BP1 phosphorylation following oral leucine gavage. Homogenates obtained from animals in Fig. 5 were analyzed for phosphorylated S6K1 (top) and proportion of 4E-BP1 in the {gamma}-form (bottom) as described in Fig. 2. Values shown are means ± SE; n = 4 in each group. S6K1: ANOVA P < 0.005, F = 9.97; 4E-BP1: ANOVA P < 0.0001, F = 130.04. {dagger}P < 0.01 vs. nonrapamycin-treated rats in each group. *P < 0.001 vs. fasting group.

 
Pretreatment of fasted rats with rapamycin reduced the proportion of 4E-BP1 in the {gamma}-form to 28% of values observed in control animals gavaged with saline. Oral leucine administration resulted in a 3.5-fold increase in 4E-BP1 phosphorylation compared with fasted animals gavaged with saline. Pretreatment of fasted animals given leucine via gavage with rapamycin lowered the proportion of 4E-BP1 in the {gamma}-form by 88% compared with animals given a leucine gavage (Fig. 6).

As was observed with meal feeding, pretreatment with the mTOR inhibitor rapamycin was without effect on the phosphorylation of PKC{varepsilon} following leucine gavage [leucine gavage 103 ± 8 arbitrary units (AU) phospho-PKC{varepsilon}/total PKC{varepsilon} vs. leucine gavage + rapamycin 96 ± 10 AU phospho-PKC{varepsilon}/total PKC{varepsilon}].

Hindlimb perfusion. The perfused hindlimb preparation allows a defined medium to be circulated through the vasculature of the gastrocnemius. Raising the concentration of leucine tenfold (10x Leu) in the perfusate increases the level of eIF4G(Ser1108) phosphorylation fourfold (Fig. 7), consistent with our previous report (5). With rapamycin present in the medium during the entire perfusion period with 10x leucine, the leucine-induced phosphorylation of eIF4G(Ser1108) was reduced by ~40% (Fig. 7). However, the extent of eIF4G(Ser1108) phosphorylation remained elevated compared with muscles perfused with medium containing 1x leucine. To assess the possibility that the concentration of rapamycin in the perfusate was insufficient to significantly reduce mTOR signaling, we examined the association of eIF4E with 4E-BP1 (Fig. 7). Raising the perfusate leucine concentration lowered the assembly of eIF4E·4E-BP1 complex by 60%. Rapamycin prevented the leucine-stimulated reduction of eIF4E association with 4E-BP1 in perfused hindlimb, as observed with meal feeding and leucine gavage.


Figure 7
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  		Fig. 7. Effect of rapamycin on the extent of eIF4G phosphorylation and eIF4E associated with 4E-BP1 in hindlimb perfused with 10x plasma leucine (LEU) concentrations. Hindlimbs were perfused with buffer containing leucine at 10 times (10x) the plasma concentration in either the presence or absence of rapamycin as described in MATERIALS AND METHODS. Homogenates were analyzed for the extent of eIF4G phosphorylation and eIF4E associated with 4E-BP1 as described in Fig. 3. Values shown are means ± SE; n = 7–21 in each group. eIF4G phosphorylation ANOVA P < 0.0001, F = 21.74; eIF4E associated with 4E-BP1 ANOVA P < 0.05, F = 3.841. *P < 0.05 vs. 1x LEU or 10x LEU + rapamycin. {dagger}P < 0.05 vs. 1x LEU.

