Insulin stimulates muscle protein synthesis in neonates during endotoxemia despite repression of...

Insulin Stimulates Muscle Protein Synthesis in Neonates During 1

Endotoxemia Despite Repression of Translation Initiation 2

3

RENAN A ORELLANA1 SCOT R KIMBALL2 AGUS SURYAWAN1 JEFFERY 4

ESCOBAR1 HANH V NGUYEN1 LEONARD S JEFFERSON2 AND TERESA A DAVIS15

1USDAARS Childrens Nutrition Research Center and Critical Care Section Department 6

of Pediatrics Baylor College of Medicine Houston Texas 77030 and the 2Department of 7

Cellular and Molecular Physiology Pennsylvania State University College of Medicine 8

Hershey PA 17033 9

10

Short title mTOR signaling in endotoxemic neonatal pigs 11

12

Correspondence and reprint requests 13

Teresa A Davis PhD 14

USDAARS Childrens Nutrition Research Center 15

Baylor College of Medicine 16

1100 Bates Street 17

Houston TX 77030 18

Phone 713-798-7169 19

Fax 713-798-7171 20

E-mail tdavisbcmtmcedu 21

22

of 34Articles in PresS Am J Physiol Endocrinol Metab (October 17 2006) doi101152ajpendo002142006

Copyright copy 2006 by the American Physiological Society

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 2 of 30 ABSTRACT 1

Skeletal muscle protein synthesis is reduced in neonatal pigs in response to endotoxemia To 2

examine the role of insulin in this response neonatal pigs were infused with endotoxin (LPS 0 3

and 10 microgmiddotkg-1middothr-1) while glucose and amino acids were maintained at fasting levels and insulin 4

was clamped at fasting or fed (2 or 10 microUml) levels Fractional rates of protein synthesis and 5

translational control mechanisms were examined in longissimus dorsi muscle and liver In the 6

presence of fasting insulin LPS reduced muscle protein synthesis (-29) and increasing insulin 7

to fed levels accelerated muscle protein synthesis in both groups (controls +44 LPS +64) 8

LPS but not insulin increased liver protein synthesis by +28 In muscle of fasting neonatal 9

pigs LPS reduced 4E-BP1 phosphorylation and eIF4E to eIF4G binding In muscle of controls 10

but not LPS pigs raising insulin to fed levels increased 4E-BP1 and S6K1 phosphorylation and 11

eIF4E to eIF4G binding In muscle and liver neither LPS nor insulin altered eIF2B activity 12

eEF2 phosphorylation decreased in response to insulin in both LPS and control animals The 13

results suggest that in endotoxemic neonatal animals the response of protein synthesis to insulin 14

is maintained despite suppression of mTOR-dependent translation initiation and eIF4E 15

availability for eIF4F assembly Maintenance of an anabolic response to the feeding-induced 16

rise in insulin likely exerts a protective effect for the neonate to the catabolic processes induced 17

by sepsis 18

19

Keywords endotoxemia translation initiation eukaryotic elongation factor 2 protein synthesis 20

Page 2 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 3 of 30 INTRODUCTION 1

Sepsis a systemic inflammatory response to infection is known to promote insulin 2

resistance for protein metabolism and elicit a profound and immediate reduction in muscle 3

protein synthesis in adult mammals (13 44) In adult rats the prominent reduction in muscle 4

protein synthesis by endotoxin (Lipopolysaccharide LPS) is associated with a marked 5

depression in the translation initiation process an effect that can be reproduced in diverse animal 6

models of sepsis (20 22 45) LPS is known to consistently reproduce some of the immune and 7

metabolic effects seen during sepsis LPS administration has been used as a surrogate to elicit a 8

systemic inflammatory response in animals that triggers the release of inflammatory mediators 9

(32 46) Some mediators of the systemic inflammatory response such as TNF-α have been 10

implicated in the depression of translation signaling (21 41) 11

Our studies using the healthy neonatal pig as an animal model have demonstrated that 12

neonates maintain elevated rates of protein synthesis in skeletal muscle as a consequence of a 13

high sensitivity and responsiveness to stimulation of protein synthesis by insulin and amino acids 14

(9 47) The higher sensitivity of muscle protein synthesis to insulin arises as a result of 15

acceleration in the rate of translation of mRNA through activation of eukaryotic translation 16

initiation factors (16 19) An early event in the translation initiation pathway is the association 17

of a ternary complex consisting of eukaryotic initiation factor (eIF) 2 the initiator methionyl-18

tRNA (met-tRNAi) and a molecule of GTP to the 40S ribosomal subunit to form the 43S 19

preinitiation complex This step is modulated by the guanine nucleotide exchange factor eIF2B 20

The 43S preinitiation complex facilitates translation through identification of the AUG start 21

codon and in some cases can trigger internal initiation by binding directly to an internal 22

ribosomal entry site (IRES) (6) A second step regulating peptide-chain initiation is the binding 23

Page 3 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 4 of 30 of mRNA to the 43S preinitiation complex and this process is mediated by the assembly of the 1

eIF4F complex of proteins Insulin and amino acids stimulate translation initiation by induction 2

of 4E-BP1 phosphorylation and 70-kD ribosomal protein S6 kinase (S6K1) phosphorylation 3

through activation of a signaling pathway involving the mammalian target of rapamycin 4

(mTOR) a signaling protein downstream of protein kinase B (PKB) in the insulin signal 5

transduction pathway 4E-BP1 is a repressor protein that competes with eIF4G for binding to 6

eIF4E The availability of eIF4E is dependent upon decreased affinity of eIF4E for 4E-BP1 7

when 4E-BP1 is phosphorylated Phosphorylation and availability of eIF4E increase the 8

association of eIF4E with eIF4G and eIF4A and the formation of the eIF4F complex which 9

mediates the binding of mRNA to the 43S ribosomal complex (12) 10

In mature animals acute inflammation elicits a profound reduction in muscle protein 11

synthesis by simultaneously suppressing translation initiation signaling downstream of mTOR 12

and eIF2B signaling pathways (22 44) although particular emphasis has been given to the 13

formation of the active eIF4E bull eIF4G complex as a crucial regulatory step affected by 14

inflammation (22 41 45) In neonatal animals acute endotoxin infusion induces only a 15

moderate decrease in muscle protein synthesis (32) but a more profound alteration in the 16

activation of many of the factors that regulate translation initiation (20) We have demonstrated 17

that LPS infusion in neonatal pigs does not alter eIF2B activity but represses the binding of 18

eIF4E to eIF4G to form the active mRNA-binding complex in muscle (20) In adult septic rats 19

insulin failed to promote an increase in protein synthesis in muscle (13 42) suggesting that 20

insulin resistance may contribute to the profound and sustained reduction in muscle protein 21

synthesis during sepsis and this phenomenon has been associated with depression of translation 22

initiation signaling However in neonatal animals we found that the response of muscle protein 23

Page 4 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 5 of 30 synthesis to insulin stimulation is largely maintained during acute endotoxin infusion (31) 1

