AJP-Endo Articles in PresS. Published on August 20, 2002 as DOI 10.1152/ajpendo.00277.2002
b-Oxidation of Free Fatty Acids is Required for Maintenance of Translational
Control of Protein Synthesis in Heart
Stephen J. Crozier, Douglas R. Bolster, Ali K. Reiter, Scot R. Kimball, and Leonard S. Jefferson
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of
Medicine, Hershey PA 17033
Abbreviated Title: Translational control of protein synthesis by FFA
Corresponding author: Dr. Leonard S. Jefferson
Department of Cellular and Molecular Physiology
The Pennsylvania State University College of Medicine
PO Box 850
Hershey, PA 17033
Telephone: (717) 531-8567
Fax: (717) 531-7667
E-mail: [email protected]
Copyright 2002 by the American Physiological Society.
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ABSTRACT
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
b-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 a-subunit of eIF2 was
enhanced, and phosphorylation of 4E-BP1 and S6K1 was reduced. Similar changes in protein
synthesis and translation initiation were not observed in the gastrocnemius following treatment
with methyl palmoxirate. In heart, repressed b-oxidation of FFA correlated, as demarcated by
changes in ATP/AMP ratio and phosphorylation of AMP-activated kinase (AMPK), with
alterations in the energy status of the tissue. Therefore, the activation state of signal transduction
pathways that are responsive to the cellular energy stress represents one mechanism whereby
translation initiation may be regulated in cardiac muscle.
Keywords: translation initiation, gastrocnemius muscle, AMP-activated protein kinase
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INTRODUCTION
Previous studies have suggested a role for free fatty acids 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 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 non-working (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 to fast-twitch skeletal muscle which predominantly depends upon
glucose to maintain energy stores.
It is well recognized that unlike non-working 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. Based on this premise, inhibition of b-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.
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As 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/AMP as well as phosphocreatine/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 5aminoimidazole-4-carboxamide 1-b-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 (tRNAiMet) to the 40S ribosomal subunit
where GTP is hydrolyzed to GDP. For subsequent rounds of initiation to occur the guanine
nucleotide exchange protein, eIF2B, must catalyze the exchange of GDP for GTP on eIF2 (27).
Phosphorylation of the a-subunit of eIF2 (eIF2a) 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
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skeletal muscle, reviewed by (1; 23) 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.
MATERIALS AND METHODS
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 (~200g) were maintained on a 12 hour light: dark cycle
with a standard diet (Harlan-Tekland Rodent Chow, Madison, WI) and water provided ad
libitum.
Inhibition of b-oxidation. b-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 hours, weighed, and randomly received either a single bolus of methyl
palmoxirate (25mg/kg body weight 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 below.
Administration of metabolic tracer and sample collection. A flooding dose (1.0 mL/100
g body weight) of L-[2,3,4,5,6-3H] phenylalanine (150 mM, containing 3.70GBq/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 1800 x g for 10 min at 4° C to obtain serum. After
excision, a portion of the gastrocnemius or heart were homogenized in seven volumes of buffer
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consisting of 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (pH 7.4), 100 mM
KC1, 0.2 mM EDTA, 2 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N’, N’-tetraacetic
acid, 1 mM dithiothreitol, 50 mM sodium fluoride, 50 mM b-glycerophosphate, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine and 0.5 mM sodium vanadate. An aliquot
(0.5mL) of the homogenate was used for the measurement of muscle protein synthesis as
described below. The remainder of the homogenate was immediately centrifuged at 10,000 x g
for 10 min at 4° C. The supernatant was used for analysis of mRNA translation initiation factors
as described below. The remaining tissue was used to assess eIF2B activity as described below.
Serum measurements. Serum free fatty acids 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, 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 (22). 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 eIF2a was evaluated in 10,000 x g supernatants by protein immunoblot analysis as
described previously (12; 13; 22). The guanine nucleotide exchange activity of eIF2B was
assessed by the exchange of [3H]GDP bound to eIF2a for non-radioactively 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 Nembutal
Sodium (Abbott Laboratories, North Chicago, IL). Adenine nucleotide concentrations were
determined as described previously (24).
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Analysis of AMP-activated Protein Kinase phosphorylation. Phosphorylation of the
catalytic subunit of AMPK was evaluated in 10,000 x g supernatants by protein immunoblot
analysis using a phosphospecific (Thr 172) AMPK antibody.
Statistical Analysis. Data are expressed as the mean ± SEM. All data were analyzed by
the InStat Version 3 statistical software package (GraphPad SoftwareInc., San Diego, CA).
Statistical significance was assessed using a two-tailed Student’s t-test unless stated otherwise. P
values of less than 0.05 were considered statistically significant.
