1 Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033; and Departments of 2 Surgery and 3 Internal Medicine, Gotenburg University, S-41345 Gotenburg, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study evaluated the ability of insulin-like growth factor I (IGF-I) complexed with IGF binding protein-3 (IGFBP-3) to modulate the sepsis-induced inhibition of protein synthesis in gastrocnemius. Beginning 16 h after the induction of sepsis, either the binary complex or saline was injected twice daily via a tail vein, with measurements made 3 and 5 days later. By day 3, sepsis had reduced plasma IGF-I concentrations ~50% in saline-treated rats. Administration of the binary complex provided exogenous IGF-I to compensate for the sepsis-induced diminished plasma IGF-I. Sepsis decreased rates of protein synthesis in gastrocnemius relative to controls by limiting translational efficiency. Treatment of septic rats with the binary complex for 5 days attenuated the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. Assessment of potential mechanisms regulating translational efficiency showed that neither the sepsis-induced change in gastrocnemius content of eukaryotic initiation factor 2B (eIF2B), the amount of eIF4E associated with 4E binding protein-1 (4E-BP1), nor the phosphorylation state of 4E-BP1 or eIF4E were altered by the binary complex. Overall, the results are consistent with the hypothesis that decreases in plasma IGF-I are partially responsible for enhanced muscle catabolism during sepsis.
gastrocnemius; eukaryotic initiation factors; translation initiation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SEPSIS CAUSES PROFOUND ALTERATIONS in protein metabolism in many tissues. The earliest recognizable alteration in protein metabolism is an excessive urea nitrogen excretion, indicative of a loss of body protein and a negative nitrogen balance. The major source of the excreted nitrogen is derived from the net catabolism of proteins in skeletal muscle. The catabolism of protein in skeletal muscle during sepsis results from an increased rate of protein degradation and a decreased rate of protein synthesis (for review, see Refs. 23, 60). When prolonged, the erosion of lean body mass has potential deleterious effects in septic patients, including sustained muscle fatigue, weakness, and poor wound healing.
Several treatment modalities have been advanced to prevent and/or modulate the sepsis-induced alterations in protein metabolism (64), including 1) provision of sufficient nutrients, 2) modulation of the cytokine/inflammatory response, or 3) administration of anabolic hormones. Provision of exogenous nutritional support is not sufficient to completely arrest muscle wasting in septic patients (5). In contrast, modulating the cytokine response (7, 8, 30) prevents sepsis-induced inhibition of protein synthesis in muscle. In these studies, anticytokine therapies were provided either prophylactically or shortly after the induction of the septic focus, a situation which is controllable in the laboratory but is of limited benefit in the clinical setting.
Information regarding the role of anabolic hormones in augmenting protein synthesis in muscle from septic patients or animals is limited. The major anabolic hormones modulating protein synthesis in skeletal muscle include insulin, growth hormone (GH), and insulin-like growth factor-I (IGF-I). The effects of insulin on protein synthesis in skeletal muscle have been well documented (35). However, skeletal muscle is resistant to the anabolic actions of insulin on protein synthesis during chronic intra-abdominal sepsis (29) or after infusion of tumor necrosis factor (19), indicative of an insulin resistance regarding protein synthesis.
Like insulin, the effects of other anabolic hormones on protein metabolism are well documented. GH promotes nitrogen retention and improves nitrogen balance in a variety of conditions, including burn and surgical postoperative patients (22, 36, 64). The anabolic actions of GH on protein metabolism are mediated primarily through an increase in protein synthesis. However, GH has been shown to have reduced effectiveness in retarding protein catabolism in septic patients, potentially limiting the usefulness of this hormone during sepsis (9, 49). Moreover, administration of GH is associated with an increased morbidity and mortality in critically ill patients (54).
The protein anabolic actions of GH are mediated indirectly by the hepatic synthesis and release of another hormone, IGF-I (44). Systemically administered IGF-I results in weight gain in normal rats, reduces weight loss during starvation or diabetes (47), and attenuates protein loss during cachetic states (55, 56). Chronic IGF-I administration improves nitrogen balance in rats injected with endotoxin (10).
Part of the anabolic action of IGF-I may be mediated through an increase in protein synthesis in muscle (3, 27, 51). In humans, intravenous infusion of IGF-I directly increases protein synthesis in skeletal muscle, provided adequate substrate supply is also present (17, 50). Recombinant human IGF-I (rhIGF-I) stimulates protein synthesis in freshly isolated myocytes from adult rats or in hearts perfused in vitro (18). In myotubes or myoblasts from the L8 or L6 cell lines in culture, IGF-I is a more potent stimulator of protein synthesis than insulin (2, 21). Likewise, IGF-I stimulates protein synthesis in vitro in either incubated muscles (51, 61) or perfused hindlimb (31).
We have previously demonstrated that the ability of IGF-I to stimulate skeletal muscle protein synthesis in vitro was unimpaired during the hypermetabolic phase of sepsis (31). Likewise, we have shown that IGF-I stimulates protein synthesis in incubated epitrochlearis muscle during either the anorexic or the hypermetabolic phase of the host's response to injection of live bacteria in vitro (61). IGF-I also stimulates protein synthesis in incubated extensor digitorum longus muscle from rats with severe acute peritonitis in vitro (26). However, there are no reports concerning the ability of IGF-I to stimulate protein synthesis in muscles of septic patients or animals in vivo.
Single intravenous injections of this growth factor may have limited therapeutic potential for several reasons. First, exogenously administered IGF-I has a short half-life in vivo (37), making a sustained elevation of IGF-I difficult. Second, IGF-I injections are associated with hypoglycemia, necessitating administration of exogenous glucose (6). Third, an intraperitoneal bolus injection of IGF-I, which transiently elevates plasma IGF-I concentrations, does not cause a clear stimulation of protein synthesis in overnight-starved mice (53). Finally, single injections of IGF-I lose their efficacy over time (41).
