TNF-alpha impairs heart and skeletal muscle protein synthesis by altering translation initiation

Charles H. Lang1, Robert A. Frost1, Angus C. Nairn2, David A. MacLean1, and Thomas C. Vary1

1 Departments of Cellular and Molecular Physiology and Surgery, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

This study examined potential mechanisms contributing to the inhibition of protein synthesis in skeletal muscle and heart after administration of tumor necrosis factor (TNF)-alpha . Rats had vascular catheters implanted, and TNF-alpha was infused continuously for 24 h. TNF-alpha decreased in vivo-determined rates of global protein synthesis in gastrocnemius (39%) and heart (25%). The TNF-alpha -induced decrease in protein synthesis in the gastrocnemius involved a reduction in the synthesis of both myofibrillar and sarcoplasmic proteins. To identify potential mechanisms responsible for regulating mRNA translation, we examined several eukaryotic initiation factors (eIFs) and elongation factors (eEFs). TNF-alpha decreased the activity of eIF-2B in muscle (39%) but not in heart. This diminished activity was not caused by a reduction in the content of eIF-2Bepsilon or the content and phosphorylation state of eIF-2alpha . Skeletal muscle and heart from TNF-alpha -treated rats demonstrated 1) an increased binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF-4E, 2) a decreased amount of eIF-4E associated with eIF-4G, and 3) a decreased content of the hyperphosphorylated gamma -form of 4E-BP1. In contrast, the infusion of TNF-alpha did not alter the content of eEF-1alpha or eEF-2, or the phosphorylation state of eEF-2. In summary, these data suggest that TNF-alpha impairs skeletal muscle and heart protein synthesis, at least in part, by decreasing mRNA translational efficiency resulting from an impairment in translation initiation associated with alterations in eIF-4E availability.

eukaryotic initiation factors-2B and -4E; 4E-binding protein-1; elongation factors-1 and -2; rats; tumor necrosis factor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha is a pleiotropic cytokine, the overexpression of which appears responsible for many of the metabolic alterations observed in response to various catabolic conditions (4, 11, 22, 58). One of the metabolic hallmarks produced by TNF-alpha is the erosion of lean body mass as evidenced by a decrease in total carcass nitrogen and a pronounced impairment in muscle protein balance (1, 37, 48, 50). This imbalance was observed in early studies where the acute administration of TNF-alpha to naive control animals or humans led to an increased urinary nitrogen excretion and efflux of amino acids from muscle (15, 16, 46, 55). Moreover, acute in vivo administration of TNF-alpha has been shown to impair amino acid uptake by muscle (2, 57). However, subsequent investigations generally failed to detect significant alterations in either the rate of protein synthesis or protein degradation in response to the acute injection of TNF-alpha (8, 25, 40, 46). More recently, Zamir et al. (58) was able to demonstrate a significant increase in proteolysis and a tendency for a decrease in protein synthesis in skeletal muscle incubated in vitro 2 h after a single injection of TNF-alpha . These investigators postulated that the apparent discrepancy between their work and earlier studies may be related to the interval between the time of cytokine injection and tissue removal. That is, TNF-alpha was shown to increase proteolysis when muscle was examined 2 h post-TNF-alpha (58) but not when protein balance was determined after 6-8 h (19). The limited ability of TNF-alpha to impair muscle protein balance at these later time points is consistent with the rapid clearance of TNF-alpha from the systemic circulation (5). However, in previous studies a primed, constant intravenous infusion of this cytokine for 24 h has been demonstrated to produce a severe insulin resistance in skeletal muscle (29) and to markedly decrease the concentration of the anabolic hormone insulin-like growth factor (IGF) I in both the circulation and muscle (12). These data suggest that a more prolonged exposure of tissues to TNF-alpha may be a prerequisite for producing detectable cytokine-induced changes in muscle protein synthesis in vivo.

The decrease in muscle protein synthesis observed in a number of cachectic conditions is caused by a decreased translation efficiency rather than a reduction in the number of ribosomes (10, 30, 45). The translation of mRNA into protein is often divided into the following three stages: initiation, elongation, and termination. In sepsis and other catabolic conditions, the decrease in muscle protein synthesis is associated with defects in mRNA translation initiation, the first rate-determining phase of protein synthesis (10, 31, 44). Translation initiation is regulated by a large number of protein factors termed eukaryotic initiation factors (eIFs). One of these initiation factors, eIF-2, mediates the first step in initiation and promotes the attachment of the initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit to form the 43S preinitiation complex (10, 42). A second critical point of translational regulation involves the binding of the 5'-end of cellular mRNA to the 43S preinitiation complex, which is mediated by the cap-binding protein complex eIF-4F (42, 44). Although the decrease in muscle protein synthesis in various catabolic conditions has been generally linked to inhibition of peptide-chain initiation, the exact site of the molecular lesion often varies (10, 31, 44).

Although translational controls operate most frequently during the initiation phase, some studies have also implicated alterations in elongation as a control point in the translation of specific mRNAs (38). Both eukaryotic elongation factor (eEF)-1 and -2 are critical for catalyzing the sequential addition of amino acid residues to the COOH-terminal end of the nascent peptide. eEF-1alpha promotes the GTP-dependent recruitment of aminoacyl-tRNAs to the A-site of ribosomes. eEF-2 is also important for normal mRNA translation and catalyzes the translocation of the peptidyl-tRNA from the A-site to the P-site on the ribosome. Alterations in the amount and/or phosphorylation state of eEF-1 and eEF-2 have been linked to changes in protein synthesis (35, 53).

The purpose of the present study was to determine whether a prolonged continuous infusion of TNF-alpha would decrease skeletal muscle protein synthesis and, if so, to determine whether this response was associated with defects in peptide-chain initiation, via alterations in eIF-2 and/or eIF-4F, or an impairment in elongation. Because defects in myocardial performance are often observed in response to TNF-alpha and various catabolic conditions (28, 41), we also examined the mechanism by which the infusion of TNF-alpha impairs protein synthesis, translation initiation, and elongation in heart. Finally, the TNF-alpha -induced change in global protein synthesis in various nonmuscle tissues was also determined and compared with that observed in different striated muscles.


