Unlike insulin, amino acids stimulate p70S6K but not GSK-3 or glycogen synthase in human skeletal muscle

Zhenqi Liu, Yangsong Wu, Edward W. Nicklas, Linda A. Jahn, Wendie J. Price, and Eugene J. Barrett

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia 22908

Submitted 4 April 2003 ; accepted in final form 25 November 2003


    ABSTRACT
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 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70S6K), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70S6K, and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70S6K. To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70S6K, and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed amino acid solution (1.26 µmol·kg-1·min-1 x 6 h, n = 9), a physiological dose of insulin (1 mU·kg-1·min-1 euglycemic hyperinsulinemic clamp x 2 h, n = 6), or a pharmacological dose of insulin (20 mU·kg-1·min-1 euglycemic hyperinsulinemic clamp x 2 h, n = 9). Whole body glucose disposal rates were assessed by calculating the steady-state glucose infusion rates, and vastus lateralis muscle was biopsied before and at the end of the infusion. Both amino acid infusion and physiological hyperinsulinemia enhanced p70S6K phosphorylation without affecting GSK-3 phosphorylation, but only physiological hyperinsulinemia also increased whole body glucose disposal and GS activity. In contrast, a pharmacological dose of insulin significantly increased whole body glucose disposal, p70S6K, GSK-3 phosphorylation, and GS activity. We conclude that amino acids at physiological concentrations mediate p70S6K but, unlike insulin, do not regulate GSK-3 and GS phosphorylation/activity in human skeletal muscle.

hyperinsulinemia; hyperaminoacidemia; signaling proteins; phosphorylation; in vivo


IN HUMANS, SKELETAL MUSCLE GLYCOGEN STORES are a major energy resource during brief and intense muscular contraction. Hormonal, nutritional, and physiological factors, including insulin, growth factors, epinephrine, glucose, muscle glycogen content, amino acids, exercise, and others, have been shown to actively regulate skeletal muscular glycogen biosynthesis. Glycogen synthase (GS) is a rate-limiting enzyme in this process, and GS activity is determined by complex multisite phosphorylation as well as allosteric factors (20, 46). GS phosphorylation at specific sites decreases, whereas dephosphorylation increases GS activity. Among the effectors modulating the phosphorylation status of GS, glycogen synthase kinase 3 (GSK-3) has been shown to play a major role in catalyzing the phosphorylation and inactivation of GS (9, 20, 46).

Insulin's action on GS and glycogen synthesis has been extensively studied in the past several decades. Insulin enhances glucose uptake, glycolysis, and glycogen synthesis in skeletal muscle, at least in part, through the activation of the phosphatidylinositol 3-kinase (PI 3-kinase)-PKB (or Akt)GSK-3 signaling pathway (7, 12, 15, 20, 25, 44). Insulin also enhances the dephosphorylation and therefore activation of GS through activation of glycogen-bound protein phosphatase 1 (PP1), again through a PI 3-kinase-dependent pathway (27). GSK-3 is a key component of the Wnt and PI 3-kinase-Akt signaling pathways and plays pivotal roles in embryonic development, protein synthesis, glycogen synthesis, cell proliferation and differentiation, and cell motility (9). In the insulin-signaling cascade, GSK-3 is downstream of Akt. Akt phosphorylates and inhibits GSK-3 activity, which leads to an increase in GS activity.

GSK-3 is ubiquitously expressed in mammalian tissues and exists as two closely related isoforms, GSK-3{alpha} and GSK-3{beta}. GSK-3{beta} phosphorylation at Ser9 and GSK-3{alpha} phosphorylation at Ser21 by Akt inactivate the enzyme, allowing activation of GS. GSK-3 can also be phosphorylated at Ser9/Ser21 by 90-kDa ribosomal protein S6 kinase, or p90RSK, a key component of the MAPK pathway, and 70-kDa ribosomal protein S6 kinase (p70S6K) (35, 36). Both kinases are regulated by insulin.

The p70S6K is a serine/threonine kinase critical both to cell cycle progression through G1 and to the translation of a subpopulation of mRNAs containing an oligopyrimidine sequence near the 5' cap (19, 29, 42). Phosphorylation of ribosomal protein S6 by p70S6K increases the translation of mRNA-encoding ribosomal proteins, initiation, and elongation factors that play important roles in protein synthesis (18, 29). Mammalian target of rapamycin (mTOR), a kinase downstream of Akt in the insulin-signaling cascade, phosphorylates p70S6K (5, 28) and is selectively inhibited by rapamycin. Recently, evidence has emerged for a rapamycin-sensitive component in insulin-stimulated GSK-3 phosphorylation and/or GS activation, suggesting a possible role of p70S6K (12, 15, 32, 34). With use of either mixed amino acids or leucine, because both are potent stimulators of p70S6K phosphorylation, several laboratories have shown that amino acids stimulate GSK-3 phosphorylation (2), diminish GSK-3 activity (2, 30), and increase the activity of GS (2) via a rapamycin-dependent fashion in cultured human muscle cells or L6 muscle cells.