The vast majority of IGF-I is bound to one of several binding proteins (IGFBPs) in vivo, the most abundant of which is IGFBP-3. Administration of IGF-I prebound to nonglycosylated IGFBP-3 is being studied as an alternative to administration of free IGF-I. Binding of IGF-I to IGFBP-3 and the acid-labile subunit (ALS) forms a ternary complex that maintains circulating levels of IGF-I by slowing its rate of clearance (37, 40). Furthermore, the IGF-I/IGFBP-3 complex is less likely to cause hypoglycemia compared with IGF-I (1, 24). The purpose of the present investigation was to establish whether a binary complex, consisting of equal molar amounts of IGF-I and IGFBP-3, could modulate the sepsis-induced inhibition in skeletal muscle protein synthesis in vivo during the anorexic (day 3 postinfection) and hypermetabolic (day 5 postinfection) phases of septic response. Furthermore, we wished to determine whether injection of the IGF-I/IGFBP-3 complex could modulate the inhibition of translation efficiency responsible for diminished rates of protein synthesis during sepsis (8, 31, 58, 60). The results provide evidence that in vivo administration of the binary complex stimulates protein synthesis in skeletal muscle during sepsis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental animals. Male Sprague-Dawley rats weighing 175-200 g were anesthetized with a combination of ketamine (110 mg/kg body wt) and acepromazine (1 mg/kg body wt). Sepsis was then induced by implanting a fecal-agar pellet inoculated with 103 colony-forming units (CFU) of Escherichia coli and 108 CFU of Bacteroides fragilis into the abdominal cavity (29-31, 57-59). After recovery from surgery, all animals were allowed free access to rat chow (Harlan Teklad) and water. Beginning the next morning after induction of sepsis, animals were randomly divided into two groups, binary complex-treated and untreated. Binary complex-treated rats were injected via a tail vein twice daily with 5 µg/g body wt of the binary complex. Untreated septic rats received an equal volume of saline. On day 3 or day 5 after induction of sepsis, binary complex-treated and untreated septic rats were anesthetized, and rates of protein synthesis were measured in vivo. In the first 72 h, animals were recovering from surgery and the implantation of fecal agar pellet. During this period, food consumption was reduced relative to ad libitum-fed controls (30). This stage is referred to as the anorexic phase of the septic response. By day 5 postinfection, septic abscess animals consumed the same amount of food as they did before the surgery but showed muscle loss and other metabolic derangements (8, 30, 57-59). This period corresponds with the hypermetabolic phase of sepsis. Food consumption in binary complex-treated and untreated septic rats was the same over the course of the experiment. Control animals underwent the implantation of a sterile agar pellet (30, 58, 59) and were pair-fed with respect to the amount of food consumed by the septic rats. The experiments described herein were performed in adherence with the National Institutes of Health Guide for Care and Use of Laboratory Animals and with the approval of the Penn State University College of Medicine Animal Care and Use Committee.
Binary complex. The recombinant human (rh) binary complex was provided by Celtrix Pharmaceuticals, Inc, (Santa Clara, CA) in a 1:1 molar ratio of rhIGF-I to rhIGFBP-3 corresponding to the naturally occurring protein complex. The recombinant proteins were purified as previously described in detail (1). The two proteins were mixed together in a 1:1 molar ratio, with the complex being further purified by ion exchange chromatography. Purity of the complex was verified by reversed-phase HPLC and SDS-PAGE and was estimated to be >95%. Bioactivity was monitored using a cell culture assay (proliferation of MG63 cells). Injections were prepared from aliquoted frozen concentrates and performed twice daily at 0900 and 1800.
Measurement of protein synthesis in vivo. Rates of protein synthesis in vivo were estimated 3 or 5 days after the implantation of the fecal agar pellet after a bolus infusion of L-[3H]phenylalanine (150 mM, 30 µCi/ml; 1 ml/100 g body wt) described by Garlick et al. (20) and used previously in our laboratory (7, 8, 30, 57, 58). Ten minutes after injection of the radioisotope, a second blood sample (3 ml) was withdrawn for measurement of phenylalanine concentrations and specific radioactivity. Soleus, gastrocnemius, liver, and kidney were excised, weighed, and frozen to the temperature of liquid nitrogen or homogenized immediately for assay of eukaryotic initiation factors (eIFs). The frozen muscle samples were subsequently powdered under liquid nitrogen.
A portion of the frozen powdered tissue (0.5 g) was homogenized in ice-cold 3.6% (wt/vol) perchloric acid (PCA) to estimate the rate of incorporation of radioactive phenylalanine into protein, as described previously (7, 8, 29-31, 57, 58). A portion of this sample was assayed for protein concentration by the Biuret method, using crystalline bovine serum albumin as a standard. Another portion of the sample was used for the measurement of radioactivity in protein by liquid scintillation spectrometry using the proper corrections for quenching (dpm). The specific radioactivity of phenylalanine was measured in deproteinized plasma samples by HPLC analysis according to the method of Drnevich and Vary (11). The specific radioactivity of the phenylalanine was then calculated by dividing the disintegrations per minute of the phenylalanine peak by the concentration of phenylalanine in the sample. Rates of protein synthesis were calculated as described earlier with the use of the specific radioactivity of the plasma phenylalanine as the precursor pool (7, 8, 29-31, 57, 58). The assumption in using this technique to estimate the rate of protein synthesis in vivo is that the intracellular phenylalanine concentration is elevated to high concentrations, thereby limiting any dilution effect of nonradioactive phenylalanine derived from proteolysis. The plasma phenylalanine concentration was increased from ~70 nmol/ml to 1,344 ± 76 nmol/ml at the time of tissue sampling (average of all conditions). At perfusate concentrations >800 nmol/ml, the specific radioactivity of tRNA-bound phenylalanine is the same as that of the extracellular and intracellular pools of free phenylalanine (4, 28). Therefore, the specific radioactivity of plasma phenylalanine provides an accurate estimate of the specific radioactivity of phenylalanine bound to the tRNA.Determination of total RNA. Total RNA was measured from homogenates of muscle samples. Briefly, 0.3 g of frozen powdered tissue was homogenized in 5 volumes of ice-cold 10% TCA. The homogenate was centrifuged at 10,000 g for 11 min at 4°C. The supernatant was discarded, and the remaining pellet was mixed in 2.5 ml of 6% (wt/vol) PCA. The sample was centrifuged at 10,000 g for 6 min at 4°C, the supernatant was discarded, and the procedure was repeated. Then, 1.5 ml of 0.3 N KOH was added to the pellet, and the samples were placed in a 50°C water bath for 1 h. Samples were then mixed with 5 ml of 4 N PCA and centrifuged at 10,000 g for 11 min. The concentration of RNA in the supernatant was determined as previously described (7, 8, 57, 58).
Determination of plasma IGF-I concentrations. Plasma samples were acid-ethanol extracted and then subjected to an additional cryoprotection step to remove IGFBPs; this procedure quantitatively removes IGFBPs from serum (15). IGF-I was assayed by RIA as described previously (14). Human and rat IGF-I in plasma were determined with either human- or rat-specific RIA (Diagnostic Systems Laboratories, Webster, TX).
