Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanisms by which insulin-like
growth factor I (IGF-I) and insulin regulate eukaryotic initiation
factor (eIF)4F formation were examined in the ovine fetus. Insulin
infusion increased phosphorylation of eIF4E-binding protein
(4E-BP1) in muscle and liver. IGF-I infusion did not alter
4E-BP1 phosphorylation in liver. In muscle, IGF-I increased
4E-BP1 phosphorylation by 27%; the percentage in the -form in
the IGF-I group was significantly lower than that in the insulin group.
In liver, only IGF-I increased eIF4G. Both IGF-I and insulin increased
eIF4E · eIF4G binding in muscle, but only insulin decreased the
amount of 4E-BP1 associated with eIF4E. In liver, only IGF-I
increased eIF4E · eIF4G binding. Insulin increased the
phosphorylation of p70 S6 kinase (p70S6k) in both muscle
and liver and protein kinase B (PKB/Akt) in muscle, two indicative
signal proteins in the phosphatidylinositol (PI) 3-kinase pathway.
IGF-I increased PKB/Akt phosphorylation in muscle but had no effect on
p70S6k phosphorylation in muscle or liver. We conclude that
insulin and IGF-I modulate eIF4F formation; however, the two hormones have different regulatory mechanisms. Insulin increases phosphorylation of 4E-BP1 and eIF4E · eIF4G binding in muscle, whereas
IGF-I regulates eIF4F formation by increasing total eIF4G. Insulin, but
not IGF-I, decreased 4E-BP1 content associated with eIF4E. Insulin
regulates translation initiation via the PI 3-kinase-p70S6k
pathway, whereas IGF-I does so mainly via mechanisms independent of the
PI 3-kinase-p70S6k pathway.
insulin; insulin-like growth factor I; fetus; eukaryotic initiation factors; p70 S6 kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INSULIN HAS LONG BEEN REGARDED as the primary fetal growth factor. Hyperinsulinemic fetuses of mothers with diabetes or the Beckwith-Weideman syndrome are usually large for gestational age. In contrast, the infant born with pancreatic agenesis and hypoinsulinemia is uniformly small for gestational age. Insulin administration to the fetus results in accelerated growth (1, 43, 52, 60) and increased protein utilization (54).
Insulin-like growth factor I (IGF-I) is one of the potent metabolic, mitogenic, and differentiative factors and is considered to be one of the important regulators of fetal growth (12, 22, 29). IGF-I is widely expressed in fetal tissues (19, 47). The circulating concentration of IGF-I has been shown to correlate directly with fetal weight in humans, and decreased concentrations of IGF-I have been found in association with fetal growth retardation (28, 41). Deletion of the IGF-I gene in mice results in high mortality, reduced birth weight, and retarded rate of postnatal growth (35).
Both insulin and IGF-I have been found to regulate protein anabolism in fetal and postnatal life (3, 13, 14, 21, 23, 48, 56). However, the cellular mechanisms by which IGF-I and insulin regulate fetal protein anabolism in vivo are largely unknown. One possible mechanism for IGF-I and insulin-induced increases in fetal protein synthesis is via alterations in the amount and/or activity of eukaryotic initiation factors (eIF). The initiation of mRNA translation is a complex process requiring several steps and more than a dozen eIFs (42, 46, 51). It is believed that the binding of mRNA to the 43S preinitiation complex is one of the rate-limiting steps in protein synthesis. The binding of mRNA to the 43S preinitiation complex is regulated by a multisubunit complex, called the eIF4F complex (2). The complex consists of three proteins: 1) eIF4A, an RNA helicase that functions to unwind secondary structure in the 5'-untranslated region of the mRNA; 2) eIF4E, a protein that binds the 7-methyl-GTP (m7GTP) cap present at the 5' end of eukaryotic mRNAs; and 3) eIF4G, a 220-kDa polypeptide that functions as a scaffold for eIF4E, eIF4A, the mRNA, and the ribosome. Presumably, increased formation of the eIF4F complex will lead to an increase of the cap-dependent mRNA translation.
Formation of an eIF4F complex may be regulated by alterations in either the phosphorylation state or the availability of eIF4E. Phosphorylation of eIF4E is suggested to stimulate translation rates through increased association with eIF4G and eIF4A (5) and/or increased mRNA cap-binding affinity (39). The availability of eIF4E appears to be regulated by a group of small acid and heat-stable proteins termed eIF4E-binding proteins (4E-BP1, 4E-BP2, and 4E-BP3). Hypophosphorylated 4E-BP1 binds to eIF4E to form an eIF4E · 4E-BP1 complex. When eIF4E is bound to 4E-BP1, eIF4E binds to mRNA but cannot form an eIF4E · eIF4G complex (18), therefore blocking the binding of mRNA to the ribosome. The binding of eIF4E to 4E-BP1 is, in turn, regulated by phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 releases eIF4E from the eIF4E · 4E-BP1 complex and allows the eIF4E · mRNA complex to bind to eIF4G and through eIF4G to the 40S ribosome (16, 49, 51).
The intracellular signal transduction pathways leading to translation initiation are beginning to be elucidated in cell culture systems. It has been demonstrated that the phosphatidylinositol (PI) 3-kinase pathway is responsible for the phosphorylation of 4E-BP1 and p70 S6 kinase (p70S6k) (45, 49). The signaling pathway that leads to phosphorylation of 4E-BP1 and p70S6k appears to bifurcate immediately upstream of the two proteins, likely at the mammalian target of rapamycin (mTOR) (57). Protein kinase B (PKB/Akt) has been implicated in the activation of the mTOR pathway and in the regulation of 4E-BP1 and p70S6k (15). However, in vivo data from intact animals are sparse, and essentially nothing is known from the fetal model regarding the signal pathways leading to activation of eIFs.