The major purpose of the present study was to examine whether amino acids, at physiological concentrations, can mimic insulin's action on p70S6K and GSK-3 phosphorylation and GS activity in human skeletal muscle in vivo. We compared the effects of amino acids and insulin on whole body glucose disposal, on the phosphorylation status of p70S6K and GSK-3, and on the activity of GS in biopsied vastus lateralis muscles. Our results indicate that the moderate enhancement in p70S6K phosphorylation induced by changes in amino acid concentrations within physiological range is not sufficient to modulate GSK-3 phosphorylation and GS activity in vivo in human skeletal muscle.


    SUBJECTS AND METHODS
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 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects

Twenty-four healthy volunteers (19 men and 5 women), ranging in age from 20 to 32 yr, were studied. No subject was taking medication, and all female participants had a negative serum pregnancy test 1-2 days before study. Each gave informed written consent before the study. The study protocol was approved by the Human Investigation Committee and the General Clinical Research Center Advisory Committee at the University of Virginia.

Study Protocol

All subjects were admitted to the University of Virginia General Clinical Research Center on the evening before study. After a 12-h overnight fast, in each subject a catheter was placed into a median deep antecubital vein for blood sampling, and another catheter was inserted into a contralateral arm vein for infusion of insulin and glucose or amino acids. After collection of baseline blood samples, a muscle biopsy of the vastus lateralis muscle was done using a Bergstrom muscle biopsy needle (Popper and Sons, New Hyde Park, NY), as described previously (22). After the muscle biopsy, each subject was studied under one of the following three protocols.

Amino acid protocol (n = 9). Each subject received a systemic amino acid infusion for 6 h at a constant rate of 0.015 ml (1.26 µmol)·kg-1·min-1. The 6-h duration was selected to allow full equilibration of amino acids with the interstitium (10). The amino acid solution (10% Travasol, Baxter Healthcare, Deerfield, IL) was a mixture of essential and nonessential amino acids, and each 100-ml infusate contained (in mg) histidine 480, isoleucine 600, leucine 730, lysine 580, methionine 400, phenylalanine 560, threonine 420, tryptophan 180, valine 580, alanine 2070, arginine 1150, glycine 1030, proline 680, serine 500, and tyrosine 40. Blood samples were obtained at the end of 3 and 6 h of amino acid infusion.

Physiological-dose insulin protocol (Low Insulin, n = 6). Each subject received a systemic infusion of regular insulin (Humulin from Eli Lilly) for 2 h at a constant rate of 1 mU·kg-1·min-1. Whole blood glucose was monitored every 5 min, and a 20% dextrose solution was infused at a variable rate to maintain blood glucose within 10% of basal (euglycemic hyperinsulinemic clamp) (8). Blood samples were taken at the end of the infusion.

Pharmacological-dose insulin protocol (High Insulin, n = 9). Regular insulin was administered systemically for 2 h at a constant rate of 20 mU·kg-1·min-1 to each subject. Whole blood glucose was monitored every 5 min, and a 20% dextrose solution was infused at a variable rate to maintain blood glucose within 10% of basal (8). A continuous infusion of amino acids (10% Travasol) at a rate of 0.007 ml (0.59 µmol)·min-1·kg-1 was simultaneously administered to prevent insulin-induced hypoaminoacidemia (14). Blood samples were obtained at the end of the insulin infusion.

A biopsy of vastus lateralis muscle was repeated in the opposite leg at the end of the infusion study. Muscle tissues were immediately frozen in liquid nitrogen and stored at -70°C for later analysis of the phosphorylation state of p70S6K and GSK-3 and of the activity of glycogen synthase.

