In the industrialized societies, clinical events, directly or indirectly consequence of the atherosclerotic processes, represent the principal cause of death and disability.1
Diabetes and related clinical conditions predispose to a fast progression of atherosclerosis. The importance of insulin-related metabolic pathways on vascular physiology is confirmed by the presence of vascular alterations in insulin resistant conditions.2,3 At the vascular level, insulin controls vascular tone, cell cycle progression, gene expression, and angiogenesis.4 Dysfunction of the insulin transduction pathway is a feature of the insulin resistant conditions. However, not all the intracellular steps involved in insulin resistance are known: insulin receptor substrate, IRS-1/2, and phosphotidylinositol 3-kinase (PI-3K) are the privileged sites of insulin resistance. Recent data indicate that also mammalian target of rapamycin (mTor) can modulate signal transduction by insulin.5,6
Amino acids control protein synthesis and other cellular functions; they are also known to modulate insulin activities, but the molecular steps involved in their actions are not completely known.7
Activation of p70/S6-K (S6-K) and PHAS-18 is considered essential for the progression of protein synthesis.9 In our previous work, we demonstrated that both insulin and amino acids activate these kinases in human endothelial cells.10
PI-3K/Akt and mitogen activated kinase (MEK)/extracellular signal-regulated protein kinase (ERK) 1/2 are two intracellular pathways involved in insulin signal transduction. They can activate mTor, which in turn activates p70/S6-K and PHAS-1. These pathways are important in the modulation of protein synthesis and of other cellular processes as cell apoptosis, cell cycle progression, vascular tone, and angiogenesis.11
In this work, we investigate in human endothelial cells the intracellular pathways that lead to insulin-induced and amino acid-induced S6-K activations.
We demonstrate that amino acids synergize with insulin in the activation of the kinases that lie downstream mTor as S6-K, whereas they inhibit the upstream kinases, Akt and ERK1/2, when activated by insulin. We demonstrate that these inhibitory effects of amino acids are mediated by mTor.
Isolation, Culture, and Stimulation of Endothelial Cells
Human umbilical vein endothelial cells (HUVEC) were isolated according to established procedures12 and were cultured under standard conditions in medium M-199 containing 15% fetal calf serum (Sigma-Aldrich, Milan, Italy), heparin (15 U/mL)(Sigma), and endothelial cell growth factor (20 μg/mL) (Boerhinger Mannheim, Mannheim, Germany) and used within the fifth passage.
After 1 day of confluence, HUVEC were starved by an overnight incubation in medium containing 0.5% of insulin-free bovine serum albumin (Sigma). Cells were then washed with phosphate-buffered saline pH 7.4 and kept in medium without amino acids for a further 1 hour. In the experimental conditions in which kinase inhibitors were used, they were added 30 minutes before the addition of the agonists. The inhibitors used were rapamycin 25 ng/mL (Calbiochem, La Jolla, CA), wortmannin 100 nM (Calbiochem), LY294002 50 μM (Calbiochem), PD98059 40 μM (Calbiochem), UO126 5 μM (Calbiochem), U7 10 μM (Calbiochem), and U89 30 μM (Calbiochem). Cells were then stimulated with insulin 100 ng/mL (Sigma) and/or with the following mixture of amino acids: L-arginin 100 μM, L-glutamin 350 μM, L-istidin 60 μM, L-lysine 300 μM, L-methionine 40 μM, L-phenilalanine 50 μM, L-threonine 180 μM, L-triptophane 70 μM, L-tyrosine, 75 μM, L-alanine 300 μM, L-asparagine 60 μM, L-aspartic acid 30 μM, L-cysteine 60 μM, L-glutammic acid 100 μM, L-prolin 100 μM, L-serine 200 μM, and the branched amino acids L-isoleucine 100 μM, L-leucine 250 μM, and L-valine 180 μM. When insulin was used alone, it was added to a medium without amino acids. After adequate time of incubation, stimulated cells were washed with phosphate-buffered saline and immediately lysed with Laemmly buffer at room temperature. The samples were collected, boiled at 95°C for 5 minutes, and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Samples were run on 12% SDS-PAGE and electro transferred to a nitrocellulose membrane. Membranes were blocked for 4 hours at 4°C in 5% dry milk Tris buffer saline. Blots were then washed twice with Tris buffer saline and incubated overnight at 4°C with the primary antibody (antiphospho-Akt (Ser-473) (1:1000, New England Biolab, Inc, Beverly, MA), anti-Akt (1:1000, New England Biolab), anti-ERK and antiphospho-ERK (1:1000, Santa Cruz Biotec. Inc, CA), anti-S6-K (1:1000, Santa Cruz), anti-PHAS-1 (1:1000, Santa Cruz), antiphospho-endothelial nitric oxide synthase (eNOS), and anti-eNOS (1:1000, Santa Cruz).