 
We also examined the ability of rapamycin to affect the phosphorylation of PKC{varepsilon} after elevating the leucine perfusion concentration. The extent of PKC{varepsilon} phosphorylation was not significantly reduced with rapamycin present in the perfusate (10x leucine –103 ± 8 AU phospho-PKC{varepsilon}/total PKC{varepsilon} vs. 10x leucine + rapamycin 97 ± 2 AU phospho-PKC{varepsilon}/total PKC{varepsilon}).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Control of mRNA translation initiation posits mTOR as a central regulator of multiple hormonal and nutritional inputs (49). In this scenario, TORC1 functions as integrator of signaling pathways from hormones as well as direct inputs from metabolites, including amino acids. As such, TORC1, but not TORC2, can then modulate rates of protein synthesis through adjustments in mRNA translation and in long-term ribosome biogenesis (11, 12, 16, 37) to respond to various stimuli enhancing cell growth, including meal feeding (41, 47, 48, 54). In the present set of studies, rapamycin largely inhibited TORC1, as evidenced by the prevention the meal-feeding induced stimulation of mTOR phosphorylation as well as the phosphorylation of two mTOR downstream effectors, S6K1 and 4EBP1. Pretreatment with rapamycin also blocked the effect of meal feeding to increase (7-fold) the phosphorylation of eIF4G. These findings would indicate that the meal feeding-induced, augmented eIF4G phosphorylation is sensitive to inhibition of mTOR by rapamycin.

Meal feeding provides as nutrients both carbohydrates and proteins (amino acids) to animals. To distinguish the consequence of amino acid-induced activation of mTOR on the level of eIF4G phosphorylation, we assessed the ability of rapamycin to prevent the increase in phosphorylation of eIF4G following oral gavage with leucine. As observed following meal feeding, rapamycin completely prevented the stimulatory effect of an oral leucine gavage to increase eIF4G phosphorylation. Thus, when animals are pretreated with rapamycin, the rise in eIF4G phosphorylation induced by meal feeding or leucine gavage is completely attenuated. In contrast, when skeletal muscle is simultaneously exposed to both leucine (10x the plasma amino acid concentration) and rapamycin, as was the case in the perfused hindlimb studies, the rise in phosphorylation of eIF4G was only partially attenuated. These observations support our earlier reports that there are two pathways potentially involved in the regulation of eIF4G phosphorylation, one that is rapamycin sensitive and one that is rapamycin insensitive (5, 47).

The rapamycin-sensitive pathway appears to predominate in vivo. A caveat to this conclusion surrounds the timing of rapamycin administration between the in vivo and in vitro studies. Rapamycin was administered prior to either meal feeding (0.25 h) or oral leucine administration (2 h) in the in vivo studies. In contrast, both rapamycin and leucine were present simultaneously from the time of the initiation of perfusion. Therefore, in the perfusion studies described herein, leucine may have induced phosphorylation of Ser1108 prior to maximal TORC1 inhibition by rapamycin. In contrast, in the in vivo studies, rapamycin-induced inhibition of mTOR prior to feeding or leucine gavage would have prevented the subsequent feeding- or leucine-induced phosphorylation of Ser1108. Such a scenario is consistent with observations in cells in culture where serum-induced phosphorylation of wild-type eIF4G on Ser1108 is blocked by rapamycin, but a variant lacking the NH2 terminus is resistant to rapamycin treatment (34). Hence, the kinase that phosphorylates Ser1108 is neither mTOR nor an mTOR-regulated protein kinase, and rapamycin-sensitive phosphorylation of residues in the NH2 terminus of eIF4G is permissive for phosphorylation of Ser1108 by a rapamycin-resistant kinase.

Increased phosphorylation of eIF4G occurs with conditions known to stimulate protein synthesis (5, 34), but the mechanism remains obscure. Changes in the extent of eIF4G phosphorylation are associated with increased formation of active eIF4G·eIF4E complex (5, 25, 34, 47). Assembly of the eIF4F complex is thought to be dependent, in part, upon both the availability of eIF4E, which is limited by its binding to the protein 4E-BP1. In the present study, rapamycin prevented the dissociation of the inactive eIF4E·4E-BP1 complex characteristic of meal feeding. This occurred despite an elevated level of 4E-BP1 in the {gamma}-form during meal feeding, indicating that factors other than 4E-BP1 phosphorylation can modify the extent of formation of eIF4E·4E-BP1 complex in skeletal muscle in vivo. Furthermore, rapamycin attenuates, but does not prevent, the leucine-induced stimulation of protein synthesis, even though the inhibitor completely prevents the leucine-induced increase in 4E-BP1 and S6K1 phosphorylation (3).