Western Immunoblotting

Pieces (~20 mg) of frozen vastus lateralis muscle tissue were weighed and powdered in frozen 25 mM Tris·HCl buffer (26 mM KF and 5 mM EDTA, pH 7.5) and then disrupted by sonication using 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. The homogenate was centrifuged at 2,000 rpm for 2 min, and the protein concentration was measured in the supernatant using the Bradford method (4). For p70S6K, one aliquot of the muscle homogenate supernatant (~100 µg protein) was diluted with an equal volume of SDS sample buffer and run on an 8% SDS-PAGE. For GSK-3, another aliquot of supernatant (~60 µg protein) was diluted with an equal volume of SDS sample buffer and electrophoresed on a 10% polyacrylamide gel. Proteins on both gels were electrophoretically transferred to nitrocellulose membranes. After blocking with 5% low-fat milk in Tris-buffered saline-Tween 20, membranes were incubated with rabbit anti-rat p70S6K antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature or rabbit anti-rat GSK-3{beta}, phospho-GSK-3{alpha} (Ser21), or phospho-GSK-3{beta} (Ser9) antibodies (New England BioLabs, Beverly, MA) overnight at 4°C. This was followed by a donkey anti-rabbit IgG coupled to horseradish peroxidase, and the blot was developed using an enhanced chemiluminescence Western blotting kit (Amersham Life Sciences, Piscataway, NJ).

Quantitation of p70S6K and GSK-3 Phosphorylation State

Autoradiographic films were scanned densitometrically (Molecular Dynamics, Piscataway, NJ) and quantitated using ImageQuant 3.3. To quantitate the phosphorylation status of p70S6K, we exploited the electrophoretic motility shift property of variously phosphorylated proteins. When p70S6K is subjected to SDS-PAGE, the most heavily phosphorylated fraction (the {gamma}-band) migrates more slowly, whereas the less phosphorylated species (the {alpha}-band) migrates faster, with the moderately phosphorylated fraction (the {beta}-band) having an intermediate mobility. The densities of all bands were measured, and the fraction analysis was presented as the ratio of protein migrating more slowly ({beta}+{gamma}) to the total ({beta}+{gamma}/total). The overall p70S6K activity is dependent on the phosphorylation of at least seven Ser/Thr residues at three separate domains (31, 42), and available data support a good correlation between biological activity and electrophoretic mobility for p70S6K (42). We have also demonstrated in our laboratory that p70S6K electrophoretic mobility correlates well with protein synthetic rates in rat skeletal muscle (data not shown). For GSK-3, the densities of phospho-GSK-3{alpha}, phospho-GSK-3{beta}, and total GSK-3{beta} bands were quantitated. The ratios of phosphorylated GSK-3 (phospho-GSK-3{alpha} + phospho-GSK-3{beta}) to total GSK-3{beta} were calculated to normalize for protein loading and transfer recovery variations. The top panels in Figs. 1 and 2 illustrate the p70S6K and GSK-3 phosphorylation status observed on Western blots of biopsied muscle samples obtained during the basal period and at the end of amino acid or insulin infusion in all three study groups.



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Fig. 1. Effects of amino acid (AA) or insulin infusion on the phosphorylation of 70-kDa protein S6 kinase (p70S6K) in human skeletal muscle. Top: SDS-PAGE gel pattern of human skeletal muscle p70S6K. The {alpha}-band is the least phosphorylated form and has the most rapid electrophoretic mobility. The {beta}-and {gamma}-bands are more phosphorylated and move more slowly. Both amino acids and physiological-dose insulin infusion (Low Insulin) significantly stimulated the phosphorylation of p70S6K; however, this effect was more dramatic in pharmacological-dose insulin-infused individuals (High Insulin). *P < 0.002, **P < 0.02, and ***P < 0.003 vs. respective baseline. #P < 0.002 vs. AA group and ##P < 0.02 vs. Low Insulin group.

 


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Fig. 2. Effects of AA or insulin infusion on human skeletal muscle glycogen synthase kinase 3 (GSK-3) phosphorylation. Top: gel patterns of phospho-GSK-3{alpha}, phospho-GSK-3{beta}, and total GSK-3{beta} on SDS-PAGE. Neither amino acid infusion nor physiological-dose insulin infusion (Low Insulin) changed the amount of GSK-3 being phosphorylated, whereas pharmacological-dose insulin infusion (High Insulin) significantly enhanced GSK-3 phosphorylation.

 

Measurement of GS Activity

GS activity was measured in cell-free homogenates of skeletal muscle samples (21, 38). The activity of the active (glucose 6-phosphate independent; GS-I) form of the enzyme is defined as the rate (µmol·g-1·min-1) of incorporation of [U-14C]uridine diphosphoglucose into glycogen at a physiological concentration (0.17 mM) of glucose 6-phosphate. Total GS (glucose 6-phosphate dependent; GS-D) activity is defined as that observed with 7.2 mM glucose 6-phosphate present.