    METHODS AND MATERIALS
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Animal preparation and experimental protocol. Adult specific pathogen-free male Sprague-Dawley rats (285-310 g; Charles River Breeding Laboratories, Cambridge, MA) were housed at a constant temperature, exposed to a 12:12-h light-dark cycle, and maintained on standard rodent chow and water ad libitum for at least 1 wk before experiments were performed. All experiments were approved by the Animal Care and Use Committee at the Pennsylvania State University College of Medicine and adhered to the National Institutes of Health guidelines for the use of experimental animals.

Rats were anesthetized with an intramuscular injection of ketamine and xylazine (90 and 9 mg/kg, respectively), and sterile surgery was performed to implant catheters in the carotid artery and jugular vein, as previously described (12, 29). Briefly, the venous catheter was passed through a tightly coiled stainless steel spring and was fixed to a freely rotating swivel (Instech, Plymouth, PA); the arterial catheter was coiled and secured with tape on the dorsal surface of the rat. The intravenous infusion of TNF-alpha or vehicle was started after the animals had regained consciousness. Recombinant human TNF-alpha (Amgen, Thousand Oaks, CA) was diluted in 0.1% human serum albumin and infused intravenously for the next 24 h at a rate of 5 µg · kg-1 · h-1 (0.35 ml/h). Time-matched control animals were infused with an equal volume of vehicle. After surgery, all rats were housed in individual cages with no food but with water ad libitum. We have previously reported that this experimental protocol elevates the blood TNF-alpha concentration to ~500 pg/ml (12). Comparable TNF-alpha values are observed in a number of inflammatory conditions, including sepsis, neoplastic disease, burn injury, and hepatic ischemia-reperfusion injury (6, 7, 9, 11), but levels are markedly lower than those seen in rats injected with nonlethal doses of endotoxin (18). In addition, this dose of TNF-alpha also produces peripheral insulin resistance (29) and decreases the IGF-I content in blood and muscle (12).

Protein synthesis. After the start of the TNF-alpha infusion (24 h), the in vivo rate of global protein synthesis was determined for various tissues using the flooding-dose technique, as originally described by Garlick et al. (17). Animals were injected intraperitoneally with L-[2,3,4,5,6-3H]phenylalanine (Phe; 150 mM, 30 µCi/ml; 1 ml/100 g body wt). Later (10 min), a blood sample was collected from the arterial catheter into heparinized syringes. The gastrocnemius, psoas, soleus, heart, diaphragm, liver, small intestine, kidney, spleen, and brain were rapidly excised and weighed. A portion of the gastrocnemius and heart from each animal was taken for measurement of eIF-2B activity and analysis of the eIF-4E system, and the remaining tissue was frozen between aluminum blocks precooled to the temperature of liquid nitrogen. The frozen tissues were later powdered under liquid nitrogen using a mortar and pestle. Blood was centrifuged (13,000 g for 1 min at 4°C), and plasma was collected. All tissue and plasma samples were stored at -70°C until analyzed.

A portion of the powdered tissue was homogenized in ice-cold perchloric acid (PCA), and the supernatant was used to estimate the rate of incorporation of [3H]Phe into protein exactly as described previously (30, 45). The fractional rate of protein synthesis (ks) represents the percentage of the tissue protein pool synthesized each day. The ks was calculated from the specific radioactivity of phenylalanine in protein (SB) and that of free phenylalanine in the tissue (Si) 10 min after injection of the radioactive phenylalanine using the following equation: ks = (SB × 100)/(Si × t), where t is the period of incorporation (days). The assumption in using this technique to estimate the rate of protein synthesis in vivo is that the tissue phenylalanine concentration is elevated to a high concentration, thereby limiting any dilution effect of nonradioactive phenylalanine derived from proteolysis on the intracellular specific radioactivity. Under the condition of elevated plasma phenylalanine concentrations (~1.3 ± 0.9 mM), the specific radioactivity of plasma phenylalanine is assumed to be equal to the specific radioactivity of tRNA-bound phenylalanine. Studies by McKee et al. (36) and Williams et al. (56) have shown in the isolated perfused heart that at a perfusate phenylalanine concentration of 0.4 mM, the perfusate, intracellular, and tRNA-bound phenylalanine have the same specific radioactivity within 10 min of the start of perfusion with radioisotopes.

A portion of frozen gastrocnemius was used to separate the myofibrillar and sarcoplasmic proteins according to the procedures described previously (54). Briefly, muscle was homogenized in ice-cold buffer containing 10 mM KH2PO4 (pH 7.4) using a motor-driven glass-on-glass homogenizer. The samples were centrifuged at 3,000 g at 4°C for 20 min. The pellet contains the myofibrillar proteins, and the supernatant contains the sarcoplasmic proteins. The pellet containing the myofibrillar proteins was washed two times with buffer containing 0.1 mM KH2PO4 (pH 7.4). The supernatant fractions from these washes were pooled and added to the nonmyofibrillar fraction. The myofibrillar pellet was dissolved in 0.6 M NaOH and centrifuged, and the supernatant was used to determine radioactivity in myofibrillar proteins. Both the myofibrillar and sarcoplasmic protein fractions were treated with an equal volume of 10% TCA. The supernatants were discarded, and the precipitated protein fractions were processed as described above for measurement of incorporation of radioactivity in total protein.

