Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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Refeeding reverses the
muscle protein loss seen with fasting. The physiological regulators and
cellular control sites responsible for this reversal are incompletely
defined. Phosphorylation of phosphorylated heat-acid stabled protein
(PHAS-I) frees eukaryotic initiation factor 4E (eIF4E) and stimulates
protein synthesis by accelerating translation initiation.
Phosphorylation of p70 S6-kinase (p70S6k) is thought to be
involved in the regulation of the synthesis of some ribosomsal proteins
and other selected proteins with polypyrimidine clusters near the
transcription start site. We examined whether phosphorylation of PHAS-I
and p70S6k was increased by feeding and determined the
separate effects of insulin and amino acids on PHAS-I and
p70S6k phosphorylation in rat skeletal muscle in vivo.
Muscle was obtained from rats fed ad libitum or fasted overnight
(n = 5 each). Other fasted rats were infused with insulin (3 µU · min1 · kg
1, euglycemic
clamp), amino acids, or the two combined. Gastrocnemius was
freeze-clamped, and PHAS-I and p70S6k phosphorylation was
measured by quantifying the several phosphorylated forms of these
proteins seen on Western blots. We observed that feeding increased
phosphorylation of both PHAS-I and p70S6k (P < 0.05). Infusion of amino acids alone reproduced the effect of feeding.
Physiological hyperinsulinemia increased p70S6K (P
< 0.05) but not PHAS-I phosphorylation (P = 0.98).
Addition of insulin to amino acid infusion was no more effective than
amino acids alone in promoting PHAS-I and p70S6k
phosphorylation. We conclude that amino acid infusion alone enhances the activation of the protein synthetic pathways in vivo in rat skeletal muscle. This effect is not dependent on increases in plasma
insulin and simulates the activation of protein synthesis that
accompanies normal feeding.
messenger ribonucleic acid translation initiation; protein synthesis; insulin clamp; mammalian target of rapamycin
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INTRODUCTION |
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BRIEF STARVATION DECREASES the rate of protein synthesis in skeletal muscle, and this is rapidly reversed on refeeding (11). The discrete contributions by nutrients themselves and by the hormonal responses that accompany feeding to the stimulation of protein synthesis with refeeding are only beginning to be unraveled. For example, recent in vitro experiments have shown that amino acids, beyond furnishing substrate for protein synthesis, perform a signaling role to enhance protein synthesis by phosphorylating a heat-acid stabled protein (PHAS-I) or eukaryotic initiation factor 4E-binding protein 1 (eIF4E-BP1) and p70 S6-kinase (p70S6k), two key regulatory proteins involved in initiation of mRNA translation. These actions, at least in the in vitro systems studied, appear independent of insulin availability (14, 26, 36).
In recent in vivo studies, refeeding mice starved for brief or more prolonged times increased p70S6k and PHAS-I phosphorylation (32). These changes are also seen in a murine model of type 1 diabetes, suggesting that a rise in plasma insulin may not be required (32). Interestingly, refeeding with a protein-deficient diet does not increase PHAS-I phosphorylation or protein synthesis (37). In aggregate, these findings suggest an effect of amino acids, independent of insulin, to stimulate muscle protein synthesis in vivo.
The signaling pathway by which insulin activates protein synthesis in a variety of cell systems has been clarified in the past decade. After insulin receptor autophosphorylation and tyrosine phosphorylation of insulin receptor substrate (IRS) proteins, the phosphatidylinositol (PI) 3-kinase pathway is activated, and protein kinase B (Akt), mammalian target of rapamycin (mTOR), eIF4E, PHAS-I, and p70S6k (among other proteins) are each phosphorylated (20, 21). Recent studies in Chinese hamster ovary-insulin receptor (CHO-IR) cells showed that amino acid withdrawal does not significantly alter insulin stimulation of receptor or IRS protein tyrosine phosphorylation, PI 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity, but that it results in rapid dephosphorylation of p70S6k and PHAS-I (2). This suggests a link between amino acid availability and phosphorylation of the more distal signaling events involved in the regulation of protein synthesis at the translational level. These findings imply that amino acids can regulate the activity of the p70S6k and PHAS-I through an insulin-independent mechanism in vitro and in vivo.