Analytic Methods

Whole blood glucose concentrations were measured in duplicate using a glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) technique (Diagnostic Systems Laboratories, Webster, TX). Plasma amino acid concentrations were measured using an automated ion exchange chromatographic technique (D-500, Dionex, Sunnyvale, CA).

Statistical Analysis

All data are presented as means ± SE. Data for glucose infusion rates are averaged over the last 30 min during the insulin infusion (100-120 min). Stochastic comparisons between the basal and infusion periods were made using a two-tailed, paired t-test and those between different groups with a two-tailed, unpaired t-test or one-way analysis of variance (ANOVA).


    RESULTS
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 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Characteristics of the Study Subjects

The postabsorptive blood glucose, plasma insulin, and amino acid concentrations and steady-state glucose infusion rates are shown in Table 1. Amino acid infusion did not significantly alter plasma glucose or insulin concentrations. Physiological-dose insulin infusion induced a 9-fold increase in plasma insulin concentrations and an average glucose infusion rate of 5.7 ± 0.8 mg·kg-1·min-1. In contrast, pharmacological-dose insulin infusion produced an >600-fold increase in the plasma insulin concentrations (P < 0.007) and an average glucose disposal rate of 13.4 ± 0.8 mg·kg-1·min-1 (P < 0.00001 when compared with low-insulin group). Amino acid infusion raised the plasma total amino acid concentrations by ~57% (P < 0.001), whereas the amino acid concentrations remained stable in pharmacological-dose insulin-infused subjects.


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Table 1. Characteristics of study subjects

 

Effects of Amino Acid or Insulin Infusion on Skeletal Muscle p70S6K Phosphorylation

To quantify the extent of phosphorylation of p70S6K, we measured the ratio of the intensity of the more slowly migrating species ({beta}+{gamma}) to that of the total integrated intensity ({alpha}+{beta}+{gamma}). The more slowly migrating bands ({beta} and {gamma}) represent the more highly phosphorylated forms of p70S6K, which generally correspond to species with greater kinase activity. Both amino acid and physiological hyperinsulinemia significantly increased the ratios of {beta}+{gamma}/{alpha}+{beta}+{gamma} (i.e., the phosphorylated portions) of p70S6K. Compared with the respective basal period, amino acid infusion induced a 70% increase (P < 0.002), whereas physiological-dose insulin induced an ~100% increase (P < 0.02) in p70S6K phosphorylation. The percentage increases were not statistically different between these two groups (P = 0.385). However, the net change is much more dramatic in pharmacological-dose insulin-infused subjects, with an average 320% increase over the basal period (P < 0.015 when compared with amino acid and low-insulin groups, ANOVA; Fig. 1).

Effects of Amino Acid or Insulin Infusion on Skeletal Muscle GSK-3 Phosphorylation

For GSK-3, we calculated the ratios of total phosphorylated proteins (phospho-GSK-3{alpha} + phospho-GSK-3{beta}) to total GSK-3{beta} to assess the extent of phosphorylation of this protein. Phosphorylation of both GSK-3 subtypes ({alpha} and {beta} isoforms) corresponds to decreased GSK-3 kinase activity (9). We calculated the GSK-3 phosphorylation as a combined function of both GSK-3{alpha} and GSK-3{beta}, as both isoforms are widely expressed in human skeletal muscles, and the overall GSK-3 activity in vivo reflects the activities of both isoforms. Neither amino acid infusion nor physiological-dose insulin infusion changed the phosphorylation status of GSK-3, whereas pharmacological-dose insulin infusion significantly increased the ratios of GSK-3 ({alpha}+{beta})/total {beta} by 54 ± 17% (P < 0.007; Fig. 2).

Effects of Amino Acid or Insulin Infusion on Skeletal Muscle GS Activity

We quantitated the activity of GS in the active form (GS-I form) and the total GS (GS-D form) activity in biopsied muscle samples obtained before and after amino acid or insulin infusion. The GS-I/GS-D ratios for the basal period were comparable among all three groups (22.6 ± 2.2, 23.4 ± 3.6 vs. 29.5 ± 3.0%, respectively, for the amino acid group, the physiological-dose insulin group, and the dose insulin group, P = 0.228, ANOVA). Amino acid infusion did not significantly increase the percentage of GS in the active form (25.6 ± 2.3%, P > 0.1; Fig. 3A). However, both low- (Fig. 3B) and high-dose insulin (Fig. 3C) infusions significantly increased the ratios of GS-I/GS-D (47.9 ± 3.7%, P = 0.02, and 65.8 ± 3.0%, P < 0.0001, respectively).



