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Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, PA 17033
* To whom correspondence should be addressed. E-mail: clang{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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The 5'-AMP-activated protein kinase (AMPK) is an important sensor for monitoring cellular energy status (11). AMPK regulates carbohydrate, lipid, and protein metabolism in muscle via its effects on multiple signaling pathways and thereby suppresses ATP-demanding processes and activates ATP-repleting pathways. The acute metabolic consequences of AMPK activation on protein balance are mediated in part by the tuberous sclerosis complex (TSC)1 gene product and TSC2, which are upstream of mTOR (12). Both increased TSC2 phosphorylation, and enhanced formation of the TSC1·TSC2 heterodimer can negatively regulate mTOR activity (12,13). In addition, AMPK activation regulates gene expression of metabolic pathways influencing protein balance (14,15).
A variety of cellular stressors, including 2-deoxyglucose, nutrient deprivation, and exercise, activate AMPK by raising the cellular AMP:ATP ratio. In addition, AMPK can also be activated without altering endogenous adenine nucleotide concentration by the pharmacological agent 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) (11). Administration of AICAR in vivo blunts the anabolic actions of electrical stimulation in skeletal muscle (16) and the cardiac hypertrophy produced by pressure overload (17). Similarly, because AMPK is an obligate effector of many cellular responses to nutrient limitation, its activation may blunt the ability of the branched-chain amino leucine to stimulate protein synthesis. In this regard, during the preparation of this manuscript, Du et al. (18) reported that incubation of C2C12 myoblasts with AICAR prevented leucine-induced phosphorylation of mTOR and S6K1.
In general, both AICAR and leucine appear to have their effect on skeletal muscle predominantly via alterations in mTOR signaling as opposed to altering the eIF2/2B system and the formation of 43S preinitiation complex (19–21). Therefore, the current in vivo study focused on the ability of AICAR to modulate the anabolic effects of leucine by regulating steps in peptide-chain initiation involving the binding of mRNA to the 43S preinitiation complex, which is mediated by a functional eIF4F complex. Accordingly, we tested the hypothesis that direct AMPK activation by AICAR attenuates or prevents the ability of leucine to stimulate in vivo protein synthesis via inhibition of mTOR activity in skeletal muscle.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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18% protein, 6% fat, and 3.8% fiber. On the morning of the study, food was removed and rats were injected subcutaneously with AICAR (1 mg/g body weight; Toronto Research Chemicals) to stimulate AMPK. Time-matched control rats were injected with an equal volume of vehicle [sterile 0.9% saline (Sal)]. Rats were orally gavaged with leucine (1.35 g/kg body weight) or an equal volume of Sal 60 min after AICAR. Hence, this study contained 4 experimental groups: Sal + Sal, Sal + Leu (leucine), AICAR + Sal, and AICAR + Leu. The average body weight did not differ among the groups (data not shown). The dose and timing for AICAR were selected, because they have been reported to decrease basal muscle protein synthesis (20) and the leucine protocol shown to maximally stimulate muscle protein synthesis via increases in mTOR activity and cap-dependent translation (3,8,19,22).
After leucine gavage, rats were anesthetized with intraperitoneal pentobarbital and a catheter inserted into the carotid artery for blood collection. Exactly 20 min after leucine gavage, protein synthesis was determined using the flooding-dose method (23). Rats were injected with L-[2,3,4,5,6-3H]phenylalanine (Phe; 150 mmol/L, 30 mCi/L; 1 mL/100 g body weight) via the jugular vein and arterial blood collected into heparinized syringes at 2, 6, and 10 min for measurement of plasma Phe-specific radioactivity. Thereafter, a portion of the representative fast-twitch gastrocnemius was freshly homogenized and the remainder freeze-clamped and stored at –70°C. Muscle was powdered under liquid nitrogen and a portion used to estimate the rate of incorporation of [3H]Phe into protein, exactly as previously described (3,5,24).