The major purpose of the present study was to assess whether phosphorylation of p70S6k and PHAS-I was increased by feeding in normal rats and to determine the separate effects of insulin and amino acids on p70S6k and PHAS-I phosphorylation in vivo. The results show that, in rat skeletal muscle, feeding promotes phosphorylation of both PHAS-I and p70S6k, and that this is mimicked by physiological increments in plasma amino acids. In contrast, physiological increases of insulin alone enhance phosphorylation of p70S6k but not PHAS-I. Furthermore, the combination of insulin and amino acids is no more effective than amino acids alone. These findings suggest that amino acids play a significant signaling role in the increased mRNA translation that accompanies feeding.
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MATERIALS AND METHODS |
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Male Sprague-Dawley rats weighing 230-300 g were studied after an overnight 14-h fast or were fed ad libitum before study. The study protocol was approved by the University of Virginia Animal Care and Use Committee. PHAS-I antibody was generated in rabbit with recombinant His-tagged rat PHAS-I and was kindly provided by Dr. J. Lawrence, Univ. of Virginia. p70S6k antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-rabbit antibody linked to horseradish peroxidase and enhanced chemiluminescence (ECL) Western blot kits were purchased from Amersham. Insulin and Travesol (a mixed amino acid solution) were obtained from Lilly and Clintec Nutrition (Deerfield, IL), respectively. All other chemicals were from commercially available sources.
Experimental protocol.
Five ad libitum-fed rats and 20 overnight-fasted rats were studied. All
rats were anesthetized with pentobarbital sodium (50 mg/kg ip). A
midline neck incision was made, and the external jugular vein, internal
carotid artery, and trachea were exposed and cannulated. The arterial
catheter was connected through a three-way stopcock to a pressure
transducer, which was in turn connected to a Transonic Systems detector
for heart rate and blood pressure monitoring. Pentobarbital was infused
intravenously at a variable rate to maintain a steady level of
anesthesia. After an ~30-min baseline period to assure hemodynamic
stability (mean arterial pressure = 100-120 mmHg) and level
of anesthesia, the fed rats received a saline infusion for a 3-h
period. The fasted rats were divided into four groups
(n = 5 each). Each of the groups then also received
separately a 3-h continuous infusion of saline, insulin (3 mU · kg1 · min
1), 10% Travesol (10 µl/min), or a combination of 10% Travesol (10 µl/min) and insulin
(3 mU · kg
1 · min
1). In rats
given insulin, 30% dextrose was infused at a variable rate to keep
blood glucose within 10% of basal, and the equivalent volume of saline
was given to the remaining rats. Blood glucose was measured every 10 min, and heart rate and mean arterial pressure were monitored
throughout the study. At the end of the infusion period, gastrocnemius
muscles were rapidly excised and freeze-clamped in liquid nitrogen and
subsequently stored at
70°C. Plasma insulin was measured by
immunoassay in plasma obtained at the beginning and completion of the study.
Western blotting.
Pieces (~20 mg) of frozen gastrocnemius muscle were weighed and
powdered in liquid nitrogen, then mixed with ice-cold 25 mM Tris
· HCl buffer (26 mM KF and 5 mM EDTA, pH 7.5), and disrupted by
sonication with a microtip probe, 0.5 s on/0.5 s off for 45 s
total, at a 3.0 power setting on the Fisher XL2020 sonicator. For
p70S6k, one aliquot of the muscle homogenate (~60 µg
protein) was diluted with an equal volume of SDS sample buffer and run
on an 8% SDS-PAGE. For PHAS-I, another aliquot was heated to
1000C for 5 min and centrifuged at 2,000 rpm × 2 min,
and an aliquot of supernatant (~60 mg protein) was diluted with an
equal volume of SDS sample buffer and electrophoresed on a 15%
polyacrylamide gel. Proteins on both gels were electrophoretically
transferred to nitrocellulose membranes (Schleicher & Schuell). After
blocking with 5% low-fat milk in Tris-buffered saline-Tween-20,
membranes were incubated with rabbit anti-rat PHAS-I or rabbit
anti-p70S6k for 1 h at room temperature. This was
followed by incubation with a donkey anti-rabbit IgG coupled to
horseradish peroxidase, and the blot was developed using an ECL Western
blotting kit. Autoradiographic film was scanned densitometrically
(Molecular Dynamics) and quantitated using ImageQuant software version
3.3. PHAS-I, a 12-kDa protein, migrates in this system anomalously at
~20 kDa. One to three bands are seen on Western blotting that correspond to the unphosphorylated and several more slowly migrating phosphorylated forms of the protein. Likewise, p70S6k in
extracts from unstimulated muscle migrates predominantly as a single
band, with several faint bands at a higher molecular weight. However,
with further phosphorylation, electrophoretic mobility is retarded, and
the intensity of these more slowly migrating forms increases. For
PHAS-I we quantified the ratio of the most rapidly migrating
(nonphosphorylated) band () to the total immunoreactive material.