Therefore we hypothesized that insulin stimulates muscle protein synthesis in neonates during 2

early endotoxemia by augmenting the translation initiation process To address this hypothesis 3

we infused neonatal pigs with Escherichia coli (E coli) endotoxin LPS while insulin was 4

maintained at fasting (2 microUml) and fed (10 microUml) plasma concentrations and plasma amino 5

acids and blood glucose levels were maintained in the fasting range To evaluate the effect of 6

insulin on translation initiation signaling we measured 4E-BP1 and S6K1 phosphorylation the 7

association of the inactive complex of 4E-BP1 bound to eIF4E and the association of the active 8

eIF4E bull eIF4G complex which are events required for eIF4F complex formation eIF2B activity 9

was measured to examine the regulation of the initiator met-tRNAi binding to the 40S ribosomal 10

complex eIF2α phosphorylation an event that mediates IRES-dependent translation P4244 11

phosphorylation an event associated with transcriptional up-regulation and eEF2 12

phosphorylation an event that is inversely correlated with rates of translation elongation were 13

also measured The results show that insulin stimulates muscle protein synthesis in neonates 14

during acute endotoxemia despite suppression of translation initiation suggesting that an mTOR-15

independent process regulates muscle protein synthesis in the neonate during acute systemic 16

inflammation 17

18

METHODS 19

Animals Two crossbred (Landrace x Yorkshire x Hampshire x Duroc) pregnant sows 20

(Agriculture Headquarters Texas Department of Criminal Justice Huntsville Texas) were 21

housed in lactation crates fed a commercial diet (5084 PMI Feeds Richmond IN) and 22

provided with water ad libitum for 1 to 2 weeks before farrowing After farrowing piglets 23

Page 5 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 6 of 30 remained with the sow Three days prior to the infusion piglets were anesthetized and 1

underwent sterile catheter insertion into a jugular vein and a carotid artery Piglets were then 2

returned to the sow and allowed to suckle freely until studied The previously described protocol 3

was approved by the Animal Care and Use Committee of Baylor College of Medicine and was 4

conducted in accordance with the National Research Councils Guide for the Care and Use of 5

Laboratory Animals 6

Experimental design Thirty piglets (5-6 days of age 19 plusmn 031 kg body weight) were 7

assigned randomly to control (n=15) and LPS (n=15) treatment groups After an overnight fast 8

the animals were placed in a sling restraint system Pancreatic glucose-amino acid clamps were 9

then performed in each animal after average basal concentrations of whole blood glucose and 10

plasma branched chain amino acids were obtained following techniques previously described 11

(31) The clamp was initiated with a primed (20 microgbullkg-1) continuous (100 microgbullkg-1bullhr-1)12

somatostatin (BACHEM Torrance CA) infusion to block endogenous insulin secretion (-60t) 13

A physiologic replacement glucagon infusion (150 ngbullkg-1bullhr-1 Eli Lilly Indianapolis IN) was 14

provided 10 minutes after the initiation of somatostatin and was continued to the end of the 15

clamp period Simultaneously with the glucagon infusion insulin was infused at 175 microUbullkg-16

066bullmin-1 to achieve plasma insulin concentrations of 2-5 microUml to simulate a fasting insulinemic 17

state Glucose and amino acids were clamped to the individual basal fasting levels achieved 18

during the first hour (-60 to 0 t) and maintained during the 8 hour infusion by monitoring the 19

blood glucose and serum BCAA at five to ten minute intervals and adjusting the infusion rates of 20

dextrose and a balanced amino acid mixture to maintain plasma BCAA and blood glucose within 21

10 of the desired level One hour after the initiation of the somatostatin infusion the LPS 22

group received a continuous infusion [10 microgbullkg-1bullhr-1] of Escherichia coli endotoxin (lyophilized 23

Page 6 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 7 of 30 E coli Serotype 0111-B4 Sigma Chemical Co St Louis MO) that was continued for 8 h while 1

the control group received an equal volume of sterile normal saline solution 2

After 6 hours of LPSsaline infusion (6 t) insulin infusion was either increased to 1000 3

microUbullkg-066bullmin-1 (LPS + Insulin n=7 Control + Insulin n=8) to replicate fed levels of insulin (10 4

microUml) or remained at replacement levels (LPS n=8 Control n=7) Blood glucose and serum 5

BCAA continued in the targeted fasting range in both groups All animals were sacrificed 8 6

hours after the LPS infusion began (8 t) ie two hours after increasing the insulin infusion rate 7

in the fed-insulin-level groups (6 t) thereby providing sufficient time to achieve a steady-state 8

for the targeted circulating glucose amino acids and insulin levels before the animals were 9

subjected to a flooding dose of the tracer 10

Measuring protein synthesis in vivo Tissue protein synthesis was measured in vivo using 11

a modification of the flooding dose technique as previously described (31) Briefly seven hours 12

and 30 min after the LPS infusion was initiated pigs were injected via the jugular vein catheter 13

with 15 mmolkg body weight (1 mCi kg body weight) of a flooding dose of L-[4-3H] 14

phenylalanine (Amersham Arlington Heights IL) Pigs were killed 8 h after the LPS infusion 15

began and longissimus dorsi (LD) muscle and liver tissue samples were rapidly removed 16

Fractional rates of protein synthesis were determined as previously described (31) The RNA-to-17

protein ratio (mg RNAg protein) was used as an estimate of the protein synthetic capacity ie 18

ribosome number Protein synthetic efficiency was estimated as the total protein synthesized per 19

total RNA (g proteinmiddotday-1 middotg RNA-1) 20

Plasma hormones and substrate assays Whole blood glucose concentrations were 21

determined by a glucose oxidase reaction (YSI 2300 STAT Plus Yellow Springs Instruments 22

Yellow Springs OH) Plasma concentrations of total BCAA were measured by analysis of 23

Page 7 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 8 of 30 leucine isoleucine and valine deamination by leucine dehydrogenase with stoichiometric 1

reduction of NAD measured by spectrophotometry (3) Plasma insulin concentrations were 2

measured using a porcine insulin RIA kit (Linco St Charles MO) 3

Measurement of eIF2B activity Fresh tissues were processed immediately after the 4

animals were euthanized to examine the activation of eIF2B signaling The eIF2B activity in 5

muscle and liver supernatants were measured as the exchange of [3H]GDP bound to eIF2 for 6

unlabeled GDP or GTP as previously described (20 30) Activity was expressed as the rate of 7

GDP exchange 8

Muscle and liver homogenates Freshly collected LD and liver tissue samples were 9

homogenized centrifuged at 10000 g for 10 min at 4degC heated at 100degC for 10 min and cooled 10

to room temperature Supernatants were diluted in sodium dodecyl sulfate (SDS) sample buffer 11

frozen in liquid nitrogen and stored at ndash70degC until protein immunoblot analyses 12

Protein immunoblot analysis Electrophoretically separated proteins in polyacrylamide 13

gels (PAGE) from LD and liver tissue homogenates were transferred to a BioTrace PVDF 14

(polyvinylidene difluoride) membrane (BioRad Hercules CA) as previously described (18) 15

The membranes were then incubated with primary antibody (Amersham Life Sciences Arlington 16

