Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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The study
described herein investigated the role of free fatty acids (FFAs) in
the maintenance of protein synthesis in vivo in rat cardiac and
skeletal muscle. Suppression of FFA -oxidation by methyl palmoxirate
caused a marked reduction in protein synthesis in the heart. The effect
on protein synthesis was mediated in part by changes in the function of
eukaryotic initiation factors (eIFs) involved in the initiation of mRNA
translation. The guanine nucleotide exchange activity of eIF2B was
repressed, phosphorylation of the
-subunit of eIF2 was enhanced, and
phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6
kinase was reduced. Similar changes in protein synthesis and
translation initiation were not observed in the gastrocnemius following
treatment with methyl palmoxirate. In heart, repressed
-oxidation of
FFA correlated, as demarcated by changes in the ATP/AMP ratio and
phosphorylation of AMP-activated kinase, with alterations in the energy
status of the tissue. Therefore, the activation state of signal
transduction pathways that are responsive to cellular energy stress
represents one mechanism whereby translation initiation may be
regulated in cardiac muscle.
translation initiation; gastrocnemius muscle; adenosine 5'-monophosphate-activated protein kinase
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INTRODUCTION |
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PREVIOUS STUDIES HAVE SUGGESTED a role for free fatty acids (FFA) in modulation of the initiation of mRNA translation in muscles composed primarily of oxidative fibers. Those studies show that protein synthesis is stimulated in association with enhanced translation initiation in rat hearts perfused with medium containing palmitate, whereas the fatty acid is without effect in perfused preparations of rat skeletal muscle (19, 31). These observations suggest that long-chain fatty acids facilitate protein synthesis solely within oxidative fibers. Support for this suggestion comes from the observation that hypophysectomized rats, which have both decreased serum insulin and decreased FFA concentrations, have ribosome profiles indicative of reduced translation initiation rates in both skeletal muscle and heart. In contrast, diabetic hypophysectomized rats, which have low serum insulin but elevated FFA concentrations, have similarly altered ribosome profiles in nonworking (i.e., not actively contracting) skeletal muscle but not in the heart (30). To explain these observations, it has been proposed that the relatively greater ability of the heart to metabolize FFA allows for maintenance of cardiac intracellular energy stores during insulin deficiency (10). This, in turn, permits protein synthesis to be maintained at or near control values in heart compared with fast-twitch skeletal muscle, which predominantly depends on glucose to maintain energy stores.
It is well recognized that, unlike nonworking glycolytic muscle
fibers, in which glucose is the preferred substrate, the working cardiac muscle utilizes FFAs as its principle substrate
(8). Moreover, FFA oxidation in the myocardium is greatly
increased during pathological conditions such as diabetes
(32). In light of these facts, one could surmise that it
is the ability of oxidative fibers, relative to glycolytic fibers, to
efficiently synthesize ATP from FFAs that allows them to maintain
protein synthetic rates in the presence of reduced glucose uptake, as
occurs with diabetes and starvation. On the basis of this premise,
inhibition of -oxidation would be expected to reduce the energy
status and thus produce an energetic stress within working oxidative
fibers but not within resting glycolytic fibers.
Because the development of energetic stress is closely mirrored by a
fall in protein synthesis, it has been suggested that energy status is
an important determinant of the protein synthetic process
(3). The protein 5'-AMP-activated protein kinase (AMPK) is
particularly sensitive to the energy status of a tissue and is
recognized as a cellular energy sensor (16). AMPK responds to changes in the ratio of ATP to AMP (ATP/AMP) as well as the ratio of
phosphocreatine to creatine, and its activation has been correlated
with the suspension of various anabolic processes (17, 20). For example, a recent study reported that skeletal muscle protein synthesis and mRNA translation initiation are inhibited in vivo
in response to the artificial activation of AMPK using the chemical
5-aminoimidazole-4-carboxamide-1--D-ribonucleoside (AICAR) (2).
Translational control of protein synthesis is mediated primarily at the
stage of initiation of mRNA translation. The proteins that mediate this
process are referred to as eukaryotic initiation factors (eIFs). For
initiation to occur in most eukaryotic systems, a complex including
eIF2-bound GTP transports an initiator methionyl-tRNA (tRNA-subunit of eIF2 (eIF2
) results in the sequestration of eIF2B and
thereby prevents guanine nucleotide exchange (18). Another
mechanism whereby translation initiation may be regulated is through
the association of mRNA to the 40S ribosomal subunit. This process
initially requires that a 7-methylguanosine-capped mRNA be bound by the
cap-binding protein eIF4E (18). Availability of eIF4E is
regulated by the eIF4E-binding proteins (4E-BPs) (14). Finally, a correlation exists between the activity of the 40S ribosomal
protein S6 kinase (S6K1) and the capacity to synthesize protein
(5, 6). This correlation stems from the fact that the
translation of transcripts that contain a terminal oligopyrimidine sequence (TOP mRNA), which often encode components of the translational apparatus such as ribosomal proteins and translation factors, is
increased when S6 is phosphorylated (15).