Blots were then washed 4 times, incubated with horseradish peroxidase-conjugate secondary antibody for 2 hours at room temperature, washed again, and finally incubated with the enhanced chemiluminescence system (ECL, Dupont, Boston, MA).
Statistical analysis was performed with the Mann-Whitney and Wilcoxon tests. The results were considered significant when P<0.05.
Amino acids activate protein synthesis and also other intracellular steps in several cellular systems.8 However, their role in the modulation of these activities at the level of endothelial cells is not completely known.
In eukariotic cells, S6-K and PHAS-1 play a key role in the progression of protein synthesis.9 In our previous work, we demonstrated that insulin and amino acids activate both these protein kinases and that branched chain amino acids are necessary but not sufficient to stimulate such activations.10
As found in our previous work,10 the effect of amino acid mixture is already maximal at 3 mM. Therefore, this concentration has been used in all the experiments described in this work. The aim of the present work was to investigate the intracellular pathways that lead to S6-K activation after insulin and/or amino acid stimulation in human endothelial cells. In particular, as the activation of S6-K is controlled by the upstream kinase mTor, we focused our investigation on 2 principal pathways that activate mTor as PI-3K/Akt and MEK/ERK1/2.
P70/S6-K Activation by Insulin and Amino Acids
The activity of S6-K is mainly regulated by a site-specific phosphorylation.13 Such phosphorylation induces an increase in its molecular weight that, in a gel mobility shift assay, induces a band shift of the molecule. We have, therefore, used this assay to evaluate S6-K activation. In a preliminary work, we confirmed that the shifted bands correspond to the activated/phosphorylated form of S6-K (data not shown).
As described in our previous work,10 in HUVEC, insulin increases S6-K phosphorylation after 5 minutes of stimulation, with a rapid decrement thereafter. In contrast, amino acids increase S6-K phosphorylation after 5 to 10 minutes to persist up to 60 minutes. The stimulation with insulin and amino acids further increases S6-K phosphorylation at all the time-points analyzed (Fig. 1A).
FRAP/mTor is the molecule that controls the activity of S6-K and PHAS-1. Accordingly, the inhibition of mTor by rapamycin leads to a complete suppression of both insulin-induced and amino acid-induced S6-K phosphorylations (Fig. 1B).
Several intracellular pathways can activate mTor. Among these, PI-3K/phosphophosphoinositide-dependent protein kinases (PDK)1/2/Akt, MEK/ERK1/2, and protein kinase C (PKC) play an important role.14 We have, therefore, evaluated their involvement by using specific inhibitors.
LY294002, a PI-3K inhibitor, totally revert both insulin-induced and amino acid-induced S6-K activations, whereas PD98059, an MEK inhibitor, reduces only insulin-induced but not amino acid-induced S6-K activation (Fig. 1B). The use of the PKC inhibitors H7 and H89 does not modify the stimulation of S6-K induced by insulin and/or amino acids (data not shown).
Similar results have been obtained when PHAS-1 activation was analyzed (data not shown).
ERK1/2 Activation by Insulin and Amino Acids
Extracellular signals activate the mitogen-activated protein kinases (MAPKs) through the receptor-coupled activation of the Ras/raf/MEK pathway.15 This finally activates MAPK through a site-specific phosphorylation. We evaluated the activation of ERK1/2 by analyzing in western blotting the levels of their phosphorylated/activated form.
In HUVEC, insulin activates ERK1/2 already at 5 minutes and increases the activation thereafter (Fig. 2A). This activation is MEK-dependent as it is totally inhibited by PD98059 and UO126. LY294002 modestly reduces such activation (Fig. 2B), whereas the use of SB203580 (p38 inhibitor), H7, and H89 (PKC inhibitors) does not modify ERK1/2 activation (data not shown).