The
-band was selected because it is this form of the protein that
binds to eIF4E and prevents the association of 4E with the initiation
complex. Conversely, for p70S6k we quantified the ratio of
the more heavily phosphorylated (more slowly migrating) forms to the
total immune reactivity, because it is the phosphorylated forms that
possess kinase activity. In preliminary experiments, we examined the
effect of varying the amount of protein loaded on the SDS-PAGE on the
measured ratios of the several phosphorylated forms of PHAS-I and
p70S6k in Western blots. Over the concentration range of
protein used in the current study, there was no significant effect.
Statistical analysis (Sigmastat 3.0) was based on one-way ANOVA with
post hoc testing, as indicated in RESULTS.
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RESULTS |
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Body weight, blood glucose, blood pressure, plasma insulin
characteristics of animals.
Table 1 shows the animal body weights,
the blood glucose concentrations, the mean arterial pressures, and the
glucose infusion rates in the five groups. There was no difference
between groups in any of these variables except for the amount of
glucose infused. The steady-state (120-to 180-min) glucose infusion
rate required to maintain euglycemia was significantly higher in the
rats given insulin and insulin plus amino acids than in animals given
amino acids or saline infusion. Although the glucose infusion rate in the rats given insulin infusion alone seemed higher than in the rats
given combination infusion, this was not statistically significant. Figure 1 illustrates the plasma insulin
concentration at baseline and at the end of the 3-h infusion period for
each of the five study groups. Basal insulin concentrations were higher
in the fed group than in each of the fasted groups. At 3 h, the
insulin concentrations in the animals receiving insulin or insulin with amino acids, and in the fed animals, were significantly higher than
those in the fasted saline-infused animals. Plasma insulin in the amino
acid-infused animals was not different from that in the saline-infused
animals.
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PHAS-I and p70S6k phosphorylation. In these studies we elected to use electrophoretic behavior on SDS-PAGE and Western blotting as indexes of the biological effect of PHAS-I and p70S6k. Inasmuch as this method allows the simultaneous quantitation of multiple forms of both proteins, as well as internal normalization for both the recovery of target proteins from tissue and for loading of gels, it is well suited to the quantitative approach required in these studies. Available data support an inverse correlation between electrophoretic mobility and kinase activity for p70S6k (29) and a direct correlation with PHAS-I bound to eIF4E (7, 27). Because the change in apparent molecular weight induced by phosphorylation of either protein exceeds that expected from simple stoichiometry of the added phosphate groups, factors other than changes in mass appear to be involved in the altered electrophoretic mobility.
Figure 2 illustrates typical patterns of PHAS-I observed on Western blots of gastrocnemius muscle at 3 h of infusion in each of the five study groups. The blots appear as three distinct bands marked
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DISCUSSION |
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The current results suggest that, in the rat as in the mouse (32), feeding promotes the phosphorylation of both PHAS-I and p70S6k. In overnight-fasted rats, infusion of insulin, at a rate that increments plasma insulin to levels that match those of fed animals while maintaining euglycemia, reproduced the phosphorylation of p70S6k seen with feeding. However, this insulin infusion did not mimic the effect of feeding on the phosphorylation of PHAS-I. It is of considerable interest that amino acid infusion alone enhanced the phosphorylation of both p70S6k and PHAS-I and appeared to fully mimic the pattern of phosphorylation of both proteins seen in muscle from the ad libitum-fed rats. This occurred despite the absence of a significant increment in plasma insulin. Indeed, plasma insulin concentrations in the amino acid-infused rats were no greater than those in the fasted animals. We cannot eliminate the possibility that small or transient increments in plasma insulin occurred during the amino acid infusion and were not captured by our blood sampling regimen, and these might account for the phosphorylation of p70S6k. However, these would not explain the phosphorylation of PHAS-I seen in these animals. Moreover, it appears clear that greater increments in plasma insulin, as seen in the insulin + amino acid-infused animals, had no further effect on the phosphorylation state of p70S6k than that seen with amino acid infusion alone. We emphasize that the insulin concentrations in all of these animals are in the low physiological range. We cannot exclude, on the basis of these studies, an effect of much greater concentrations of insulin on the degree of phosphorylation of PHAS-I. Indeed, in vivo, large doses of insulin (5 U given to <300-g rats) increase phosphorylation of PHAS-I and promote its dissociation from eIF4E (18). Thus the dose-response relationship between insulin and PHAS-I phosphorylation in vivo remains to be defined. Suffice it to say that the low insulin concentrations used here differentially stimulate the phosphorylation of p70S6k.