The present study was designed to test the hypotheses that IGF-I and insulin increase the formation of the eIF4F complex. In addition, the possible pathways involved in insulin- and IGF-I-induced alterations in eIFs were explored by measuring the phosphorylation of the indicative signal proteins of specific signaling pathways. The results show that both insulin and IGF-I alter the eIF4F complex formation in the fetus but that the response of component initiation factors to insulin infusion is organ specific. The results also show that the signal pathway of insulin-induced alterations in initiation factors is different from that of IGF-I.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and surgical procedures. Thirty-three ewes of 115-120 days of gestation were utilized for this study. Animal care was in strict compliance with National Institutes of Health guidelines within an American Association for Laboratory Animal Care-certified facility, and the study protocols were approved by the Institutional Animal Care and Use Committee.
Surgical procedures were performed aseptically under general anesthesia. Anesthesia was induced with intravenous ketamine and maintained with isoflurane inhalation. Fetal catheters were placed in the inferior vena cava and abdominal aorta. Catheters were exteriorized and irrigated daily with 0.9% saline containing 50 U heparin/ml. All ewes consumed ad libitum a diet consisting of hay and pelletized alfalfa and had constant access to water and a salt lick.Study design. The animals were allowed a minimum of 5 days of recovery from operative stress before the study. Complete recovery and fetal health were assessed by monitoring maternal food intake, fetal and maternal glucose concentrations, and acid-base status. The animals were fed ad libitum before and throughout the study.
On the day of the study, baseline blood samples were obtained for amino acid, glucose, and hormone concentration assay. Then, the fetus was infused with one of the following four infusates: 1) saline, 2) recombinant human (rh)IGF-I (gift of Eli Lilly Research Laboratories, Indianapolis, IN) plus a replacement dose of insulin (40 nmol IGF-I/h + 16 mIU insulin/h), 3) insulin (890 mIU/h), and 4) IGF-I plus insulin (40 nmol IGF-I/h + 890 mIU insulin/h). The infusion rates of insulin and IGF-I are similar to those of our previous work, have been shown to result in phamacological concentrations of insulin and IGF-I, and have been shown to affect amino acid and protein kinetics in the ovine fetus (4, 30, 31). The dose of insulin infused in group 2 was expected to maintain plasma insulin concentrations at the baseline level, counteracting the inhibition of insulin secretion during the rhIGF-I infusion. During the hormone infusion, fetal whole blood glucose was clamped at the baseline level by frequent glucose concentration measurement with a glucose analyzer (YSI 2300, Yellow Springs Instrument, Yellow Springs, OH) and a variable infusion of 20% glucose. Likewise, the total fetal plasma branched-chain amino acid (BCAA) concentrations were measured at 15-min intervals (58), and a balanced amino acid solution (AminoSyn II, Abbott Laboratories, Abbott Park, IL) was infused at a variable rate to maintain plasma BCAA at the baseline level. Seven hours after initiation of infusion, the ewe was anesthetized, and fetal biopsy samples (muscle and liver) were taken. Finally, the ewe underwent euthanasia, and fetal size and fetal weight were recorded. All catheter placements were confirmed at autopsy, and fetal autopsy samples were taken. Tissue samples were snap-frozen in liquid nitrogen and stored atMeasurements for leucine, IGF-I, and insulin. During the hormone infusion, total BCAA concentrations were monitored by spectrophotometry as described in the previous paragraph. The fetal leucine concentrations in plasma and whole blood from representative samples were measured with an automated amino acid analyzer (Beckman 6300, Beckman-Coulter, Palo Alto, CA).
Insulin concentrations in fetal plasma were determined in duplicate by a double-antibody RIA using ovine insulin to construct the standard curve (kit no. SRI-13K, Linco Research, St. Charles, MO). Total IGF-I in fetal plasma was determined by a validated competitive RIA that employed formic acid-acetone as the IGF-I extraction procedure (30).Measurements of eIF4E, eIF4G, and 4E-BP1.
Frozen tissue (muscle or liver) was homogenized using a PowerGen125
(Fisher Scientific, Pittsburgh, PA) in 7 ml/g tissue ice-cold buffer A (in mM: 20 HEPES-NaOH, pH 7.4, 100 KCl, 0.2 EDTA, 2 EGTA, 1 dithiothreitol, 50 NaF, 50 -glycerophosphate, 1 benzamidine, 0.5 sodium vanadate). Phenylmethylsulfonyl fluoride (PMSF; 0.5 mM), 1%
phosphatase inhibitor cocktail (Sigma, St. Louis, MO), and 1% protease
inhibitor cocktail (Sigma) were added immediately before use. The
homogenate was centrifuged at 13,000 g at 4°C for 30 min.
The protein concentration of the supernatant was measured by the Lowry
method (36). The supernatant was stored at
70°C until
further analysis. Our preliminary study showed that, in the
13,000-g pellets, less than 5% of eIF4E was found, and
eIF4G and 4E-BP1 were undetectable, suggesting that the eIF
extraction protocol works well.
Quantification of 4E-BP1 phosphorylation and eIF4E
phosphorylation.
Previous experiments had established that phosphorylation of 4E-BP1
retards the protein migration rate on SDS-polyacrylamide gels
(25, 32, 33). Consequently, when the tissue or cell extract is subjected to SDS-PAGE, multiple electrophoretic forms may be
resolved. These forms have been identified as (least phosphorylated
and fastest migrating),
(intermediate), and
(most
phosphorylated and slowest migrating). The phosphorylation of
4E-BP1 was expressed as the percentage of the
-form in the total content (
+
+
).