Alterations in the function of eIFs can occur in response to environmental, nutritional, and/or hormonal signals. Insulin, for example, has been shown to modulate several eIFs in skeletal muscle (reviewed by Refs. 1 and 22), but surprisingly, research focusing on the role of FFAs in the translational control of protein synthesis in heart or skeletal muscle, or any other tissue, is extremely limited. Therefore, the objective of the study described herein was to investigate the role of FFA oxidation in the control of protein synthesis in heart and skeletal muscle in vivo.
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MATERIALS AND METHODS |
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Animal care. The animal facilities and protocol were reviewed and approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine. Male Sprague-Dawley rats (~200 g) were maintained on a 12:12-h light-dark cycle with a standard diet (Harlan-Teklad Rodent Chow, Madison, WI) and water provided ad libitum.
Inhibition of -oxidation.
-Oxidation of FFAs was inhibited through the administration of
methyl palmoxirate (R. W. Johnson Pharmaceutical Research Institute, Spring House, PA), a known inhibitor of carnitine
palmitoyltransferase I (CPT I) (35). The protocol for
methyl palmoxirate administration has been described previously
(35) and was modified slightly. To ensure that fatty acids
were being used as the major energy substrate, animals were food
deprived for 21 h, weighed, and then randomly received either a
single bolus of methyl palmoxirate (25 mg/kg body wt suspended in 0.5%
carboxymethylcellulose) or 0.5% carboxymethylcellulose via oral
gavage. Animals were returned to their cages and allowed free access to
water only until administration of metabolic tracer as described in the
next section.
Administration of metabolic tracer and sample collection.
A flooding dose (1.0 ml/100 g body wt) of
L-[2,3,4,5,6-3H]phenylalanine (150 mM
containing 3.70 GBq/l) was administered via tail vein injection 170 min
after methyl palmoxirate administration for the measurement of protein
synthesis (11). Animals were killed by decapitation 10 min
later. Trunk blood was collected and centrifuged at 1,800 g
for 10 min at 4°C to obtain serum. After excision, a portion of the
gastrocnemius or heart was homogenized in 7 volumes of buffer
consisting of (in mM) 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 dithiothreitol, 50 sodium fluoride, 50
-glycerophosphate, 0.1 phenylmethylsulfonyl
fluoride, 1 benzamidine, and 0.5 sodium vanadate. An aliquot (0.5 ml)
of the homogenate was used for the measurement of muscle protein synthesis as described in Measurement of muscle protein
synthesis. The remainder of the homogenate was immediately
centrifuged at 10,000 g for 10 min at 4°C. The supernatant
was used for analysis of mRNA translation initiation factors as
described in Analysis of translation control regulatory
mechanisms. The remaining tissue was used to assess eIF2B
activity as described in Measurement of muscle protein
synthesis.
Serum measurements. Serum FFAs were analyzed using a commercial colorimetric kit (Wako Chemicals, Richmond, VA). Serum insulin concentrations were analyzed using a commercial RIA kit for rat insulin (Linco Research, St. Charles, MO). Serum glucose concentrations were measured using an automated glucose oxidase-peroxidase method (YSI model 2300 analyzer, Yellow Springs Instrument, Yellow Springs, OH).
Measurement of muscle protein synthesis. Fractional rates of protein synthesis were estimated from the rate of incorporation of radioactive phenylalanine into total mixed muscle protein, as described previously (23). The elapsed time from injection of the metabolic tracer until homogenization of the muscle was recorded as the actual time for radiolabeled phenylalanine incorporation.
Analysis of translation control regulatory mechanisms.
Phosphorylation of 4E-BP1, S6K1, and eIF2 was evaluated in
10,000-g supernatants by protein immunoblot analysis, as
described previously (12, 13, 23). The guanine nucleotide
exchange activity of eIF2B was assessed by the exchange of
[3H]GDP bound to eIF2
for nonradioactively labeled
GDP, as described previously (21).