Total RNA. Because some catabolic conditions can lead to a decrease in total RNA within 24 h (43), the total RNA was measured from muscle homogenates, as previously described (30). Briefly, frozen powdered tissue was homogenized in 5 vol of ice-cold 10% TCA. After centrifugation, the supernatant was discarded, and the remaining pellet was mixed with 6% PCA. The sample was centrifuged at 10,000 g for 6 min at 4°C. The supernatant was discarded, and the procedure was repeated. Next, 0.3 N KOH was added to the pellet, and the samples were placed in a 50°C water bath for 1 h. Samples were then mixed with 4 N PCA and centrifuged. The concentration of RNA in the supernatant was determined by measuring the absorbance at 260 nm and correcting for the absorbance at 232 nm. These data were then used to calculate translational efficiency, which equals the ks for a particular tissue divided by the RNA content for that tissue.

Isolation of ribosomal subunits. Fresh muscle tissue (psoas and heart) was used to isolate 40S and 60S ribosomal subunits by sucrose density gradient centrifugation (51). Briefly, muscles were homogenized in a motor-driven glass-on-glass homogenizer in 4 vol of homogenization buffer [14 mM triethanolamine (pH 7.0), 2 mM magnesium acetate, 250 mM KCl, 0.5 mM dithiothreitol (DTT), 0.08 mM EDTA, 5 mM EGTA, 250 mM sucrose, and 1 mg nagarse (protease, type XXVII); Sigma Chemical, St. Louis, MO]. The homogenate was centrifuged at 10,000 g for 15 min, and the supernatant was recovered. Aliquots of the samples (0.7 ml), to which 0.1 vol of 10% (wt/vol) Triton X-100 and deoxycholate solution had been added, were then layered on 0.44-2.0 M exponential sucrose gradients. The samples were centrifuged at 167,000 g in a SW41 rotor (Beckman Instruments) for 20 h to resolve the 40S and 60S ribosomal subunits. The absorbance of the gradients was monitored at 254 nm, and fractions were collected using a density gradient fractionator (Instrumentation Specialties, Lincoln, NE). These data provide information pertaining to changes in peptide-chain initiation relative to changes in elongation/termination.

Amount of eIF-2 and eIF-2B. The relative amounts of the alpha -subunit of eIF-2 (eIF-2alpha ), the phosphorylated form of eIF-2alpha , and the epsilon -subunit of eIF-2B (eIF2-Bepsilon ) in gastrocnemius and heart were estimated by protein immunoblot analysis, as described previously (30,32, 54). eIF-2 and eIF-2B were chosen because change in the content and/or activity of these initiation factors correlates with alterations in protein synthesis (10, 42). eIF-2 consists of three subunits of which the alpha -subunit appears important in regulating protein synthesis (42). Likewise, eIF-2B is a multimeric protein consisting of five subunits, with the epsilon -subunit being the catalytic subunit (42). Previous studies have established that the expression of the epsilon -subunit is representative of other subunits (54). Therefore, the relative abundance of eIF-2Bepsilon is believed to be representative of the eIF-2B holoenzyme. Briefly, tissue was homogenized in 7 vol of buffer composed of (in mM) 20 Tris (pH 7.4), 250 sucrose, 100 KCl, 0.2 EDTA, 1 DTT, 50 NaF, 50 beta -glycerolphosphate, 1 phenylmethylsulfonyl fluoride (PMSF), 1 benzamidine, and 0.5 sodium vanadate. The samples were mixed with 2× Laemmli SDS buffer, boiled, and centrifuged. Equal amounts of protein from tissue homogenates were electrophoresed in a 12.5% polyacrylamide gel. After electrophoresis, proteins in the gel were transferred to nitrocellulose. After blocking for 30 min with nonfat milk (5% wt/vol) in 25 mM Tris (pH 7.6)-0.9% saline containing 0.01% Tween 20 (Tris-NaCl-Tween), the membranes were washed extensively in Tris-NaCl-Tween. The nitrocellulose was incubated for 1 h at room temperature with antibodies specific for either eIF-2alpha , Ser51-phosphorylated eIF-2alpha , or eIF-2Bepsilon . Antibodies were visualized using an enhanced chemiluminescence (ECL) procedure with the secondary antibody linked to horseradish peroxidase (Amersham). The blots were 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 National Institutes of Health Image 1.6 software.

Determination of eIF-2B activity. eIF-2B activity in tissue was measured in postmitochondrial supernatants using a [3H]GDP-GDP exchange assay, as previously described (30, 32). Fresh tissue was homogenized in buffer consisting of (in mM) 20 triethanolamine (pH 7.0), 2 magnesium acetate, 150 KCl, 0.5 DTT, 0.1 EDTA, 250 sucrose, 5 EGTA, and 50 beta -glycerolphosphate. The homogenate was centrifuged at 15,000 g for 15 min at 4°C. The supernatant was assayed immediately for eIF-2B activity by measuring the decrease in the eIF-2 · [3H]GDP complex bound to nitrocellulose filters. The rate of exchange was linear over the time points measured (data not shown). Under these conditions, ~50% (0.3 pmol) of the [3H]GDP was released from eIF-2 · [3H]GDP during the assay procedure.

Measurement of phosphorylation state of eIF-2alpha . The relative amount of eIF-2alpha present in the phosphorylated form, designated eIF-2alpha (P), was estimated by immunological visualization of proteins after separation using slab-gel isoelectric focusing (IEF; see Refs. 30, 32, and 54). Tissues were homogenized in the same buffer as described above for eIF-2. A 75-µl aliquot of the homogenate was mixed with 42.9 mg of urea and 300 µl of IEF sample buffer [9.5 M urea, 2% Nonidet P-40, ampholytes (BHD Resolyte 4-8), and 0.7 M beta -mercaptoethanol]. The samples were electrofocused and then transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Bio-Rad Laboratories, Hercules, CA). The membranes were subsequently incubated with an eIF-2alpha monoclonal antibody that recognizes both phosphorylated and nonphosphorylated forms of the protein. eIF-2alpha was visualized as described above. The proportion of eIF-2alpha present in the phosphorylated state was measured by densitometric scanning of the membranes and was expressed as a percentage of the total eIF-2alpha content (i.e., phosphorylated + unphosphorylated).