Measurement of plasma IGFBP-3 by ligand and Western blot techniques. Relative amounts of the various IGFBPs in plasma were determined by ligand blot analysis as previously described. Plasma samples were electrophoresed on 10% denaturing polyacrylamide gels, as described previously (15). Proteins were transferred electrophoretically to nitrocellulose membranes with a semidry blotter (Bio-Rad Laboaratories, Melville, NY). The membranes were blocked with 5% nonfat dry milk for 1.5 h and subsequently incubated with antibodies against human IGFBP-3 (Upstate Biotechnology, Lake Placid, NY), ALS (Diagnostic Systems Laboratories, Webster, TX), or [125I]IGF-I (Amersham, Arlington Heights, IL). After several washes, the membranes were then incubated with anti-rabbit immunoglobulin conjugated to horseradish peroxidase. The blots were then developed by use of an ECL Western blotting kit as per the manufacturer's (Amersham) instructions. Proteins were visualized by exposing X-ray film to the blots.
Separation of ternary complexes by Sephadex chromatography of plasma. To establish that injection of the binary complex formed a ternary complex in vivo, plasma samples (100 µl) were mixed with a tracer amount of [125I]IGF-I and fractionated on a Sephadex G100 column equilibrated with 0.1 M Tris-buffered saline. The radioactivity in each fraction was determined with a gamma counter, and an aliquot of each fraction was subjected to Western blot analysis of IGFBP-3 and ALS after electrophoresis with a SDS-PAGE gel. The radioactivity of the fractions was measured, and selected fractions were electrophoresed on a 12% polyacrylamide gel for Western blot analysis of human IGFBP-3, as described above.
Quantification of IGF-I, IGF-I receptor, and GH receptor mRNA.
Anti-sense IGF-I [35S]UTP-labeled RNA was synthesized
from an EcoR I linearized pSP64 plasmid carrying a 153-bp
mouse genomic subclone corresponding to exon 3 by analogy to the human
IGF-I gene and thus recognizing all reported variants of IGF-I mRNAs (44). The probe gives a 147-bp protected band in RNAse
protection assays (46). Anti-sense IGF-I receptor (IGF-IR)
[35S]UTP-labeled RNA was synthesized from an
EcoR I linearized pSP64 plasmid carrying a 265-bp
BamH I fragment of the rat IGF-IR cDNA (63).
The pGEM 3 plasmid contains a 265-bp BamH I fragment of the
rat IGF-I receptor cDNA that encodes the putative signal peptide and
the first 53 amino acids of the -subunit (63).
Anti-sense GH receptor (GH-R) [35S]UTP-labeled RNA was
synthesized from an EcoR I linearized pT7T3 18U plasmid
pSP64 plasmid carrying a 560-bp BamH I fragment of the rat
GH-R cDNA (43). The GH-R cDNA fragment corresponds to a
part of the extracellular domain of the GH-R.
Quantification of 4E-BP1 · eIF4E complex.
The association of eIF4E with 4E-BP1 was determined as previously
described in our laboratory (34, 52, 65). Briefly, the
gastrocnemius was rapidly removed, immediately weighed, and homogenized
in 7 volumes of buffer A [20 mM HEPES, pH 7.4, 100 mM KCl,
0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 50 mM
-glycerolphosphate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM microcystin LR]
using a Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. eIF4E and 4E-BP1 · eIF4E complexes were immunoprecipitated from
aliquots of 10,000-g supernatants using an anti-eIF4E
monoclonal antibody. The antibody-antigen complex was collected by
incubation for 1 h with goat anti-mouse Biomag IgG beads
(PerSeptive Diagnostics, Cambridge, MA). Before use, the beads were
washed in 1% nonfat dry milk in buffer B (50 mM
Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1%
-mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM
-glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium
vanadate). The beads were captured by means of a magnetic stand and
were washed twice with buffer B and once with buffer
B containing 500 rather than 150 mM NaCl. Resuspension in
SDS-sample buffer and boiling for 5 min eluted the protein bound to the
beads. The beads were collected by centrifugation, and the supernatants
were subjected to electrophoresis on a 15% polyacrylamide gel for
quantitation of 4E-BP1 and eIF-4E. Proteins were then
electrophoretically transferred to a polyvinylidene difluoride membrane
as previously described (34, 52, 65). The membranes were
incubated with a mouse anti-human eIF4E antibody or a rabbit anti-rat
4E-BP1 antibody for 1 h at room temperature. The blots were then
developed with an ECL Western blotting kit. Films were scanned by a
Microtek ScanMaker III scanner equipped with a transparent media
adaptor connected to a Macintosh computer. Images were obtained with
the ScanWizard Plugin (Microtek) for Adobe Photoshop and were
quantitated by means of NIH Image 1.60 software.
Determination of phosphorylation state of eIF4E. Phosphorylated and unphosphorylated forms of eIF4E in extracts of gastrocnemius were separated by isoelectric focusing on a slab gel and were quantitated by protein immunoblot analysis, as previously described (34, 52, 65).
Determination of phosphorylation state of 4E-BP1. The various phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from muscle homogenates after centrifugation at 10,000 g (34, 52). 4E-BP1 was immunoprecipitated from the supernatants as described in the previous section for immunoprecipitation of eIF4E. The immunoprecipitates were solubilized with SDS sample buffer. The various phosphorylated forms of 4E-BP1 were separated by electrophoresis and quantitated by protein immunoblot analysis as described previously (32-34, 65).
Biochemical analyses of blood. Blood samples were collected into heparinized syringes and centrifuged, and plasma was separated from cells and frozen until analyses. Insulin was measured with a commercially available RIA kit (Diagnostic Products, Los Angeles, CA). Plasma amino acids were analyzed by HPLC chromatography (Waters Associates Liquid Chromatographic System) with a precolumn derivatization method (25). Plasma glucose concentrations were determined with a model GL5 glucose analyzer (Analox Instruments, Lunenburg, MA).
Statistical analysis. Experimental data for each condition are summarized as means ± SE. Statistical evaluation of the data was performed using ANOVA to test for overall differences among groups, followed by the Sidak test for multiple comparisons to determine significance between means only when ANOVA indicated a significant difference among the group means. Differences among means were considered significant when P < 0.05. The number of animals in each group is indicated in the table and figure legends.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we examined the responses to treatment with
the binary complex during the anorexic (day 3 postinfection) and hypermetabolic (day 5 postinfection) phases of sepsis.
There were no significant differences in the mortality rates between the treated and untreated septic groups (data not shown). Changes in
body weights in the different groups are shown in Fig.
1. Pair-fed control animals showed a loss
of body weight during the first 48 h, when food restriction was
greatest. After that time, control rats gained weight. Both binary
complex-treated and untreated septic rats began to show weight gain by
day 4 postinfection. Beginning on day 3 after
induction of sepsis, the weight of the animals treated with the binary
complex tended to be higher than that of the untreated animals,
although the increase did not reach statistical significance.
|
Plasma IGF-I, insulin, and glucose concentrations.