RESULTS
To examine the contribution of b-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 CPT1. The effectiveness of the treatment was assessed by
measuring plasma FFA concentrations. As shown in Table 1, methyl palmoxirate treatment
resulted in an almost 3-fold increase in plasma FFAs. These results are consistent with methyl
palmoxirate inhibiting the b-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).
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 using 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 using 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 eIF2a was measured by protein immunoblot analysis using 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 eIF2a 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 eIF2a.
The most well 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 by 34). In addition to 4E-BP1, mTOR also controls the
activity of the ribosomal protein S6 kinase, 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
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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.
The most well 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 using 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 Figure 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.
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 on 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
indicates an impairment in the transport of met-tRNAiMet to the 40S ribosomal subunit. As the
phosphorylation of eIF2a 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 have also 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+/NADPH ratios are altered in the hearts, but not the gastrocnemius, of methyl
palmoxirate-treated animals (data not shown).
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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 is inhibited when FFA
oxidation is inhibited. Furthermore, as 4E-BP1 and S6K1 are downstream targets of the kinase
mTOR, the results indicate that an mTOR-dependant 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 b-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 b-oxidation. These
results are in keeping with the fact that FFAs are the primary energy source of the heart, while
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-dependant
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 eIF2a was actually decreased.
These results are in contrast to those presented here with regards to the heart and suggest that
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methyl palmoxirate may induce eIF2a 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. Based on 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, independent of changes in
circulating insulin, following a 5 hour lipid infusion (28). Therefore, it is possible that FFAmediated 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 b-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 b-oxidation in working oxidative fibers
result in the development of cellular stress . Both AMPK-dependant 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
The authors sincerely thank Lynne Hugendubler and Sharon Rannels for their invaluable
assistance with sample collection and guanine nucleotide exchange activity measurements and
Dr. Joshua Anthony for his expert advice with tracer administration. We would also like to
thank Dr. David MacLean, Teresa Markle, and Kristine Rice for their help with protein synthesis
measurements and Dr. Kathryn LaNoue and Deborah Berkich for their help with determination
of adenine nucleotide concentrations. Finally, we would like to thank the R.W. Johnson
Pharmaceutical Research Institute for providing the methyl palmoxirate utilized in these studies.
This research was supported by National Institutes of Health grants DK15658 (L.S.J.) and
GM08619 (supports S.J.C.).
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FIGURE LEGENDS
Figure 1. Rates of protein synthesis in cardiac and gastrocnemius muscles of control and
methyl palmoxirate-treated rats. Protein synthesis was measured as the incorporation of
[3H]phenylalanine into protein as described under “Materials and Methods”. Light bars, control
animals; dark bars, animals administered methyl palmoxirate. Values represent means ± SEM; n
= 5-13. Means not sharing a superscript are significantly different, p < 0.05.
Figure 2. eIF2B activity in cardiac and gastrocnemius muscles of control and methyl
palmoxirate-treated rats. The guanine nucleotide activity of eIF2B was measured as the
exchange of nonradiolabeled GDP in an eIF2•GDP binary complex for free GTP as described
under “Materials and Methods”. Light bars, control animals; dark bars, animals administered
methyl palmoxirate. Values represent means ± SEM; n = 5-9. Means not sharing a superscript
are significantly different, p < 0.05.
Figure 3. Phosphorylation of eIF2a on Serine 51 in cardiac and gastrocnemius muscles
of control and methyl palmoxirate-treated rats. Phosphorylation of the a-subunit of eIF2 was
assessed by Western blot analysis using an anti-phospho-eIF2a that specifically recognizes the
protein when it is phosphorylated on Ser51. Total eIF2 content was measured by Western blot
analysis using a monoclonal antibody that recognizes phosphorylated and unphosphorylated
forms of the protein. Results of typical blots are shown as an insert to the figure. Gastroc,
gastrocnemius muscle; C, control rats; MP, methyl palmoxirate-treated rats; eIF2a(P), eIF2
phosphorylated on Ser51; eIF2, total eIF2a content. Light bars, control animals; dark bars,
animals administered methyl palmoxirate. Values represent means ± SEM; n = 7-21. Means not
sharing a superscript are significantly different, p< 0.05.
19
Figure 4. Phosphorylation of 4E-BP1 in cardiac and gastrocnemius muscles of control
and methyl palmoxirate-treated rats. When subjected to SDS-PAGE, 4E-BP1 is resolved into
multiple electrophoretic forms whereby the hypophosphorylated forms exhibit the greatest
mobility and hyperphosphorylated forms the least. Results of a typical blot are shown as an
insert to the figure. Gastroc, gastrocnemius muscle; C, control rats; MP, methyl palmoxiratetreated rats; a, 4E-BP1a ; b, 4E-BP1b; g, 4E-BP1g. Results are expressed as the ratio of
hyperphosphorylated 4E-BP1 (b and g forms) to total 4E-BP1. Light bars, control animals; dark
bars, animals administered methyl palmoxirate. Values represent means ± SEM; n = 5–13.