Analysis of 4E-binding protein-1 · eIF-4E and eIF-4G · eIF-4E complexes. The association of eIF-4E with either 4E-binding protein-1 (BP1) or eIF-4G was determined as previously described (30, 32, 45). Briefly, tissue was homogenized in 7 vol 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 alpha -glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM microcystin LR) using a Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. eIF-4E, 4E-BP1 · eIF-4E, and eIF-4G · eIF-4E complexes were immunoprecipitated from aliquots of 10,000-g supernatants using an anti-eIF-4E monoclonal antibody. The antibody-antigen complex was collected by incubation for 1 h with BioMag goat anti-mouse IgG beads (Perseptive Biosystems, Framingham, MA). Before use, the beads were washed in 1% nonfat dry milk in buffer B (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% alpha -mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM alpha -glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium vanadate). The beads were captured using a magnetic sample rack and were washed two times with buffer B and one time with buffer B containing 500 mM NaCl rather than 150 mM. Protein bound to the beads was eluted by boiling in SDS-sample buffer for 5 min. The beads were collected by centrifugation, and the supernatants were subjected to electrophoresis either on a 7.5% polyacrylamide gel for analysis of eIF-4G or on a 15% polyacrylamide gel for quantitation of 4E-BP1 and eIF-4E. Proteins were then electrophoretically transferred to nitrocellulose. The membranes were incubated with a mouse anti-human eIF-4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-eIF-4G antibody for 1 h at room temperature. The blots were developed using ECL, and autoradiographs were scanned and analyzed as described above.

Phosphorylation state of eIF-4E and 4E-BP1. The phosphorylated and nonphosphorylated forms of eIF-4E in tissue extracts were separated by IEF on a slab gel and analyzed by protein immunoblot analysis, as previously described (30, 32). The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from tissue homogenates after centrifugation at 10,000 g. 4E-BP1 was immunoprecipitated as described above for immunoprecipitation of eIF-4E. The various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting as described above.

Analysis of elongation factors eEF-1 and eEF-2. Muscles were homogenized in 7 vol of buffer A. The samples were mixed with equal volumes of 2× Laemmli SDS sample buffer (60°C), boiled for 3 min, and centrifuged. Equal amounts of protein were subjected to SDS-PAGE using Criterion Precast 10-20% Tris · HCl gradient gels (Bio-Rad Laboratories) followed by transfer of proteins to PVDF membranes as described previously (53). After blocking with nonfat dry milk, the membranes were incubated with antibodies specific for eEF-1A (Santa Cruz Antibodies, Santa Cruz, CA) or eEF-2 (35, 53). The phosphorylation status of eEF-2 in the tissue homogenate was analyzed by sequential immunoblotting first with an antibody that specifically recognized eEF-2 phosphorylated on Thr56 (35). After development of the blot as described above, the membranes were treated with SDS as per the manufacture's instruction to remove antibodies. The membranes were then blocked with milk and probed with an antibody that recognizes eEF-2 independent of its phosphorylation state. The blots were developed using ECL, and the autoradiographs were scanned and analyzed as described above.

Hemodynamic and metabolic determinations. Mean arterial blood pressure (MABP) and heart rate (HR) were determined by a pressure transducer attached to the arterial catheter before the injection of [3H]Phe. Colonic temperature was determined immediately after determination of MABP and HR, as previously described (29). The plasma concentrations of insulin, IGF-I, and corticosterone were determined by RIA (12, 29). Leptin concentrations were also determined by RIA (Linco Research, St. Charles, MO). Amino acid concentrations were determined using reverse-phase HPLC after precolumn derivatization of amino acids with phenylisothicyanate, as previously described (34). Plasma free fatty acid (FFA) concentrations were determined colorimetrically (Waco Chemicals, Richmond, VA).

Statistics. Values are presented as means ± SE. The number of rats in each group is indicated in Figs. 1-6 and Tables 1-4. Data were analyzed by Student's t-test to determine the treatment effect. Statistical significance was set at P < 0.05. 


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Fig. 1.   Effect of tumor necrosis factor (TNF)-alpha on the fractional rate of protein synthesis (ks; A), RNA content (B), and translational efficiency (C) in various striated muscles. Tissues were collected 24 h after starting a primed continuous infusion of human recombinant TNF-alpha or from time-matched control rats. Values are means ± SE; n = 7 rats/group. *P < 0.05 compared with control values for the same tissue.



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Fig. 2.   Effect of TNF-alpha on the fractional synthesis rate (FSR) of myofibrillar (A) and sarcoplasmic (B) proteins in gastrocnemius. Values are means ± SE; n = 7/group. *P < 0.05 compared with control values for the same tissue.



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Fig. 3.   Effect of TNF-alpha on levels of free ribosomal subunits in psoas muscle (A) and heart (B). Units are arbitrary units (AU)/g muscle wet wt. Muscles were obtained from either 24-h TNF-alpha -infused rats or time-matched control rats. The psoas muscle was used as a representative fast-twitch muscle in place of the gastrocnemius because it yields a better polysome profile. Values are means ± SE; n = 7/group. *P < 0.05 compared with control values for the same tissue.



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Fig. 4.   Effect of TNF-alpha on eukaryotic initiation factor-2B (eIF-2B) activity (A) and the phosphorylation of eIF-2alpha (B) in gastrocnemius and heart. Both phosphorylated and unphosphorylated forms of eIF-2alpha are noted on the representative autoradiographs. Tissues were obtained from either time-matched control rats or rats infused with TNF-alpha for 24 h. Values are means ± SE; n = 7/group. *P < 0.05 compared with control values for the same tissue.