Sepsis caused a diminution in plasma IGF-I concentrations by 58% on
day 3 and by 47% on day 5 compared with pair-fed
control animals (Fig. 2). Injection of
the binary complex into septic rats raised the plasma IGF-I on both
days compared with untreated septic rats, such that values were not
significantly different from nonseptic control rats. As illustrated in
Fig. 2, increases in plasma IGF-I in the binary complex-injected rats
was entirely the result of an increase in human IGF-I. The plasma
concentration of rat IGF-I in binary complex-treated rats was not
significantly different from that observed in untreated septic rats.
|
IGFBP-3 complex.
It was also important to determine whether the IGFBP-3 was retained in
plasma from septic animals after injection of the binary complex. Analysis of the ligand blot (Fig.
3 top) shows the appearance of
a 29-kDa band only in the plasma from animals treated with the binary
complex. Western blot analysis indicated that this 29-kDa protein
was rhIGFBP-3 (Fig. 3 bottom).
|
|
Hepatic and muscle IGF-I, IGF-IR, and GH-R mRNA.
The hepatic and gastrocnemius IGF-I mRNA content in control, untreated
septic, and binary complex-treated septic rats is shown in Table
1. In association with the fall in plasma
IGF-I concentrations on day 3 postinfection, hepatic and
muscle IGF-I mRNA content fell 34% and 23%, respectively, compared
with control. Treatment of septic rats with the binary complex did not
affect the hepatic IGF-I mRNA content but did restore the muscle IGF-I
mRNA to control values. The expression of hepatic IGF-I mRNA in control
rats was augmented on day 5 compared with day 3 in control rats, presumably because food intake was increased.
Likewise, the hepatic IGF-I mRNA content was increased on day
5 compared with day 3 in untreated septic rats or in
septic rats treated with the binary complex. However, the hepatic IGF-I
mRNA contents remained lower in both treated and untreated septic rats
compared with controls on day 5. Unlike liver, the muscle
IGF-I mRNA content returned to control values by day
5.
|
|
Organ weights and protein content.
The organ weights and protein content in muscle and visceral tissues in
the presence and absence of the binary complex on day 3 after induction of sepsis are shown in Table 2. Weights of
gastrocnemius (46%), soleus (
38%), and kidney (
13%) were all
significantly reduced by sepsis. Treating rats with the binary complex
significantly increased the weights of the gastrocnemius and soleus but
had no effect on kidney. The magnitude of the increase in weight was
similar in both gastrocnemius (19%) and soleus (22%) after treatment
with the binary complex. Despite the increase in muscle mass, weights
of gastrocnemius and soleus remained significantly lower than those of controls.
|
Protein synthesis.
To further investigate the factors responsible for the changes in
tissue weights and protein content, rates of protein synthesis were
measured in vivo by the incorporation of radioactive phenylalanine into
total mixed proteins. In vivo rates of protein synthesis in
gastrocnemius and soleus are shown in Fig.
5. Sepsis caused an inhibition of protein
synthesis in gastrocnemius on day 3. Treatment with
the binary complex significantly increased protein synthesis by
~50%. Similarly, sepsis inhibited protein synthesis in gastrocnemius
on day 5. The reduction in gastrocnemius protein synthesis
was significantly ameliorated by treatment of septic animals with
binary complex. Unlike day 3, the rate of gastrocnemius protein synthesis was not significantly different in control and septic
rats treated with the binary complex on day 5. In contrast to gastrocnemius, sepsis was without significant effect on rates of
protein synthesis in soleus. Moreover, there was no significant change
in the protein synthesis in soleus in response to the binary complex
administration.
|
|
Plasma amino acids.
The ability of IGF-I to modulate protein synthesis can be dependent on
the plasma amino acid concentration. To test the possibility that amino
acids limited the ability of IGF-I to stimulate protein synthesis in
different tissues, plasma amino acid concentrations were measured by
HPLC (Table 4). On day 3 postinfection,
total amino acids in plasma were reduced by 25% compared with
controls. Plasma concentrations of alanine, arginine, asparagine,
glutamine, tryptophan, threonine, taurine, and serine were
significantly reduced compared with controls. Treatment of septic rats
with the binary complex restored the total plasma amino acid content, primarily by augmenting the plasma concentrations of alanine, glutamine, histidine, and serine compared with untreated septic rats.
The concentrations of the other amino acids were not significantly different from untreated septic rats.
|
Translation efficiency.
Because administration of the binary complex improved rates of protein
synthesis in gastrocnemius, we investigated several potential
mechanisms responsible for the stimulation of protein synthesis during
sepsis. Changes in the number of ribosomes or changes in the efficiency
of mRNA translation may be responsible for the sepsis-induced reduction
in gastrocnemius protein synthesis. The number of ribosomes cannot be
measured directly, but it is usually estimated from the RNA content of
the tissue, where rRNA constitutes ~85% of the total RNA. Therefore,
changes in total RNA content presumably reflect changes in the number
of ribosomes. There was no significant difference in RNA content in any
of the conditions examined (Fig. 7
top). Therefore, alterations in the relative abundance of
ribosomes were not responsible for changes in gastrocnemius protein
synthesis in either untreated or binary complex-treated septic rats.
|
Effect of binary complex on eIF2B protein content during sepsis.
We have previously shown that the relative abundance of the -subunit
of eIF2B (eIF2B
) falls during the first 5 days after the septic
insult (8, 62). Therefore, we measured the effect of binary complex
administration on the expression of eIF2B
. The amount of eIF2B
on
day 3 postinfection tended to be diminished, although this
change did not reach statistical significance (Table 5). In contrast to day 3, the
amount of eIF2B
on day 5 postinfection was significantly
reduced by 37% compared with control values (Table 5). Injection of
the binary complex did not alter the amount of eIF2B
at either time
point examined.
|
Regulation of eIF4E.
To investigate the effect of sepsis and treatment with binary complex
on the association of 4E-BP1 with eIF4E, eIF4E immunoprecipitates were
analyzed for 4E-BP1 content (Fig. 8
top). Compared with control animals, sepsis increased the
amount of 4E-BP1 associated with eIF4E ~4-fold on day 3 postinfection. Treatment of septic rats with the binary complex had no
significant effect on the amount of 4E-BP1 associated with eIF4E.