Plasma hormones and tissue adenine nucleotides. Prior to the injection of [3H]Phe, arterial blood was collected and the plasma used to determine insulin (Linco Research), total insulin-like growth factor-I (Immunodiagnostic Systems Limited), and testosterone (DSL) by ELISA. Branched-chain amino acids were determined using reverse-phase HPLC after precolumn derivatization of amino acids with phenylisothiocyanate. Powdered gastrocnemius was extracted in cold perchloric acid, neutralized, and used for the determination of ATP, AMP, and creatine phosphate (CP) by standard fluorometric methods (25).
Immunoprecipitation and Western blot analysis.
The tissue preparation was the same as described by our laboratories (1–3,6,8,26). A muscle homogenate was prepared using a 1:5 ratio of ice-cold homogenization buffer consisting of (in mmol/L) 20 HEPES, pH 7.4, 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50 β-glycerophosphate, 1 dithiothreitol, 0.1 phenylmethylsulphonyl fluoride, 1 benzamidine, and 0.5 sodium vanadate, plus a protease inhibitor cocktail table from Roche, and clarified by centrifugation. The samples were subjected to SDS-PAGE and the proteins electrophoretically transferred to polyvinylidene difluoride membranes. The blots were incubated with either primary antibodies (unless otherwise noted from Cell Signaling) to total (C-20) and Thr1462-phosphorylated TSC2, total G protein β-subunit-like protein [GβL; mLST8], total and phosphorylated (Ser792) raptor, total S6K1 (Santa Cruz Biotechnology), phospho-specific S6K1 (Thr389;), total 4E-BP1 (Bethyl Laboratories), phospho-specific 4E-BP1 (Thr37/46), total and phosphorylated (Ser1108) eIF4G, total and phosphorylated-S6 (Ser240/Ser244), total and phosphorylated (Thr56) eukaryotic elongation factor (eEF)-2, and total and phosphorylated (Thr246) proline-rich Akt substrate 40 (PRAS40) (Biosource). In addition, to determine activation of AMPK, total and phosphorylated AMPK
(Thr172) and acetyl-CoA carboxylase (Ser79) were used (14). Blots were washed with 1x Tris-buffered Sal including 0.1% Tween-20 and incubated with secondary antibody (horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit) IgG at room temperature. The blots were developed with enhanced chemiluminescence Western blotting reagents, as per the manufacturer's (Amersham) instructions, and exposed to X-ray film in a cassette equipped with a DuPont Lightning Plus intensifying screen. After development, the film was scanned (Microtek ScanMaker IV) and analyzed using NIH Image 1.6 software.
The eIF4E·4EBP1and eIF4E·eIF4G complexes were quantified as described (3,6,8,26). From an aliquot of supernatant, eIF4E was immunoprecipitated using an anti-eIF4E monoclonal antibody (kindly provided by Drs. Jefferson and Kimball, Pennsylvania State College of Medicine). Antibody-antigen complexes were collected using magnetic beads, subjected to SDS-PAGE, and proteins transferred to a PVDF membrane. Blots were incubated with a mouse anti-human eIF4E antibody, rabbit anti-rat 4E-BP1 antibody, or rabbit anti-eIF4G antibody.
To maintain protein-protein interaction, muscle samples were homogenized in CHAPS buffer consisting of (in mmol/L) 40 HEPES, pH 7.5, 120 NaCl, 1 EDTA, 10 pyrophosphate, 10 β-glycerol phosphate, 50 NaF, 1.5 sodium vanadate, 0.3% CHAPS, and 1 protease inhibitor cocktail tablet. The homogenate was mixed on a platform rocker and clarified by centrifugation. An aliquot of the resulting supernatant was combined with either anti-TSC2, anti-mTOR, or anti-raptor antibody and immune complexes isolated with goat anti-rabbit BioMag IgG beads (PerSeptive Diagnostics). Beads were collected, washed with CHAPS buffer, precipitated by centrifugation (14,000 x g; 10 min), and subjected to SDS-PAGE and analysis as above.
Statistical analysis. Data for each condition are summarized as means ± SE, where the number of rats per treatment group is indicated in the legends of the figure or table. Unless otherwise indicated, statistical evaluation of the data was performed using 2-way ANOVA with post hoc Student-Newman-Keuls test when the interaction was significant. When necessary, data were logarithmically transformed to normalize the distribution of residuals and to obtain variance homogeneity. Differences were considered significant when P < 0.05.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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AICAR alone decreased the plasma insulin concentration by 45% compared with time-matched values from control rats (Table 1). Conversely, leucine alone increased insulin 49% compared with control values. In contrast, no hyperinsulinemia was detected in rats in the AICAR + Leu group. Neither AICAR nor leucine significantly altered the plasma concentration of either insulin-like growth factor-I or testosterone at the time point assessed (data not shown).