Amino acids alone do not activate ERK1/2 at all the time-points observed (Fig. 3A). When associated with insulin, amino acids inhibit insulin-induced ERK1/2 activation. This inhibition is reverted by LY294002 and by rapamycin (Fig. 3B).
Akt Activation Induced by Insulin and Amino Acids
Akt activity is regulated by the binding of its plecstrin homology domain to the membrane-bound lipid products of PI-3K and by its phosphorylation at Thr-308 and Ser-473 residues by 2 phosphoinositide-dependent protein kinases, PDK1 and PDK2.16
It is known that insulin activates Akt in endothelial cell and that through this kinase it controls several cellular functions as angiogenesis and vascular tone.17 However, the actions of amino acids on Akt activity have not been fully evaluated in human endothelial cells.
Akt activation has been evaluated in western blot by using a specific antibody recognizing the phosphorylated/activated form of Akt (phospho-Ser-473). As expected, also in our experimental condition, insulin activates Akt already at 5 minutes to further increase at 15-minute and 30-minute time-points analysis (Fig. 4A). Differently, amino acids do not modify Akt phosphorylation, and when they are associated with insulin, they inhibit insulin-induced Akt phosphorylation (Fig. 4B).
Insulin-induced Akt phosphorylation is inhibited by LY294002 and by wortmannin but is not modified by rapamycin, H7, and H89. A modest reduction is observed with the MEK inhibitor PD98059 (data not shown).
The inhibition of the insulin-induced Akt phosphorylation by amino acids is reverted by the mTor inhibitor rapamycin (Fig. 4B).
To assess whether the activities of other molecules that lie downstream Akt are inhibited by amino acids, we evaluated the level of eNOS phosphorylation (in Ser-1177). Such phosphorylation is regulated by Akt.18 In our experimental conditions, both insulin and amino acids increase eNOS phosphorylation (Fig. 5).
In our previous work, we have demonstrated that in human endothelial cells, both insulin and/or amino acids activate the mTor downstream kinase S6-K. We have shown that this activation presents a different time course according to the stimulus used; insulin induces an early and short activation of S6-K, whereas amino acids induce a greater and prolonged S6-K activation. The association of the 2 stimuli further augmented the effect of insulin or amino acids alone.
Many steps of the insulin transduction pathway are known, as well as the actions of insulin and amino acids in different cell types. Indeed, both in vivo and in vitro, insulin synergizes with amino acids in the control of protein synthesis through the activation of 2 related kinases, S6-K and PHAS-1. Our previous work confirms these actions also in human endothelial cells.14,19–23
In the present work, we demonstrate that insulin activates S6-K in an mTor-dependent and PI-3K-dependent way. We also demonstrate that the MAPKs are partially involved. Accordingly, insulin activates both Akt and ERK1/2. The transduction pathways used by amino acids in the activation of protein synthesis are not completely known. Some data suggested the existence of an intracellular amino acid sensor;19 however, the specific intracellular receptor has not been identified. A role of the tRNA aminoacylation has been demonstrated in the process that leads to S6-K activation.24 Several reports indicated in mTOR the intracellular molecule activated by amino acids, which in turn is responsible of the activation of the downstream kinases S6-K and PHAS-1. Accordingly, in our work amino acids activate S6-K in an mTor-dependent way.
Several intracellular pathways, as PI-3K/PDK/Akt, ERK1/2, and PKC, can activate mTor through its phosphorylation in Ser-2448.14,15,25,26 Our data indicate that (a) the phosphorylation of S6-K induced by insulin and amino acids is totally mediated by mTor, (b) PI-3K and MEK are involved in insulin stimulation, whereas (c) PI-3K but not MEK is involved in S6-K activation induced by amino acids.
However, the possible involvement of other kinases in the activation of S6-K induced by amino acids is suggested by the different degrees of inhibition observed with LY294002 and wortmannin. Indeed, although these 2 inhibitors equally inhibit insulin-induced S6-K activation, only LY294002 suppresses such activation induced by amino acids, whereas wortmannin exerts only a lower inhibitory effect. Wortmannin is a nonspecific PI-3K inhibitor, and inhibits other kinases as phospholipase A2, PI-4K, and MLCK.27,28 These data support the hypothesis that other kinases can be activated by amino acids with respect to insulin.