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Fig. 3. Effects of AA or insulin infusion on glycogen synthase (GS) activity in human skeletal muscle. AA infusion did not increase the percentage of GS in the active form (A). However, both physiological-dose insulin (Low Insulin) and pharmacological-dose insulin (High Insulin) infusion dramatically activated GS (B and C).

 


    DISCUSSION
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we compared the effects of pharmacological-dose insulin, physiological-dose insulin, and physiological increases of plasma amino acids on p70S6K and GSK-3 phosphorylation and GS activity in human skeletal muscle. We used pharmacological-dose insulin to mimic that used in vitro (2, 30), when actions of insulin and amino acids were compared, and physiological concentrations of insulin to allow more realistic assessment of the physiological significance of changes induced by physiological hyperaminoacidemia (40). Our current results indicate that pharmacological-dose insulin dramatically enhanced the phosphorylation of both p70S6K and GSK-3, activated GS, and increased glucose disposal. Physiological-dose insulin or amino acids likewise induced significant increases in the phosphorylation of p70S6K (albeit less marked than pharmacological-dose insulin) but no concurrent hyperphosphorylation of GSK-3. However, physiological-dose insulin significantly activated GS and glucose disposal, whereas amino acids had no significant impact on GS, glucose disposal, or forearm arteriovenous glucose balance (data not shown). Because the extent of enhanced p70S6K phosphorylation was comparable between the amino acid and physiological-dose insulin groups, our findings suggest that the activation of p70S6K provoked by physiological concentrations of amino acids is insufficient to contribute significantly to the regulation of GSK-3 and/or GS in human skeletal muscle.

In muscle, insulin enhances the phosphorylation of Akt via the PI 3-kinase cascade. Activation of Akt not only enhances the phosphorylation of GSK-3 but also activates mTOR, which in turn phosphorylates p70S6K (6, 26, 33, 39). Previous evidence has suggested that p70S6K is also involved in the regulation of GSK-3 activity by modulating its phosphorylation status (35, 36). Our results showed a dramatic increase in the phosphorylation status of p70S6K with concurrent phosphorylation of GSK-3 and activation of GS in subjects who received pharmacological-dose insulin infusion, also suggesting a possible role of p70S6K in the regulation of these two proteins by insulin in human skeletal muscle. Indeed, insulin has been shown to regulate GSK-3 and/or GS in vitro partially through the rapamycin-sensitive pathway (12, 15, 32, 34). However, caution is needed in interpreting the physiological significance of this finding, inasmuch as all previous studies have been performed using cell culture systems and pharmacological insulin concentrations. High doses of insulin not only maximally activate the PI 3-kinase pathway but also stimulate the MAPK pathway and PP1 activity (20, 27, 37). All of these modulate the phosphorylation and/or activity of GSK-3 and GS, leaving uncertain the physiological role of rapamycin-dependent regulation of GSK-3/GS in vivo in human skeletal muscle.

We and others (1, 11, 13, 16, 23, 41) have shown that amino acids activate p70S6K through an Akt-independent and rapamycin-dependent pathway. This action of amino acids is more specific than that of insulin. These findings make amino acids an attractive option for examining the role of p70S6K phosphorylation in the physiological regulation of GSK-3 and GS in human skeletal muscle. In the current study, we observed a moderate increase in the ratios of p70S6K ({beta}+{gamma}) to the total and no enhancement in GSK-3 phosphorylation after amino acid infusion or physiological-dose insulin infusion. However, GS was activated only by physiological-dose insulin infusion, not by amino acid infusion. Pharmacological-dose insulin increased both GSK-3 phosphorylation and GS activity. The explanation for these differences is likely multifaceted and would include the following observations. 1) The extent of p70S6K phosphorylation is much higher with pharmacological-dose insulin than with amino acids or physiological-dose insulin, and overstimulated p70S6K may be able to regulate GSK-3 and/or GS. 2) We have observed in our laboratory that pharmacological-dose insulin stimulates Akt phosphorylation much more potently than physiological-dose insulin, whereas amino acids have no effect, and Akt directly phosphorylates GSK-3. 3) Insulin stimulates the MAPK pathway, which can also regulate GSK-3 phosphorylation/activity. 4) Insulin activates PP1, which dephosphorylates and activates GS. As previously noted, there is no evidence that amino acids activate either Akt or the MAPK pathway.