Determination of eIF4G and 4E-BP1 associated with eIF4E. The association of 4E-BP1 or eIF4G with eIF4E was assessed by determining how much eIF4G or 4E-BP1 was recovered when eIF4E was extracted with m7GTP-Sepharose 4B (Amersham Pharmacia Biotech). In addition to free eIF4E, eIF4E associated with eIF4G, or eIF4E associated with 4E-BP1, is presumably to bind the m7GTP-Sepharose 4B as well (25, 33). One hundred twenty microliters of prewashed and preequilibrated (with buffer A) suspension of m7GTP-Sepharose 4B were added to a minicolumn. Tissue extract of equal protein concentration (4 mg of muscle protein or 10 mg of liver protein) was subsequently added and mixed for 1 h at 4°C. After incubation, the resin was washed three times with ice-cold buffer A (1.5 ml/wash) with the use of a Vac-Man vacuum manifold to pull the buffer through the column. The resin was then resuspended in 300 µl of 100 µM m7GTP (Sigma) in buffer A and incubated for 5 min on ice. The eluate was then collected. The eluate was reapplied to the column, and the final eluate was collected. The eluate was diluted with an equal volume of the SDS sampling buffer and subjected to electrophoresis. Proteins were then electrophoretically transferred to PVDF membranes, and eIF4G and 4E-BP1 were quantified as described. The amount of eIF4G and 4E-BP1 detected in the eluate represents eIF4G or 4E-BP1 associated with eIF4E in the muscle and liver. Then, the membranes were stripped and reprobed for total eIF4E. The results were normalized to the amount of eIF4E.
Determination of p70S6k and PKB/Akt phosphorylation. Muscle or liver homogenates were combined with an equal volume of SDS sample buffer, and the diluted samples were subjected to electrophoresis on a 7.5% polyacrylamide gel. The samples were then analyzed by protein immunoblot analysis by use of rabbit anti-rat p70S6k polyclonal antibody (1:1,500; Santa Cruz Biotechnology). Previous studies have demonstrated that p70S6k resolves into multiple electrophoretic forms, with increased phosphorylation corresponding to decreased electrophoretic mobility (7, 24). The slowest migrating electrophoretic forms represent p70S6k phosphorylated on multiple residues.
PKB/Akt phosphorylated at Ser473 and total PKB/Akt were determined by immunoblotting by means of phosphospecific (Ser473) PKB/Akt antibody (1:1,000) and PKB/Akt antibody (phosphorylation state independent; 1:1,000), respectively (Cell Signaling Technology).Statistics. The data were analyzed by two-way ANOVA, with insulin and IGF-I as independent factors (JMP, SAS, Cary, NC). All values are expressed as means ± SE. Main effects were taken to be significantly different if the F-test resulted in a P < 0.05. In all figures, the P values for the main effects and interactions between main effects are given at the top left. Tukey's Honestly Significant Difference (HSD) test was used for post hoc analysis of group differences. In the figures, the results of the post hoc analysis are given by letters a-d, denoting group differences as detected by post hoc analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glucose, leucine, IGF-I, and insulin concentration.
The fetal weights and baseline concentrations of glucose and leucine in
the fetal arterial circulation are given in Table 1. The infusion protocol was determined
randomly on the day of the experiment, before fetal weights were known.
There were large individual variations in fetal weight. All metabolic
data were normalized to fetal weight to minimize the effect of
variability in fetal weight. The baseline levels of glucose and leucine
varied among animals; however, all of the measurements were within
normal ranges.
|
Glucose and amino acid infusion rates for clamp.
During the experimental infusions, arterial glucose and
BCAA were held constant by infusing a variable amount of glucose
and a mixture of amino acids into the fetal inferior vena cava (Table 1). In the IGF-I group, an average glucose infusion rate of 14 µmol · kg1 · min
1 was
needed to keep glucose concentration constant. In the insulin infusion
group, 24 µmol · kg
1 · min
1 of
exogenous glucose was needed to keep fetal glucose concentrations constant. As expected, more exogenous glucose (33 µmol · kg
1 · min
1) was
needed for the purpose of the "clamping" when insulin and IGF-I
were infused in combination. To keep fetal arterial amino acids
constant, exogenous amino acids were infused in the IGF/Ins group. When
both hormones were used in combination, a significantly higher amino
acid infusion rate was required to maintain amino acid concentrations.
Initiation factors.
eIF4E, one of the subunits of the eIF4F complex, plays a crucial role
in the binding of mRNA to the 43S preinitiation complex. The total
amount of eIF4E was not affected by insulin or IGF-I infusion in ovine
skeletal muscle (Fig. 1A), and
only IGF-I altered eIF4E phosphorylation (Fig. 1B).
Phospho-eIF4E varied little among individuals in the control group. In
the IGF, Insulin, or IGF/Ins groups, however, phospho-eIF4E varied
greatly among individuals, from a level similar to that of the controls
to a level more than threefold that of the controls. This can be seen
by the large increase in the SE bars in the IGF, Insulin, and IGF/Ins
groups.
|
4E-BP1.
The availability of eIF4E can be regulated through changes in the
amount of eIF4E bound to 4E-BP1. The association of 4E-BP1 with
eIF4E is regulated by the phosphorylation of 4E-BP1. In skeletal muscle, both insulin and IGF-I infusion increased the phosphorylation of 4E-BP1, but the insulin effect was much more
pronounced (Fig. 2A).