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Fig. 5.   Effect of TNF-alpha on the amount of eIF-4E associated with 4E-binding protein 1 (4E-BP1; A) or eIF-4G (B) in either gastrocnemius or heart muscle. Insets: representative immunoblots. In A, alpha - and beta -forms of 4E-BP1 in the immunoprecipitate are identified. Bar graphs show densitometric analysis of 4E-BP1 bound to eIF-4E (A) or eIF-4G bound to eIF-4E (B) in each tissue. Tissues were obtained from either time-matched control rats (C) or rats infused with TNF-alpha for 24 h (T). Values are means ± SE; n = 7/group. *P < 0.05 compared with control values for the same tissue.



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Fig. 6.   Effect of TNF-alpha on the phosphorylation status of 4E-BP1 and eIF-4E in gastrocnemius and heart. Insets: representative immunoblots with phosphorylated and unphosphorylated forms noted. Bar graphs show densitometric analysis of the percentage of 4E-BP1 in the hyperphosphorylated gamma -form (A) and the percentage of phosphorylated eIF-4E (B). Tissues were obtained from either time-matched control rats or rats infused with TNF-alpha for 24 h. Values are means ± SE; n = 7/group. *P < 0.05 compared with control values for the same tissue.


                              
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Table 1.   Effect of 24-h TNF-alpha infusion on ks and translational efficiency in various nonmuscle tissues


                              
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Table 2.   TNF-alpha -induced changes in eEF content in skeletal muscle and heart


                              
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Table 3.   Hemodynamic and metabolic characteristics of TNF-alpha -infused rats


                              
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Table 4.   TNF-alpha -induced changes in plasma amino acid concentrations


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

In vivo rates of protein synthesis and translational efficiency. Figure 1A shows that after 24 h of TNF-alpha infusion the in vivo rate of protein synthesis was decreased in the predominantly fast-twitch gastrocnemius by 39% compared with values from time-matched control animals. Similarly, TNF-alpha also decreased ks in the slow-twitch soleus muscle (23%) and in heart (ventricle only) by 25%. In contrast to other muscles, there was no difference in the rate of protein synthesis for the diaphragm between TNF-alpha -infused and time-matched control animals. Changes in the number of ribosomes or in the efficiency of mRNA translation may cause a reduction in tissue protein synthesis (10). To determine which mechanism was responsible for the TNF-alpha -induced decrease in ks, the RNA content and translational efficiency were determined. Because ~85% of the RNA is ribosomal RNA, changes in total RNA content reflect changes in the number of ribosomes. We could not detect any significant TNF-alpha -induced change in total RNA in any muscle examined (Fig. 1B). These data suggest that an alteration in the relative abundance of ribosomes was not responsible for the TNF-alpha -induced reduction in muscle ks. Hence, the efficiency of translation, which provides an index of how rapidly the existing ribosomes are synthesizing protein, was also decreased in gastrocnemius, soleus, and heart in response to the infusion of TNF-alpha (Fig. 1C).

The gastrocnemius was also used to determine the effect of TNF-alpha on the synthetic rate of sarcoplasmic and myofibrillar (contractile) proteins. As shown in Fig. 2, TNF-alpha decreased the synthesis in both protein fractions, but the decrease appeared to be greater in the sarcoplasmic fraction (50%) compared with that produced in the myofibrillar fraction (31%).

The fractional synthetic rate was also determined in a number of nonmuscle tissues (Table 1). TNF-alpha decreased ks in the small intestine (23%), failed to significantly alter ks in the kidney, lung, and brain, and increased ks in the liver (25%) and spleen (35%). Moreover, TNF-alpha did not alter the RNA content in any of these tissues but did produce proportional changes in the calculated translational efficiency.

Polysome profiles. Insight into whether alterations in initiation or elongation-termination were mediating the TNF-alpha -induced decrease in protein synthesis was obtained by examining polysome profiles from skeletal muscle and heart. The relative rate of mRNA translation initiation vs. elongation-termination can be assessed by isolating nonpolysome-associated ("free") 40S and 60S ribosomal subunits using sucrose gradient centrifugation. Figure 3A shows that TNF-alpha significantly increased the RNA content of fractions containing both the free 40S and 60S subunits isolated from the psoas (fast-twitch) muscle by ~35%. In general, the distribution of 40S and 60S ribosomal subunits between polysome and nonpolysome fractions is indicative of the balance between the rates of mRNA translation initiation and elongation-termination. That is, when the rate of initiation is decreased relative to elongation-termination, free ribosomal subunits are binding to mRNA at a slower rate (initiation) than they are moving along mRNA (elongation) and exiting (termination). The net result of this defect is an increased abundance of free 40S and 60S ribosomal subunits in skeletal muscle in response to TNF-alpha . In contrast, there was no difference in free ribosomal subunits in cardiac muscle from TNF-alpha -infused rats compared with control animals (Fig. 3B).

Relative amounts of eIF-2 and eIF-2B and eIF-2B activity. One possible mechanism for the TNF-alpha -induced decrease in translational efficiency is altering the amount, availability, or activity of specific eIF proteins. The effect of TNF-alpha on eIF-2B activity was measured in postmitochondrial supernatant of muscles from control and experimental rats. eIF-2B activity was decreased 39% in gastrocnemius from TNF-alpha -infused rats compared with control values (Fig. 4A). However, this decreased activity could not be explained by a concomitant decrease in the amount of eIF-2Bepsilon protein (control = 224 ± 28 vs. TNF-alpha  = 266 ± 35 AU; P = not significant). In contrast to skeletal muscle, there was no detectable difference in eIF-2B activity in the heart between control and TNF-alpha -infused rats (Fig. 4).

Western blot analysis indicated that TNF-alpha did not significantly alter the cellular content of eIF-2alpha in either gastrocnemius or heart (Fig. 4B). In both tissues eIF-2alpha was largely in the dephosphorylated state regardless of treatment. The percentage of phosphorylated eIF-2alpha , as determined by IEF and Western blotting, averaged 10 ± 2% in gastrocnemius and 7 ± 2% in heart from control rats. The phosphorylation of Ser51 on the alpha -subunit by eIF-2alpha kinases plays a central role in regulating eIF-2 activity (24). However, the infusion of TNF-alpha did not significantly change eIF-2alpha phosphorylation in either gastrocnemius or heart (Fig. 4B).

