Endotoxin Induces Differential Regulation of mTOR-Dependent Signaling in Skeletal Muscle and Liver of Neonatal Pigs

Endotoxin Induces Differential Regulation of mTOR-Dependent Signaling inSkeletal Muscle and Liver of Neonatal Pigs

Scot R. Kimball1, Renán A. Orellana2, Pamela M.J. O'Connor2, Agus Suryawan2, Jill A. Bush2,

Hanh V. Nguyen2, M. Carole Thivierge2, Leonard S. Jefferson1, and Teresa A. Davis2

1Department of Cellular and Molecular Physiology, The Pennsylvania State University College

of Medicine, Hershey, PA 17033; 2 United States Department of Agriculture/Agricultural

Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor

College of Medicine, Houston, TX 77030

Short title: mTOR-Dependent Signaling in the Septic Neonate

Correspondence and reprint requests:

Scot R. Kimball, Ph.D.

Department of Cellular and Molecular Physiology (H166)

The Pennsylvania State University College of Medicine

500 University Drive

Hershey, PA 17033

Telephone: 717-531-8970

FAX: 717-531-7667

Email: [email protected]

Copyright (c) 2003 by the American Physiological Society.

Articles in PresS. Am J Physiol Endocrinol Metab (May 28, 2003). 10.1152/ajpendo.00340.2002

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ABSTRACT

In the present study, differential responses of regulatory proteins involved in translation initiation

in skeletal muscle and liver during sepsis were studied in neonatal pigs treated with

lipopolysaccharide (LPS). LPS did not alter eukaryotic initiation factor (eIF)2B activity in either

tissue. In contrast, binding of eIF4G to eIF4E to form the active mRNA binding complex was

repressed in muscle and enhanced in liver. Phosphorylation of eIF4E binding protein, 4E-BP1,

and ribosomal protein S6 kinase, S6K1, was reduced in muscle during sepsis, but increased in

liver. Finally, changes in 4E-BP1 and S6K1 phosphorylation were associated with altered

phosphorylation of the protein kinase, mTOR. Overall, the results suggest that translation

initiation in both skeletal muscle and liver is altered during neonatal sepsis by modulation of the

mRNA binding step through changes in mTOR activation. Moreover, the LPS-induced changes

in factors that regulate translation initiation are more profound than previously reported changes

in global rates of protein synthesis in the neonate. This finding suggests that the met-tRNAi,

rather than mRNA, binding step in translation initiation may play a more critical role in

maintaining protein synthesis rates in the neonate during sepsis.

Keywords: eukaryotic initiation factor eIF2B, eIF4G, eIF4E, lipopolysaccharide

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INTRODUCTION

Previous studies (48, 49) have shown that in adult rodents, the function of three proteins

that play important regulatory roles in translation initiation, i.e. eukaryotic initiation factors

eIF2B and eIF4F and the ribosomal protein S6 kinase S6K1, are downregulated in skeletal

muscle of septic rats. eIF2B is a guanine nucleotide exchange factor whose substrate, eIF2,

mediates the binding of initiator methionyl-tRNAi (met-tRNAi) to the 40S ribosomal subunit

(reviewed in 16). Thus, eIF2B modulates the first step in translation initiation and changes in its

activity result in corresponding alterations in global rates of protein synthesis. Binding of

mRNA to the 40S ribosomal subunit is mediated by a heterotrimeric complex referred to as

eIF4F. The heterotrimeric complex referred to as eIF4F mediates the binding of mRNA to the

40S ribosomal subunit. The three proteins that comprise the eIF4F complex are eIF4A, an RNA

helicase, eIF4E, the protein that binds to the m7GTP cap structure at the 5’-end of the mRNA,

and eIF4G, a scaffolding protein that, in addition to eIF4A and eIF4E, also binds to the eIF3•40S

ribosomal subunit complex. Thus, mRNA binds to the 40S ribosomal subunit through the

association of eIF4E with eIF4G. The binding of eIF4E to eIF4G is reversible and regulated by

the association of eIF4E with the eIF4E binding proteins (e.g., 4E-BP1). eIF4E complexed with