Measurement of tissue adenine nucleotides. Adenine nucleotides were measured in tissue samples that had been frozen in situ from animals administered a lethal dose of pentobarbital sodium (Nembutal) (Abbott Laboratories, North Chicago, IL). Adenine nucleotide concentrations were determined as described previously (24).
Analysis of AMPK phosphorylation. Phosphorylation of the catalytic subunit of AMPK was evaluated in 10,000-g supernatants by protein immunoblot analysis by use of a phosphospecific (Thr172) AMPK antibody.
Statistical analysis. Data are expressed as means ± SE. All data were analyzed by the InStat version 3 statistical software package (GraphPad Software, San Diego, CA). Statistical significance was assessed using a two-tailed Student's t-test unless stated otherwise. P values <0.05 were considered statistically significant.
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RESULTS |
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To examine the contribution of -oxidation of FFAs to the
maintenance of protein synthesis in cardiac and skeletal muscle, male
Sprague-Dawley rats were treated with methyl palmoxirate, a known
inhibitor of CPT I. The effectiveness of the treatment was assessed by
measuring plasma FFA concentrations. As shown in Table
1, methyl palmoxirate treatment resulted
in an almost threefold increase in plasma FFAs. These results are
consistent with methyl palmoxirate inhibiting the
-oxidation of FFAs
and thus reducing their clearance from the blood. As reported
previously (26), serum glucose, but not serum insulin,
concentrations were significantly decreased following methyl
palmoxirate administration. This decrease in serum glucose
concentrations is consistent with Randle's hypothesis that FFA
oxidation reduces glucose oxidation in the heart (29).
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The effect of treatment with methyl palmoxirate on protein synthesis in
cardiac and skeletal muscle was examined by measuring in vivo rates of
protein synthesis by means of the flooding dose method
(11). In animals treated with methyl palmoxirate, cardiac protein synthesis was reduced to 64% of the control value (Fig. 1). In contrast, protein synthesis was
unaltered in the gastrocnemius following methyl palmoxirate
administration. These results support the concept that FFAs are
positive regulators of protein synthesis specifically within working
muscles composed primarily of oxidative fibers.
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Regulation of mRNA translation can occur through changes in the rate of
translation initiation, translation elongation, or both. Multiple
mechanisms exist for regulating translation initiation, including
modulation of eIF2B activity, assembly of the eIF4F complex, and S6K1
activity. To examine whether or not methyl palmoxirate treatment
altered eIF2B activity, tissue extracts were assayed for guanine
nucleotide exchange activity with the use of
eIF2 · [3H]GDP as a substrate. As
shown in Fig. 2, eIF2B activity was
significantly repressed following methyl palmoxirate administration in
the heart but not in the gastrocnemius. Thus eIF2B activity paralleled
the changes in protein synthesis observed following treatment with methyl palmoxirate. To gain further insight into the regulation of
eIF2B activity under these conditions, the phosphorylation status of
eIF2 was measured by protein immunoblot analysis with the use of an
antibody that recognizes the protein only when it is phosphorylated on
Ser51. As shown in Fig. 3,
treatment with methyl palmoxirate resulted in a significant increase in
the relative phosphorylation of eIF2
specifically within the heart.
These results suggest that the decrease in eIF2B activity observed in
rats treated with methyl palmoxirate was due, at least in part, to
increased phosphorylation of eIF2
.
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The best characterized mechanism for regulating assembly of the eIF4F
complex involves changes in eIF4E association with 4E-BP1, an event
that is modulated by the phosphorylation status of 4E-BP1. Thus
hyperphosphorylation of 4E-BP1 prevents it from associating with eIF4E,
allowing eIF4E to bind to eIF4G and form the active eIF4F complex
(13). As shown in Fig. 4,
treatment with methyl palmoxirate resulted in a marked reduction in the
phosphorylation status of 4E-BP1 in the heart. There was, however, no
significant change in 4E-BP1 phosphorylation in the gastrocnemius.
Phosphorylation of 4E-BP1 in response to nutrients or hormones depends
on the activity of a protein kinase referred to as the mammalian target of rapamycin (mTOR; reviewed in Ref. 34). In addition to
4E-BP1, mTOR also controls the activity of the ribosomal protein S6K1 by promoting its hyperphosphorylation (7). As observed for 4E-BP1, treatment with methyl palmoxirate reduced the amount of S6K1
present in hyperphosphorylated forms in the heart but not in the
gastrocnemius (Fig. 5). Thus, in addition
to a reduction in eIF2B activity, the reduction in protein synthesis in
the heart by treatment with methyl palmoxirate was also associated with a redistribution of 4E-BP1 and S6K1 into hypophosphorylated forms.