The current results emphasize the potentially very important role of direct amino acid signaling on skeletal muscle protein synthesis. These findings extend results of a recent study by Yoshizawa et al. (38), which demonstrated that refeeding a protein-rich, but not a protein-free, diet reverses the inhibition of protein synthesis seen with fasting. Those authors further showed that refeeding the protein-rich, but not the protein-free, diet was associated with increased phosphorylation of PHAS-I and the predicted increase in the formation of the active eIF4G · eIF4E complex involved in translation initiation, strongly suggesting a role for the protein component of the diet (i.e., amino acids). Together with the current results, these findings appear to complement several recent in vitro studies that demonstrated a direct effect of amino acids to stimulate phosphorylation of both PHAS-I and p70S6k. Thus, with use of either CHO cells (14, 36) or a hepatoma cell line (26), amino acid withdrawal led to the dephosphorylation of both proteins, and, conversely, subsequent replacement of amino acids resulted in significant stimulation in the phosphorylation of both. These findings, however, do not exclude a role for other humoral signals [e.g., insulin-like growth factor I (IGF-I) or growth hormone] in the physiological regulation of message translation.
We (13, 22, 39) and many other laboratories (2, 6, 8, 23-25, 30, 35) have previously demonstrated that physiological hyperinsulinemia does not enhance protein synthesis in adult rats or humans. One contrary result has been reported (3). All of these studies are based on the use of tracer methods and are to an uncertain extent compromised by the inaccessibility to repeated sampling of the aminoacyl-tRNA pool that is used for protein synthesis. However, available data in rats (39) and very recently in humans (1), in which aminoacyl-tRNA labeling was measured, confirm that physiological hyperinsulinemia alone suppresses proteolysis in muscle without increasing protein synthesis. This lack of action of insulin on protein synthesis in vivo appears to be the case even when amino acid concentrations in plasma are maintained at basal (13). In contrast, a number of studies have indicated that increasing the plasma concentration of amino acids can enhance whole body (5, 33) and muscle (4, 10, 28) protein synthesis. However, these studies are not without ambiguity, because an effect of amino acid infusion (and raised plasma amino acid concentration) to alter the relationship between tracer enrichment in plasma and in the aminoacyl-tRNA pool generally cannot be excluded.
In light of this, the differential effect of insulin on the phosphorylation of p70S6k but not PHAS-I in the current studies is of particular interest. In a variety of studies, phosphorylation of PHAS-I has consistently been observed to accompany increases in protein synthesis (12, 32, 37, 38). Recent in vitro studies in which p70S6k is deleted by a homologous recombination have emphasized that protein synthesis can still be stimulated by insulin and other growth factors (17). p70S6k appears to play a particular role in promoting the translation of messenger RNA with polypyrimidine sequences near the cap site on messenger RNA. These messages code for specific proteins, and among them are a number of ribosomal proteins, as well as initiation and the elongation factors involved in mRNA translation. It is attractive to consider that insulin, by stimulating p70S6k selectively, allows for priming of the protein synthetic apparatus, but initiation of translation requires the additional phosphorylation of PHAS-I, which may respond more sensitively to nutrient signaling. In this manner, specific nutrient (amino acid) signaling is integrated with the more general feeding signal (insulin) to allow message translation to commence. Against this construct is the observation that high doses of insulin in vivo in rats (19) and humans (15) can stimulate muscle protein synthesis. However, it is difficult to exclude an effect of high-dose insulin acting via the IGF-I receptor. The latter hormone has been demonstrated to increase muscle protein synthesis in both humans (9, 31) and animals (16, 34).
In summary, the current studies provide evidence that, in the rat, feeding regular chow sustains PHAS-I and p70S6k in a highly phosphorylated form, and this is lost during fasting but can be fully restored by amino acid infusion. Physiological increments in plasma insulin in fasted animals, by contrast, can restore only the p70S6k phosphorylation. These findings suggest that amino acids per se may play a signaling role in activating PHAS-I, and this is needed for the normal recovery of protein synthesis after refeeding.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. J. Lawrence for providing the PHAS-I antibody.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38978 and DK-54058.
