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Amino acids and leucine allow insulin activation of the PKB/mTOR pathway in normal adipocytes treated with wortmannin and in adipocytes from db/db mice

CHARLOTTE HINAULT^*, ISABELLE MOTHE-SATNEY^*,1, NADINE GAUTIER^*, JOHN C. LAWRENCE, JR and EMMANUEL VAN OBBERGHEN^*

^* INSERM, Unité 145, Institut Fédératif de Recherche, Nice, Cédex 02, France; and
Departments of Pharmacology and Medicine, University of Virginia Health System, Charlottesville, Virginia, USA.

¹ Correspondence: INSERM, Unité 145, Institut Fédératif de Recherche (IFR 50), 06107 Nice, Cédex 02, France. E-mail: satney{at}unice.fr

SPECIFIC AIMS

Amino acids are nutrients involved in mammalian target of rapamycin (mTOR) regulation and mainly known to potentiate insulin-stimulated protein synthesis in mammalian cells. The aim of the present study was to define in rat adipocytes the effect of amino acids on insulin-stimulated mTOR pathway and on insulin-mediated metabolic responses.

PRINCIPAL FINDINGS

1. Amino acids allow insulin to activate PKB/mTOR when PI 3-kinase is inhibited by wortmannin
We first investigated mTOR phosphorylation on serine 2448 residue, a PKB consensus phosphorylation site. Studies have shown that PKB can phosphorylate mTOR on this residue in vitro, thus PKB may be involved in mTOR activation by insulin in cells. However, the precise role of this serine 2448 phosphorylation is still unknown. We show here that in freshly isolated rat adipocytes, insulin and amino acids stimulate mTOR phosphorylation on serine 2448 via two independent pathways. Moreover, our data suggest that mTOR serine 2448 phosphorylation does not modulate its kinase activity. In parallel, we analyzed amino acids effects on PKB and PI 3-kinase, since both are localized upstream of mTOR in insulin signaling. We observe that amino acids are unable to activate PKB and do not significantly modulate insulin’s stimulatory action. As expected, wortmannin, a pharmacological PI 3-kinase inhibitor, blocks insulin-stimulated serine 473 and threonine 308 phosphorylation of PKB, as well as its activity. Surprisingly, this inhibition is reversed by amino acids. They also allow mTOR, p70S6k, and PHAS-I phosphorylation to be recovered in wortmannin-treated adipocytes. Concerning PI 3-kinase, we verified that, in our conditions, its activity is indeed inhibited by wortmannin. We show that amino acids decrease by half insulin-stimulated PI 3-kinase activity, which is correlated with IRS-1 hyperphosphorylation on serine/threonine residues. Thus, the positive effect of amino acids on PKB activity and on the mTOR pathway is independent of PI 3-kinase. Moreover, this newly identified pathway requires both insulin and amino acids to be activated.

2. Amino acids partially rescue glucose transport and metabolism when insulin signaling is inhibited
We next studied the effects of amino acids on glucose transport and lipogenesis directly related to glucose metabolism (Fig. 1 ). We show that amino acids either alone or with rapamycin, a specific mTOR inhibitor, have no effect on insulin-stimulated glucose transport in freshly isolated rat adipocytes. However, rapamycin completely inhibits p70S6k phosphorylation induced by insulin and amino acids showing that mTOR signaling is blocked. Insulin-induced glucose transport is totally inhibited by wortmannin, but this inhibition is decreased by half in the presence of amino acids. The partial rescue of glucose transport is correlated with PKB and p70S6k phosphorylation. Moreover, we show that amino acids inhibit by 20% insulin-stimulated lipogenesis. This inhibitory effect is not prevented by treatment with rapamycin, suggesting it is not mediated through the mTOR pathway. Despite wortmannin inhibition, amino acids allow insulin to stimulate lipogenesis. In summary, we found that under conditions in which PI 3-kinase is inhibited, amino acids restore the insulin pathway at the level of PKB and partially rescue glucose transport and metabolism.

View larger version (32K): [in this window] [in a new window]   Figure 1. Effect of amino acids on insulin-stimulated glucose transport and lipogenesis. Adipocytes were treated with or without vehicle (0.005% Me2SO), rapamycin (50 nM), or wortmannin (100 nM). After 30 min, adipocyte suspensions were incubated with or without insulin (100 nM) or/and amino acids (1x) for 80 min. A) Glucose transport was assessed by the uptake of 2-deoxy-[3H]glucose. 3H incorporated in DOG was counted and expressed as a % of 2-DOG uptake in the presence of insulin. Means ± SE from 3 individual experiments, each performed in triplicate, are shown. *P < 0.025 vs. wortmannin and insulin. B) In parallel, cell lysates were separated by SDS-PAGE and immunoblotted with phosphoPKB (Ser 473) and p70S6k antibodies. Representative immunoblots of 3 individual experiments are presented. C) Lipogenesis was measured by the incorporation of [2-3H]glucose into lipids. 3H incorporated in lipids was counted and expressed as a % of lipogenesis stimulated by insulin. Means ± SE from 4 individual experiments, each performed in triplicate, are shown. *P < 0.01 vs. insulin. **P < 0.005 vs. wortmannin and insulin.