Unlike on day 3, there was no significant difference in the
amount of 4E-BP1 associated with eIF4E between control and septic rats
on day 5. There was no significant difference in the amount
of 4E-BP1 bound to eIF4E between untreated or the binary
complex-treated septic rats on day 5; however, the amount of
4E-BP1 bound to eIF4E was significantly reduced by ~50% in either
treated or untreated septic rats on day 5 compared with day 3.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present results indicate that the sepsis-induced inhibition in protein synthesis in gastrocnemius can be modulated by the in vivo administration of a binary complex consisting of equimolar concentrations of IGF-I and IGFBP-3. Administration of the binary complex-stimulated protein synthesis in gastrocnemius 3 days after initiation of the septic focus but did not restore rates of protein synthesis to values observed in control animals. By day 5 postinfection, administration of the binary complex raised gastrocnemius protein synthesis to a rate comparable with that of control rats. Injection of the binary complex restored the plasma concentrations of IGF-I in septic rats to control values without alterations in either plasma insulin or glucose concentrations. These findings are consistent with reports in human burn patients, when hypoglycemia was not observed after injection of the binary complex (24), and are in contrast to the hypoglycemia observed after injection of free IGF-I (41).
The augmentation of plasma IGF-I after administration of the binary complex was entirely the result of an increase in human IGF-I. There was no detectable depression of endogenous rat IGF-I by administration of the binary complex. These observations concerning rat plasma IGF-I concentrations imply that injection of the binary complex did not downregulate the ability of the liver to synthesize IGF-I. These data are consistent with the comparable expression of hepatic IGF-I mRNA in binary complex-treated and untreated septic rats. Furthermore, exogenous rhIGFBP-3 binary complex formed a ternary complex with rat ALS in vivo. Sephadex chromatography confirmed that infused rhIGFBP-3, which was subsequently isolated from plasma, co-eluted with ALS and IGF-I in a 150-kDa complex. It is likely that complex formation extends the half-life of injected IGF-I beyond that which is seen for the free peptide. Thus binary complex administration most likely accelerates protein synthesis in gastrocnemius by prolonging the availability of IGF-I, although we cannot exclude the possibility of a ligand-independent IGFBP-3 effect.
The enhanced rates of protein synthesis in vivo after elevation of plasma IGF-I concentrations with the binary complex extends our previous findings, whereby IGF-I stimulates the synthesis of mixed muscle proteins in either perfused hindlimb (31) or incubated muscle preparations (26, 61) from septic animals. In the present study, the restoration of protein synthesis was associated with anabolic effects on gastrocnemius weight and protein content on both days 3 and 5 postinfection. In contrast to muscle, administration of the binary complex did not alter protein synthesis or protein content in liver or kidney. These observations are consistent with reports whereby increased rates of protein synthesis after an acute administration of IGF-I were localized to striated muscle in mice fasted overnight (3). Furthermore, the findings are consistent with improved net protein balance and stimulation of muscle protein fractional synthetic rates in severely burned children treated with the binary complex (24).
In contrast to gastrocnemius, administration of the binary complex was without effect in soleus. Thus muscles with predominantly fast-twitch fibers (gastrocnemius) were more sensitive to the administration of binary complex, whereas the muscles with slow-twitch fibers (soleus) were relatively unresponsive during sepsis. This finding is consistent with two observations. First, the ability of IGF-I to stimulate protein synthesis was greatest in gastrocnemius and plantaris, and least in soleus and heart, after infusion of IGF-I in mice (3). Second, sepsis preferentially inhibits protein synthesis in muscles composed primarily of fast-twitch fibers (e.g., gastrocnemius), whereas it is without effect in muscles composed primarily of slow-twitch fibers (e.g., soleus) (58). Hence, one might expect a greater effect of the binary complex in gastrocnemius compared with soleus during sepsis.
Synthesis of protein in eukaryotic cells is achieved through a complex series of discrete reactions. The process involves the association of the 40S and 60S ribosomal subunits, mRNA, initiator methionyl-tRNA (met-tRNAimet), other amino acyl-tRNAs, cofactors (i.e., GTP and ATP), and protein factors (collectively known as eIF, elongation factors, and releasing factors) through a series of reactions resulting in the translation of mRNA into proteins. Translation of mRNA on the ribosome is composed of three phases: 1) initiation, whereby met-tRNAimet associates with mRNA bound to 40S ribosomal subunit and subsequent binding of the 40S ribosomal subunit to the 60S subunit to form a ribosome complex capable of translation; 2) elongation, during which tRNA-bound amino acids are incorporated in growing polypeptide chains according to the mRNA template; and 3) termination, when the completed protein is released from the ribosome. Regulation of protein synthesis occurs predominantly through changes in the abundance of ribosomes, translational efficiency, and/or translatable mRNA.
To determine the potential mechanisms responsible for the increased rates of protein synthesis in septic rats treated with the binary complex, we examined the total muscle RNA content and translational efficiency. Because ~85% of the total cellular RNA is ribosomal, alterations in the muscle RNA content reflect changes in the relative amount of ribosomes. Treatment of septic rats with the binary complex did not increase the total RNA content in muscle. Therefore, the restoration of protein synthesis in the binary complex-treated septic rats did not result from an increased number of ribosomes; instead, the binary complex prevented the sepsis-induced inhibition of gastrocnemius protein synthesis by increasing translational efficiency. These observations are consistent with our previous reports whereby IGF-I was shown to acutely stimulate translational efficiency by enhancing translation initiation in perfused hindlimb of septic rats (31).
The relative abundance of the eIF2B decreases during the first 5 days after the septic insult (8, 62). We have previously suggested that
this is one mechanism by which sepsis can reduce protein synthesis in
skeletal muscle. Injection of the binary complex did not alter the
amount of eIF2B
in septic rats at either time point examined. Thus
it is unlikely that the binary complex enhanced protein synthesis
during sepsis by increasing the muscle content of eIF2B
. This
finding is in contrast to the ability of anticytokine modalities to
enhance protein synthesis during sepsis by preventing the fall in
skeletal muscle eIF2B
content (7, 8, 62). Thus the
binary complex was able to enhance protein synthesis despite a
diminished eIF2B
content in skeletal muscle. This observation is
consistent with our observations in perfused hindlimb, whereby IGF-I
stimulated protein synthesis in gastrocnemius without increasing the
expression of eIF2B (31). Furthermore, the data suggest
that factors regulating eIF2B expression during sepsis are independent
of IGF-I.
We also investigated the ability of the binary complex to modulate the binding of eIF4E to the translation repressor protein 4E-BP1, a crucial step controlling translation initiation in skeletal muscle. The inhibition of translation initiation during diabetes and starvation correlates with an increased amount of eIF4E found in the inactive 4E-BP1 · eIF4E complex in skeletal muscle (33, 34). Conversely, feeding starved rats or insulin treatment of diabetic rats causes a dissociation of the 4E-BP1 · eIF4E complex, thereby promoting translation initiation (33, 65). In the present set of experiments, sepsis increased the amount of 4E-BP1 bound to eIF4E at both time points, albeit to a lesser extent on day 5 postinfection. The binary complex did not diminish the abundance of eIF4E associated with 4E-BP1 in septic rats. Thus we did not observe a reciprocal relationship between eIF4E found in the inactive 4E-BP1 · eIF4E complex and protein synthesis in muscle. Hence, protein synthesis appears to be augmented by the binary complex in septic rats without reductions in the abundance of eIF4E · 4E-BP1 complex.