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The best characterized mechanism for regulating translation elongation involves modulation of the phosphorylation status of translation elongation factor eEF2. In this regard, phosphorylation of eEF2 is associated with reduced rates of protein synthesis under a variety of conditions (4). To assess whether or not treatment with methyl palmoxirate altered protein synthesis in the heart through the step in translation elongation mediated by eEF2, the phosphorylation status of the protein was examined by protein immunoblot analysis by use of an antibody that recognizes the protein only when it is phosphorylated on Thr56. The relative phosphorylation of eEF2 on Thr56 was unaffected by treatment with methyl palmoxirate (data not shown). Although possible effects on the activity of eEF1 cannot be excluded, the results suggest that methyl palmoxirate treatment does not regulate translation elongation through the step involving eEF2.
It has been proposed that FFAs maintain protein synthetic rates in the
heart by acting as oxidative substrates. Thus deprivation of this
energy source, as occurs during methyl palmoxirate-mediated inhibition
of FFA oxidation, would be expected to lower the energy status of the
heart. As depicted in Fig. 6A,
the energy status in the heart, as monitored by the ATP/AMP ratio, was
significantly reduced following methyl palmoxirate administration,
whereas the nucleotide ratio was unchanged in the gastrocnemius. AMPK
has been shown to be activated during energy deficit conditions
(17, 20), and its activation is demarcated by
phosphorylation at Thr172 (16). As shown in
Fig. 6B, there was a significant increase in the
phosphorylation of AMPK on Thr172 in the heart following
treatment with methyl palmoxirate. In contrast, there was no
significant change in AMPK phosphorylation in the gastrocnemius.
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DISCUSSION |
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Previous studies have suggested a role for FFAs in modulation of the initiation of mRNA translation. Furthermore, those studies indicate that the role is tissue specific, being limited to working muscles composed primarily of oxidative fibers. However, it is only recently, with the development of pharmacological agents that inhibit fat metabolism, that it has become possible to investigate the role of FFAs in the translational control of protein synthesis in vivo.
To our knowledge, this study is the first to demonstrate in vivo that protein synthetic rates in the heart are markedly reduced in response to decreased FFA oxidation. This suggests that an inhibition of mRNA translation occurs at the stage of initiation and/or elongation, when FFA oxidation is inhibited or availability of FFAs becomes limiting. As eEF2 phosphorylation was unaltered following methyl palmoxirate administration, FFA oxidation would appear not to modulate translation elongation. This result is in keeping with previous in vitro studies demonstrating that a decrease in free (i.e., nonpolysome associated) ribosomal subunit content, indicative of inhibited elongation/termination, does not occur in hearts perfused without FFAs (30). In the aforementioned perfusion studies, the authors observed an accumulation of free ribosomal subunits, suggestive of an inhibition of translation initiation, which led them to hypothesize that FFAs modulate translation initiation in the heart.
Our results support the hypothesis that FFAs modulate translation
initiation in the heart and, to our knowledge, are the first to
demonstrate that multiple mechanisms underlie this modulation in vivo.
The inhibition of eIF2B activity in the hearts of methyl palmoxirate-treated animals indicates an impairment in the transport of
met-tRNA was enhanced under these conditions, the
decrease in eIF2B activity most certainly results, at least in part,
from competitive inhibition mediated by phosphorylated eIF2. Changes in
allosteric regulation may also have contributed to the aforementioned
changes in eIF2B activity in this study. It has been demonstrated
previously that eIF2B may be regulated allosterically by
NADP+ (9), and initial observations from our
laboratory indicate that NADP+-to-NADPH ratios are altered
in the hearts, but not the gastrocnemius, of methyl palmoxirate-treated
animals (data not shown).
The phosphorylation of both 4E-BP1 and S6K1 was decreased in the hearts of methyl palmoxirate-treated animals. These respective changes indicate that transport of mRNA to the 40S ribosomal subunit and synthesis of the translational apparatus itself are inhibited when FFA oxidation is inhibited. Furthermore, because 4E-BP1 and S6K1 are downstream targets of the kinase mTOR, the results indicate that an mTOR-dependent signaling pathway is modulated by FFA oxidation.