Activation of AMPK, adenine nucleotide concentration, and protein synthesis in muscle. AICAR administered in vivo activated AMPK in gastrocnemius as indicated by the increased Thr172 phosphorylation (control = 1.00 ± 0.07-fold vs. AICAR = 2.33 ± 0.36-fold of Sal control; P < 0.05). Furthermore, AICAR increased Ser79 phosphorylation of acetyl-CoA carboxylase, a well-established substrate of AMPK (control = 1.00 ± 0.11-fold vs. AICAR = 1.79 ± 0.21-fold of Sal control; P < 0.05).
The in vivo-determined rate of muscle protein synthesis was reduced 34% by AICAR (Fig. 1). Protein synthesis increased 28% in the Sal + Leu group compared with that of the Sal + Sal group. In contrast, leucine did not increase protein synthesis in gastrocnemius from AICAR-treated rats.
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AICAR alone decreased the active eIF4E·eIF4G complex by 65% in muscle from rats in the Sal + Sal group (Fig. 3A,D). Contrary to expectations, the inactive eIF4E·4EBP1 complex did not significantly increase (Fig. 3B,E). Whereas leucine increased eIF4E·eIF4G and produced a reciprocal decrease in the formation of the eIF4E·4EBP1 complex in control rats, AICAR completely prevented the leucine-induced redistribution of eIF4E between the inactive eIF4e·4EBP1 and active eIF4E·eIF4G complex.
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eEF2 phosphorylation. Select catabolic agents may also impair protein synthesis via enhanced phosphorylation (Thr56) of eEF and inhibition of translation elongation (30). However, at the time point assessed, the phosphorylation of eEF2 in muscle was not significantly altered by AICAR (control = 1.00 ± 0.11 arbitrary units vs. AICAR = 0.94 ± 0.08 arbitrary units). The amount of total eEF2 was also not altered by either AICAR and/or leucine (data not shown).
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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A possible mechanism by which AMPK might cause mTOR dysregulation involves its inhibition of AKT-dependent phosphorylation of TSC2 and/or the binding of TSC2 with its homolog TSC1, which increases the binding of GDP to Ras homolog enriched in brain (Rheb) and thereby inhibits mTOR activity. Previous studies using cultured myocytes have demonstrated such changes in TSC2 (13). However, under in vivo conditions, AMPK activation in muscle resulted in a relatively small decrease in TSC2 phosphorylation under basal conditions and this change was not associated with a change in either TSC1·TSC2 binding or the total amount of TSC1 or TSC2 protein.
Alternatively, mTOR activity can be regulated by its binding to various proteins, including raptor, which is sensitive to nutrient and hormonal regulation (29). Incubation of cultured myocytes with AICAR increases the binding of raptor to mTOR (13), which may imply AICAR promotes a "closed conformation" in the mTOR·raptor complex, rendering it less active. However, in other cells, AICAR does not alter mTOR·raptor complex function (35) and whether raptor enhances or represses mTOR activity remains controversial (36,37). Regardless, our present data fail to confirm any change in mTOR·raptor binding in skeletal muscle in response to AICAR. Furthermore, there was no AICAR-induced change in either the total amount of GβL, a positive regulator of mTOR activity (38), or its binding to mTORC1.
The mTORC1 complex also binds PRAS40 via its interaction with raptor (39,40). Overexpression of PRAS40 inhibits mTOR autophosphorylation and kinase activity. However, the ability of in vivo administered AICAR to inhibit basal muscle protein synthesis was not associated with a change in either the total amount of PRAS40 or its binding with raptor in the mTORC1. PRAS40 is also a phospho-protein and the extent of its phosphorylation is inversely proportional to the concentration of PRAS40 (39,40), suggesting PRAS40 is also a downstream substrate for mTOR. Our data showing that AICAR decreases basal PRAS40 phosphorylation are consistent with this view.