4E-BP1 can bind mRNA, but cannot bind to eIF4G such that when the eIF4E•mRNA complex is

associated with 4E-BP1, the complex cannot bind to the 40S ribosomal subunit. The association

of eIF4E with 4E-BP1 is regulated by phosphorylation of 4E-BP1 whereby eIF4E will bind to

hypophosphorylated, but not hyperphosphorylated, 4E-BP1. Hyperphosphorylation of 4E-BP1

that occurs in response to treatment with growth factors or amino acids is mediated through a

signal transduction pathway involving a protein kinase referred to as the mammalian target of

rapamycin (mTOR; aka FRAP or RAFT) (13). mTOR also phosphorylates, and thereby

activates, the 70 kDa ribosomal protein S6 kinase, S6K1. Both phosphorylation events are,

under some circumstances, associated with changes in global rates of protein synthesis, but more

importantly they lead to a preferential increase in translation of mRNAs encoding particular

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proteins, particularly those mRNAs containing extensive secondary structure in their 5’-

untranslated region (reviewed in 13) or those mRNAs containing a terminal oligopyrimidine

(TOP) tract adjacent to the 5’-cap structure (29).

A recent study (25) demonstrated that in adult rats, the LPS-induced repression of global

rates of protein synthesis observed in gastrocnemius muscle is associated with both a decrease in

eIF2B activity and decreased assembly of the active eIF4F complex. Such changes in translation

initiation factors are quantitatively similar to those observed in a longer term model of chronic

abdominal sepsis (48, 49) as well as in rats treated with tumor necrosis factor (TNF)-a (26).

Which of these steps, the met-tRNAi binding or the mRNA binding step, is rate controlling for

global rates of protein synthesis in muscle during sepsis is unknown. In contrast to muscle,

considerably less is known about the mechanism(s) involved in the increase in hepatic protein

synthesis that occurs during sepsis. However, it has been reported that during chronic abdominal

sepsis, phosphorylation of S6K1 is enhanced in liver (4). The signal transduction pathway(s)

that mediates the effects of LPS or TNF-a on eIF4F assembly and S6K1 phosphorylation have

not been identified in either muscle or liver.

Little is known about the effects of sepsis in the neonate, a population whose protein

synthesis rates are relatively high and uniquely sensitive to anabolic agents (5, 7-9, 11). In a

recent study (35), lipopolysaccharide (LPS) administration to neonatal pigs to promote cytokine

production repressed protein synthesis in skeletal muscle and stimulated protein synthesis in

liver. However, the magnitude of the changes in both tissues was smaller than previously

reported for adult animals (35). In fact, in longissimus dorsi skeletal muscle, the decrease in

protein synthesis was proportional to the decline in muscle RNA content, suggesting that a fall in

ribosome number is causative in the effect of LPS on protein synthesis in that tissue. The

relative insensitivity of protein synthesis to LPS treatment in neonates thus may provide an

unique opportunity to examine the mechanisms by which LPS differentially regulates protein

synthesis in skeletal muscle and liver. Overall, the results indicate that LPS administration

results in enhanced signaling through mTOR in liver, but repressed signaling in skeletal muscle.

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Moreover, the results suggest that eIF2B activity, rather than eIF4F assembly or S6K1

phosphorylation, may be rate controlling for global rates of protein synthesis in response to LPS

administration.

MATERIALS AND METHODS

Materials. The anti–phospho-Thr389 S6K1 antibody was purchased from Santa Cruz

Biotechnology. The anti-phospho-Ser2448 mTOR antibody was obtained from Cell Signaling

Technology. The anti-mTOR antibody was purchased from Calbiochem.

Animals. Pigs (Landrace x Yorkshire x Hampshire x Duroc) were studied at 5-6 d of age.

Three days before the study was performed, piglets were anesthetized and catheters were

surgically inserted into a jugular vein and a carotid artery as described previously (5). Twenty

piglets (5-6 days of age; 2.2 ± 0.37 kg) from two litters were assigned randomly to control

(n=10) and LPS (n=10) treatment groups. To minimize variability in the nutritional status of

pigs at the time of study, pigs were fasted for 18 h, and one hour prior to the start of the study, a

constant infusion of dextrose (800 mg/(kg•hr)) and an amino acid mixture (1.8 mmol total amino

acids/(kg•hr)) (7) was begun to simulate a fed state. At t=0, animals were administered LPS (10

µg/kg/h) or an equal volume of saline (control) and 7.5 h later, a bolus dose of [3H]phenylalanine

was administered intravenously. Eight hours after the LPS infusion was initiated, pigs were

euthanized with an intravenous dose of pentobarbital sodium (50 mg/kg body weight), and all

tissue samples were rapidly removed, frozen in liquid nitrogen, and stored at -70° C until

analysis. The protocol, previously described in Orellana et al. (35), was approved by the

Animal Care and Use Committee of Baylor College of Medicine and was conducted in

accordance with the National Research Council’s Guide for the Care and Use of Laboratory

Animals.