Heights IL) for 1 hr at room temperature Blots were developed using an enhanced 17

chemiluminescence (ECL) Western blotting kit (Amersham Life Sciences Arlington Heights 18

IL) visualized using a GeneGnome bioimaging system and analyzed using Genetools software 19

(Syngene) Site-specific phosphorylation and total protein content were determined and the 20

results in this report are expressed as arbitrary units which represent the band intensity of the 21

integrated pixels being analyzed 22

Page 8 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 9 of 30

Determination of the 4E-BP1middoteIF4E and eIF4GmiddoteIF4E complexes The association of 1

eIF4E with 4E-BP1 was determined as previously described (18 20) The obtained supernatants 2

were subjected to an overnight immunoprecipitation at 4degC using a monoclonal antibody against 3

eIF4E Proteins in the immunoprecipitate were resolved by SDS-PAGE and then transferred to 4

polyvinylidene difluoride membranes The membranes were then probed with either anti-4E-5

BP1 or anti-eIF4G antibodies (Bethyl Laboratories Montgomery TX) or the mouse monoclonal 6

anti-eIF4E antibody Values obtained using the anti-4E-BP1 and anti-eIF4G antibodies were 7

normalized for the amount of eIF4E present in the sample 8

Determination of PKB phosphorylation Muscle and liver homogenates were incubated 9

with an anti-PKB antibody (New England BioLabs Beverly MA) The second membrane was 10

incubated with a rabbit polyclonal antibody that recognizes the phosphorylation of PKB on Ser473 11

(New England BioLabs Beverly MA) Results were normalized for the total amount of PKB on 12

the blot 13

Determination of 4E-BP1 phosphorylation Muscle and liver homogenates were 14

subjected to protein immunoblot analysis using an Amersham enhanced chemiluminescence 15

(ECL) Western blotting kit as previously described (18) 4E-BP1 present in tissue extracts was 16

separated into multiple electrophoretic forms by a rabbit polyclonal antibody that recognizes 17

phosphorylation at Thr70 (Cell Signaling Technology Beverly MA) during SDS-PAGE with the 18

more slowly migrating forms representing more highly phosphorylated 4E-BP1 19

Phosphorylation of 4E-BP1 was corrected for total 4E-BP1 20

Determination of S6K1 phosphorylation Muscle and liver homogenates were subjected 21

to protein immunoblot analysis as previously described (18) using a rabbit polyclonal antibody 22

that recognizes S6K1 when it is phosphorylated at the activating residue Thr389 (Santa Cruz 23

Page 9 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 10 of 30 Biotechnology Santa Cruz CA) Results were normalized for the total amount of PKB on the 1

blot 2

Determination of eukaryotic elongation factor-2 (eEF2) eIF2α and p4244 3

phosphorylation Aliquots of muscle and liver homogenates were subjected to protein 4

immunoblot analysis using a rabbit polyclonal antibody that recognizes the site-specific 5

phosphorylation of eEF2 at Thr56 or total eEF2 Phosphorylation of eEF2 was corrected for total 6

eEF2 Rabbit polyclonal antibodies were used for protein immunoblot analysis to determine 7

eIF2α phosphorylation and dual p4244 phosphorylation The results were normalized for the 8

total amount of eIF2α and total p4244 on the blot respectively 9

Data analysis Treatment with insulin was the grouping factor for different parameters 10

A general linear model of ANOVA was used to determine the effect of LPS insulin and their 11

interaction on protein synthesis and translation initiation factor activation using statistical 12

software (Minitab Inc for Windows) T-tests were also performed to examine the specific 13

effects of each treatment group Results are presented as means plusmn SE Probability values of lt 14

005 were considered statistically significant 15

16

RESULTS 17

Hormones and substrates during pancreatic-substrate clamps C-peptide was not 18

detectable in plasma of either LPS or control animals indicating endogenous insulin blockade 19

Baseline fasting insulin levels were obtained for each individual animal (controls 3 plusmn 06 LPS 3 20

plusmn 04 microUmL P gt 005) and those fasting levels were maintained during the period of LPS 21

infusion in both control and experimental animals (P gt 005 baseline vs fasting insulin levels) 22

In the insulin-stimulated group insulin levels similar to those observed in the fed state were 23

Page 10 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 11 of 30 obtained by providing exogenous insulin in both control and LPS-infused animals during the last 1

2 hours of LPS infusion (controls 12 plusmn 3 LPS 13 plusmn 08 microUmL P gt 005) and they were 2

different from those in the fasting condition in both control and LPS-infused animals (P lt 005 3

baseline vs fed insulin levels) In controls and LPS-infused animals whole blood glucose was 4

maintained at fasting levels during the entire experimentation (controls 75 plusmn 4 LPS 82 plusmn 45

mgdL P gt 005) even when exogenous insulin was supplemented (P gt 005) In fasting LPS-6

infused animals plasma BCAA were maintained at baseline levels during the entire experimental 7

period (controls 515 plusmn 34 LPS 509 plusmn 36 mmolmL P gt 005) When exogenous insulin was 8

supplemented LPS-infused animals maintained baseline levels of blood glucose and plasma 9

BCAA (P gt 005) 10

Protein synthesis in muscle and liver during endotoxemic pancreatic-substrate clamps 11

In the presence of fasting insulin levels and fasting blood glucose and plasma amino acid levels 12

LPS reduced protein synthesis in the LD (-29 P lt 005 Fig 1A) a muscle composed of 13

primarily fast-twitch muscle fibers Raising insulin to fed levels while maintaining fasting 14

blood glucose and plasma amino acid levels accelerated muscle protein synthesis in both groups 15

(controls +44 LPS +64 P lt 005 Fig 1A) The protein synthetic capacity of the tissue 16

measured by the RNA-to-protein ratio was not affected by LPS or insulin in muscle (P gt 005 17

Fig 1B) In animals maintained at fasting insulin levels the protein synthetic efficiency as 18

indicated by the total protein synthesized per total RNA was significantly decreased by LPS in 19

LD muscle (-28 P lt 005 Fig 1C) Increasing insulin to fed levels enhanced protein synthetic 20

efficiency in muscle (controls by 38 LPS by 60 P lt 005 Fig 1C) The effect of insulin on 21

protein synthesis in muscle was proportionally similar in LPS-infused and control animals and 22

no interaction between LPS and insulin was detected LPS increased protein synthesis in liver by 23

Page 11 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 12 of 30 +28 when compared to controls (P lt 005 Fig 1D) Raising insulin to fed levels did not alter 1

protein synthesis in liver of either group (P gt 005 Fig 1D) LPS increased the protein synthetic 2

efficiency but not the protein synthetic capacity of the liver of neonatal animals (P lt 005 Fig 3

1E and 1F) 4

Effects of insulin on PKB signaling and biomarkers of mRNA translation in muscle and 5

liver during endotoxemic pancreatic-substrate clamps In skeletal muscle insulin but not LPS 6

increased the phosphorylation of PKB (P lt 005 Fig 2A) In liver neither LPS or insulin 7

altered PKB phosphorylation (P gt 005 Fig 2D) In muscle of control animals insulin increased 8