The activity of Akt is regulated by the binding of its plecstrin homology domain to the membrane-bound lipid products of PI-3K and by its phosphorylation at Thr-308 and Ser-473 residues by 2 phosphoinositide-dependent protein kinases, PDK1 and PDK2.16,29–31 Accordingly, in our work insulin activates Akt by a PI-3K-dependent way. Differently, amino acids phosphorylate S6-K in a PI-3K-dependent manner without the activation the interposed Akt. This suggests that PI-3K activates mTor/S6-K directly or indirectly by a nonidentified pathway, or that, as suggested by other authors,19 amino acids directly stimulate mTor. In this case, yet, it must be explained the involvement of PI-3K in S6-K activation.
Our data are in agreement with several reports that demonstrate the ability of amino acids to activate S6-K/PHAS-1 in a PI-3K/mTor-dependent pathway, and with authors who have observed an inhibition of Akt by amino acids in different cellular systems.19
The fact that Akt plays a central role in the modulation of several cellular processes as angiogenesis and vascular tone16 rises the question whether the inhibitory activity exerted by amino acids on Akt might also interfere with vascular functions. However, in our experimental conditions, Akt has been evaluated only for 30 minutes and therefore our data are not exhaustive of all the Akt activities. Moreover, a downstream molecule of Akt, eNOS, is not inhibited by amino acids. The fact that pathways other than Akt (ie, calcium/calmodulin-dependent) can induce the phosphorylation of eNOS18 suggests that their involvement in eNOS activation and that the activation is not inhibited by amino acids.
The observation that rapamycin reverts the inhibitions exerted by amino acids on insulin-induced Akt and ERK activation identifies in mTor the molecule responsible for this effect.
We also showed an activation of Akt after 30 minutes of insulin stimulation, whereas the downstream kinase S6-K is activated only for 5 minutes. These data suggest the existence of an inhibitory mechanism blocking the insulin signal at the level of S6-K.
Inside the insulin transduction pathway, several negative feedbacks have been described.32 Among these, a recently individuated mechanism has in mTor a possible candidate.21 In skeletal muscle cells, amino acids inhibit insulin signaling via the mTor/p70/S6-K, through an increased serine/threonine phosphorylation of IRS-1. This in turn inhibits PI-3K and Akt. However, in our experimental conditions, Akt inhibition was PI-3K independent, suggesting a direct role of mTor on Akt activation.
Therefore, in our experimental conditions, mTor represents an important target of amino acids. In fact, it is involved in S6-K phosphorylation and inhibits the phosphorylation of Akt and ERK1/2.
It is known that insulin stimulates ras/raf/MEK/ERK1/2 in an insulin-receptor-dependent and a PI-3K-independent pathway.15 This effect has been demonstrated in several cell types and also in HUVEC. In our experimental condition, we confirm these previous data and confirm that this activation is totally MEK dependent. The fact that the inhibition of PI-3K by LY294002 and wortmannin induces only a small reduction of ERK1/2 phosphorylation confirms that in the activation of ERK1/2 the signal bifurcates at the level of IRS. In contrast, amino acids do not activate ERK1/2 and inhibit insulin-induced ERK1/2 activation, by a PI-3K-dependent and mTor-dependent mechanism.
A fine cross talk exists between insulin transduction pathway and MAPK.33 In particular, a double way of interaction is present. Insulin can induce an early Ras-dependent ERK1/2 stimulation and a later Akt/raf-dependent ERK1/2 inhibition. The final result of the interaction between these 2 pathways is different according to cell type, cell cycle, and agonist used. In our experimental conditions we do not observe a later ERK1/2 inhibition by insulin. On the other hand, the observed inhibition exerted by amino acids on ERK1/2 pathway cannot be explained by this mechanism because Akt is not activated by amino acids.
The protein kinases belonging to the PKC family can exert important and different effects on insulin transduction pathway.34–36 In our work, we have not observed a significant involvement of PKC on both insulin-dependent and/or amino acid-dependent S6-K activation(s).
In conclusion, we demonstrate that amino acids cooperate with insulin in the stimulation of the protein kinase that lies downstream mTor as S6-K, but they inhibit the insulin-induced activation of the kinases that lie upstream mTor as Akt and ERK1/2.
We show the existence of a negative feedback in the insulin transduction pathway at the level of S6-K, which is removed by amino acids.
Our data confirm in endothelial cells the central role played by mTor in amino acid action, and suggest the existence of mTor-dependent mechanisms by which amino acids can interfere with insulin action at the level of endothelial cells.
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