Address for reprint requests and other correspondence: E. J. Barrett, Dept. of Internal Medicine, MR-4 Box 5116, Univ. of Virginia Health Sciences Center, Charlottesville, VA 22908 (E-mail: EJB8X{at}virginia.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. §1734 solely to indicate this fact.
Received 25 October 1999; accepted in final form 8 March 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albright, R,
Joyner M,
Dietz N,
and
Nair KS.
Does insulin stimulate muscle protein synthesis in humans (Abstract).
Diabetes
48, Suppl1:
A69,
1999[ISI].
2.
Baillie, AGS,
and
Garlick PJ.
Attenuated responses of muscle protein synthesis to fasting and insulin in adult female rats.
Am J Physiol Endocrinol Metab
262:
E1-E5,
1992
3.
Biolo, G,
Fleming RYD,
and
Wolfe RR.
Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle.
J Clin Invest
95:
811-819,
1995[ISI][Medline].
4.
Biolo, G,
Tipton KD,
Klein S,
and
Wolfe RR.
An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.
Am J Physiol Endocrinol Metab
273:
E122-E129,
1997
5.
Castellino, P,
Luzi L,
Simonson DC,
Haymond M,
and
DeFronzo RA.
Effect of insulin and plasma amino acid concentrations on leucine metabolism in man.
J Clin Invest
80:
1784-1793,
1987[ISI][Medline].
6.
Dardevet, D,
Sornet C,
Attaix D,
Baracos VE,
and
Grizard J.
Insulin-like growth factor-1 and insulin resistance in skeletal muscles of adult and old rats.
Endocrinology
134:
1475-1484,
1994[Abstract].
7.
Fadden, P,
Haystead TAJ,
and
Lawrence JC.
Identification of phosphorylation sites in the translational regulator, Phas-I, that are controlled by insulin and rapamycin in rat adipocytes.
J Biol Chem
272:
10240-10247,
1997
8.
Fluckey, JD,
Vary TC,
Jefferson LS,
Evans WJ,
and
Farrell P.
Insulin stimlulation of protein synthesis in rat skeletal muscle following resistance exercise is maintained with advancing age.
J Gerontol
51:
B323-B330,
1996[ISI].
9.
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
10.
Fryburg, DA,
Jahn LA,
Hill SA,
Oliveras DM,
and
Barrett EJ.
Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms.
J Clin Invest
96:
1722-1729,
1995[ISI][Medline].
11.
Garlick, PJ,
Fern M,
and
Preedy VR.
The effect of insulin infusion and food intake on muscle protein synthesis in postabsorptive rats.
Biochem J
210:
669-676,
1983[ISI][Medline].
12.
Gautsch, TA,
Anthony JC,
Kimball SR,
Paul GL,
Layman DK,
and
Jefferson LS.
Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise.
Am J Physiol Cell Physiol
274:
C406-C414,
1998
13.
Gelfand, RA,
and
Barrett EJ.
Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man.
J Clin Invest
80:
1-6,
1987[ISI][Medline].
14.
Hara, K,
Yonezawa K,
Weng QP,
Kozlowski MT,
Belham C,
and
Avruch J.
Amino acid sufficiency and mTOR regulate P70 S6 kinase and eIF-4E Bp1 through a common effector mechanism.
J Biol Chem
273:
14484-14494,
1998
15.
Hillier, TA,
Fryburg DA,
Jahn LA,
and
Barrett EJ.
Extreme hyperinsulinemia unmasks insulin's effect to stimulate protein synthesis in the human forearm.
Am J Physiol Endocrinol Metab
274:
E1067-E1074,
1998
16.
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
17.
Kawasome, H,
Papst P,
Webb S,
Keller GM,
Johnson GL,
Gelfand EW,
and
Terada N.
Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity.
Proc Natl Acad Sci USA
95:
5033-5038,
1998
18.
Kimball, SR,
Jefferson LS,
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
19.
Kimball, SR,
Jurasinski CV,
Lawrence JC,
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
20.
Kimball, SR,
Vary TC,
and
Jefferson LS.
Regulation of protein synthesis by insulin.
Ann Rev Physiol
56:
321-348,
1994[ISI][Medline].
21.
Lawrence, JC,
and
Abraham RT.
Phas/4e-Bps Aa regulators of mRNA translation and cell proliferation.
Trends Biochem Sci
22:
345-349,
1997[ISI][Medline].