4E-BP1 and S6K1 phosphorylation decreased the association of the inactive eIF4E bull 4E-BP1 9

complex and increased the abundance of the active eIF4E bull eIF4G complex (P lt 005 Figs 2B 10

and 3B 2C and 3A) In muscle LPS reduced 4E-BP1 phosphorylation and the formation of the 11

active eIF4E bull eIF4G complex (P lt 005 Figs 2B and 3A) but did not alter S6K1 12

phosphorylation or the abundance of the inactive eIF4E bull 4E-BP1 complex (P gt 005 Figs 3B 13

and 2C) Insulin failed to augment 4E-BP1 or S6K1 phosphorylation in muscle of LPS-infused 14

animals (P gt 005 Figs 2B and 3B) In addition insulin failed to decrease the abundance of the 15

inactive eIF4E bull 4E-BP1 complex or to increase the abundance of the active eIF4E bull eIF4G 16

complex in LPS-infused animals (P gt 005 Figs 2C and 3A) In liver LPS but not insulin 17

increased 4E-BP1 and S6K1 phosphorylation and decreased the formation of the inactive eIF4E 18

bull 4EBP1 complex (P lt 005 Figs 2D and 3E and 2F) 19

In muscle of controls insulin decreased eIF2α and eEF2 phosphorylation (P lt 005 Figs 20

4A and 3C) and tended to increase the relative p42p44 phosphorylation (P gt 005 Fig 4C) 21

eIF2α and eEF2 phosphorylation did not change in response to LPS infusion (P gt 005 Figs 4A 22

and 3C) and p42p44 phosphorylation in muscle of fasting animals increased in response to LPS 23

Page 12 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 13 of 30 (P lt 005 Fig 4C) Raising insulin to fed levels in LPS-infused animals did not affect eIF2α1

phosphorylation (P gt 005 Fig 4A) did not decrease p42p44 phosphorylation (P gt 005 Fig 2

4C) and decreased eEF2 phosphorylation in muscle (P lt 005 Fig 3C) In liver LPS but not 3

insulin decreased eIF2α phosphorylation (P lt 005 Fig 4D) decreased eEF2 phosphorylation 4

(P lt 005 Fig 3F) and increased p42p44 phosphorylation (P lt 005 Fig 4F) eIF2B activity 5

was not affected by insulin or LPS infusion in muscle or liver (P gt 005 Fig 4B and 4E) 6

7

DISCUSSION 8

Recently the beneficial role of insulin therapy in minimizing the effects of critical illness 9

on muscle wasting has been reported (40) It has also been recognized that insulin resistance 10

plays a major role in the metabolic dysregulation during sepsis (1 35) However the 11

mechanisms that regulate the beneficial effects of insulin in the critically ill are unknown (7) and 12

few reports address the effect of insulin on muscle metabolism during sepsis (13 22 42) LPS 13

infusion elicits a consistent response that imitates some of the clinical signs and cytokine 14

production seen in sepsis and this response has been validated and reliably reproduced in adult 15

and young swine (32 46) We have demonstrated that the muscle of neonatal pigs retains the 16

ability to respond to the stimulatory action of insulin by increasing protein synthesis after LPS 17

administration (31) Because the regulation of muscle protein synthesis in the neonate is 18

mediated by key translation initiation factors (16) we wished to define the mechanisms that 19

regulate the effect of insulin on translation initiation during neonatal endotoxemia As a 20

comparison we examined the activation of the translation initiation process in the liver of LPS-21

infused neonatal pigs since we have demonstrated previously that liver protein synthesis is 22

stimulated by endotoxin but not by insulin infusion (29 31) 23

Page 13 of 34


Effects of endotoxin on protein synthesis in muscle and liver in neonatal pigs Sepsis a 1

systemic inflammatory response syndrome profoundly decreases protein synthesis in skeletal 2

muscle (22 44) and represses the insulin-induced stimulation of protein synthesis in mature 3

animals (13 42) By contrast we have demonstrated that LPS infusion elicits only a modest 4

reduction in muscle protein synthesis in neonatal animals in the presence of insulin and amino 5

acid stimulation (20 30) in part because insulin-stimulated muscle protein synthesis is 6

maintained in neonates faced with acute inflammation generated by LPS administration (31) 7

Similar to adult rats we found that the decrease in muscle protein synthesis is accompanied by a 8

decrease in translational efficiency rather than changes in the protein synthetic capacity ie 9

ribosome number (45) In the current study raising insulin to fed levels increased protein 10

synthesis rates as a result of augmenting the protein synthetic efficiency in muscle similar to our 11

previous report (32) This suggests that insulin stimulates translational efficiency in the muscle 12

of growing organisms even when faced with systemic inflammation enabling protein deposition 13

in muscle 14

In the healthy neonatal pig insulin does not stimulate hepatic protein synthesis (29) and 15

the production of proteins by the liver (ie retinol-binding protein and albumin) responds to 16

amino acid rather than hormonal stimulation (29) In our study liver protein synthesis in 17

neonatal pigs was enhanced by LPS but not by insulin likely because LPS stimulates the hepatic 18

synthesis of acute phase proteins during sepsis (11) Even though we did not identify the 19

proteins that were synthesized by the liver in our model our data suggests that the stimulation of 20

liver protein synthesis during endotoxin infusion is dependent on the inflammatory response 21

triggered by LPS and this response is not influenced by insulin levels (31 44) We conclude that 22

Page 14 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 15 of 30 maintaining either fasting or fed levels of insulin will not alter the LPS-induced increase in liver 1

protein synthesis 2

Effects of endotoxin on translation initiation during fasting Previously we have shown 3

that muscle protein synthesis decreases only modestly (-11) in response to LPS infusion if 4

insulin glucose and amino acids are maintained at levels similar to those seen in the fed state 5

(32) and this is associated with substantial reductions in the phosphorylation of 4E-BP1 and 6

S6K1 and a decrease in the binding of eIF4E to eIF4G (20 32) The fasting animals in the 7

current study had a more profound reduction in protein synthesis (ie -29) than those in our 8

previous study in which insulin glucose and amino acid levels of LPS-infused animals were 9

maintained in the fed range The decrease in muscle protein synthesis with LPS in our fasting 10

neonatal pigs was associated with depression in 4E-BP1 phosphorylation and decreased 11

abundance of the active eIF4E bull eIF4G complex However we did not find an effect of LPS on 12

other events that also alter the mRNA binding step in translation initiation such as S6K1 13

phosphorylation and the assembly of the inactive eIF4E bull 4E-BP1 complex We suspect that the 14

LPS-induced decrease in muscle protein synthesis in our study is enhanced by the restriction in 15

substrate and hormonal stimulation ie insulin glucose and amino acids which occurs during 16

fasting However the decrease in the abundance of phosphorylated initiation factors in response 17

to fasting may mask a further LPS-induced reduction in translation initiation Although insulin 18

stimulates translation factor signaling activation the response of 4E-BP1 phosphorylation and 19

eIF4G bull eIF4E formation to insulin stimulation is also diminished in ovine fetuses during 20

maternal fasting (36) Similar studies in neonatal pigs have shown that insulin does not affect 21