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Measurement of eIF2B Activity. eIF2B activity in muscle and liver homogenates was

measured by the exchange of [3H]GDP bound to eIF2 for nonradioactively labeled GDP, as

described previously (22). Activity is expressed as the rate of GDP exchange.

Quantitation of 4E-BP1•eIF4E and eIF4G•eIF4E complexes. The association of eIF4E

with 4E-BP1 or eIF4G was quantitated as described previously (21). Briefly, eIF4E was

immunoprecipitated from muscle or liver homogenates using a monoclonal anti-eIF4E antibody.

Proteins in the immunoprecipitate were resolved by SDS-polyacrylamide gel electrophoresis and

then transferred to PVDF membranes. The membranes were then probed with either anti-4E-

BP1 or anti-eIF4G antibodies and then developed using an ECL Western Blotting kit (Amersham

Pharmacia Biotech). The horseradish peroxidase coupled to the anti-rabbit secondary antibody

was then inactivated by incubating the blot in 15% hydrogen peroxide for 30 min at room

temperature and the membranes were reprobed with the monoclonal anti-eIF4E antibody.

Values obtained using the anti-4E-BP1 and anti-eIF4G antibodies were normalized for the

amount of eIF4E present in the sample.

Measurement of site specific phosphorylation of S6K1 and mTOR. Phosphorylation of

ribosomal protein S6 kinase S6K1 was assessed by Western blot analysis using an antibody

specific for S6K1 when it is phosphorylated at the activating residue, Thr389. Phosphorylation

of mTOR on Ser2448, a residue located in a repressor domain, was assessed by Western blot

analysis using an antibody specific for mTOR phosphorylated on Ser2448. The membranes were

then treated with hydrogen peroxide as described above and reprobed with an anti-mTOR

antibody that recognizes both the phosphorylated and unphosphorylated forms of the protein.

Values obtained using the anti-phospho-mTOR antibody were normalized for the total amount of

mTOR present in the sample

Phosphorylation of the eIF4E binding protein 4E-BP1. During SDS-polyacrylamide gel

electrophoresis, 4E-BP1 resolves into multiple isoelectric forms based on its phosphorylation

state. The hyperphosphorylated g-form is the slowest migrating form and does not bind to

eIF4E. Phosphorylation of 4E-BP1 was assessed by Western blot analysis using an antibody that

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recognizes all three forms of 4E-BP1 resolved during electrophoresis. Results are expressed as

the amount of the protein present in the g-form as a percentage of total 4E-BP1.

Statistics. Values shown are means ± SE. Statistical evaluation of the data was

performed using an unpaired, two-tailed t-test. Differences between means were considered

significant at p<0.05.

RESULTS

Because a change in eIF2B activity is the primary mechanism through which the met-

tRNAi binding step is regulated (reviewed in 17), the guanine nucleotide exchange activity of

eIF2B was measured in extracts of longissimus dorsi and liver from control and LPS-treated

pigs. The difference in eIF2B activity between muscle and liver (Fig. 1) was directly

proportional to the previously reported difference in protein synthesis (35). Thus, eIF2B activity

was greater in liver compared to muscle. However, no change in eIF2B activity was observed in

either skeletal muscle or liver of LPS-treated compared to control pigs.

The best characterized mechanism through which the binding of mRNA to the 40S

ribosomal subunit is regulated involves the reversible binding of eIF4G to eIF4E (reviewed in

38). In the present study, the amount of eIF4G bound to eIF4E was measured by

immunoprecipitation of eIF4E followed by protein immunoblot analysis for eIF4G present in the

immunoprecipitate. As shown in Fig. 2, the amount of eIF4G present in the eIF4G•eIF4E

complex in muscle was significantly reduced (-79%) by LPS treatment. In contrast, the binding

of eIF4G to eIF4E in liver was enhanced (+77%) in response to LPS treatment.