Recently, AICAR-induced activation of AMPK in myocytes and other cells has been reported to increase the phosphorylation of raptor via a TSC2-independent mechanism and thereby impair mTORC1 kinase activity (35). In the current study, the leucine-induced stimulation of muscle protein synthesis was not associated with a change in raptor phosphorylation. However, AICAR markedly increased Ser792 phosphorylation of raptor in muscle of control rats. Because raptor phosphorylation was similarly increased in both control + AICAR and Leu + AICAR rats, the enhanced phosphorylation of this mTORC1 complex protein remains a possible mechanism by which AICAR produces leucine resistance in skeletal muscle.
In addition to its role as a precursor molecule, leucine also regulates protein synthesis by activating intracellular signaling pathways stimulating translation initiation (40). Our current data, which show increased phosphorylation of 4E-BP1, S6K1, S6, and eIF4G as well as increased eIF4E·eIF4G complex after leucine, are consistent with reports indicating stimulation in cap-dependent translation and mTOR activity (21). However, increased mTOR activity could not be ascribed to a change in mTOR·raptor binding, phosphorylation of raptor, or the association of PRAS40 with raptor. The inability of amino acids to alter PRAS40·raptor binding in cultured cells has been reported and differs from the marked decrease in PRAS40·raptor seen in insulin-treated cells (39). Furthermore, oral administration of leucine to rats also did not alter TSC2 phosphorylation or its binding with TSC1, results consistent with studies showing that nutrients can activate mTOR by a TSC-independent pathway (41).
Several catabolic conditions, including sepsis, alcohol abuse, and glucocorticoid excess in aged rats, produce a leucine resistance in striated muscle (3,6–10). AICAR also prevents mTOR activation and hypertrophy in cardiac muscle in response to pressure overload (17) and in skeletal muscle following electrical stimulation (16). Our results indicate that AICAR-induced activation of AMPK also antagonizes the normal protein anabolic response to leucine. The ability of AICAR to produce leucine resistance is associated with inhibition of mTOR activity, as evidenced by the failure of leucine to enhance 4E-BP1 and S6K1 phosphorylation. Further, this AICAR-induced defect in 4E-BP1 phosphorylation seems the likely cause for the inability of leucine to alter the distribution of eIF4E between the active and inactive complexes. Moreover, AICAR essentially prevented the ability of leucine to increase eIF4G phosphorylation. A similar reduction in leucine-stimulated eIF4G phosphorylation has been reported in muscle of rats given the mTOR inhibitor rapamycin (42). Conversely, the AICAR-induced decrement in mTOR activity could not be attributed to a change in TSC2 phosphorylation, the binding of TSC2 with TSC1, or to the amount of mTOR·raptor or PRAS40·raptor complex. It remains possible that AICAR caused leucine resistance by impairing the subsequent movement of the mTOR·raptor complex to the eIF3-preinitiation complex or via activation of human vacuolar protein sorting (hvps)-34, which is a class III phosphatidylinositol 3-kinase (43); however, these possibilities were not pursed in the current investigation.
The ability of AICAR to inhibit protein synthesis appears to be mediated primarily by inhibition of translation initiation, because in this study, AICAR did not alter the expression or phosphorylation of eEF2. Hyperphosphorylation of eEF2 renders this protein inactive and thereby inhibits protein synthesis (30). However, our data are in apparent contrast to those reported in rat muscle 40 min after AICAR administration (16). The difference between our study and this earlier report may be related to the relatively transient effect of AICAR on this particular parameter. This is exemplified by in vitro studies showing AICAR increases eEF2 phosphorylation time dependently, with eEF2 phosphorylation increased at 15–30 min but comparable to basal levels at 60 min (13).