4E-BP1 and S6K1 phosphorylation or eIF4E bull 4E-BP1 and eIF4G bull eIF4E content when plasma 22

insulin was increased from 0 to 2 microUml (28) Furthermore most studies in adult septic animals 23

Page 15 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 16 of 30 have allowed the subjects to feed ad libitum but they become anorectic and it is possible that the 1

lack of substrate availability contributes to enhanced protein catabolism (4) and a pronounced 2

decrease in muscle protein synthesis 3

Effects of insulin on biomarkers of mRNA translation in skeletal muscle of LPS-infused 4

pigs Although both glucose and amino acids are required for insulin to stimulate the activation 5

of translation initiation factor signaling in vitro insulin exerts an independent and profound 6

stimulatory effect on muscle protein synthesis and translation initiation signaling in neonatal 7

animals (8 16) when levels are above the fasting range The effects of sepsis on the insulin 8

signaling cascade that leads to translation initiation occurs downstream of mTOR in mature 9

animals (20-22 30 44 45) However we have demonstrated using rapamycin as an inhibitor of 10

mTOR that both mTOR-dependent and mTOR-independent pathways are involved in the 11

regulation of protein synthesis in muscle of neonatal animals (19) In the current study in 12

contrast to reports in adult rats (2 13) raising insulin to fed levels in control neonatal pigs was 13

associated with an increase in fractional protein synthesis rates in muscle even though glucose 14

and amino acids were maintained in the fasting range and this response was coupled with 15

increased phosphorylation of PKB 4E-BP1 and S6K1 reduced binding of eIF4E to 4E-BP1 and 16

increased assembly of the active eIF4E bull eIF4G However in LPS-infused animals the response 17

to insulin was abrogated 18

The eIF4F complex of proteins and S6K1 activation have been considered important 19

regulators of muscle protein synthesis in adult animals (24 45) Upon activation of apoptotic 20

pathways eIF4E and eIF4G undergo cleavage and degradation (5) and these events may play a 21

role in the development of restrictions in translation signaling and its lack of response to insulin 22

(22 42) Although S6K1-induced phosphorylation of ribosomal protein S6 has been reported to 23

Page 16 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 17 of 30 be impaired in rats subjected to experimental intrabdominal sepsis (42) it has been suggested 1

that S6K1 may not be a key regulator for the translation of most mRNAs but regulates the 2

translation of TOP mRNAs that encode proteins that are essential for cell growth (18) In our 3

study LPS restrained the expected increase in eIF4E binding to eIF4G and S6K1 4

phosphorylation despite an increase in protein synthesis rates in skeletal muscle in response to 5

insulin administration Inconsistent effects of insulin on signal transduction pathways have been 6

reported (15) but the effect of acute inflammation in different animal models of experimental 7

sepsis suggest a depression in eIF4E to eIF4G binding and S6K1 phosphorylation in muscle (45) 8

In our study in LPS infused neonatal animals the stimulation of muscle protein synthesis by 9

insulin occurred independent of change in eIF4E to eIF4G binding and S6K1 phosphorylation 10

while studies in mature animals report a restriction in protein synthesis in response to insulin 11

stimulation (13 42 44) Since the stimulation of muscle protein synthesis by insulin during LPS 12

infusion in neonatal animals cannot be explained by changes in the translation initiation events 13

that lead to protein synthesis we conclude the response of muscle protein synthesis to insulin 14

during neonatal endotoxemia may be regulated by a process other than translation initiation 15

In our study eIF2B a translation initiation factor that does not depend on mTOR 16

activation was not affected by either LPS or insulin infusion Previously we have shown that the 17

lack of effect of LPS on eIF2B activity may account for the near maintenance of global rates of 18

protein synthesis in LPS-infused neonatal pigs with fed insulin and amino acid levels (20) It has 19

been suggested that eIF2 complex of proteins influence global rates of protein synthesis (20) and 20

the phosphorylation of eIF2α coincides with the inhibition of translation followed by activation 21

of apoptotic pathways (5) In adult rat models eIF2B activity in skeletal muscle is reduced 22

following infusion of TNF-α experimentally-induced sepsis with LPS and peritoneal infection 23

Page 17 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 18 of 30 (21 23 43) and thus eIF2B appears to have a key role in regulating global rates of protein 1

synthesis during inflammation (45) In normal fasted neonates eIF2B activity in muscle is not 2

affected by insulin in contrast to the insulin-stimulated increase in 4E-BP1 phosphorylation 3

eIF4EmiddoteIF4G content and S6K1 phosphorylation in these animals (28) In the current study we 4

found that modulation of eIF2B activity is not involved in the regulation of muscle protein 5

synthesis by insulin during acute endotoxemia in neonates 6

eIF2α phosphorylation an event that can mediate IRES-dependent translation has been 7

reported to regulate in vitro global rates of protein synthesis and offers an appealing link between 8

the regulation of translation initiation control of gene expression and apoptosis (34 38) 9

Phosphorylation of the α-subunit of eIF2 has been reported to occur during oxidative stress and 10

apoptosis (5 39) Enhanced IRES-dependent translation is believed to function as a protective 11

homeostatic mechanism through increased synthesis of several proteins critically involved in 12

apoptosis cell cycle development amino acid availability and endoplasmic reticulum stress 13

and IRESrsquos are found preferentially in the mRNAs of genes involved in the control of cellular 14

proliferation survival and death (27) In the current study insulin stimulated muscle protein 15

synthesis concomitant with a decrease in eIF2α phosphorylation in control animals However 16

LPS blunted an insulin-induced reduction in eIF2α phosphorylation despite an increase in protein 17

synthesis in response to insulin stimulation In response to cellular stress hepatic GADD34 18

(growth arrest and DNA damage-inducible protein 34) promotes dephosphorylation of eIF2α19

suggesting a protective role for the recovery of protein synthesis after stress (33) Since eIF2B 20

exhibits little or no binding to the α-subunit of eIF2 regardless of whether or not the α-subunit of 21

eIF2 has been phosphorylated and in rat muscle only about 2-4 of eIF2α is basally 22

Page 18 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 19 of 30 phosphorylated (14) we suggest that a decrease in eIF2α phosphorylation would have little if 1

any effect on eIF2B activity (17) 2

The translation of mRNA to protein occurs in three distinct phases initiation elongation 3

and termination (26) All three of these phases can undergo regulation but initiation and 4

elongation seem to be the most tightly controlled (37) The elongation process occurs after 5

translation initiation and requires that eEF2 couples to GTP hydrolysis (26) and thus the 6

phosphorylation of eEF2 is associated with decreased elongation In our study insulin in the 7

presence of fasting levels of amino acids and glucose reduced the phosphorylation of eEF2 8

This effect of insulin on eEF2 phosphorylation was present in both control and LPS animals 9