Availability of eIF-4E. A second potential site for control of peptide-chain initiation involves the regulation of eIF-4E (10, 42). Western blot analysis indicated there was no significant difference in the tissue content of eIF-4E between control and TNF-alpha -infused rats for either gastrocnemius (control = 1,124 ± 68 AU vs. TNF-alpha  = 1,089 ± 102 AU) or heart (control = 667 ± 45 vs. 713 ± 61 AU). However, eIF-4E can be sequestered into an inactive complex through binding of the translational repressor molecule 4E-BP1. This is visualized on an immunoblot as an increase in the amount of 4E-BP1 present in the eIF-4E immunoprecipitate. Therefore, we examined the ability of TNF-alpha to modify the distribution of eIF-4E in both gastrocnemius and heart. Figure 5A shows that the alpha - and beta -forms of 4E-BP1 were detected in immunoprecipitates of both tissues from all groups. TNF-alpha increased the relative content of the inactive eIF-4E · 4E-BP1 complex by 46% in gastrocnemius and by 33% in heart. When eIF-4E is bound to 4E-BP1, it is unable to interact with eIF-4G to form the active eIF-4E · eIF-4G complex. Therefore, we determined whether the TNF-alpha -induced increase in eIF-4E · 4E-BP1 complex formation resulted in a decreased association of eIF-4E with eIF-4G. As shown in Fig. 5B, TNF-alpha also dramatically decreased the tissue content of the active eIF-4E · eIF-4G complex in muscle and heart (66 and 43%, respectively).

Phosphorylation of 4E-BP1 and eIF-4E. 4E-BP1 has at least five potential phosphorylation sites, and the various phosphorylated forms of the protein are resolved into three bands by one-dimensional SDS-PAGE (44). These bands have been designated alpha , beta , and gamma  in order of their decreasing electrophoretic mobility (Fig. 6A, inset). For each tissue, the total amount of all three bands combined did not differ between control and TNF-alpha -infused rats (data not shown), indicating that the total amount of 4E-BP1 was not altered. Hyperphosphorylation of 4E-BP1 is known to decrease the association of the binding protein with eIF-4E and increase translation. In the present study, the amount of 4E-BP1 in the hyperphosphorylated gamma -form was decreased in both gastrocnemius (64%) and heart (50%) from TNF-alpha -treated rats compared with tissues from time-matched control animals (Fig. 6A).

The influence of TNF-alpha on the phosphorylation state of eIF-4E was also assessed because phosphorylation of the cap-binding protein eIF-4E has been demonstrated to be increased by certain stressors and to regulate eIF-4E function (39). Figure 6B indicates that the infusion of TNF-alpha resulted in a small, but statistically significant, increase in eIF-4E phosphorylation in gastrocnemius (21%) but not in heart.

eEF-1 and -2. Compared with time-matched control values, the infusion of TNF-alpha did not significantly alter the content of eEF-1alpha , eEF-2, or phosphorylated eEF-2 in either gastrocnemius or heart (Table 2).

Hemodynamic and metabolic characteristics. TNF-alpha -infused rats had an MABP and body temperature that was not significantly different from control values (Table 3). TNF-alpha -treated rats demonstrated a significant tachycardia (21%). These data indicate that TNF-alpha -infused rats were hemodynamicly stable and not preterminal at the time measurements of protein synthesis and translation control were performed.

The infusion of TNF-alpha altered the circulating concentrations of various hormones that might potentially influence protein balance (Table 3). The plasma corticosterone concentration was increased almost threefold in TNF-alpha -infused rats. In contrast, the circulating concentration of IGF-I was reduced 44% by TNF-alpha compared with control values, whereas the plasma insulin concentrations were not changed significantly. Plasma leptin concentrations were increased more than fivefold by TNF-alpha . Finally, TNF-alpha did not significantly alter plasma FFA concentrations (Table 3).

The effect of TNF-alpha infusion on the concentration of individual plasma amino acids is presented is Table 4. Overall, there was no significant difference in the concentration of total amino acids between control and TNF-alpha -treated rats. However, several individual amino acids showed significant decreases in response to TNF-alpha ; these included valine (-22%), isoleucine (-27%), threonine (-23%), serine (-43%), and glycine (-25%). In contrast, the concentrations of several amino acids were elevated in response to TNF-alpha , including tryptophan (150%), histidine (36%), and 3-methylhistidine (185%).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

In the present study, TNF-alpha decreased the rate of protein synthesis in the gastrocnemius, soleus, and heart between 20 and 40%. Moreover, at least for gastrocnemius, TNF-alpha decreased synthesis of both myofibrillar and sarcoplasmic proteins. The rate of total mixed protein synthesis was approximately equal to that of the myofibrillar proteins. The relative abundance of contractile proteins in muscle may in part explain this observation. A similar response pattern occurs in septic rats (54). In contrast, no alteration in protein synthesis was observed in diaphragms from TNF-alpha -infused rats. The ability of TNF-alpha to decrease protein synthesis in both the gastrocnemius and soleus is in contrast to the selective decrease in synthesis observed in fast-twitch skeletal muscle (e.g., gastrocnemius) in other catabolic conditions, including sepsis, endotoxemia, alcohol intoxication, and glucocorticoid excess (30, 32, 43, 45). Previous studies have generally failed to detect consistent alterations in the in vivo rate of muscle protein synthesis in response to a bolus injection of TNF-alpha (1, 8, 25, 46, 58). This difference between the current investigation and these previous studies may be related to the sustained increase in blood TNF-alpha levels produced by the primed constant infusion of the cytokine for 24 h as opposed to the transient elevations produced by protocols using bolus injections. Additionally, even though our control animals underwent the same degree of surgical trauma (e.g., vascular catheterization) as the TNF-alpha -infused rats, we cannot exclude the possibility that this preceding trauma may have sensitized or primed animals to the catabolic effects of TNF-alpha . A similar situation as been reported whereby implantation of vascular catheters augmented the metabolic effects induced by endotoxin (3).