To determine whether the changes in eIF4G binding to eIF4E were due to altered

association of eIF4E with the eIF4E binding proteins, the eIF4E immunoprecipitates used for the

analyses shown in Fig. 2 were also analyzed for 4E-BP1 content. In muscle, the amount of 4E-

BP1 associated with eIF4E was increased (+53%) in LPS-treated compared to control pigs (Fig.

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3A). In contrast, in liver, the amount of 4E-BP1 bound to eIF4E was reduced (-44%) in response

to LPS treatment.

Binding of 4E-BP1 to eIF4E is regulated by phosphorylation of 4E-BP1. When 4E-BP1

is resolved by SDS-polyacrylamide gel electrophoresis, it separates into three isoforms where the

fastest migrating form (referred to as 4E-BP1a) is the least phosphorylated and the slowest

migrating form (referred to as 4E-BP1g) is the most highly phosphorylated form of the protein

(15, 36). Because the g-form is the only one that does not bind to eIF4E, the phosphorylation

state of 4E-BP1 is presented as the proportion of the protein present in the g-form. As shown in

Fig. 3B, the amount of 4E-BP1 present in the hyperphosphorylated g-form was reduced (-60%)

in muscle, but increased (+70%) in liver, of LPS-treated compared to control pigs.

Like 4E-BP1, S6K1 is downstream of mTOR (reviewed in 38). In in vitro studies,

mTOR phosphorylates S6K1 on Thr389 (3). To determine whether phosphorylation of

S6K1(Thr389) is altered by LPS, muscle and liver extracts were analyzed by protein immunoblot

analysis using an antibody that binds to S6K1 only when it is phosphorylated on Thr389. In the

present study, phosphorylation of S6K1 on Thr389 was reduced (-74%) in muscle and increased

(+387%) in liver of LPS-treated pigs compared to control animals (Fig. 4).

Phosphorylation of both 4E-BP1 and S6K1 are dependent upon the activity of mTOR

(reviewed in 38). One mechanism that has been proposed for regulation of mTOR activity

involves phosphorylation of the protein on Ser2448, a residue that is present in a putative

repressor domain (43). In the present study, the phosphorylation state of mTOR on Ser2448 was

assessed by protein immunoblot analysis using an antibody that specifically recognizes the

phosphorylated form of the protein. As shown in Fig. 5, the relative phosphorylation of mTOR

on Ser2448 was reduced (-51%) in muscle and enhanced (+83%) in liver of LPS-treated

compared to control pigs. Interestingly, the changes in mTOR phosphorylation were similar in

magnitude to those observed for 4E-BP1 and S6K1 phosphorylation (Fig. 3B and 5,

respectively).

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DISCUSSION

In the present study, no change in eIF2B activity was observed in either longissimus dorsi

or liver of neonatal pigs after 8 hours of LPS infusion, suggesting that the met-tRNAi binding

step in translation initiation is unaffected under such conditions. This finding is in contrast to

previous results showing that in adult rats, either TNF-a (26) or LPS (25) administration or

chronic abdominal sepsis (48) reduces eIF2B activity in gastrocnemius muscle. The basis for the

differential response observed in neonatal pigs compared to adult rats may be due to species

differences, duration of sepsis, or in the methods used to induce a septic-like response.

Alternatively, the difference may be due to the age of the animals being studied. For example,

skeletal muscle protein synthesis is stimulated by insulin in 7 day old pigs, an effect that is

severely attenuated by 28 days of age (6). Moreover, in cells in culture, insulin stimulates eIF2B

by modulating phosphorylation of the e-subunit of eIF2B (50). Because plasma insulin

concentrations are elevated in LPS-treated pigs (35) and protein synthesis exhibits heightened

sensitivity to insulin in neonatal pigs (6), it may be that insulin is better able to maintain eIF2B

activity in muscle of neonatal compared to adult animals.

In contrast to the lack of effect on eIF2B activity in neonatal pigs, LPS-treatment

decreased the amount of eIF4G associated with eIF4E and increased the binding of 4E-BP1 to

eIF4E in skeletal muscle. It is interesting to note that the magnitude of the change in eIF4G

binding to eIF4E was greater than the change in 4E-BP1 binding. In part, this result may be due

to alterations in eIF4E binding to other eIF4E binding proteins, such as 4E-BP2 and/or 4E-BP3.