22.
McNulty, PH,
Young LH,
and
Barrett EJ.
Response of rat heart and skeletal muscle protein in vivo to insulin and amino acid infusion.
Am J Physiol Endocrinol Metab
264:
E958-E965,
1993
23.
McNurlan, MA,
Essén P,
Thorell A,
Calder AG,
Anderson SE,
Ljungquist O,
Sandgren A,
Grant I,
Tjöder I,
Ballmer PE,
Wernerman J,
and
Garlick PJ.
Response of protein synthesis in human skeletal muscle to insulin: an investigation with L-[2H5]phenylalanine.
Am J Physiol Endocrinol Metab
267:
E102-E108,
1994
24.
Mosoni, L,
Houlier M-L,
Mirand PP,
Bayle G,
and
Grizard J.
Effect of amino acids alone or with insulin on muscle and liver protein synthesis in adult and old rats.
Am J Physiol Endocrinol Metab
264:
E614-E620,
1993
25.
Pacy, P,
Nair K,
Ford C,
and
Halliday D.
Failure of insulin infusion to stimulate fractional muscle protein synthesis in type I diabetic patients, anabolic effect of insulin and decreased proteolysis.
Diabetes
38:
618-624,
1989[Abstract].
26.
Patti, ME,
Brambilla E,
Luzi L,
Landaker EJ,
and
Kahn CR.
Bidirectional modulation of insulin action by amino acids.
J Clin Invest
101:
1519-1529,
1998
27.
Pause, A,
Belsham G,
Gingras AC,
Donze O,
Lin TA,
Lawrence JC,
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].
28.
Preedy, VR,
and
Garlick PJ.
Effects of intravenous infusions of branched-chain amino acids and amino acid mixtures on muscle protein synthesis in rats.
Clin Sci (Colch)
72:
1-19,
1987[Medline].
29.
Quing-Pong, W,
Kozlowski M,
Belham C,
Zhang A,
Comb M,
and
Avruch J.
Regulation of p70 S6 kinase by phosphorylation in vivo.
J Biol Chem
273:
16621-16629,
1998
30.
Rooyackers, OE,
and
Nair KS.
Hormonal regulation of human muscle protein metabolism.
Ann Rev Nutr
17:
457-485,
1997[ISI][Medline].
31.
Russell-Jones, DL,
Umpleby AM,
Hennessy TR,
Bowes SB,
Shojaee-Moradie F,
Hopkins KD,
Jackson NC,
Kelly JM,
Jones RH,
and
Sonksen 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
32.
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
33.
Tessari, P,
Inchiostro S,
Biolo G,
Trevisan R,
Fantin G,
Marescotti MC,
Iori E,
Tiengo A,
and
Crepaldi G.
Differential effects of hyperinsulinemia and hyperaminoacidemia on leucine-carbon metabolism in vivo. Evidence for distinct mechanisms in ragulation of net amino acid deposition.
J Clin Invest
79:
1062-1069,
1987[ISI][Medline].
34.
Umpleby, AM,
Shojaee-Moradie F,
Thomason MJ,
Kelly JM,
Skottner A,
Sonksen PH,
and
Jones RH.
Effects of insulin-like growth factor-I (IGF-I), insulin and combined IGF-I-insulin infusions on protein metabolism in dogs.
Eur J Clin Invest
24:
337-344,
1994[ISI][Medline].
35.
Vogiatzi, MG,
Nair KS,
Beckett PR,
and
Copeland KC.
Insulin does not stimulate protein synthesis acutely in prepubertal children with insulin-dependent diabetes mellitus.
J Clin Endocrinol Metab
82:
4083-4087,
1997
36.
Wang, X,
Campbell LE,
Miller CM,
and
Proud CG.
Amino acid availability regulates p70 S6 kinase and multiple translation factors.
Biochem J
334:
261-267,
1998[ISI][Medline].
37.
Yoshizawa, F,
Kimball SR,
and
Jefferson LS.
Modulation of translation initiation in rat skeletal muscle and liver in response to food intake.
Biochem Biophys Res Comm
240:
825-831,
1997[ISI][Medline].
38.
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
39.
Young, LH,
Stirewalt W,
McNulty PH,
Revkin JH,
and
Barrett EJ.
Effect of insulin on rat heart and skeletal muscle phenylalanyl-tRNA labeling and protein synthesis in vivo.
Am J Physiol Endocrinol Metab
267:
E337-E342,
1994