Consistent with a number of studies showing that physiological hyperinsulinemia activates GS in human muscle (3, 24, 45), physiological hyperinsulinemia significantly activated GS in the current study. Although we did not observe significant change in GSK-3 phosphorylation after the physiological hyperinsulinemic clamp, it is possible that insulin may have transiently increased GSK-3 phosphorylation and inactivated GSK-3 kinase activity. Studies using a 50% higher insulin infusion rate (1.5 mU·kg-1·min-1) resulted in significant increases in GSK-3 phosphorylation and decreases in its kinase activity (43). As both amino acids and physiological-dose insulin induced similar increment in p70S6K phosphorylation and amino acids failed to activate GS, it appears that moderate enhancement in p70S6K phosphorylation is insufficient to regulate GSK-3 phosphorylation, and/or that the GS activity and insulin at physiological concentrations must have activated GS via p70S6K-independent mechanisms.

It is of interest that incubating either cultured human muscle cells or L-6 muscle cells with amino acids increases p70S6K phosphorylation (2) or activity (30) nearly to the extent seen with pharmacological doses of insulin. It may be important to note that, in these in vitro studies, cells were deprived of amino acids for several hours before being incubated with amino acids at concentrations ranging from 2 to 12 mM. In isolated cell systems, amino acid deficiency results in reversible inactivation of p70S6K, whereas restoration of amino acids promptly restores the activity toward normal (13, 41). Leucine deprivation alone was also associated with decreased phosphorylation of p70S6K in cultured L6 myoblasts (17). Clearly, in humans there is no circumstance when amino acid concentrations fall to negligible in the extracellular fluid, as in those in vitro studies. In our study, the plasma total amino acid concentrations after overnight fast averaged 2.3-2.6 mM. It is therefore likely that, under normal physiological conditions, the p70S6K receives some tonic baseline stimulation by amino acids. The amino acid infusion rate we used in the current study resulted in an average of 57% increase in the plasma amino acid concentrations, from 2.61 ± 0.77 to 4.1 ± 1.56 mM, after 6 h, a difference of only ~1.5 mM. Our current results underscore that changes in amino acid concentrations within the physiological range are sufficient to trigger an effect on the phosphorylation of p70S6K, but that this extent of p70S6K activation is not sufficient to inactivate GSK-3 and activate GS in human skeletal muscle. Also, when results from studies using cultured muscle cells are compared with those from human in vivo studies, caution is needed, because the cultured cells in the artificial culture milieu may not respond to anabolic stimuli in the same way as the in vivo muscle cells do.

When cultured, amino acid-deprived human muscle or L6 muscle cells are suddenly exposed to high concentrations of amino acids, and GSK-3 is transiently inactivated within the first 2-10 min (2, 30). Given that our amino acid infusion lasted for 6 h, we cannot exclude the possibility that amino acid infusion transiently increased the phosphorylation of GSK-3 and activated GS in our study subjects soon after we started amino acid infusion. However, this appears unlikely in the current study, as GS activity was not changed, blood glucose remained stable throughout, and no exogenous glucose infusion was required in the amino acid group. Moreover, even though low-dose insulin and amino acids had similar effects on p70S6K, only insulin persistently increased glucose disposal. With the infusion rate we employed, we estimate that it takes nearly 5 h to raise extracellular amino acid concentration by 1.5 mM. Within the first 2-10 min, the extracellular amino acid concentrations may have increased by only 3-15 µM, which is unlikely to have significant impact on the GSK-3 and GS phosphorylation/activity.

In conclusion, our findings suggest that insulin may regulate GSK-3 and GS through both p70S6K-dependent and -independent pathways. However, the phosphorylation/activation of p70S6K alone, as occurs with physiological increments of plasma amino acid concentrations, does not contribute a significant signaling role to the regulation of GSK-3 and GS in vivo in human skeletal muscle.


    GRANTS
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 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institutes of Health Grants RR-15540 (to Z. Liu), DK-38578 and DK-54058 (to E. J. Barrett), and RR-0847 to the University of Virginia General Clinical Research Center.


    ACKNOWLEDGMENTS
 
We thank Liping Wei for excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Liu, Division of Endocrinology and Metabolism, Dept. of Internal Medicine, Univ. of Virginia Health System, PO Box 801410, Charlottesville, VA 22908-1410 (E-mail zl3e{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. Section 1734 solely to indicate this fact.


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 ABSTRACT
 SUBJECTS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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