It is also noteworthy that the magnitude of the change in the association of eIF4G with eIF4E in

skeletal muscle of the neonate is similar to that reported in models of adult sepsis (26, 49),

despite the small changes in muscle protein synthesis in the neonatal model (35). This suggests

that other factors may be involved in maintaining the relatively high protein synthetic rates in

skeletal muscle of the neonate during catabolic conditions such as sepsis and endotoxemia. In

fact, it was previously reported that RNA content in both skeletal muscle and liver of neonatal

10

pigs tended to change during LPS infusion such that, when normalized for RNA content, protein

synthesis in longissimus dorsi is unchanged and in liver the magnitude of the change is reduced

to 14%. Thus, much of the change in protein synthesis in LPS-treated neonates is a result of

changes in RNA content.

The finding that the magnitude of the changes in eIF4F assembly and S6K1

phosphorylation are much larger than the changes in global protein synthesis suggests that in the

neonate, such changes may play a relatively more important role in regulating the translation of

specific mRNAs compared to be mediating global changes in protein synthesis. In this regard,

numerous studies have shown that S6K1-induced phosphorylation of rpS6 has little if any effect

on the translation of most mRNAs, but instead promotes the translation of TOP mRNAs, i.e

mRNAs that encode proteins important for cell growth (reviewed in 46). For example, during

liver regrowth after partial hepatectomy, TOP mRNA translation correlates with increased

activity of S6K1 and phosphorylation of rpS6 (1, 14, 18, 32). In contrast, cell cycle arrest is

associated with selective translational arrest of TOP mRNA translation (12, 19, 28). Finally, in

both flies (30) and mice (44) disruption of the gene encoding S6K1 results in organisms that

have the same number of cells as do wild-type animals, but with smaller cells. In part, the small

cell phenotype observed in response to S6K1 deletion is a result of impaired regulation of TOP

mRNA translation. Thus, in contrast to wild-type cells, in S6K1-/- cells serum does not promote

recruitment of TOP mRNAs into polysomes (20). One model that could account for the

preferential recruitment of TOP mRNAs into polysomes is that phosphorylated, but not

unphosphorylated, rpS6 might exhibit increased affinity for the TOP structure. The observation

that rpS6 is located near the mRNA binding site on the 40S ribosomal subunit (33, 34) makes

such a model feasible. Thus, it is not surprising that in the present study, large changes in S6K1

phosphorylation are not associated with similar changes in global protein synthesis, because TOP

mRNAs represent only a fraction of total mRNA.

Similarly, increased availability of eIF4E results in preferential translation of a subset of

mRNAs. For example, addition of either eIF4E or eIF4F to cell-free translation systems results

11

in a differential enhancement of mRNA translation; i.e. certain mRNAs are preferentially

translation when eIF4F content is increased (39, 41, 45). This finding has been extended in

recent years to show that overexpression of eIF4E in cells in culture leads to cell transformation

as a result of increased translation of mRNAs encoding proteins important in the transformation

process (reviewed in 10). One model that has been proposed to account for the observed

changes in mRNA translation is that mRNAs with short, relatively unstructured 5’-untranslated

regions (UTR) are translated preferentially compared to mRNAs with long, highly structured 5’-

UTRs (reviewed in 13). In this model, increasing eIF4F availability upregulates the translation

of mRNAs with highly structured 5’-UTRs. Thus, in the present study, the observed changes in

eIF4G•eIF4E association may result in a preferential change in the translation of specific groups

of mRNAs, an effect that might not be detected by measuring the incorporation of radioactive

amino acid into total protein over a 30 min period. It is thus interesting to note that during

sepsis, the pattern of proteins secreted by the liver changes such that secretion of normally

expressed proteins like albumin and transferrin falls dramatically (reviewed in 47). In contrast,

synthesis and secretion of the acute phase proteins from liver is enhanced. The importance of the

translational control mechanisms described above in mediating the reported changes in liver

protein synthesis is unknown. However, it is tempting to speculate that the changes in

eIF4G•eIF4E complex formation and/or S6K1 phosphorylation that occur during sepsis might

play an important role in modulating gene expression. In this regard, in unpublished studies, we

have found that the distribution of the mRNA encoding ribosomal protein S8 is predominantly

polysomal in livers from neonatal pigs treated with LPS compared to a primarily nonpolysomal

distribution in livers from control animals.