In the control group, three phosphorylation forms of 4E-BP1 were
typically detected, and the percentage of the -form was 41%. In
contrast, only
- and
-forms were usually detected in the insulin
group, and 85% of 4E-BP1 was in the
-form. In the IGF group,
4E-BP1 phosphorylation was increased by 27% (P < 0.05), but the percentage of the highly phosphorylated form (
) in
the IGF-I group was lower than that in the insulin group
(P < 0.05). When insulin and IGF-I were infused
simultaneously, a substantial increase in the hyperphosphorylated form,
similar to that seen in the insulin infusion alone, was observed.
|
eIF4G.
As illustrated in Fig. 3A,
IGF-I and insulin infusion increased muscle total eIF4G by 21 and 9%,
respectively, but this was not a significant difference when compared
with the control group. When IGF-I and insulin were infused
simultaneously, eIF4G in skeletal muscle increased significantly
(P < 0.05). In contrast, liver eIF4G content was
markedly increased by IGF-I infusion (Fig. 3B). Insulin
infusion did not alter eIF4G content and in fact appeared to have an
inhibitory effect on IGF-induced increase in the eIF4G content when
both hormones were infused simultaneously.
|
Association of eIF4G or 4E-BP1 with eIF4E.
In skeletal muscle, insulin decreased the association of eIF4E with the
repressor protein 4E-BP1 as well as the expected reciprocal increase in eIF4G associated with eIF4E (Fig.
4). The degree of eIF4E binding to eIF4G
is reflective of eIF4F formation. IGF-I had no effect on the
association of eIF4E with 4E-BP1, consistent with its moderate
effect on 4E-BP1 phosphorylation. However, it did result in an
increased association of eIF4E with eIF4G (P < 0.01).
In addition, there was a statistically significant interaction effect
between insulin and IGF-I, and the degree of association of the eIF4E
and eIF4G was no greater when the hormones were given simultaneously
than when either was given alone. Thus the significant interaction
effect may indicate that one or both hormones have a negative impact on
the other's ability to stimulate eIF4E association with eIF4G.
|
|
Phosphorylation of p70S6k.
p70S6k is resolved into several bands on SDS-polyacrylamide
gels. Incubation of muscle or liver extracts with calf alkaline
phosphatase at 37°C for 45 min converted the slow migration species
to fast migration species, confirming that increased phosphorylation
corresponds to decreased electrophoretic mobility. The results showed
that insulin significantly stimulated p70S6k
phosphorylation in skeletal muscle of the ovine fetus (Fig.
6). However, IGF-I did not
exhibit effects on p70S6k phosphorylation. Similar results
were found in fetal liver (data not shown).
|
Phosphorylation of PKB/Akt.
Neither IGF-I nor insulin had any effect on total PKB/Akt content in
muscle (Fig. 7A). Insulin
significantly increased the phosphorylation of PKB/Akt in the muscle
(Fig. 7B). The two-way ANOVA P value for the
IGF-I effect was 0.16, but there was a significant difference between
the control group and the IGF-I group by post hoc analysis. There was
also a significant interaction effect between insulin and IGF-I, which
suggests that IGF-I may inhibit the insulin effect on PKB/Akt
phosphorylation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The roles of insulin and IGF-I in promoting mRNA translation initiation have not been previously explored in the fetus. Our present results demonstrate that, although both hormones increase eIF4F complex formation, only insulin does so by altering phosphorylation of 4E-BP1. Conversely, only IGF-I infusion increases the amount of eIF4G. Thus insulin and IGF-I, both fetal anabolic hormones, appear to modulate the eukaryotic initiation factors by distinct cellular mechanisms.
Amino acid and glucose concentrations decrease with insulin or IGF-I infusion because of inhibitory effects of the peptides on protein breakdown (6, 13, 21, 38, 48). It has been well documented that amino acids, specifically leucine, and glucose can regulate protein translation initiation (2, 55, 59). To minimize this uncertainty and assess an independent effect of the insulin or IGF-I infusion on protein translation initiation, a glucose-amino acid clamp technique was introduced to keep fetal glucose and amino acid concentrations at baseline levels during IGF-I and insulin infusion. In addition, to assess the independent role of IGF-I in regulating translation initiation, a replacement dose of insulin was administered to counteract the inhibition of insulin secretion by IGF-I during the IGF-I infusion. The replacement dose of insulin used in the IGF group was optimal in counteracting the inhibition of insulin secretion during IGF-I infusion. The amino acid clamping procedure was likewise successful at preventing the usual 30-50% decline in amino acid concentrations during insulin or IGF-I infusion (30, 31). There were some fluctuations in leucine concentrations due to individual variations in their response to exogenous amino acid infusion. However, individual data analysis within a group demonstrated minor fluctuation (within 10% of the baseline level) in amino acid concentrations. We do not believe that these fluctuations would cause the alterations in eIF4F formation observed in this study. Rather, the alterations in initiation factors in the study were directly due to the insulin or IGF-I infusion.
Insulin stimulates eIF4F formation with a reciprocal decrease of 4E-BP1 associated with eIF4E. The association of eIF4E with eIF4G is reflective of eIF4F formation. Our results show that insulin infusion increases 4E-BP1 phosphorylation (Fig. 2) and decreases eIF4E · 4E-BP1 binding while increasing the eIF4E · eIF4G binding significantly in fetal skeletal muscle (Fig. 4).