PKC{varepsilon} is a phosphatidyl serine/diacylglyceride-dependent, calcium-independent PKC isoform. PKC{varepsilon} is controlled via phosphorylation at three sites in the catalytic domain (Thr566 in the activation loop, Thr710 in the turn motif, and Ser729 in COOH-terminal hydrophobic motif). Phosphorylation of these sites is required for binding to and activation by diacylglyerol (6, 8, 15). The pathway(s) responsible for phosphorylation of the hydrophobic COOH-terminal site in the novel PKCs (PKC{delta} and PKC{varepsilon}) is not well understood, and the physiological kinase(s) remains obscure (28). In assessing the role of potential kinases controlling the phosphorylation of the COOH-terminal hydrophobic sites in nPKC, their phosphorylation is inhibited by the treatment of cells in culture with rapamycin (55). The phosphorylation is sensitive to the action of mTOR, as rapamycin inhibits serum-induced phosphorylation at this site in HEK293 cells in culture (27). These observations identify the nPKC isotypes as potential additional downstream targets of the mTOR pathway, and mTOR plays a selective role in controlling phosphorylation in the hydrophobic COOH-terminal site following serum starvation in cells in culture, consistent with a role for mTOR in nutrient sensing.

A role for mTOR in modifying PKC{varepsilon} phosphorylation does not appear to be supported in skeletal muscle in vivo. The phosphorylation of PKC{varepsilon} on the catalytic domain autophosphorylation site (Ser729) becomes elevated during meal feeding (41). Furthermore, activation of mTOR signaling pathway above basal conditions does not appear to be necessary to cause an increased phosphorylation PKC{varepsilon} following meal feeding. In the present study, rapamycin did not lower the extent of PKC{varepsilon} phosphorylation following meal feeding, leucine gavage, or perfusion of hindlimb with buffer containing 10x plasma leucine concentrations. Our previous report also indicated that rapamycin does not reduce the extent of phosphorylation of PKC{varepsilon} in hindlimb muscles perfused with elevated (10x) plasma concentrations of leucine (41). Thus, PKC{varepsilon} phosphorylation seems independent of TORC1, suggesting that signaling pathways other than mTOR are likely to be responsible for the increase in the extent of PKC{varepsilon} phosphorylation during meal feeding. In this regard, the atypical PKC, PKC{zeta}/{lambda} does not appear to be important in mediating the phosphorylation of PKC{varepsilon} following meal feeding, because no change in the extent of PKC{zeta}/{lambda} phosphorylation with meal feeding or with pretreatment with rapamycin was observed.

In summary, the studies provide evidence that rapamycin can partially limit the rise in the phosphorylation of eIF4G in response to either meal feeding or leucine administration. These findings suggest that changes in the extent of phosphorylation of eIF4G occur through both rapamycin-dependent and rapamycin-independent mechanisms in skeletal muscle. The effects of rapamycin on eIF4G phosphorylation are observed in both in vivo and in situ perfused muscles, suggesting that rapamycin directly modulates cell-signaling mechanisms responsible for eIF4G phosphorylation. In contrast, rapamycin was without effect on the nutrient-stimulated increase in the level of PKC{varepsilon} phosphorylation. Thus, phosphorylation of PKC{varepsilon} in response to meal feeding occurs in a rapamycin-independent manner, in contrast to eIF4G phosphorylation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institute of General Medical Sciences Grant GM-39277 (T. C. Vary) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-015658 (L. S. Jefferson), DK-053843, and DK-062880 (C. J. Lynch).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Vary, Dept. of Cellular and Molecular Physiology, Rm. C4710, Penn State University College of Medicine, H166, 500 University Drive, Hershey, PA 17033 (e-mail:tvary{at}psu.edu)

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.

* Current address of J. C. Anthony: Mead Johnson & Company, Evansville, IN 47721. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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