The interaction between 4E-BP1 and eIF4E is regulated by the extent of
4E-BP1 phosphorylation. Phosphorylation of 4E-BP1 releases eIF4E from
the 4E-BP1 · eIF4E complex (48). In the present
study, sepsis caused a marked reduction in the amount of 4E-BP1 in the -phosphorylated form compared with controls. Furthermore, the amount
of 4E-BP1 in the
-phosphorylated form increased from day 3 to day 5 postinfection, whereas no differences were
observed in controls between the two days. A reciprocal relationship
existed between the extent of binding of 4E-BP1 to eIF4E and the amount of 4E-BP1 in the
-phosphorylated form during sepsis. Treatment of
septic rats with the binary complex did not significantly modulate the
phosphorylation status of 4E-BP1. Because 4E-BP1 phosphorylation was
unaltered, it is not surprising that the binary complex did not affect
abundance of eIF4E associated with 4E-BP1.
Phosphorylation of eIF4E enhances affinity of the factor for m7GTP cap analogs of mRNA (45). Reduced phosphorylation of eIF4E correlates with an inhibition of protein synthesis during heat shock or serum depletion (12). Both phosphorylated and nonphosphorylated forms of eIF4E bind to the mRNA cap structure (45). However, the phosphorylated form possesses a fourfold greater affinity for cap analogs and mRNA than does the unphosphorylated form, providing a potential explanation for the correlation between phosphorylation of eIF4E and rates of protein synthesis (45). Sepsis decreased phosphorylation of eIF4E on both day 3 and day 5. Moreover, we were unable to detect a significant increase in phosphorylation of eIF4E at a time when protein synthesis was stimulated by the binary complex. Thus it is unlikely that the stimulation of protein synthesis in gastrocnemius of septic rats induced by binary complex administration resulted from an enhanced phosphorylation of eIF4E. This observation may not be surprising, because insulin failed to increase the phosphorylation of eIF4E in perfused hindlimb at a time when protein synthesis was enhanced (34).
In summary, these results indicate that injection of the binary complex minimizes the loss of muscle protein during sepsis, in part by promoting protein synthesis. The acceleration of protein synthesis occurred secondary to stimulating translational efficiency, rather than to increasing the number of ribosomes. The increase in muscle protein synthesis induced by administration of the binary complex appears to be independent of changes in eIF2B expression or 4E-BP1 regulation. The importance of these findings lies in the observation that the binary complex has a beneficial effect on protein synthesis, even when administered after initiation of the septic focus.
![]() |
ACKNOWLEDGEMENTS |
---|
We greatly appreciate the expert technical assistance afforded by Rebecca Eckman, Tracie Gilpin, Sharon Rannels, and Britt-Marie Iresjo.
![]() |
FOOTNOTES |
---|
This work was supported by Grants GM-39277 (T. C. Vary), GM-38032 (C. H. Lang), and DK-15658 (L. S. Jefferson), awarded by the United States Public Health Service National Institutes of Health, and by Swedish Cancer Society Grant 2014-B93-07XDD (E. Svanberg), Swedish Medical Research Council Grants 08712 and 13159 (E. Svanberg), the Tore Nilsson Foundation (E. Svanberg), the Knut and Alice Wallenberg Foundation (E. Svanberg), and by the Swedish and Goteborg Medical Societies (E. Svanberg).
Address for reprint requests and other correspondence: T. C. Vary, Dept. Cellular and Molecular Physiology (H166) Penn State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: tvary{at}psu.edu).
Present address of E. Svanberg: Ares Serano International, 15 bis Chemin des Mines, 1211 Geneva, Switzerland.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 November 1999; accepted in final form 12 June 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bagi, C,
Brommage R,
DeLeon E,
Adams S,
Rosen D,
and
Sommer A.
Benefit of systemically administered rhIGF-I and rhIGF-I/IGFBP-3 on cancellous bone in ovarectomized rats.
J Bone Miner Res
9:
1301-1312,
1994[ISI][Medline].
2.
Ballard, FJ,
Read LC,
Francis GL,
Bagley CJ,
and
Wallace JC.
Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts.
Biochem J
233:
223-230,
1986[ISI][Medline].
3.
Bark, TH,
McNurlan MA,
Lang CH,
and
Garlick PJ.
Increased protein synthesis after acute IGF-I or insulin infusion is localized to muscle in mice.
Am J Physiol Endocrinol Metab
275:
E118-E123,
1998
4.
Bylund-Fellenius, A-C,
Ojamaa KM,
Flaim KE,
Li JB,
Wassner SJ,
and
Jefferson LS.
Protein synthesis versus energy state in contracting muscle of perfused rat hindlimb.
Am J Physiol Endocrinol Metab
246:
E297-E305,
1984
5.
Cerra, FB,
Siegel JH,
Coleman B,
Border JR,
and
McMenamy R.
Septic autocannibalism: a failure of exogenous nutritional support.
Ann Surg
192:
570-580,
1980[ISI][Medline].
6.
Cioffi, WG,
Gore DC,
Rue LW, III,
Carrougher G,
Guler P-H,
McNanus WF,
and
Pruitt BA.
Insulin-like growth factor-1 lowers protein oxidation in patients with thermal injury.
Ann Surg
220:
310-319,
1994[ISI][Medline].
7.
Cooney, R,
Kimball SR,
Eckman R,
Maish G, III,
Shumate M,
and
Vary TC.
TNF binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis.
Am J Physiol Endocrinol Metab
276:
E611-E619,
1999
8.
Cooney, R,
Owens E,
Jurasinski C,
Gray K,
Vannice J,
and
Vary T.
Interleukin-1 receptor antagonist prevents sepsis-induced inhibition of protein synthesis.
Am J Physiol Endocrinol Metab
267:
E636-E641,
1994.
9.
Dahn, MS,
Lange MP,
and
Jacobs LA.
Insulin-like growth factor-1 production is inhibited in human sepsis.
Arch Surg
123:
1409-1414,
1988[Abstract].
10.
Dickerson, RN,
Manzo CB,
Charland SL,
Settle RG,
Stein TP,
Kuhl DA,
and
Rajter J-J.
The effects of insulin-like growth factor-1 on protein metabolism and hepatic response to endotoxin in parenterally fed rats.
J Surg Res
58:
260-266,
1995[ISI][Medline].
11.
Drnevich, D,
and
Vary T.
Analsyis of physiological amino acids using dabsyl derivatization and reverse phase liquid chromatogrphy.
J Chromatogr A
613:
137-144,
1993[ISI].
12.