This suggests that the response of muscle protein synthesis to insulin during endotoxemia may be 10

mediated by the elongation step in peptide formation 11

In vitro TNF-α has been reported to stimulate P4244 phosphorylation an event 12

associated with transcriptional alterations (10 41) and activation of P4244 MAPK has been 13

associated with cell growth arrest (25) LPS has been shown to elicit a predictable TNF-α14

response in swine even in a neonatal model (32) In the adult rat liver Maitra et al reported an 15

increase in p4244 activation following cecal ligation and puncture and insulin pretreatment 16

appeared to amplify this response (25) It has been postulated that events that mediate 17

transcription such as p4244 activation are related to suppression of translation initiation 18

signaling by decreasing availability of eIF4E for eIF4F complex assembly (41) In the present 19

study we found that LPS increased P4244 phosphorylation in muscle during fasting and this 20

response was reduced by insulin suggesting that insulin may modify protein metabolism in 21

muscle during endotoxemia by affecting some transcriptional processes which in turn may 22

regulate protein synthesis and translational events 23

Page 19 of 34


Effects of insulin on biomarkers of mRNA translation in liver of LPS-infused pigs The 1

results of the current study demonstrated that liver protein synthesis was stimulated by 2

endotoxin but not by insulin infusion The LPS-induced increase in liver protein synthesis was 3

associated with an increase in S6K1 phosphorylation and reductions in the abundance of the 4

inactive eIF4E bull 4E-BP1 complex phosphorylation of eIF2α and activation of p4244 These 5

events suggest a differential regulation of protein synthesis by insulin during acute inflammation 6

in different organs and that the effects of LPS in the liver occur independent of insulin 7

administration 8

PERSPECTIVES 9

The results of the current study suggest that insulin stimulates muscle protein synthesis in 10

LPS-infused neonates in spite of a suppression of the insulin-stimulated mTOR-dependent 11

translation initiation signaling In our study insulin appears to modulate protein synthesis in 12

muscle of neonates during endotoxemia by altering the elongation process It is possible that 13

age and the duration and intensity of the septic model may result in different alterations in the 14

translational signaling pathway and other processes that regulate cell growth such as 15

transcription and gene expression In spite of the profound changes in cellular signaling in 16

muscle neonatal animals appear to maintain an anabolic response to insulin during LPS infusion 17

which may exert a protective effect for the neonate to the catabolic processes induced by sepsis 18

Understanding of the interface between insulin and the molecular mechanisms that govern amino 19

acid metabolism during acute inflammation will facilitate the design of appropriate nutritional 20

and hormonal therapies for use during neonatal sepsis 21

Page 20 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 21 of 30 ACKNOWLEDGMENTS 1

The authors thank Marta L Fiorotto for helpful discussion William Liu and Judy 2

Rosenberger for technical assistance Jerome Stubblefield for assistance with care of animals 3

Susan Nguyen for laboratory assistance and Linda Weiser for secretarial assistance 4

This work is a publication of the USDAARS Childrens Nutrition Research Center 5

Department of Pediatrics Baylor College of Medicine and Texas Childrens Hospital Houston 6

Texas This project has been funded in part by National Institute of Arthritis and 7

Musculoskeletal and Skin Diseases Institute Grant R01 AR 44474 and the US Department of 8

Agriculture Agricultural Research Service under Cooperative Agreement number 58-6250-6-9

001 This research was also supported in part by NIH training grant T32 HD074451 NIH K12 10

HD41648 and NIH mentored award K08 AR51563-01A1 The contents of this publication do 11

not necessarily reflect the views or policies of the US Department of Agriculture nor does 12

mention of trade names commercial products or organizations imply endorsement by the US 13

Government The contents of this publication do not necessarily reflect the views or policies of 14

the US Department of Agriculture nor does mention of trade names commercial products or 15

organizations imply endorsement by the US Government 16

17

Page 21 of 34


1

Reference List 23

1 Agwunobi AO Reid C Maycock P Little RA and Carlson GL Insulin resistance and 4

substrate utilization in human endotoxemia J Clin Endocrinol Metab 85 3770-3778 5

2000 6

2 Balage M Sinaud S Prodhomme M Dardevet D Vary TC Kimball SR Jefferson 7

LS and Grizard J Amino acids and insulin are both required to regulate assembly of the 8

eIF4E eIF4G complex in rat skeletal muscle Am J Physiol Endocrinol Metab 281 E565-9

E574 2001 10

3 Beckett PR Hardin DS Davis TA Nguyen HV Wray-Cahen D and Copeland KC11

Spectrophometric assay for measuring branched-chain amino acid concentrations 12

application for measuring the sensitivity of protein metabolism to insulin Anal Biochem 13

240 48-53 1996 14

4 Breuille D Arnal M Rambourdin F Bayle G Levieux D and Obled C Sustained 15

modifications of protein metabolism in various tissues in a rat model of long-lasting 16

sepsis Clin Sci (Lond) 94 413-423 1998 17

5 Clemens MJ Translational regulation in cell stress and apoptosis Roles of the eIF4E 18

binding proteins J Cell Mol Med 5 221-239 2001 19

Page 22 of 34


6 Clemens MJ Bushell M Jeffrey IW Pain VM and Morley SJ Translation initiation 1

factor modifications and the regulation of protein synthesis in apoptotic cells Cell Death 2

Differ 7 603-615 2000 3

7 Das UN Current advances in sepsis and septic shock with particular emphasis on the role 4

of insulin Med Sci Monit 9 RA181-RA192 2003 5

8 Davis TA Fiorotto ML Burrin DG Reeds PJ Nguyen HV Beckett PR Vann RC 6

and OConnor PM Stimulation of protein synthesis by both insulin and amino acids is 7

unique to skeletal muscle in neonatal pigs Am J Physiol Endocrinol Metab 282 E880-8

E890 2002 9

9 Davis TA Nguyen HV Suryawan A Bush JA Jefferson LS and Kimball SR10

Developmental changes in the feeding-induced stimulation of translation initiation in 11

muscle of neonatal pigs Am J Physiol Endocrinol Metab 279 E1226-E1234 2000 12

10 de Alvaro C Teruel T Hernandez R and Lorenzo M Tumor necrosis factor alpha 13

produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in 14

a p38 MAPK-dependent manner J Biol Chem 279 17070-17078 2004 15

11 Hasselgren PO Pedersen P Sax HC Warner BW and Fischer JE Current concepts 16

of protein turnover and amino acid transport in liver and skeletal muscle during sepsis 17

Arch Surg 123 992-999 1988 18

Page 23 of 34


12 Hinnebusch AG Mechanism and regulation of initiator methionyl-tRNA binding to 1

ribosomes In Translational Control of Gene Expression edited by Nahum Sonenberg 2

JWBHMBM Cold Spring Harbor NY Cold Spring Harbor Laboratory Press 2000 3

13 Jurasinski C Gray K and Vary TC Modulation of skeletal muscle protein synthesis by 4

amino acids and insulin during sepsis Metabolism 44 1130-1138 1995 5

14 Karinch AM Kimball SR Vary TC and Jefferson LS Regulation of eukaryotic 6

initiation factor-2B activity in muscle of diabetic rats Am J Physiol 264 E101-E108 7