Direct support for a model wherein eIF2B is rate controlling for changes in global rates

of protein synthesis whereas eIF4F assembly and S6K1 activation are not, is provided by studies

where L6 myoblasts were deprived of either leucine or histidine (21, 24). Deprivation of either

amino acid was shown to cause decreased global rates of protein synthesis and eIF2B activity,

but only deprivation of leucine repressed eIF4F assembly and S6K1 phosphorylation. Moreover,

12

addition of insulin to leucine deprived cells restored eIF4F assembly and S6K1 phosphorylation

to values observed in control cells or histidine-deprived cells, but had no effect on either global

rates of protein synthesis or eIF2B activity. Thus, in that study changes in eIF4F assembly and

S6K1 phosphorylation had no effect on global rates of protein synthesis. Instead, changes in

eIF4F assembly and S6K1 phosphorylation were shown to promote preferentially the translation

of mRNAs encoding ornithine decarboxylase and elongation factor 1A (24).

The changes in 4E-BP1 binding to eIF4E observed in the present study were associated

with alterations in 4E-BP1 phosphorylation, whereby the proportion of the protein in the

hyperphosphorylated g-form was reduced in skeletal muscle but enhanced in liver of LPS-treated

pigs. Previous studies have shown that the feeding-induced increase in 4E-BP1

hyperphosphorylation that occurs in skeletal muscle is dependent upon the activity of mTOR (2,

23). Thus, treatment with the mTOR inhibitor rapamycin prior to feeding prevents the

subsequent phosphorylation of 4E-BP1 and S6K1 in skeletal muscle. Moreover, in cells in

culture, the phosphorylation of 4E-BP1 that occurs in response to a variety of stimuli, including

insulin, IGF-I, and amino acids, is blocked by pre-treatment with rapamycin (reviewed in 27,

37). One mechanism that has been proposed for regulation of the protein kinase activity of

mTOR involves phosphorylation of Ser2448, a residue in a domain that has been characterized

as a repressor of mTOR function (43). In this regard, PKB phosphorylates Ser2448 on mTOR in

vitro (31, 40), and activation of PKB in cells in culture is associated with enhanced

phosphorylation of Ser2448 on mTOR as well as phosphorylation of proteins downstream of

mTOR, such as 4E-BP1 and S6K1 (31, 42, 43). In the present study, we demonstrate for the

first time that changes in 4E-BP1 and S6K1 phosphorylation during sepsis were mirrored by

alterations in phosphorylation of Ser2448 on mTOR. Thus, in skeletal muscle of the neonate,

phosphorylation of all three proteins is reduced, whereas in liver, phosphorylation is enhanced by

LPS-treatment.

Perspectives. Overall, the results of the present study demonstrate for the first time that

LPS-treatment of neonatal pigs represses signaling through the mTOR pathway in muscle while

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stimulating signaling through this pathway in liver. The observed changes in mTOR

phosphorylation are associated with modulation of both S6K1 and 4E-BP1 phosphorylation, as

well as alterations in eIF4G binding to eIF4E. In contrast, no change in eIF2B activity is

observed in either muscle or liver of neonatal pigs in response to LPS-treatment. The results

therefore suggest that in neonatal pigs, the lack of effect of LPS treatment on eIF2B activity

likely accounts for the observed maintenance of global rates of protein synthesis.

The magnitude of the changes in the phosphorylation of 4E-BP1 and the association of

eIF4E with eIF4G in skeletal muscle were similar to that reported in models of adult sepsis. In

contrast, the magnitude of the reduction in muscle protein synthesis rates in neonatal sepsis in

our recent paper (35) was less profound than that in adult sepsis models. This suggests that the

relatively high synthesis rates of skeletal muscle proteins in the neonate may in part be

maintained during neonatal sepsis through a mechanism that is distinct from eIF4F complex

formation.

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ACKNOWLEDGEMENTS

The authors thank S. Rannels, S. Nguyen, and W. Liu for technical assistance and J.

Cunningham and F. Biggs for care of animals. We also thank Drs. C.H. Lang and T.C. Vary for

critical reading of the manuscript prior to submission.