Our observation that methyl palmoxirate does not have a significant effect on protein synthetic rates in the gastrocnemius supports the hypothesis that FFAs modulate mRNA translation initiation specifically within oxidative fibers. It should be noted, however, that whole gastrocnemius muscle was utilized in these experiments and that this muscle is composed primarily of glycolytic fibers but does contain mixed fibers as well as a small proportion of purely oxidative fibers (25). Therefore, it is not surprising that the mean synthesis value from the gastrocnemius of methyl palmoxirate-treated animals was slightly below that of controls.
Our results indicate that methyl palmoxirate-induced inhibition of FFA
-oxidation culminates in decreased ATP/AMP ratios in the heart but
not in the gastrocnemius. Accordingly, AMPK phosphorylation is
increased in the heart but not in the gastrocnemius following methyl
palmoxirate administration. Thus an energetic stress develops within
the heart, but not the gastrocnemius, following methyl palmoxirate-induced inhibition of FFA
-oxidation. These results are
in keeping with the fact that FFAs are the primary energy source of the
heart, whereas glucose is the principle energy source of the
gastrocnemius at rest. Experiments performed in our laboratory suggest
that AMPK signals through PKB to mTOR and its downstream effectors
(2). Therefore, we believe that the phosphorylation of
AMPK, stemming from reduced FFA oxidation, in the heart represents a
causal event in the inhibition of the mTOR-dependent signaling pathway
and the subsequent inhibition of translation initiation. It should also
be noted that, in our previous studies involving the artificial
activation of AMPK with the chemical AICAR, eIF2B activity was
unaltered and the phosphorylation of eIF2
was actually decreased.
These results are in contrast to those presented here with regard to
the heart and suggest that methyl palmoxirate may induce eIF2
phosphorylation through a mechanism distinct from AMPK.
Although unexplored in these studies, FFAs may also modulate protein synthetic rates in oxidative fibers in a manner that is independent of their oxidation. On the basis of the finding that the stimulation of protein synthesis in hearts perfused with palmitate is significantly blunted in the presence of the transcriptional inhibitor actinomycin D, Rannels et al. (30) speculated that FFAs function as transcriptional regulators, and recent in vivo studies support this hypothesis. For example, it has been demonstrated that the increased expression of various mRNAs that occurs in skeletal muscle with fasting can be prevented by inhibiting fasting-induced elevations in circulating FFAs (a relevant observation from these studies was that this inhibition is specific to slow-twitch oxidative muscles) (33). It has also been demonstrated that the expression of certain mRNAs involved in substrate metabolism is elevated in fat biopsies, independently of changes in circulating insulin, following a 5-h lipid infusion (28). Therefore, it is possible that FFA-mediated transcriptional events also contribute to changes in protein synthesis within the heart following methyl palmoxirate administration.
In summary, the results of this study indicate that the contribution of
-oxidation of FFAs to the maintenance of mRNA translation initiation
differs in a tissue-specific manner, thus offering one explanation for
the tissue-specific effects of metabolic disorders like diabetes on
protein synthesis. We propose that plasma FFAs play a key role in the
modulation of mRNA translation specifically within muscles composed
primarily of oxidative fibers. More specifically, we hypothesize that
alterations in FFA
-oxidation in working oxidative fibers result in
the development of cellular stress. Both AMPK-dependent and
-independent signaling pathways are activated by this stress,
culminating in the inhibition of translation initiation. Further
research will be required to explore the mechanisms whereby FFA
oxidation activates intracellular signaling pathways and to elucidate
the relationship between cellular stress, FFA-mediated transcriptional
events, and the translational control of protein synthesis.
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ACKNOWLEDGEMENTS |
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We sincerely thank Lynne Hugendubler and Sharon Rannels for invaluable assistance with sample collection and guanine nucleotide exchange activity measurements, and Dr. Joshua Anthony for expert advice with tracer administration. We also thank Dr. David MacLean, Teresa Markle, and Kristine Rice for help with protein synthesis measurements, and Dr. Kathryn LaNoue and Deborah Berkich for help with determination of adenine nucleotide concentrations. Finally, we thank the R. W. Johnson Pharmaceutical Research Institute for providing the methyl palmoxirate utilized in these studies.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grants DK-15658 (L. S. Jefferson) and GM-08619 (S. J. Crozier).
Address for reprint requests and other correspondence: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, The Pennsylvania State Univ. College of Medicine, PO Box 850, Hershey, PA 17033 (E-mail: jjefferson{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.
August 20, 2002;10.1152/ajpendo.00277.2002
Received 24 June 2002; accepted in final form 4 August 2002.
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