Stimulation of 4E-BP1 phosphorylation and eIF4E · eIF4G binding by insulin has been previously demonstrated in vitro and in postnatal animals. Insulin increased 4E-BP1 phosphorylation and decreased association of eIF4E to 4E-BP1 in 3T3-L1 adipocytes in DMEM medium (33). In a hindlimb model, insulin perfusion (1 mU/ml) increased protein synthesis, increased release of eIF4E from the eIF4E · 4E-BP1 complex secondary to increased 4E-BP1 phosphorylation, and increased eIF4E · eIF4G binding (26). In diabetic animals, insulin treatment increased 4E-BP1 phosphorylation and released eIF4E from the eIF4E · 4E-BP1 complex (25). Our present results suggest that insulin may regulate protein translation initiation factors in fetal skeletal muscle by the same mechanism as that in vitro or in postnatal life. Our results also suggest that the turnover of eIF4E phosphorylation is altered after insulin infusion in both skeletal muscle (Fig. 1B) and liver (results not shown). The physiological significance of eIF4E phosphorylation is still controversial (5, 11, 16, 26, 37, 39). When it is considered that translation initiation on the cap structure is a cyclic event, the turnover rate of eIF4E phosphorylation, rather than its static phosphorylation state measured at a single time point, may be most important in regulating the rate of protein synthesis. This principle may well explain the disparity of previous studies of eIF4E phosphorylation. These insulin-induced alterations in the translation initiation factors are organ specific in the fetus. After insulin infusion, eIF4E · eIF4G binding increased significantly in skeletal muscle (Fig. 4) but not in liver (Fig. 5). There are several possible explanations for this apparent organ-specific function of insulin. First, there could be differences in the abundance and/or distribution of insulin receptors among different tissues. However, insulin receptors have been found in the fetal liver of humans and many animal species (40). Also, we found that insulin stimulated the phosphorylation of eIF4E and 4E-BP1/p70S6k, the downstream effectors of extracellular signal-regulated kinase-1/2-mitogen-activated protein kinase (MAPK) and PI 3-kinase, respectively, in fetal liver. Presumably, the activation of both cascades is initiated by interaction of insulin with insulin receptors. Alternatively, uncharacterized component(s) required for eIF4E · eIF4G binding might not be expressed in fetal liver. However, this is also unlikely, as IGF-I increased eIF4E · eIF4G binding in fetal liver in similar experiments (Fig. 5). Increased 4E-BP1 phosphorylation is the best characterized regulatory mechanism for releasing eIF4E. Therefore, our results run counter to the prevailing hypothesis that increases in 4E-BP1 phosphorylation necessarily lead to increased eIF4F formation. However, the 4E-BP1 amount in the liver was only ~0.05% of that in skeletal muscle. Although more than one mechanism may be involved in the regulation of eIF4E · eIF4G binding after insulin treatment, we think that the low expression of 4E-BP1 in the liver may be responsible. Even though insulin stimulated 4E-BP1 phosphorylation in fetal liver, it would be less likely for 4E-BP1 to play an important role in releasing eIF4E in the liver due to the low overall expression level. Furthermore, the fact that hepatic eIF4E · eIF4G binding increased after IGF-I treatment alone, but not when IGF-I was administered with insulin, suggests that IGF-I may be acting by a mechansim which is antagonized by insulin. Organ-specific regulation of eIF4F formation parallels organ-specific stimulation of protein synthesis by insulin, which has been previously implicated in mice, lambs, and pigs. Intravenous infusion of insulin (4 mIU · kgIGF-I regulates eIF4F formation without altering 4E-BP1 bound to eIF4E. Our results showed that IGF-I increased eIF4G associated with eIF4E despite not altering 4E-BP1 phosphorylation in liver (Figs. 2 and 5). In skeletal muscle, IGF-I increased eIF4G bound to eIF4E without alteration in the amount of 4E-BP1 associated with eIF4E (Fig. 4). Our findings are consistent with those of Vary et al. (56) and Svanberg et al. (53). Using a perfused hind limb model, Vary et al. (56) found that IGF-I increased binding of eIF4G to eIF4E in adult rats but had no effect on eIF4E · 4E-BP1 binding.
The mechanism by which IGF-I increases eIF4E · eIF4G binding in the fetus is unclear. It has been speculated that both eIF4E availability (51) and eIF4E phosphorylation (5) can regulate eIF4E · eIF4G binding. Our data suggest that increased availability of eIF4E is an unlikely explanation. The eIF4E amount was constant after IGF-I treatment in skeletal muscle and liver (Fig. 1). Although IGF-I increased 4E-BP1 phosphorylation in skeletal muscle (Fig. 2), the response was moderate compared with insulin, and IGF-I-induced alterations in 4E-BP1 phosphorylation did not result in a decrease in eIF4E · 4E-BP1 binding in skeletal muscle (Fig. 4). Therefore, it is less likely that the release of eIF4E from the eIF4E · 4E-BP1 complex increased after IGF-I infusion. Our data also suggest that the net amount of phosphorylated eIF4E cannot totally explain our results. Although the overall level of eIF4E phosphorylation increased after IGF-I infusion, eIF4E phosphorylation was extremely variable from one animal to another, whereas eIF4E associated with eIF4G was consistently twofold higher than that in the controls. Furthermore, we could not find a correlation between eIF4E phosphorylation and eIF4E · eIF4G binding. Similar results have been reported in other studies. In rat skeletal muscle, eIF4E · eIF4G binding has been shown to increase without alterations in eIF4E phosphorylation after IGF-I treatment (56). In cell culture, Knauf et al. (27) have demonstrated that eIF4E phosphorylation is not crucial to the formation of the eIF4F complex. However, a poor correlation between the net amount of