Duncan, R,
Milburn SC,
and
Hershey JWB
Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects.
J Biol Chem
262:
380-388,
1987
13.
Durnam, D,
and
Palmiter R.
A practical approach for quantitating specific mRNAs by solution hybridization.
Anal Biochem
131:
385-393,
1983[ISI][Medline].
14.
Fan, J,
Molina P,
Gelato M,
and
Lang C.
Differential tissue regulation of insulin-like growth factor-I content and binding proteins after endotoxin.
Endocrinology
134:
1685-1692,
1994[Abstract].
15.
Frost, R,
Fuhrer J,
Steigbigel R,
Mariuz P,
Lang C,
and
Gelato M.
Wasting in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulin-like growth factor system.
Clin Endocrinol (Oxf)
44:
501-514,
1996[ISI][Medline].
16.
Frost, R,
Nachman S,
Lang C,
and
Gelato M.
Proteolysis of insulin-like growth factor-binding protein-3 in human immunodeficiency virus-positive children who fail to thrive.
J Clin Endocrinol Metab
81:
2957-2962,
1996[Abstract].
17.
Fryburg, D.
Insulin-like growth factor I exerts growth hormone- and insulin-like actions on human muscle protein metabolism.
Am J Physiol Endocrinol Metab
267:
E331-E336,
1994
18.
Fuller, SJ,
Mynett JR,
and
Sugden PH.
Stimulation of cardiac protein synthesis by insulin-like growth factors.
Biochem J
282:
85-90,
1992[ISI][Medline].
19.
Garcia-Martinez, C,
Lopez-Soriano J,
and
Argiles JM.
Acute treatment with tumor necrosis factor- induces changes in protein metabolism.
Mol Cell Biochem
125:
11-18,
1993[ISI][Medline].
20.
Garlick, PJ,
McNurlan MA,
and
Preedy VR.
A rapid and convenient technique for measuring the rate of protein synthesis in tissue by injection of [3H]phenylalanine.
Biochem J
192:
719-723,
1980[ISI][Medline].
21.
Gulve, EA,
and
Dice JF.
Regulation of protein synthesis and degradation in L8 myotubes: effects of serum, insulin, and insulin-like-growth factors.
Biochem J
260:
377-387,
1989[ISI][Medline].
22.
Hammerqvist, F,
Stromberg C,
von der Decken A,
Vinnars E,
and
Wernerman J.
Biosynthetic human growth hormone preserves both muscle protein synthesis and the decrease in muscle-free glutamine, and improves whole-body nitrogen economy after operation.
Ann Surg
216:
185-191,
1992.
23.
Hasselgren, P-O,
and
Fischer JE.
Sepsis: stimulation of energy-dependent protein breakdown resulting in protein loss in skeletal muscle.
World J Surg
22:
203-208,
1998[ISI][Medline].
24.
Herndon, D,
Ramzy P,
DebRoy M,
Zheng M,
Ferrando A,
Chinkes D,
Barret J,
Wolfe R,
and
Wolf S.
Muscle protein catabolism after severe burn: effects of IGF-1/IGFBP-3 treatment.
Ann Surg
229:
713-722,
1999[ISI][Medline].
25.
Hill, DW,
Walters F,
Wilson T,
and
Stuart J.
High performance liquid chromatographic determination of amino acids in the picomole range.
Anal Chem
51:
1338-1341,
1979[ISI][Medline].
26.
Hobler, S,
Williams A,
Fischer F,
and
Hasselgren P-O.
IGF-I stimulates protein synthesis but does not inhibit protein breakdown in muscle from septic rats.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R571-R576,
1998
27.
Jacob, R,
Hu X,
Niederstock D,
Hasan S,
McNulty PH,
Sherwin RS,
and
Young LH.
IGF-I stimulation of muscle protein synthesis in the awake rat: permissive role of insulin and amino acids.
Am J Physiol Endocrinol Metab
270:
E60-E66,
1996
28.
Jefferson, LS,
Li JB,
and
Rannels SR.
Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus.
J Biol Chem
252:
1476-148,
1977[Abstract].
29.
Jurasinski, C,
Gray K,
and
Vary TC.
Modulation of skeletal muscle protein synthesis by amino acids and insulin during sepsis.
Metabolism
44:
1130-1138,
1995[ISI][Medline].
30.
Jurasinski, CV,
Kilpatrick L,
and
Vary TC.
Amrinone prevents muscle protein wasting during chronic sepsis.
Am J Physiol Endocrinol Metab
268:
E491-E500,
1995
31.
Jurasinski, CV,
and
Vary TC.
Insulin-like growth factor I accelerates protein synthesis in skeletal muscle during sepsis.
Am J Physiol Endocrinol Metab
269:
E977-E981,
1995
32.
Kimball, SR,
Horetsky RL,
and
Jefferson LS.
Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts.
Am J Physiol Cell Physiol
274:
C221-C228,
1998
33.
Kimball, SR,
Jefferson L,
Fadden P,
Haystead TAJ,
and
Lawrence JC, Jr.
Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle.
Am J Physiol Cell Physiol
270:
C705-C709,
1996
34.
Kimball, SR,
Jurasinski CV,
Lawrence JC, Jr,
and
Jefferson LS.
Insulin stimulates protein synthesis in skeletal muscle by enhancing the association of eIF-4E and eIF-4G.
Am J Physiol Cell Physiol
272:
C754-C759,
1997
35.
Kimball, SR,
Vary TC,
and
Jefferson LS.
Regulation of protein synthesis by insulin.
Annu Rev Physiol
56:
321-348,
1994[ISI][Medline].
36.
Koea, JB,
Breier BH,
Douglas RG,
Gluckman PD,
and
Shaw JH.
Anabolic and cardiovascular effects of recombinant human growth hormone in surgical patients with sepsis.
Br J Surg
83:
196-202,
1996[ISI][Medline].
37.
Kritsch, KR,
Huss DJ,
and
Ney DM.
Greater potency of IGF-I than IGF-I/BP-3 complex in catabolic parenterally fed rats.
Am J Physiol Endocrinol Metab
278:
E252-E262,
2000
38.
Labarca, C,
and
Paigen K.
A simple, rapid and sensitive DNA assay procedure.
Anal Biochem
102:
344-352,
1980[ISI][Medline].
39.
Lang, C,
Fan J,
Frost R,
Gelato M,
Sakurai Y,
Herdon D,
and
Wolfe R.
Regulation of the insulin-like growth factor system by insulin in burn patients.
J Clin Endocrinol Metab
81:
2474-2480,
1996[Abstract].
40.
Lewitt, MS,
Saunders H,
Phuyal JL,
and
Baxter RC.
Complex formation by human insulin-like growth factor-binding protein-3.
Endocrinology
134:
2404-2409,
1994[Abstract].