3. Leucine is sufficient to partially prevent inhibition of insulin signaling by wortmannin
Since we showed that amino acids mixture prevents PI 3-kinase blockade by wortmannin, we determined which amino acid could be involved in this unexpected effect. We tested different amino acids such as leucine, lysine and cysteine. Leucine is able to permit insulin to phosphorylate PKB on serine 473 and threonine 308 despite the presence of wortmannin. This effect is observed at 4 mM as well as at a 10-fold lower concentration, which corresponds to the leucine concentration in the amino acids mixture. Moreover, in these conditions, leucine partially restores insulin-induced glucose transport. In contrast, even at high concentrations lysine or cysteine is unable to prevent wortmannin inhibition. We also show that rapamycin does not modulate the stimulation of leucine or amino acids on PKB when insulin signaling is inhibited, indicating that this effect is not mediated via the mTOR pathway. To conclude, leucine appears to be sufficient to potentiate insulin action when PI 3-kinase is blocked by wortmannin in freshly isolated adipocytes.

4. Leucine improves insulin-induced PKB phosphorylation in db/db mice
Finally, we wondered whether the effect of amino acids—notably leucine—occurs in the setting of a physiopathologic impairment of insulin signaling. To investigate the possible involvement of amino acids in insulin resistance associated with obesity and type 2 diabetes, we analyzed their effect on insulin-stimulated PKB phosphorylation in adipose tissue of db/db mice (Fig. 2 ). Insulin stimulates PKB phosphorylation in db/+ and db/db adipose tissue explants, but insulin sensitivity is slightly reduced in db/db mice (Fig. 2 , lanes 3, 4). Amino acids, either alone or in the presence of insulin, have no significant effect on PKB phosphorylation in db/+ and db/db adipose tissue explants (Fig. 2 , lanes 5–8). Leucine alone does not or only weakly phosphorylates PKB in both strains (Fig. 2 , lanes 9, 10). Leucine seems to potentiate insulin-stimulated PKB phosphorylation in db/db and db/+ adipose tissue explants (Fig. 2 : compare lanes 3, 11 and lanes 4, 12). However, quantification of several experiments shows that only the increase observed for db/db adipose tissue explants is significant (1.7-fold; Fig. 2 , histogram). Thus, leucine alone is able to improve insulin sensitivity in the genetic db/db insulin-resistant mouse model.

View larger version (33K): [in this window] [in a new window]   Figure 2. Leucine improves insulin-induced PKB phosphorylation in adipose tissue explants of db/db mice. Adipose tissue explants from male db/+ or db/db mice at 8–10 wk of age were incubated with or without insulin or/and amino acids mixture or/and leucine (4 mM) for 80 min, rinsed twice with ice-cold PBS, and lysed. Samples were separated by SDS-PAGE and immunoblotted with phosphoPKB (Ser 473) antibody, immunoblots were then stripped and reprobed with PKB antibody. The results of phosphoPKB bound were quantified from 5 individual experiments of which 1 representative immunoblot is shown. The values were corrected for differences in the total amount of PKB recovered and expressed as a % of basal in db/+ or db/db mice. **P < 0.01 vs. insulin of db/db.

CONCLUSIONS

During recent years the involvement of amino acids in the regulation of cell function in mammals has received considerable attention. Amino acids are now well known to improve insulin stimulation of protein synthesis through the mTOR pathway. Our study brings new insights into the role of amino acids, notably for leucine, in other insulin actions in adipocytes. First, we show that amino acids and insulin have an additive effect on mTOR serine 2448 phosphorylation, a PKB consensus phosphorylation site. Since mTOR phosphorylation on this residue does not regulate its kinase activity in adipocytes, additional studies are needed to establish the role of serine 2448 phosphorylation in mTOR function. In freshly isolated adipocytes, we do not observe any modification by amino acids of insulin-stimulated glucose transport. These results, apparently in contradiction with results obtained in 3T3-L1 adipocytes, can be explained by the difference in the cellular models used. Indeed, it has been proposed that the mechanism of action of amino acids is different in freshly isolated adipocytes in comparison with cell lines. Nevertheless, amino acids negatively regulate insulin-induced lipogenesis. Rapamycin does not prevent this inhibition of amino acids, suggesting it is not mediated through mTOR signaling. The most remarkable finding of our study is the demonstration that amino acids, notably leucine, allow insulin to activate PKB when PI 3-kinase is inhibited by wortmannin (Fig. 3 ). mTOR is activated and p70S6k and PHAS-I are phosphorylated. Moreover, glucose transport and lipogenesis are partially restored. In most situations of insulin resistance, impaired activation of insulin-signaling molecules has been reported, including the PI 3-kinase/PKB module. Thus, we looked at the effect of amino acids and leucine on PKB phosphorylation in the insulin-resistant db/db mouse model. We show that leucine, but not amino acids mixture, is sufficient to potentiate insulin-stimulated PKB phosphorylation in adipose tissue of these mice vs. their control littermates.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram of potential role of amino acids contribution in insulin signaling.

This newly identified effect of amino acids, that is likely pre-existing in situations with normal insulin response, but occurring at an imperceptible level, would become necessary when the naturally predominant insulin signaling pathway is impaired. Further investigations are needed to determine the precise molecular mechanism of this rescue pathway and to define the actual importance of leucine and amino acids in insulin action by using other models of insulin resistance.

Taken as a whole, our data shed new light on the action of amino acids in adipocyte insulin signaling, and disclose, especially for leucine, an intriguing role in disease situations associated with decreased insulin action. Leucine, a "physiological" molecule, may turn out to be a propitious agent for treatment and/or prevention of insulin resistance in humans.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1409fje;

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