eIF4E phosphorylation and eIF4F formations at the sampling time does not rule out the possibility of involvement of eIF4E phosphorylation in the eIF4F formation. As we discussed earlier, it is more important to determine the turnover of eIF4E phosphorylation. Unfortunately, we were unable to measure the turnover in the present study. The finding that the amount of eIF4G bound to eIF4E changes in response to IGF-I infusion in the absence of changes in the amount of 4E-BP1 bound to eIF4E has raised several possibilities regarding the mechanisms of eIF4E · eIF4G binding. First, the eIF4E amount may not be a limiting factor in eIF4E · eIF4G binding. Second, the other two components of the eIF4F complex, eIF4G and eIF4A, could regulate the formation of the eIF4F complex. In the present study, we found that IGF-I increased the total amount of eIF4G with a magnitude similar to that of the increase in eIF4E · eIF4G binding in fetal liver (Figs. 3B and 5). In skeletal muscle, although no significant difference was detected in the total amount of eIF4G, mean eIF4G was 21% higher in the IGF-I group than that in the control group (Fig. 3A). We speculate that the increased eIF4G amount could contribute to increased eIF4E · eIF4G binding in both skeletal muscle and liver after IGF-I infusion. In addition, it has been suggested that alterations in the phosphorylation state of eIF4G might modulate the interaction of eIF4G and eIF4E (44). Third, the other components of 48S preinitiation complex, such as eIF3, the poly(A)-binding protein, may directly or indirectly modulate eIF4E · eIF4G binding. Finally, although the amount of eIF4E associated with 4E-BP1 did not change, the other two eIF4E-binding proteins, 4E-BP2 and 4E-BP3, might regulate eIF4E availability independently. This is especially possible in liver, where 4E-BP2 is highly expressed (34).Interaction of insulin and IGF-I in regulation of initiation factors. The overall effect of simultaneous infusion of insulin and IGF seems to be the same as that of insulin alone in regulating eIF4F assembly. Our previous studies suggest that IGF-I does not act simply through the insulin receptor in the ovine fetus (4, 31). We initially hypothesized that insulin and IGF-I act via distinct mechanisms to promote protein accretion in the fetus, possibly resulting in synergistic activity. The present studies show that insulin and IGF-I regulate translation initiation via different mechanisms, therefore supporting the initial hypothesis. However, the effects of the two peptides in regulating the initiation factors seem in many cases to be antagonistic rather than synergistic. It appears that, in the fetus, the insulin effect predominates when both hormones are administered simultaneously. It is possible that there is an overlap interaction between insulin/IGF-I and insulin receptors/IGF type 1 receptors when insulin and IGF-I are present in pharmacological concentrations. It has been speculated that the effects of both insulin and IGF-I are biphasic (4, 13, 20). At low concentrations, insulin specifically binds to insulin receptors and IGF-I binds to IGF type 1 receptors. However, at high concentrations, insulin cross-reacts with IGF type 1 receptors and IGF-I cross-reacts with insulin receptors. We speculate that, in the IGF/Ins group, pharmacological concentrations of insulin may occupy IGF type 1 receptors and block the function of IGF-I.
Insulin, but not IGF-I, activates translation initiation via the PI
3-kinase-p70S6k pathway.
Our results show that insulin stimulates the phosphorylation of
PKB/Akt, p70S6k, and 4E-BP1 in the skeletal muscle and
liver, suggesting that insulin stimulates protein
translation initiation via the PI 3-kinase PKB/Akt
mTOR
p70S6k pathway, which is
consistent with the in vitro model (24) and in postnatal life.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the assistance of Sara Lecklitner, Brenda Van Der Pol, and Larry Auble in the performance of these studies.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants RO1 HD-19089 and PH-60-20542 and the James Whitcomb Riley Memorial Association.
Address for reprint requests and other correspondence: E. A. Liechty, Riley R208, 699 West Dr., Indianapolis, IN 46202 (E-mail: eliecht{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 15, 2002;10.1152/ajpendo.00570.2001
Received 28 December 2001; accepted in final form 4 May 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Angervall, L,
Karlsson K,
and
Martinson A.
Effects on rat fetuses of intrauterine injections of insulin.
Diabetologia
20:
558-562,
1981[ISI][Medline].
2.
Anthony, JC,
Anthony TG,
Kimball SR,
Vary TC,
and
Jefferson LS.
Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation.
J Nutr
130:
139-145,
2000
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.
Boyle, DW,
Denne SC,
Moorehead H,
Lee WH,
Bowsher RR,
and
Liechty EA.
Effect of rhIGF-I infusion on whole fetal and fetal skeletal muscle protein metabolism in sheep.
Am J Physiol Endocrinol Metab
275:
E1082-E1091,
1998
5.
Bu, X,
Haas DW,
and
Hagedorn CH.
Novel phosphorylation sites of eukaryotic initiation factor-4E and evidence that phosphorylation stabilizes interactions of the p25 and p220 subunits.
J Biol Chem
268:
4975-4978,
1993
6.
Castellino, P,
Luzi L,
Simonson DC,
Haymond M,
and
DeFronzo RA.
Effect of insulin and plasma amino acid concentrations on leucine metabolism in man. Role of substrate availability on estimates of whole body protein synthesis.
J Clin Invest
80:
1784-1793,
1987[ISI][Medline].
7.
Dardevet, D,
Sornet C,
Vary T,
and
Grizard J.
Phosphatidylinositol 3-kinase and p70 S6 kinase participate in the regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor I.
Endocrinology
137:
4087-4094,
1996[Abstract].
8.
Davis, TA,
Burrin DG,
Fiorotto ML,
Reeds PJ,
and
Jahoor F.
Roles of insulin and amino acids in the regulation of protein synthesis in the neonate.
J Nutr
128, Suppl:
347S-350S,
1998[ISI][Medline].