41.
Lieberman, SA,
Butterfield GE,
Harrison D,
and
Hoffman AR.
Anabolic effects of recombinant insulin-like growth factor-I in cachetic patients with the acquired immunodeficiency syndrome.
J Clin Endocrinol Metab
78:
404-410,
1994[Abstract].
42.
Lin, TA,
Kong X,
Haystead TAJ,
Pause A,
Belsham G,
Sonnenberg N,
and
Lawrence JC, Jr.
PHAS-I as a link between mitogen activated protein kinase and translation initiation.
Science
266:
653-656,
1994[ISI][Medline].
43.
Matthews, L,
Enberg B,
and
Norstedt G.
Regulation of rat growth hormone receptor gene expression.
J Biol Chem
264:
9905-9910,
1989
44.
Matthews, L,
Norstedt G,
and
Palmiter R.
Regulation of insulin-like growth factor 1 gene expression by growth hormone.
Proc Natl Acad Sci USA
83:
9343-9347,
1986[Abstract].
45.
Minich, WB,
Balasta ML,
Goss DJ,
and
Rhoads RE.
Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic initiation factor eIF-4E: Increased cap affinity of the phoshphorylated form.
Proc Natl Acad Sci USA
91:
7668-772,
1994[Abstract].
46.
Möller, C,
Arner P,
Sonnenfeldt T,
and
Norstedt G.
Quantitative comparison of insulin-like growth factor mRNA levels in human and rat tissues analysed by a solution hybridization assay.
J Mol Endocrinol
7:
213-222,
1991[Abstract].
47.
O'Sullivan, U,
Gluckman P,
Breier B,
Woodhall S,
Siddiqui R,
and
McCutcheon S.
Insulin-like growth factor I (IGF-I) reduces weight loss in mice during starvation.
Endocrinology
125:
2793-2794,
1989[Abstract].
48.
Pause, A,
Belsham GJ,
Gingras A-C,
Donze O,
Lin T-A,
Lawrence JC, Jr,
and
Sonenberg N.
Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function.
Nature
371:
762-767,
1994[ISI][Medline].
49.
Ross, RJ,
Miell JP,
Freeman E,
Jones J,
Matthews D,
Preece M,
and
Buchanan C.
Critically ill patients have high basal growth hormone levels with attenuated oscillatory activity associated with low levels of insulin-like growth factor-I.
Clin Endocrinol (Oxf)
35:
47-54,
1991[ISI][Medline].
50.
Russell-Jones, DL,
Umpleby A,
Hennessey T,
Bowes S,
Shojaee-Moradies F,
Hopkins K,
Jackson N,
Kelly J,
Jones R,
and
Sonksen P.
Use of a leucine clamp to demostrate that IGF-I actively stimulates protein synthesis in normal humans.
Am J Physiol Endocrinol Metab
267:
E591-E598,
1994
51.
Sandstrom, R,
Svanberg E,
Hyltander A,
Haglind E,
Ohlsson C,
Zachrisson H,
Berglund B,
Lindholm E,
Brevinge H,
and
Lundholm K.
The effect of recombinant human IGF-I on protein metabolism in postoperative patients without nutrition compared with effects in experimental animals.
Eur J Clin Invest
25:
784-792,
1995[ISI][Medline].
52.
Svanberg, E,
Jefferson LS,
Lundholm K,
and
Kimball SR.
Postprandial stimulation of muscle protein synthesis is independent of changes in insulin.
Am J Physiol Endocrinol Metab
272:
E841-E847,
1997
53.
Svanberg, E,
Zachrisson H,
Ohlsson C,
Iresjo B-M,
and
Lindholm KG.
Role of insulin and IGF-I in activation of muscle protein synthesis after oral feeding.
Am J Physiol Endocrinol Metab
270:
E614-E620,
1996
54.
Takala, J,
Ruokonen E,
Webster NR,
Nielsen MS,
Zandstra DF,
Vundelinckx G,
and
Hinds CJ.
Increased mortality associated with growth hormone treatment in critically ill adults.
N Engl J Med
341:
785-792,
1999
55.
Tomas, FM,
Knowles SE,
Owens PC,
Chandler CS,
Francis GL,
Read LC,
and
Ballard FJ.
Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are anabolic in dexamethasone-treated rats.
Biochem J
282:
91-97,
1992[ISI][Medline].
56.
Tomas, F,
Knowles S,
Owens P,
Read L,
Chandler C,
Gargosky S,
and
Ballard F.
Increased weight gain, nitrogen retention, and muscle protein synthesis after treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1-3)IGF-I.
Biochem J
276:
547-554,
1991[ISI][Medline].
57.
Vary, T,
and
Kimball S.
Regulation of hepatic protein synthesis in chronic inflammation and sepsis.
Am J Physiol Cell Physiol
262:
C445-C452,
1992
58.
Vary, T,
and
Kimball S.
Sepsis-induced changes in protein synthesis: Differential effects on fast- and slow-twitch muscles.
Am J Physiol Cell Physiol
262:
C1513-C1518,
1992
59.
Vary, T,
Siegel J,
Placko R,
Tall B,
and
Morris J.
Effect of dichloroacetate on plasma and hepatic amino acids in sterile inflammation and sepsis.
Arch Surg
124:
1071-1077,
1989[Abstract].
60.
Vary, TC.
Regulation of skeletal muscle protein turnover in sepsis.
Curr Opin Clin Nutr Metab Care
1:
217-224,
1998[Medline].
61.
Vary, TC,
Dardevet D,
Grizard J,
Voisin L,
Buffiere C,
Denis P,
Brueille D,
and
Obled C.
Differential regulation of skeletal muscle protein turnover by insulin and IGF-I after bacteremia.
Am J Physiol Endocrinol Metab
275:
E584-E593,
1998
62.
Voisin, L,
Gray K,
Flowers K,
Kimball S,
Jefferson L,
and
Vary T.
Altered expression of eukaryotic initiation factor 2B in skeletal muscle during sepsis.
Am J Physiol Endocrinol Metab
270:
E43-E50,
1996
63.
Werner, H,
Woloschak M,
Adamo M,
Shen-Orr Z,
Roberts C, Jr,
and
LeRoith D.
Developmental regulation of the rat insulin-like growth factor I receptor gene.
Proc Natl Acad Sci USA
19:
7451-7455,
1989.
64.
Wilmore, D.
Catabolic illnessstrategies for enhancing recovery.
N Engl J Med
325:
695-802,
1991[Abstract].
65.
Yoshizawa, F,
Kimball SR,
Vary TC,
and
Jefferson LS.
Effect of dietary protein on translation initiation in rat skeletal muscle and liver.
Am J Physiol Endocrinol Metab
275:
E814-E820,
1998