The AICAR-induced decrease in muscle protein synthesis also could not be attributed to a difference in the circulating concentrations of the branched-chain amino acids. Leucine, isoleucine, and valine concentrations were as high in AICAR + Leu rats as in the Sal + Leu rats. However, AICAR attenuated the leucine-induced hyperinsulinemia. Therefore, the lack of a permissive effect of insulin with leucine in the AICAR + Leu-treated rats might partially explain the inability of muscle protein synthesis to increase after AICAR. The role of the transient leucine-induced hyperinsulinemia in mediating the anabolic effect of leucine on muscle protein synthesis is currently unclear [see discussion in (44)] and the relative importance of this diminished insulin response in AICAR-treated rats compared with a direct effect on muscle per se will need to be defined in future studies. However, it is noteworthy that AICAR has been shown to inhibit both basal and leucine-stimulated mTOR activity in cultured myocytes (18), suggesting that at least a portion of the AICAR effect is mediated directly within muscle.
Finally, previous studies in myoblasts suggested the ability of leucine to stimulate protein synthesis is mediated by decreased AMPK activity resulting from a concomitant elevation in the ATP concentration (18). However, such an effect of leucine on AMPK activity and ATP concentration in skeletal muscle under in vivo conditions was not observed in the current study. Therefore, whereas the anabolic effect of leucine on mTOR-stimulated protein synthesis appears mediated via a suppression of AMPK activity in myoblasts incubated under conditions of low serum and energy substrates, such a mechanism is clearly not operational in skeletal muscle under in vivo conditions.
In conclusion, we describe herein that stimulation of AMPK activity by the chemical activator AICAR produces a muscle leucine resistance under in vivo conditions. The diminution of the anabolic response to leucine by AICAR is associated with a reduction in mTOR activity (evidenced by the decreased phosphorylation of 4E-BP1, S6K1, eIF4G, and PRAS40), maintained enhanced raptor phosphorylation, and a reduction in the active eIF4E·eIF4G complex. Moreover, these changes were independent of changes in TSC2 phosphorylation, TSC1·TSC2 heterodimer formation, and the binding of either mTOR or PRAS40 with raptor. Finally, our data suggest that some mechanisms regulating mTOR activity in cell culture may not necessarily be operational under in vivo conditions.
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FOOTNOTES |
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2 Author disclosures: A. M. Pruznak, A. A. Kazi, R. A. Frost, T. C. Vary, and C. H. Lang, no conflicts of interest.
3 Abbreviations used: AICAR, 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside; AMPK, 5'-AMP-activated protein kinase; CP, creatine phosphate; 4E-BP1, eukaryotic initiation factor 4E binding protein-1; eEF, eukaryotic elongation factor; eIF, eukaryotic initiation factor; GβL, G protein β-subunit-like protein; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; PRAS40, proline-rich Akt substrate 40; Sal; saline; S6K1, 70-kDa ribosomal protein S6 kinase; TSC, tuberous sclerosis complex.
Manuscript received 9 June 2008. Initial review completed 27 June 2008. Revision accepted 30 July 2008.
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LITERATURE CITED |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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1. Lang CH, Frost RA, Vary TC. Regulation of muscle protein synthesis during sepsis and inflammation. Am J Physiol Endocrinol Metab. 2007;293:E453–9.
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3. Lang CH, Frost RA. Endotoxin disrupts the leucine-signaling pathway involving phosphorylation of mTOR, 4E–BP1, and S6K1 in skeletal muscle. J Cell Physiol. 2005;203:144–55.[Medline]
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5. Svanberg E, Frost RA, Lang CH, Isgaard J, Jefferson LS, Kimball SR, Vary TC. IGF-I/IGFBP-3 binary complex modulates sepsis-induced inhibition of protein synthesis in skeletal muscle. Am J Physiol Endocrinol Metab. 2000;279:E1145–58.
6. Lang CH, Frost RA. Differential effect of sepsis on ability of leucine and IGF-I to stimulate muscle translation initiation. Am J Physiol Endocrinol Metab. 2004;287:E721–30.
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- (top band) isoforms are identified. The total amount of S6 protein was unchanged by the various treatments and indicates equal protein loading per lane (D). Total 4E-BP1 and S6K1 did not differ between groups (data not shown). Bar graphs quantitating the densitometric analysis of all immunoblots for phosphorylated 4E-BP1 (E) and S6K1 (F). The value from the Sal + Sal group was set at 1.0. Values in bar graph are means ± SEM; n = 8–10. Means without a common letter differ, P < 0.05.