Means not sharing a superscript are significantly different, p< 0.05.
Figure 5. Hyperphosphorylated S6K1 in cardiac and gastrocnemius muscles of control
and methyl palmoxirate-treated rats. When subjected to SDS-PAGE, S6K1 is resolved into
multiple electrophoretic forms whereby the most highly phosphorylated forms exhibit the
slowest electrophoretic mobility. Results of a typical blot are shown as an insert to the figure.
Gastroc, gastrocnemius muscle; C, control rats; MP, methyl palmoxirate-treated rats; a, S6K1a;
b, S6K1b; g, S6K1g.
Results are expressed as the ratio of hyperphosphorylated S6K1 (b and g
forms) to total S6K1. Light bars, control animals; dark bars, animals administered methyl
palmoxirate. Values represent means ± SEM; n = 5–13. Means not sharing a superscript are
significantly different, p< 0.05.
Figure 6. Adenosine nucleotide ratios and AMPK phosphorylation in cardiac and
gastrocnemius muscles of control and methyl palmoxirate-treated rats.
(a) Heart and
gastrocnemius AMP and ATP contents were measured as described under “Materials and
Methods”. Light bars, control animals; dark bars, animals administered methyl palmoxirate.
Values represent means ± SEM; n = 4-5. (b) Phosphorylation of the AMPK was assessed by
Western blot analysis using an anti-phospho-AMPK antibody that specifically recognizes the
protein when it is phosphorylated on Thr177. Total AMPK content was measured by Western
20
blot analysis using a monoclonal antibody that recognizes phosphorylated and unphosphorylated
forms of the protein. Results of typical blots are shown as an insert to the figure. Gastroc,
gastrocnemius muscle; C, control rats; MP, methyl palmoxirate-treated rats; AMPK(P), AMPK
phosphorylated on Thr177; AMPK, total AMPK content. Means not sharing a superscript are
significantly different using a one-tailed Student’s t-test, P < 0.05.
21
Table I. Plasma Concentrations of Free Fatty Acids, Insulin, and Glucose in Control and Methyl
Palmoxirate-Treated Rats
Plasma Concentration
Group
FFA
(mM)
Insulin
(ng/mL)
Control
0.52 ± 0.03
Methyl Palmoxirate
1.45 ± 0.10
a
b
Glucose
(mM)
a
0.58 ± 0.05
a
0.46 ± 0.04
a
7.51 ± 0.16
b
6.72 ± 0.40
Serum free fatty acid (FFA), insulin, and glucose concentrations of control and methyl
palmoxirate-treated rats were measured as described under “Materials and Methods”. Values
represent means ± SEM; n = 10-13. Means not sharing a superscript are significantly different,
p< 0.05.
Figure 1
125
a
a’
Protein Synthesis
(% control value)
100
a’
75
b
50
25
0
Heart
Gastrocnemius
Figure 2
0.08
eIF2B Activity
(pmol GDP exchanged / min)
0.07
a
a’
a’
0.06
0.05
b
0.04
0.03
0.02
0.01
0.00
Heart
Gastrocnemius
Figure 3
2.00
Heart
b
Gastroc
– eIF2α(P)
eIF2α Phosphorylation on Ser51
(fraction of control)
1.75
– eIF2α
C
MP
C
MP
1.50
1.25
a
a’
a’
1.00
0.75
0.50
0.25
0.00
Heart
Gastrocnemius
Figure 4
4E-BP1 Phosphorylatoin
(fraction in hyperphophorylated form)
8
7
Heart
a
Gastroc
−γ
−β
−α
6
C
MP
C
MP
a’
a’
5
4
b
3
2
1
0
Heart
Gastrocnemius
Figure 5
0.6
S6K1 Phosphorylation
(hyperphosphorylated / total)
a
Heart
Gastroc
−γ
−β
−α
0.5
C
MP
C
MP
b
0.4
a’
0.3
a’
0.2
0.1
0.0
Heart
Gastrocnemius
Figure 6
15.0
A
a’
a’
ATP : AMP Ratio
12.5
10.0
a
7 .5
b
5 .0
2 .5
0 .0
AMPK Phosphorylation on Thr172
(fraction of control)
1 .75
Heart
b
B
Gastroc
– AMPK(P)
– AMPK
C
MP
C
MP
1 .50
a’
1 .25
a’
a
1 .00
0 .75
0 .50
0 .25
0 .00
Heart
Gastrocnemius