This project has been funded in part by National Institute Health Grant R01 AR44474

and the U.S. Department of Agriculture, Agricultural Research Service under Cooperative

Agreement number 58-6250-6-001 (TAD) and National Institutes of Health Grants DK13499

and DK15658 (LSJ). The contents of this publication do not necessarily reflect the views or

policies of the U.S. Department of Agriculture nor does mention of trade names, commercial

products or organization imply endorsements by the U.S. Government.

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FIGURE LEGENDS

Fig. 1. Effect of LPS treatment on eIF2B activity in skeletal muscle and liver of neonatal

pigs. Neonatal pigs were treated with LPS and the rate of exchange of [3H]GDP bound to eIF2

for nonradiolabeled GDP was measured as described under “Materials and Methods”. The

results represent the mean ± SE for 9-10 animals per condition.

Fig. 2. Effect of LPS treatment on eIF4G association with eIF4E in skeletal muscle and

liver of neonatal pigs. Neonatal pigs were treated with LPS as described under “Materials and

Methods”. The amount of eIF4G bound to eIF4E was measured as described under "Materials

and Methods". Values for eIF4G were normalized for the recovery of eIF4E in the

immunoprecipitate. The results represent the mean ± SE for 10 animals per condition. The

results of typical blots are shown as an inset to the panel. Muscle and liver samples were

analyzed on separate immunoblots and therefore the results from the two tissues cannot be

directly compared. C, control pigs; L, LPS-treated pigs. * p<0.02 vs. control muscle; † p<0.025

vs. control liver.

Fig. 3. Effect of LPS treatment on eIF4E association with 4E-BP1 and 4E-BP1

phosphorylation in skeletal muscle and liver of neonatal pigs. Neonatal pigs were treated with

LPS as described under “Materials and Methods”. (A) The amount of 4E-BP1 bound to eIF4E

was measured in the same immunoprecipitates used for the analysis shown in Fig. 2. Values for

4E-BP1 were normalized for eIF4E recovery. The results represent the mean ± SE for 9-10

animals per condition. The results of typical blots are shown as an inset to the panel. Muscle

and liver samples were analyzed on separate immunoblots and therefore the results from the two

tissues cannot be directly compared. C, control pigs; L, LPS-treated pigs. * p<0.05 vs. control

muscle; † p<0.01 vs. control liver. (B) Phosphorylation of 4E-BP1 was measured as described

under "Materials and Methods" and is expressed as the percentage of the protein present in the

22

hyperphosphorylated g-form. The results represent the mean ± SE for 9-10 animals per

condition. The results of typical blots are shown as an inset to the panel. The location of the a,

b, and g-forms of 4E-BP1 are noted to the left of the inset. C, control pigs; L, LPS-treated pigs.

* p<0.001 vs. control muscle; †p<0.002 vs. control liver.

Fig. 4. Effect of LPS treatment on S6K1(Thr389) phosphorylation in skeletal muscle and

liver of neonatal pigs. Neonatal pigs were treated with LPS as described under ‘Materials and

Methods”. Phosphorylation of S6K1 on Thr389 was measured by Western blot analysis using

an anti-phospho-Thr389 antibody as described under “Materials and Methods”. The results

represent the mean ± SE for 9 animals per condition. The results of typical blots are shown as an

inset to the panel. Muscle and liver samples were analyzed on separate immunoblots and

therefore the results from the two tissues cannot be directly compared. C, control pigs; L, LPS-

treated pigs. * p<0.05 vs. control muscle; † p<0.05 vs. control liver.

Fig. 5. Effect of LPS treatment on mTOR(Ser2448) phosphorylation in skeletal muscle

and liver of neonatal pigs. Neonatal pigs were treated with LPS as described under “Materials

and Methods”. Phosphorylation of mTOR on Ser2448 was measured by Western blot analysis

using an anti-phospho-Ser2448 antibody as described under “Materials and Methods”. Values

for phosphorylated mTOR [mTOR(P)] were normalized for total mTOR (mTOR) content. The

results represent the mean ± SE for 10 animals per condition. The results of typical blots are

shown as an inset to the panel. Muscle and liver samples were analyzed on separate

immunoblots and therefore the results from the two tissues cannot be directly compared. C,

control pigs; L, LPS-treated pigs. * p<0.01 vs. control muscle; † p<0.01 vs. control liver