The infusion of TNF-alpha also decreased protein synthesis in the small intestine, but whether this reduction was restricted to the smooth muscle layer and/or the mucosa was not assessed. This TNF-alpha -induced decrease in protein synthesis in muscle and gut is not the result of a generalized suppression of synthesis in all tissues. In this regard, rates of protein synthesis in the kidney, lung, and brain were unaltered by TNF-alpha . Moreover, TNF-alpha increased protein synthesis in liver and spleen by 25-35%. A stimulation of hepatic and splenic protein synthesis has been previously reported in response to a short-term (~3 h) infusion of TNF-alpha (21).

The cellular content of ribosomes may be rate limiting for protein synthesis under some conditions (10, 31). Thus the TNF-alpha -induced decrease in tissue protein synthesis may occur through a reduction in the number of ribosomes and/or a decrease in translational efficiency (10, 31). In the present study, the abundance of ribosomes, as estimated by the total RNA content, was not significantly altered by TNF-alpha in any of the tissues examined. Therefore, our data suggest that the TNF-alpha -induced decrease in muscle protein synthesis results primarily from an impairment in translational efficiency and not from a diminished capacity for protein synthesis. Likewise, the TNF-alpha -induced increase in hepatic and splenic protein synthesis appears to result from a corresponding increase in translational efficiency.

The impaired translational efficiency observed in muscle from TNF-alpha -infused rats may result from a defect in either peptide-chain initiation and/or elongation-termination. In the present study, analysis of the distribution of ribosomal subunits between free subunits and polysomes was used to estimate the rate of initiation relative to elongation. TNF-alpha increased the number of nonpolysome-associated 40S and 60S subunits in psoas muscle. A comparable increase has been previously observed in other catabolic states, such as infection, diabetes, and starvation (20, 27, 52). This increase indicates a decrease in peptide-chain initiation relative to elongation-termination in skeletal muscle. This conclusion is consistent with our current data demonstrating a TNF-alpha -induced change in eIF-4E availability but no change in either eEF-1alpha or eEF-2 content. In contrast to skeletal muscle, TNF-alpha did not alter the distribution in ribosomal subunits in heart.

The first step in initiation is the formation of a ternary complex consisting of eIF-2, GTP, and met-tRNAi (42). The binding of met-tRNAi to the 40S subunit to form the 43S preinitiation complex is mediated by eIF-2, which is a heterotrimer consisting of alpha -, beta - and gamma -subunits. Previous studies have indicated that a reduction in eIF-2 is associated with a concomitant reduction in global rates of protein synthesis (27). However, the relative content of eIF-2alpha was not altered in either skeletal muscle or heart of TNF-alpha -infused rats. Similarly, the wasting that accompanies sepsis, endotoxemia, and alcohol ingestion was also not associated with a change in eIF-2alpha content (10, 30, 44). The ability of eIF-2 to form a ternary complex can also be decreased via a reduction in the activity of another eukaryotic initiation factor, eIF-2B (42). eIF-2 is bound to GDP as an inactive complex when it is released from the ribosome, and this GDP must be exchanged for GTP before a new round of initiation may proceed. eIF-2B catalyzes this guanine nucleotide exchange and is required to regenerate the active eIF-2 · GTP complex. Under certain conditions, the rate of protein synthesis is directly proportional to eIF-2B activity in muscle (20, 24). In the present study, TNF-alpha decreased eIF-2B activity is skeletal muscle but not in heart. Hence, in skeletal muscle, the decrease in eIF-2B activity would be expected to reduce the amount of eIF-2 · GTP that is available to bind to met-tRNAi, thereby decreasing protein synthesis by impairing translation initiation. A similar mechanism does not appear to be responsible for the TNF-alpha -induced decrease in myocardial protein synthesis.

The activity of eIF-2B can be regulated by the amount of eIF-2B protein. However, we detected no significant change in the relative amount of eIF-2Bepsilon , which represents the catalytic subunit of this protein. Alternatively, eIF-2B activity can be regulated by changing the phosphorylation state of the alpha -subunit of eIF-2, which increases the affinity of eIF-2 for eIF-2B (42). Phosphorylation of Ser51 on eIF-2alpha converts eIF-2 from a substrate into a competitive inhibitor limiting the regeneration of the eIF-2 · GDP complex. Hence, initiation is impaired by the formation of the highly stable eIF-2alpha (P) · eIF-2B complex that sequesters available eIF-2B. Although eIF-2B activity and initiation can be strongly inhibited by the phosphorylation of only a fraction of the eIF-2, we were unable to detect a significant alteration in the phosphorylation state of eIF-2alpha in either gastrocnemius or heart in response to TNF-alpha . Therefore, the exact mechanism by which TNF-alpha decreases eIF-2B activity in gastrocnemius remains to be elucidated but is independent of changes of the amount of eIF-2B protein and the phosphorylation state of eIF-2. These data are consistent with the lack of a change in these eIFs in skeletal muscle and heart in response to other types of relatively acute (<= 24 h) catabolic stimuli (24, 30, 32).