9.
Davis, TA,
Fiorotto ML,
Beckett PR,
Burrin DG,
Reeds PJ,
Wray-Cahen D,
and
Nguyen HV.
Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs.
Am J Physiol Endocrinol Metab
280:
E770-E779,
2001
10.
Davis, TA,
Fiorotto ML,
Burrin DG,
Reeds PJ,
Nguyen HV,
Beckett PR,
Vann RC,
and
O'Connor PMJ
Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs.
Am J Physiol Endocrinol Metab
282:
E880-E890,
2002
11.
Davis, TA,
Nguyen HV,
Suryawan A,
Bush JA,
Jefferson LS,
and
Kimball SR.
Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs.
Am J Physiol Endocrinol Metab
279:
E1226-E1234,
2000
12.
D'Ercole, AJ.
The insulin-like growth factors and fetal growth.
In: Modern Concepts of Insulin-like Growth Factors, edited by Spencer EM.. New York: Elsevier Science, 1991, p. 9.
13.
Fryburg, DA.
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
14.
Fuller, S,
Mynett J,
and
Sugden P.
Stimulation of cardiac protein synthesis by insulin-like growth factors.
Biochem J
282:
85-90,
1992[ISI][Medline].
15.
Gingras, AC,
Kennedy SG,
O'Leary MA,
Sonenberg N,
and
Hay N.
4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt (PKB) signaling pathway.
Genes Dev
12:
502-513,
1998
16.
Gingras, AC,
Raught B,
and
Sonenberg N.
eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.
Annu Rev Biochem
68:
913-963,
1999[ISI][Medline].
17.
Graves, LM,
Bornfeldt KE,
Argast GM,
Krebs EG,
Kong X,
Lin TA,
and
Lawrence JC.
cAMP- and rapamycin-sensitive regulation of the association of eukaryotic factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.
Proc Natl Acad Sci USA
92:
7222-7226,
1995[Abstract].
18.
Haghighat, A,
Mader S,
Pause A,
and
Sonenberg N.
Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.
EMBO J
14:
5701-5709,
1995[Abstract].
19.
Han, VKM,
D'Ercole AJ,
and
Lund P.
Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus.
Science
236:
193-197,
1987[ISI][Medline].
20.
Hill, DJ,
Petrik J,
and
Arany E.
Growth factors and the regulation of fetal growth.
Diabetes Care
21, Suppl2:
B60-B69,
1998[ISI][Medline].
21.
Horn, J,
Stern M,
Young M,
and
Noakes D.
Influence of insulin and substrate concentration on protein synthetic rate in fetal tissues.
Res Vet Sci
35:
35-41,
1983[ISI][Medline].
22.
Iwamoto, HS,
Murray MA,
and
Chernausek SD.
Effects of acute hypoxemia on insulin-like growth factors and their binding proteins in fetal sheep.
Am J Physiol Endocrinol Metab
263:
E1151-E1156,
1992.
23.
Johnson, J,
Dunham T,
Wogenrich F,
Greenberg R,
Loftfield R,
and
Skipper B.
Fetal hyperinsulinemia and protein turnover in fetal rat tissues.
Diabetes
39:
541-548,
1990[Abstract].
24.
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
25.
Kimball, SR,
Jefferson LS,
Fadden P,
Haystead TA,
and
Lawrence JC, Jr.
Insulin and diabetes cause reciprocal changes in the association of eIF4E and PHAS-I in rat skeletal muscle.
Am J Physiol Cell Physiol
270:
C705-C709,
1996
26.
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
27.
Knauf, U,
Tschopp C,
and
Gram H.
Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2.
Mol Cell Biol
21:
5500-5511,
2001
28.
Lassarre, C,
Hardouin S,
Daffos F,
Forestier F,
Frankenne F,
and
Binoux M.
Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation.
Pediatr Res
29:
219-225,
1991[ISI][Medline].
29.
Liechty, EA,
Boyle DW,
Lee WH,
Bowsher RR,
and
Denne SC.
Effects of circulating IGF-I on glucose and amino acid kinetics in the ovine fetus.
Am J Physiol Endocrinol Metab
271:
E177-E185,
1996
30.
Liechty, EA,
Boyle DW,
Moorehead H,
Lee WH,
Yang XL,
and
Denne SC.
Glucose and amino acid kinetic response to graded infusion of rhIGF-I in the late gestation ovine fetus.
Am J Physiol Endocrinol Metab
277:
E537-E543,
1999
31.
Liechty, EA,
Boyle DW,
Moorehead H,
Liu YM,
and
Denne SC.
Effect of hyperinsulinemia on ovine fetal leucine kinetics during prolonged maternal fasting.
Am J Physiol Endocrinol Metab
263:
E696-E702,
1992
32.
Lin, TA,
Kong X,
Haystead TA,
Pause A,
Belsham G,
Sonenberg 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].
33.
Lin, TA,
Kong X,
Saltiel AR,
Blackshear PJ,
and
Lawrence JC, Jr.
Control of PHAS-I by insulin in 3T3-L1 adipocytes.
J Biol Chem
270:
18531-18538,
1995
34.
Lin, TA,
and
Lawrence JC, Jr.
Control of the translational regulators PHAS-I and PHAS-II by insulin and cAMP in 3T3-L1 adipocytes.
J Biol Chem
271:
30199-30204,
1996
35.
Liu, J-P,
Baker J,
Perkins AS,
Robertson EJ,
and
Efstratiadis A.
Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r).
Cell
75:
59-72,
1993[ISI][Medline].
36.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem
192:
265-275,
1951.
37.
McKendrick, L,
Morley SJ,
Pain VM,
Jagus R,
and
Joshi B.
Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.
Eur J Biochem
268:
5375-5385,
2001
38.
Milley, JR.
Effects of insulin on ovine fetal leucine kinetics and protein metabolism.
J Clin Invest
93:
1616-1624,
1994[ISI][Medline].
39.
Minich, WB,
Balasta ML,
Goss DJ,
and
Rhoads RE.
Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form.
Proc Natl Acad Sci USA
91:
7668-7672,
1994[Abstract].
40.
Newfeld, ND,
Scott M,
and
Kaplan SA.
Ontogeny of the mammalian insulin receptor.
Dev Biol
78:
151-160,
1980[ISI][Medline].
41.
Owens, JA,
Kind KL,
Carbone F,
Robinson JS,
and
Owens PC.
Circulating insulin-like growth factors-I and -II and substrates in fetal sheep following restriction of placental growth.
J Endocrinol
140:
5-13,
1994[Abstract].
42.
Pain, VM.
Initiation of protein synthesis in eukaryotic cells.
Eur J Biochem
236:
747-771,
1996[Abstract].
43.
Picon, L.
Effect of insulin on growth and biochemical composition of the rat fetus.
Endocrinology
81:
1419-1421,
1967[ISI][Medline].
44.
Raught, B,
Gingras AC,
Gygi SP,
Imataka H,
Morino S,
Gradi A,
Aebersold R,
and
Sonenberg N.
Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.
EMBO J
19:
434-444,
2000
45.
Rhoads, RE.
Signal transduction pathways that regulate eukaryotic protein synthesis.
J Biol Chem
274:
30337-30340,
1999
46.
Rhoads, RE,
Joshi B,
and
Minich WB.
Participation of initiation factors in the recruitment of mRNA to ribosomes.
Biochimie
76:
831-838,
1994[ISI][Medline].
47.
Rotwein, P,
Pollock K,
Watson M,
and
Milbrandt J.
Insulin-like growth factor gene expression during rat embryonic development.
Endocrinology
121:
2141-2144,
1987[Abstract].
48.
Russell-Jones, DL,
Umpleby AM,
Hennessy TR,
Bowes SB,
Shojaee-Moradie F,
Hopkins KD,
Jackson NC,
Kelly JM,
Jones RH,
and
Sönksen PH.
Use of a leucine clamp to demonstrate that IGF-I actively stimulates protein synthesis in normal humans.
Am J Physiol Endocrinol Metab
267:
E591-E598,
1994
49.
Shah, OJ,
Anthony JC,
Kimball SR,
and
Jefferson LS.
4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle.
Am J Physiol Endocrinol Metab
279:
E715-E729,
2000
50.
Shen, WH,
Yang X,
Boyle DW,
Lee WH,
and
Liechty EA.
Effect of intravenous insulin-like growth factor-I and insulin administration on insulin-like growth factor-binding proteins in the ovine fetus.
J Endocrinol
171:
143-151,
2001
51.
Sonenberg, N.
Regulation of translation and cell growth by eIF-4E.
Biochimie
76:
839-846,
1994[ISI][Medline].
52.
Susa, JB,
McCormick KL,
Widness JA,
Singer DB,
Oh W,
Adamsons K,
and
Schwartz R.
Chronic hyperinsulinemia in the fetal rhesus monkey: effects on fetal growth and composition.
Diabetes
28:
1058-1063,
1979[ISI][Medline].
53.
Svanberg, E,
Ohlsson C,
Kimball SR,
and
Lundholm K.
rhIGF-I/IGFBP-3 complex, but not free rhIGF-I, supports muscle protein biosynthesis in rats during semistarvation.
Eur J Clin Invest
30:
438-446,
2000[ISI][Medline].
54.
Thureen, PJ,
Scheer B,
Anderson SM,
Tooze JA,
Young DA,
and
Hay WW, Jr.
Effect of hyperinsulinemia on amino acid utilization in the ovine fetus.
Am J Physiol Endocrinol Metab
279:
E1294-E1304,
2000
55.
Vary, TC,
Jefferson LS,
and
Kimball SR.
Amino acid-induced stimulation of translation initiation in rat skeletal muscle.
Am J Physiol Endocrinol Metab
277:
E1077-E1086,
1999
56.
Vary, TC,
Jefferson LS,
and
Kimball SR.
Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle.
Am J Physiol Endocrinol Metab
278:
E58-E64,
2000
57.
Von Manteuffel, SR,
Dennis PB,
Pullen N,
Gingras AC,
Sonenberg N,
and
Thomas G.
The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rampamycin-sensitive point immediately upstram of p70(S6K).
Mol Cell Biol
17:
5426-5436,
1997[Abstract].
58.
Wray-Cahen, D,
Nguyen HV,
Burrin DG,
Beckett PR,
Fiorotto ML,
Reeds PJ,
Wester TJ,
and
Davis TA.
Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development.
Am J Physiol Endocrinol Metab
275:
E602-E609,
1998
59.
Xu, G,
Marshall CA,
Lin TA,
Kwon G,
Munivenkatappa RB,
Hill JR,
Lawrence JC, Jr,
and
McDaniel ML.
Insulin mediates glucose-stimulated phosphorylation of PHAS-I by pancreatic beta cells. An insulin-receptor mechanism for autoregulation of protein synthesis by translation.
J Biol Chem
273:
4485-4491,
1998
60.
Young, M,
Horn J,
and
Noakes DL.
Protein turnover rate in fetal organs: the influence of insulin.
In: Nutrition and Metabolism of the Fetus and Infant, edited by Visser HKA. The Hague: Martinus Nijhoff, 1979, p. 19-27.