1993 8

15 Keeton AB Amsler MO Venable DY and Messina JL Insulin signal transduction 9

pathways and insulin-induced gene expression J Biol Chem 277 48565-48573 2002 10

16 Kimball SR Farrell PA Nguyen HV Jefferson LS and Davis TA Developmental 11

decline in components of signal transduction pathways regulating protein synthesis in pig 12

muscle Am J Physiol Endocrinol Metab 282 E585-E592 2002 13

17 Kimball SR Heinzinger NK Horetsky RL and Jefferson LS Identification of 14

interprotein interactions between the subunits of eukaryotic initiation factors eIF2 and 15

eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34


18 Kimball SR and Jefferson LS Regulation of global and specific mRNA translation by 1

oral administration of branched-chain amino acids Biochem Biophys Res Commun 3132

423-427 2004 3

19 Kimball SR Jefferson LS Nguyen HV Suryawan A Bush JA and Davis TA4

Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an 5

mTOR-dependent process Am J Physiol Endocrinol Metab 279 E1080-E1087 2000 6

20 Kimball SR Orellana RA OConnor PM Suryawan A Bush JA Nguyen HV 7

Thivierge MC Jefferson LS and Davis TA Endotoxin induces differential regulation 8

of mTOR-dependent signaling in skeletal muscle and liver of neonatal pigs Am J Physiol 9

Endocrinol Metab 285 E637-E644 2003 10

21 Lang CH and Frost RA Endotoxin disrupts the leucine-signaling pathway involving 11

phosphorylation of mTOR 4E-BP1 and S6K1 in skeletal muscle J Cell Physiol 20312

144-155 2005 13

22 Lang CH Frost RA Jefferson LS Kimball SR and Vary TC Endotoxin-induced 14

decrease in muscle protein synthesis is associated with changes in eIF2B eIF4E and 15

IGF-I Am J Physiol Endocrinol Metab 278 E1133-E1143 2000 16

23 Lang CH Frost RA Nairn AC MacLean DA and Vary TC TNF-alpha impairs heart 17

and skeletal muscle protein synthesis by altering translation initiation Am J Physiol 18


Page 25 of 34


24 Lang CH Pruznak AM and Frost RA TNFalpha mediates sepsis-induced impairment 1

of basal and leucine-stimulated signaling via S6K1 and eIF4E in cardiac muscle J Cell 2

Biochem 94 419-431 2005 3

25 Maitra SR Chen E Rosa D Valane PD El Maghrabi MR and Brathwaite CE4

Modulations of signal transduction pathways during sepsis and the effects of insulin and 5

mifepristone Acad Emerg Med 10 1-8 2003 6

26 Nader GA Hornberger TA and Esser KA Translational control implications for 7

skeletal muscle hypertrophy Clin Orthop Relat Res S178-S187 2002 8

27 Nevins TA Harder ZM Korneluk RG and Holcik M Distinct regulation of internal 9

ribosome entry site-mediated translation following cellular stress is mediated by 10

apoptotic fragments of eIF4G translation initiation factor family members eIF4GI and 11

p97DAP5NAT1 J Biol Chem 278 3572-3579 2003 12

28 OConnor PM Kimball SR Suryawan A Bush JA Nguyen HV Jefferson LS and 13

Davis TA Regulation of translation initiation by insulin and amino acids in skeletal 14



Davis TA Regulation of neonatal liver protein synthesis by insulin and amino acids in 17

pigs Am J Physiol Endocrinol Metab 286 E994-E1003 2004 18

Page 26 of 34


30 Orellana RA Kimball SR Nguyen HV Bush JA Suryawan A Thivierge MC 1

Jefferson LS and Davis TA Regulation of muscle protein synthesis in neonatal pigs 2

during prolonged endotoxemia Pediatr Res 55 442-449 2004 3

31 Orellana RA OConnor PM Bush JA Suryawan A Thivierge MC Nguyen HV 4

Fiorotto ML and Davis TA Modulation of muscle protein synthesis by insulin is 5

maintained during neonatal endotoxemia Am J Physiol Endocrinol Metab 291 E159-6

E166 2006 7

32 Orellana RA OConnor PM Nguyen HV Bush JA Suryawan A Thivierge MC 8

Fiorotto ML and Davis TA Endotoxemia reduces skeletal muscle protein synthesis in 9

neonates Am J Physiol Endocrinol Metab 283 E909-E916 2002 10

33 Proud CG eIF2 and the control of cell physiology Semin Cell Dev Biol 16 3-12 2005 11

34 Ray PS Grover R and Das S Two internal ribosome entry sites mediate the translation 12

of p53 isoforms EMBO Rep 200613

35 Shangraw RE Jahoor F Miyoshi H Neff WA Stuart CA Herndon DN and Wolfe 14

RR Differentiation between septic and postburn insulin resistance Metabolism 38 983-15

989 1989 16

Page 27 of 34


36 Shen W Boyle DW and Liechty EA Changes in 4E-BP1 and p70S6K phosphorylation 1

in skeletal muscle of the ovine fetus after prolonged maternal fasting effects of insulin 2

and IGF-I Pediatr Res 58 833-839 2005 3

37 Sonenberg N HJMM Translation Control of Gene Expression Cold Spring Harbor 4

NY Cold Spring Harbor Laboratory Press 2006 5

38 Svitkin YV Herdy B Costa-Mattioli M Gingras AC Raught B and Sonenberg N6

Eukaryotic translation initiation factor 4E availability controls the switch between cap-7

dependent and internal ribosomal entry site-mediated translation Mol Cell Biol 258

10556-10565 2005 9

39 Tan S Somia N Maher P and Schubert D Regulation of antioxidant metabolism by 10

translation initiation factor 2alpha J Cell Biol 152 997-1006 2001 11

40 Van den BG Wouters P Weekers F Verwaest C Bruyninckx F Schetz M 12

Vlasselaers D Ferdinande P Lauwers P and Bouillon R Intensive insulin therapy in 13

the critically ill patients N Engl J Med 345 1359-1367 2001 14

41 Vary TC Deiter G and Lang CH Diminished ERK 12 and p38 MAPK 15

phosphorylation in skeletal muscle during sepsis Shock 22 548-554 2004 16

Page 28 of 34


42 Vary TC Jefferson LS and Kimball SR Insulin fails to stimulate muscle protein 1

synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1 Am J Physiol 2


43 Vary TC Jurasinski CV Karinch AM and Kimball SR Regulation of eukaryotic 4

initiation factor-2 expression during sepsis Am J Physiol 266 E193-E201 1994 5

44 Vary TC and Kimball SR Sepsis-induced changes in protein synthesis differential 6

effects on fast- and slow-twitch muscles Am J Physiol 262 C1513-C1519 1992 7

45 Vary TC and Kimball SR Effect of sepsis on eIE4E availability in skeletal muscle Am 8

J Physiol Endocrinol Metab 279 E1178-E1184 2000 9

46 Webel DM Finck BN Baker DH and Johnson RW Time course of increased plasma 10

cytokines cortisol and urea nitrogen in pigs following intraperitoneal injection of 11

lipopolysaccharide J Anim Sci 75 1514-1520 1997 12

47 Wray-Cahen D Beckett PR Nguyen HV and Davis TA Insulin-stimulated amino acid 13

utilization during glucose and amino acid clamps decreases with development Am J 14

Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 30 of 30 FIGURE LEGENDS 1

2Fig 1 Effects of insulin on fractional protein synthesis rates (A and D) and protein 3

synthetic capacities (B and E) and efficiencies (C and F) in longissimus dorsi muscle and liver of 4

LPS and control pigs during endotoxemic pancreatic-substrate clamps Values are means +- SE 5

n=7-8group abcd values with different superscript differ significantly (Plt005) 6

Fig 2 Effects of insulin on PKB (A and D) and 4E-BP1 phosphorylation (B and E) and 7

eIF4E bull 4E-BP1 association (C and F) in longissimus dorsi muscle and liver of LPS-infused 8

neonatal pigs The phosphorylated forms were normalized to the total content Values are 9

means +- SE n=7-8group abcd values with different superscript differ significantly 10

(Plt005) 11

Fig 3 Effects of insulin on the abundance of the active eIF4E bull eIF4G complex (A and 12

D) and phosphorylation of S6K1 (B and E) and eEF2 (C and F) in longissimus dorsi muscle and 13

liver of LPS-infused neonatal pigs The phosphorylated forms were normalized for the total 14

content Values are means +- SE n=7-8group abcd values with different superscript differ 15

significantly (Plt005) 16

Fig 4 Effects of insulin on eIF2α phosphorylation (A and D) eIF2B activity (B and E) 17

and p4244 phosphorylation (C and F) in longissimus dorsi muscle and liver of LPS-infused 18

neonatal pigs The phosphorylated forms were normalized for the total content Values are 19


(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 ABSTRACT 1

Skeletal muscle protein synthesis is reduced in neonatal pigs in response to endotoxemia To 2

examine the role of insulin in this response neonatal pigs were infused with endotoxin (LPS 0 3

and 10 microgmiddotkg-1middothr-1) while glucose and amino acids were maintained at fasting levels and insulin 4

was clamped at fasting or fed (2 or 10 microUml) levels Fractional rates of protein synthesis and 5

translational control mechanisms were examined in longissimus dorsi muscle and liver In the 6

presence of fasting insulin LPS reduced muscle protein synthesis (-29) and increasing insulin 7

to fed levels accelerated muscle protein synthesis in both groups (controls +44 LPS +64) 8

LPS but not insulin increased liver protein synthesis by +28 In muscle of fasting neonatal 9

pigs LPS reduced 4E-BP1 phosphorylation and eIF4E to eIF4G binding In muscle of controls 10

but not LPS pigs raising insulin to fed levels increased 4E-BP1 and S6K1 phosphorylation and 11

eIF4E to eIF4G binding In muscle and liver neither LPS nor insulin altered eIF2B activity 12

eEF2 phosphorylation decreased in response to insulin in both LPS and control animals The 13

results suggest that in endotoxemic neonatal animals the response of protein synthesis to insulin 14

is maintained despite suppression of mTOR-dependent translation initiation and eIF4E 15

availability for eIF4F assembly Maintenance of an anabolic response to the feeding-induced 16

rise in insulin likely exerts a protective effect for the neonate to the catabolic processes induced 17

by sepsis 18

19

Keywords endotoxemia translation initiation eukaryotic elongation factor 2 protein synthesis 20

Page 2 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final Page 3 of 30 INTRODUCTION 1























Page 3 of 34
























Page 4 of 34

















inflammation 17

18

METHODS 19





Page 5 of 34
























Page 6 of 34
























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 INTRODUCTION 1























Page 3 of 34
























Page 4 of 34

















inflammation 17

18

METHODS 19





Page 5 of 34
























Page 6 of 34
























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 of mRNA to the 43S preinitiation complex and this process is mediated by the assembly of the 1























Page 4 of 34

















inflammation 17

18

METHODS 19





Page 5 of 34
























Page 6 of 34
























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 synthesis to insulin stimulation is largely maintained during acute endotoxin infusion (31) 1
















inflammation 17

18

METHODS 19





Page 5 of 34
























Page 6 of 34
























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 remained with the sow Three days prior to the infusion piglets were anesthetized and 1























Page 6 of 34
























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 E coli Serotype 0111-B4 Sigma Chemical Co St Louis MO) that was continued for 8 h while 1























Page 7 of 34








GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 leucine isoleucine and valine deamination by leucine dehydrogenase with stoichiometric 1







GDP exchange 8















Page 8 of 34














the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30













the blot 13











Page 9 of 34


blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 Biotechnology Santa Cruz CA) Results were normalized for the total amount of PKB on the 1

blot 2














16

RESULTS 17







Page 10 of 34










BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 obtained by providing exogenous insulin in both control and LPS-infused animals during the last 1









BCAA (P gt 005) 10














Page 11 of 34




1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 +28 when compared to controls (P lt 005 Fig 1D) Raising insulin to fed levels did not alter 1



1E and 1F) 4




















Page 12 of 34







7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 (P lt 005 Fig 4C) Raising insulin to fed levels in LPS-infused animals did not affect eIF2α1






7

DISCUSSION 8
















Page 13 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34















in muscle 14









Page 14 of 34


protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 maintaining either fasting or fed levels of insulin will not alter the LPS-induced increase in liver 1

protein synthesis 2






















Page 15 of 34
























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 have allowed the subjects to feed ad libitum but they become anorectic and it is possible that the 1























Page 16 of 34
























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 be impaired in rats subjected to experimental intrabdominal sepsis (42) it has been suggested 1























Page 17 of 34























Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 (21 23 43) and thus eIF2B appears to have a key role in regulating global rates of protein 1






















Page 18 of 34
























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 phosphorylated (14) we suggest that a decrease in eIF2α phosphorylation would have little if 1























Page 19 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34









administration 8

PERSPECTIVES 9













Page 20 of 34

















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 ACKNOWLEDGMENTS 1
















17

Page 21 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34


1

Reference List 23



2000 6




E574 2001 10




240 48-53 1996 14






Page 22 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34




Differ 7 603-615 2000 3






E890 2002 9









Arch Surg 123 992-999 1988 18

Page 23 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34









1993 8








eIF2B J Biol Chem 273 3039-3044 1998 16

Page 24 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34




423-427 2004 3










144-155 2005 13







Page 25 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34




Biochem 94 419-431 2005 3
















Page 26 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34








E166 2006 7









989 1989 16

Page 27 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34










10556-10565 2005 9








Page 28 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34
















Physiol 273 E305-E314 1997 15

16

17

Page 29 of 34










(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

mTOR Signaling in Endotoxemic Neonatal Pigs Final of 30 FIGURE LEGENDS 1









(Plt005) 11










(Plt005) 21

Page 30 of 34

Page 31 of 34

Page 32 of 34

Page 33 of 34

Page 34 of 34

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Insulin stimulates muscle protein synthesis in neonates during endotoxemia despite repression of...

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Transcript of Insulin stimulates muscle protein synthesis in neonates during endotoxemia despite repression of...