A second rate control point in translation initiation is mediated by eIF-4F, which regulates the binding of mRNA to the 43S preinitiation complex (42). The eIF-4F holoprotein is a three-subunit complex composed of eIF-4A, eIF-4E, and eIF-4G. One of these protein components, eIF-4E, binds directly to the m7GTP cap structure present at the 5'-end of the large majority of eukaryotic mRNA and plays a critical role in maintaining protein synthesis. During translation initiation, the eIF-4E · mRNA complex binds to eIF-4G and eIF-4A to form the active eIF-4F complex. One mechanism for modulating the formation of the eIF-4F complex is by regulating the relative distribution of eIF-4E between inactive and active complexes with other proteins. The eIF-4E · eIF-4G interaction can be inhibited by small regulatory proteins, eIF-4E binding proteins, that bind to eIF-4E (33). In the present study, the total amount of eIF-4E was not altered by TNF-alpha ; however, cytokine infusion did increase the amount of eIF-4E bound to 4E-BP1 in both gastrocnemius and heart. The binding of 4E-BP1 to eIF-4E is known to inhibit cap-dependent translation (42, 44). Because both 4E-BP1 and eIF-4G contain a similar eIF-4E binding site, the binding of these two proteins to eIF-4E is mutually exclusive. In this regard, there was a pronounced concomitant decrease in the amount of eIF-4E bound to eIF-4G in muscle and heart from TNF-alpha -infused rats. These data strongly suggest that TNF-alpha decreases initiation, at least in part, by an impairment in eIF-4F function secondary to decreased eIF-4E availability. Similar changes in the relative distribution of eIF-4E have been seen in other wasting conditions (30, 31, 44, 45).

The function of eIF-4E can also be regulated by phosphorylation of 4E-BP1 or eIF-4E. Phosphorylation of 4E-BP1 greatly decreases the affinity of 4E-BP1 for eIF-4E and leads to dissociation of the 4E-BP1 · eIF-4E complex (23). This permits the formation of a competent eIF-4F complex and the stimulation of initiation. Therefore, stimuli that impair initiation and protein synthesis are often associated with a decreased amount of the hyperphosphorylated gamma -form of this protein (26, 31, 45). Consistent with the decrease in protein synthesis, TNF-alpha markedly decreased the percentage of 4E-BP1 in the phosphorylated gamma -form in both gastrocnemius and heart. A comparable decrease in the phosphorylation state of 4E-BP1 has previously been described in other catabolic states (26, 30, 31). In addition, changes in the phosphorylation state of eIF-4E itself can also influence eIF-4E availability. Although both phosphorylated and nonphosphorylated eIF-4E bind to the mRNA cap structure, phosphorylation of eIF-4E enhances the affinity of the factor for the m7GTP cap by severalfold (39). Furthermore, in vitro studies have demonstrated that increases in eIF-4E phosphorylation are proportional to increases in the rate of translation (23). Conversely, a decrease in eIF-4E phosphorylation has been correlated with a reduction in protein synthesis after viral infection (13). In this regard, quite unexpectedly, TNF-alpha resulted in a small, but statistically significant, increase in the phosphorylation of eIF-4E in the gastrocnemius.

Elongation is the phase of the protein synthetic pathway that is responsible for the growth of nascent polypeptide chains (38). Previous studies have demonstrated that an impairment in elongation is, at least in part, responsible for the decreased rate of protein synthesis in various muscle and nonmuscle tissues under certain situations (35, 53). In mammals, the elongation process is facilitated by two protein factors, eEF-1 and eEF-2. Our data indicate that the relative content of these two elongation factors in both skeletal muscle and heart was not significantly altered by the infusion TNF-alpha . Furthermore, the extent of eEF-2 phosphorylation, which has been demonstrated to be inversely proportional to the rate of protein synthesis (35), was also not affected by TNF-alpha . These results are consistent with data from a previous study that indicated rates of peptide-chain elongation were not altered in skeletal muscle in response to polymicrobial peritonitis (52). Collectively, these data suggest that the abovementioned changes in translation initiation are the primary cause for the TNF-alpha -induced decrease in muscle protein synthesis.

The physiological mediators of the TNF-alpha -induced decrease in translation have not been established. The decreased muscle protein synthesis could not be ascribed to a reduction in the plasma insulin or total amino acid concentrations in the blood. Euaminoacidemia has also been reported in rats infused with TNF-alpha for only 3.5 h (21). However, we cannot exclude the possibility that a resistant state to insulin and/or amino acids produced by TNF-alpha was at least partially responsible for the diminished rate of synthesis (29). Elevations in plasma FFAs have been shown to be capable of modulating protein balance in vivo (47). However, no significant alterations in plasma FFA concentrations were detected in TNF-alpha -infused rats. Our data extend previous work indicating that the acute injection of TNF-alpha increases plasma leptin concentrations (14). Because leptin is known to impair insulin signaling, we cannot exclude the hyperleptinemia as a possible mediator for the TNF-alpha -induced decrease in muscle protein synthesis. Finally, this study confirms our earlier work by demonstrating that TNF-alpha markedly decreases circulating levels of the anabolic hormone IGF-I (18). Alterations in muscle protein synthesis and initiation (e.g., eIF-4E availability) have been reported to be proportional to the prevailing concentration of IGF-I (44, 45).

In summary, our data show that a relative sustained infusion of TNF-alpha is capable of decreasing the rate of protein synthesis in heart and skeletal muscle. This inflammatory insult decreases both myofibrillar and sarcoplasmic protein synthesis via a reduction in translational efficiency. The TNF-alpha -induced impairment in mRNA translation appears to result primarily from a redistribution of eIF-4E into the inactive eIF-4E · 4E-BP1 complex, but not from a change in peptide-chain elongation.


    ACKNOWLEDGEMENTS

We thank Xiaoli Liu, Duanqing Wu, and Gina Deiter for technical assistance. We also acknowledge Drs. Leonard S. Jefferson and Scot R. Kimball for supplying some of the reagents used in this study.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants AA-11290, AA-12814, GM-38032, GM-39277, and HL-66443.

Address for reprint requests and other correspondence: C. H. Lang, Dept. of Cell Molec. Physiology (H166), Penn. State College of Medicine, 500 Univ. Dr., Hershey, PA 17033 (E-mail: clang{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.

10.1152/ajpendo.00366.2001

Received 16 August 2001; accepted in final form 18 September 2001.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 282(2):E336-E347
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